Conditions: Adenosine Deaminase Deficiency Severe Combined Immunodeficiencies (SCID)
Keywords: Gene therapy Hematopoietic stem and progenitor cells Lentiviral vector ADA-SCID
Allocation: NON_RANDOMIZED | Intervention model: PARALLEL
Enrollment: 36
Healthy volunteers: no

Brief summary:
This is a historically controlled, non-randomized Phase I/II clinical trial to assess the safety and efficacy of autologous transplantation of CD34+ hematopoietic stem/progenitor cells (HSPCs), obtained from infants affected by ADA-SCID, following transduction of the HSPCs with a lentiviral vector (LV) carrying the human ADA complementary DNA (cDNA) under the control of the elongation factor 1 alpha shortened (EFS) promoter. Subjects treated in the trial receive the infusion of autologous, transduced cells following marrow cytoreduction with busulfan. The outcomes are compared to those observed in a historical control group of patients who received an allogeneic hematopoietic stem cell transplant (HSCT).

This Phase I/II clinical trial will be performed at Great Ormond Street Hospital (GOSH), London, United Kingdom.

Detailed description:

Arms:
arm 1: Infusion of autologous EFS-ADA LV CD34+ cells arm 2: Historical data from ADA-SCID patients who were treated with Hematopoietic Stem Cell Transplantation (HSCT)

Interventions: intervention 1: Infusion of autologous EFS-ADA LV CD34+ cells intervention 2: Haematopoietic Stem Cell Transplantation (HSCT) intervention 3: Busulfan intervention 4: Peg-Ada
Intervention descriptions: intervention 1: Autologous EFS-ADA LV CD34+ cells (OTL-101\*) are infused intravenously intervention 2: Historical data from a database of ADA-SCID patients treated with allogeneic HSCT from GOSH will be collected as comparator group. intervention 3: Busulfan is used for non-myeloablative conditioning intervention 4: Peg-Ada enzyme replacement therapy is discontinued at Day +3- (-3/+15 days) after successful engraftment

Protocol document:
Prot_000.pdf: 
COVER PAGE
Official Study Title: Phase I/II, Historical 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
10-MI-29 Protocol v17.0. dated 28-Jun-2017
NCT01380990


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610 
Protocol Version 17.0, dated 28 June 2017
Full title of trial 
Phase I/II, historical 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  
Short title 
LV Gene therapy for ADA deficiency 
Version and date of protocol 
Version 17.0, 28 June 2017 
Sponsor: 
Great Ormond Street Hospital for Children NHS 
Foundation Trust 
Sponsor protocol number 
10-MI-29
Funder (s) : 
Department of Health, 
Internal funder 
EudraCT no 
2010-024253-36 
Active IMP(s): 
EF1αS-ADA lentiviral vector transduced patient 
CD34+ cells 
Placebo IMP(s): 
N/A 
Phase of trial 
Phase I/II 
Trial sites(s) 
Great Ormond Street Hospital for Children NHS 
Foundation Trust (single-site) 


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Table of contents 
 
 
2 
SUMMARY .................................................................................................................. 16 
3 
INTRODUCTION AND BACKGROUND ...................................................................... 21 
3.1  
Adenosine deaminase deficient – severe combined immunodeficiency (ADA-SCID) ..... 21 
3.1.1 
Adenosine deaminase deficiency ...................................................................................... 21 
3.1.2 
ADA gene: structure and function ..................................................................................... 22 
3.1.3 
Mutations in ADA-SCID patients ....................................................................................... 23 
3.1.4 
The pattern of clinical disease ........................................................................................... 24 
3.1.4.1 
Classical presentations ................................................................................................. 24 
3.1.4.2 
Non-immunological manifestations in ADA-SCID ......................................................... 25 
3.1.4.3 
Delayed/late onset disease ........................................................................................... 25 
3.1.4.4 
Partial ADA deficiency ................................................................................................... 26 
3.1.4.5 
Reversion mutations in ADA-SCID ............................................................................... 26 
3.1.5 
Diagnosis of ADA deficiency ............................................................................................. 26 
3.1.6 
Prognosis .......................................................................................................................... 27 
3.2 
Management options for ADA-SCID ...................................................................................... 27 
3.2.1 
Haematopoietic stem cell transplantation for ADA-SCID .................................................. 27 
3.2.2 
ADA enzyme replacement therapy ................................................................................... 29 
3.2.2.1 PEG-ADA treatment and long-term outcome.................................................................... 32 
3.2.2.2 PEG-ADA and neurological outcome ................................................................................ 34 
3.2.3 
Alternative therapy for ADA-SCID based on somatic gene transfer ................................. 35 
3.2.4 
Clinical trials of gene therapy for ADA-SCID .................................................................... 36 
3.2.4.1 T lymphocyte gene therapy ............................................................................................... 36 
3.2.4.2 Peripheral blood lymphocyte and T cell depleted bone marrow gene therapy ................. 36 
3.2.4.3 CD34+ stem cell gene therapy.......................................................................................... 37 
3.2.4.4 PEG-ADA withdrawal leads to proliferation of gene transduced cells .............................. 38 
3.2.5 
ADA-SCID gene therapy without PEG-ADA therapy and with the use of a mild 
conditioning regime ........................................................................................................................... 39 
3.2.6 
Safety of ADA-SCID gene therapy using a gammaretroviral vector ................................. 41 
3.3 
Lentiviral vectors for gene therapy of ADA SCID ................................................................ 43 
3.3.1 
Lentiviral vectors ............................................................................................................... 43 
3.3.2 
Rationale for using a lentiviral vector for gene delivery .................................................... 44 
3.3.3 
The EF1αS-ADA lentiviral vector ...................................................................................... 44 
3.4 
Pre-clinical data ....................................................................................................................... 45 
3.4.1 
Transduction and expression of ADA in cell lines ............................................................. 45 


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3.4.2 
Transduction and expression of ADA in CD34+ cells ....................................................... 47 
3.4.3 
Transduction of CD34+ cells and expression of ADA in vivo ............................................ 49 
3.4.4 
SIN lentiviral vector promotes immune recovery in a murine model of ADA deficiency ... 50 
3.4.5 
Genotoxicity of the EF1αS-ADA lentiviral vector............................................................... 52 
3.4.5.1 
SIN Lentiviral vector constructs are safer than LTR intact gammaretroviral constructs52 
3.4.5.2 
The EF1αS promoter is safer than the viral LTR .......................................................... 53 
3.4.5.3 
Data from other similar LV constructs ........................................................................... 54 
3.4.5.4 
IVIM assay to test genotoxic potential of the EF1αS-ADA lentiviral vector .................. 54 
3.4.6 
Clinical grade vector is able to effectively transduce human ADA-/- CD34+ stem cells ... 56 
3.5 
Continuation of PEG-ADA for 1 month after infusion of gene modified cells .................. 57 
3.6 
Busulfan dosage ..................................................................................................................... 57 
3.7 
Summary and rationale for use of the EF1αS-ADA lentiviral vector for gene therapy .... 58 
4 
TRIAL DESIGN............................................................................................................ 59 
4.1 
Patients treated with gene therapy (on study) ..................................................................... 59 
4.2 
Trial objectives ........................................................................................................................ 62 
4.2.1 
Primary objectives ............................................................................................................. 62 
4.2.2 
Secondary objectives ........................................................................................................ 63 
4.3 
Study endpoints ...................................................................................................................... 63 
4.3.1 
Primary endpoints ............................................................................................................. 63 
4.3.2 
Secondary endpoints ........................................................................................................ 64 
5  
PATIENT SELECTION AND RECRUITMENT ............................................................. 65 
5.1 Patients treated with gene therapy (on trial) .............................................................................. 65 
5.1.1 
Inclusion criteria ................................................................................................................ 65 
5.1.2 
Exclusion criteria ............................................................................................................... 65 
5.1.3 
Justification for enrolment criteria ..................................................................................... 65 
5.2 Patients treated with gene therapy (off trial): CUP group ......................................................... 66 
5.3 Historical Control group: HSCT group ....................................................................................... 66 
5.4 
Concomitant medication ........................................................................................................ 66 
6 
ENROLMENT OF SUBJECTS .................................................................................... 67 
7 
TREATMENT PROTOCOL .......................................................................................... 68 
7.1 
Informed consent procedure ................................................................................................. 68 
7.2 
Randomisation procedures .................................................................................................... 68 
7.3 
Emergency unblinding ........................................................................................................... 68 
7.4 
Screening assessments ......................................................................................................... 68 
7.5 
Treatment procedures ............................................................................................................ 69 
7.5.1 
Harvest of a back-up autologous graft .............................................................................. 69 


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7.5.2 
CD34+ cells for gene therapy ........................................................................................... 69 
7.5.3 
CD34+ cell purification ...................................................................................................... 70 
7.5.4 
CD34+ cell culture and transduction ................................................................................. 70 
7.5.5 
Testing prior to patient re-infusion ..................................................................................... 70 
7.5.6 
Patient conditioning regimen ............................................................................................. 70 
7.5.7 
Infusion of transduced cells ............................................................................................... 71 
7.5.8 
Discontinuation of PEG-ADA one month after infusion of gene modified cells ................ 71 
8 
EVALUATION AND FOLLOW-UP............................................................................... 72 
8.1 
Assessment of safety and efficacy ....................................................................................... 72 
8.1.1 
Immunological reconstitution ............................................................................................. 72 
8.1.2 
Metabolic correction .......................................................................................................... 72 
8.1.3 
Molecular characterisation of gene transfer ...................................................................... 73 
8.2 
Investigations and monitoring schedule .............................................................................. 73 
8.2.1 
Pre-treatment investigations ............................................................................................. 73 
8.2.2 
Post-infusion monitoring .................................................................................................... 73 
8.2.3 
Safety monitoring .............................................................................................................. 73 
8.2.4. 
Neurological and behavioural monitoring of patients ........................................................ 74 
8.3 
Treatment failure/lack of response ....................................................................................... 74 
8.4 
Withdrawal of individual subjects ......................................................................................... 74 
8.5 
Off study criteria ..................................................................................................................... 75 
8.6 
Follow-up of withdrawn subjects .......................................................................................... 76 
8.7 
Definition of end of trial .......................................................................................................... 76 
8.8 
Trial stopping criteria ............................................................................................................. 76 
8.9 
Statistical analysis .................................................................................................................. 77 
8.9.1 
Analysis populations ......................................................................................................... 77 
8.9.2 
Data analysis/description .................................................................................................. 78 
Safety ..................................................................................................................................................... 78 
Efficacy ................................................................................................................................................... 78 
9 
SAFETY ISSUES RELATED TO THE GENE THERAPY PROTOCOL ....................... 79 
9.1 
Risks ......................................................................................................................................... 79 
9.1.1 
Venepuncture .................................................................................................................... 79 
9.1.2 
Infection ............................................................................................................................. 79 
9.1.3 
Busulfan toxicity ................................................................................................................ 80 
9.1.4 
Bone marrow harvest ........................................................................................................ 80 
9.1.5 
Peripheral blood stem cell harvest .................................................................................... 81 
9.1.6 
CD34+ cell infusion ........................................................................................................... 82 
9.2 
Risks associated with the use of lentiviral vector ............................................................... 82 
9.2.1 
Insertional mutagenesis .................................................................................................... 82 


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9.2.2 
Replication-competent lentivirus (RCL) exposure ............................................................. 83 
9.2.3 
Germ-line transmission of vector sequences .................................................................... 83 
9.2.4 
Allergic/immunological responses to cell processing excipients ....................................... 83 
9.2.5 
Other adverse clinical consequences ............................................................................... 84 
10 
NAME DESCRIPTION OF THE IMP AND CRITICAL RAW MATERIALS ............... 84 
10.1 
Name and description of each IMP ....................................................................................... 84 
10.2 
Production, supply and release of the IMP .......................................................................... 84 
10.3 
IMP accountability ................................................................................................................... 85 
10.4 
Description and justification of the trial treatment and dosage ......................................... 85 
10.5 
Dose modifications ................................................................................................................. 85 
10.6 
Assessment of compliance .................................................................................................... 86 
10.7 
Post-trial IMP arrangements .................................................................................................. 86 
10.8 
Name of each NIMP ................................................................................................................. 86 
11 
RECORDING AND REPORTING OF ADVERSE EVENTS AND REACTIONS ....... 86 
11.1 
Definitions ................................................................................................................................ 86 
11.2 
Recording adverse events ..................................................................................................... 87 
11.3 
Expected disease adverse events ......................................................................................... 89 
11.4 
IMP expected adverse events ................................................................................................ 89 
11.5 
Procedures for recording and reporting Serious Adverse Events .................................... 89 
11.4.1 
Notification of deaths ......................................................................................................... 90 
11.4.2 
Reporting SUSARs............................................................................................................ 90 
11.4.3 
Medically important events considered critical to the safety evaluation of the study ........ 90 
11.4.4 
Long term follow-up ........................................................................................................... 91 
11.4.5 
Annual safety reports ........................................................................................................ 91 
11.4.6 
Annual progress reports .................................................................................................... 91 
11.4.7 
Pregnancy ......................................................................................................................... 91 
11.4.8 
Reporting urgent safety measures .................................................................................... 91 
11.4.9 
Notification of serious breaches to GCP and/or the protocol ............................................ 91 
12 
ADMINISTRATIVE ASPECTS ................................................................................. 92 
12.1 Data handling, record keeping and sample storage ............................................................... 92 
12.1.1 
eCRF completion............................................................................................................... 92 
12.1.2 
Sample storage ................................................................................................................. 92 
12.1.3 
Record retention ................................................................................................................ 92 
12.2 Patient confidentiality ................................................................................................................. 92 
12.3 Monitoring and audit ................................................................................................................... 93 
12.4 Direct access to source data/documents ................................................................................. 93 


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13 
ETHICS AND REGULATORY REQUIREMENTS .................................................... 93 
14 
FINANCE ................................................................................................................. 93 
15 
INSURANCE ............................................................................................................ 94 
16 
PUBLICATION POLICY ........................................................................................... 94 
17 
STATEMENT OF COMPLIANCE ............................................................................. 94 
18 
REFERENCES ......................................................................................................... 95 
APPENDIX 1: CONSTRUCTION OF EF1ΑS-ADA LENTIVIRAL VECTOR ................... 107 
 
 
 
APPENDIX 3: TRANSDUCTION PROTOCOL SCHEMATIC ......................................... 113 
 
 
 
APPENDIX 5: ADA-SCID PATIENT MONITORING PROTOCOL .................................. 120 
APPENDIX 6: UNDESIRABLE EFFECTS OF BUSILVEX (BUSULFAN) ......................... 125 
APPENDIX 7: WHO CLASSIFICATION OF ACUTE/SUBACUTE TOXIC EFFECTS .... 128 
APPENDIX 8: DETAILS OF THE HISTORICAL HSCT CONTROL GROUP .................... 131 
APPENDIX 9: DETAILS OF THE CUP GROUP ............................................................... 133 
 
 
 
 


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List of abbreviations 
ADA 
 
Adenosine deaminase 
AE 
 
Adverse Event  
AR  
 
Adverse Reaction 
BCG  
Bacillus Calmette–Guérin 
cDNA  
complementary Deoxyribonucleic Acid 
CI 
 
Chief Investigator 
CTA   
Clinical trial authorisation  
CUP 
 
Compassionate Use Program 
dAdo  
Deoxyadenosine  
dATP  
DeoxyATP  
dCydK 
Deoxycytidine kinase 
EBMT  
European Society for Blood and Marrow Transplantation 
eCRF  
electronic Case Report Form 
EFS 
 
Event-Free Survival 
ERT 
 
Enzyme Replacement Therapy 
GCP 
 
Good Clinical Practice 
GMP  
Good Manufacturing Practice 
GOSH  
Great Ormond Street Hospital 
GTAC   
Gene Therapy Advisory Committee  
GvHD  
Graft versus host disease 
HAPLO 
Haploidentical 
HIV 
 
Human Immunodeficiency Virus 
HLA 
 
Human Leukocyte Antigen 
HSC 
 
Haematopoietic stem cells  
HSCT  
Haematopoietic stem cell transplantation  
ICH 
 
Institute of Child Health 
IgRT  
Immunoglobulin Replacement Therapy 
IMP  
 
Investigational Medicinal Product 
LSS 
 
Lymphocytes subset 
LTR 
 
Long Terminal Repeats 
LTR 
 
Long Terminal Repeats 
LV 
 
Lentiviral vector 
MFD 
 
Matched Family Donor 
MHRA  
Medicines and Healthcare products Regulatory Agency  
MMUD  
Mismatched Unrelated Donor 
MSD  
Matched Sibling Donor 


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MUD   
Matched unrelated donor 
OKT3  
anti-CD3 complex antibody 
OS 
 
Overall Survival 
PBSC  
Peripheral Blood Stem Cells 
PCR 
 
Polymerase Chain Reaction 
PI 
 
Principal Investigator 
PID 
 
Primary Immune deficiency  
PIL 
 
Patient Information Leaflet  
R&D   
Research and development 
RCL   
Replication-competent lentivirus 
rIL-2 
 
recombinant interleukin 2 
RRE 
 
Rev responsive element 
SAE 
 
Serious Adverse Event 
SAHH   
S-adenosylhomocysteinehydrolase 
SCID  
Severe combined immune deficiency  
SFFV  
Spleen Focus Forming Virus 
SIN 
 
Self INactivating 
SRC   
SCID-repopulating cells  
SSAR  
Suspected Serious Adverse Reaction 
 
 
SUSAR 
Suspected Unexpected Serious Adverse Reaction 
TCR   
T-cell Receptor 
TRECs 
T Cell receptor excision circles 
UCL 
 
University College London 
UCLA  
Children’s Hospital Los Angeles 
VSV 
 
Vesicular Stomatitis Virus 
WPRE  
Woodchuck Post-transcriptional Regulatory Element 
γc  
 
Common cytokine receptor gamma chain 
 


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2 
Summary 
Title: Phase I/II, historical 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 
Short Title: LV gene therapy for ADA deficiency 
Investigational Medicinal Product (IMP): EF1αS-ADA lentiviral vector transduced patient 
CD34+ cells 
Phase of trial: Phase I/II 
Objectives: 
The primary objective is to assess the safety and efficacy of EFS-ADA LV mediated gene 
therapy for treatment of ADA-SCID patients. This will be achieved via the following specific 
objectives: 
Safety 
1. To assess clinical, haematological and immunological progress of patients. 
2. To assess vector integration sites and clonal proliferation.  
Efficacy 
1. To assess overall survival (OS) at 1 year for patients treated with IMP 
2. To assess event free survival (EFS) at 1 year for patients treated with IMP 
overall and event free survival at 1 year between patients treated with IMP and 
patients treated with allogeneic HSCT 
3. To assess at each visit engraftment success and resulting immunological and 
metabolic effects in IMP treated patients using:  
a. Vector copy numbers in peripheral blood leukocytes 
b. Cellular and humoral immune system recovery 
c. ADA activity and reduction in dATP in peripheral blood cells 
The secondary objectives are to: 
• 
Compare survival and event free survival outcomes at 2 and 3 years between patients 
treated with IMP and patients treated with allogeneic Hematopoietic Stem Cell 
Transplantation (HSCT) 


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• 
Determine the percentage of IMP treated patients requiring immunoglobulin 
replacement therapy at all evaluation points from 18 months onwards  
• 
Compare frequency of infections, and growth of pathogenic microorganisms over 3 
years 
• 
Evaluate the longitudinal clinical effect in terms of improved immunity 
• 
Evaluate tolerability of conditioning regimen 
• 
Evaluate feasibility of the transduction procedure  
Type of trial: Phase I/II, historical controlled, open-label, non-randomised, single-centre trial 
 


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Trial design and methods: Summary flowchart of the patients treated with 
gene therapy 
 
 


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Recruitment period: 5 years 
Trial duration per participant: From consent until 3 years post-infusion of transduced CD34+ 
cells 
Estimated total trial duration: 8 years 
Planned trial sites: Great Ormond Street Hospital for Children NHS Foundation Trust (single-
site trial) 
Total number of participants planned:  
Patients treated with gene therapy on trial: Up to 10 treated patients  
The trial will include two other sub-groups: patients treated with gene therapy off trial under 
GOSH Special License constituting the Compassionate Use Program (CUP) and patients 
treated with Haematopoietic Stem Cell Transplantation (HSCT) constituting the historical 
control group. 
Inclusion and Exclusion Criteria 
1. Patients treated with gene therapy(on trial) 
Inclusion criteria  
1. Diagnosis of ADA-SCID confirmed by DNA sequencing OR by confirmed absence 
of <3% of ADA enzymatic activity in peripheral blood or (for neonates) in umbilical cord 
blood erythrocytes and/or leucocytes or in cultured foetal cells derived from either 
 


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chorionic villus biopsy or amniocentesis, prior to institution of PEG-ADA replacement 
therapy 
2. Patients who lack a fully Human leukocyte antigen (HLA)-matched family donor 
3.  Patients (male or female) <5 years of age OR 
Patients (male or female) ≥ 5 years to 15 years of age who have preserved thymic 
function as evidenced by presence of >10 % naïve T cells (CD4+45RA+27+ cells)  
4. Parental/guardian signed informed consent 
Exclusion criteria 
1. Cytogenetic abnormalities on peripheral blood 
2. Evidence of active malignant disease 
3. Known sensitivity to busulfan 
4. If applicable, confirmed pregnancy (to be tested in patients above 12 years old) 
2. Patients treated with HSCT, historical control group 
Inclusion criteria 
1. Diagnosis of ADA-SCID confirmed by DNA sequencing OR by confirmed absence of 
<3% of ADA enzymatic activity in peripheral blood or (for neonates) in umbilical cord 
blood erythrocytes and/or leucocytes or in cultured fetal cells derived from either 
chorionic villus biopsy or amniocentesis, prior to institution of PEG-ADA replacement 
therapy 
2. Patients (male or female) between 0-18 years at time of treatment 
3. Patient treated with an allogeneic haematopoietic stem cell transplantation since 2000 
3. Compassionate Use Patients (CUP) 
Another sub-group of patients have been also treated off-trial with the IMP under a 
compassionate basis under GOSH Special License. They were treated following the same 
protocol schedule and procedures as the on-trial patients and they will be included in the 
analysis (details in the SAP) 
 


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3 
Introduction and Background 
3.1  
Adenosine deaminase deficient – severe combined immunodeficiency (ADA-
SCID) 
Severe combined immunodeficiencies (SCID) are a heterogeneous group of inherited 
disorders characterised by a profound reduction or absence of T lymphocyte function. As a 
result, children with SCID are susceptible to recurrent and severe infection with pneumonia, 
diarrhoea and failure to thrive being the most common clinical manifestations. Without 
treatment most children die in the first year of life from overwhelming infection. 
SCID arises from a variety of molecular defects which affect lymphocyte development and 
function (Fischer et al., 1997). Adenosine Deaminase (ADA) deficient SCID accounts for 
approximately 10-20% of all cases of SCID and was the first form of SCID in which the 
underlying defect was identified (Giblett et al., 1972). Over 18 different genetic defects have 
now been shown to give rise to a clinical and immunological phenotype of SCID and these 
include 1) defects in the lymphocyte specific signalling molecules, common  chain (Noguchi 
et al., 1993), JAK-3 (Macchi et al., 1995), and IL-7 receptor  (Puel et al., 1998), 2) in 
molecules that control immunoglobulin gene rearrangement, RAG-1/2  (Schwarz et al., 1996), 
Artemis (Moshous et al., 2001) and Cernunnos (] and 3) in subunits of the CD3 receptor 
complex (Rieux-Laucat et al., 2006; Soudais et al., 1993) amongst others. The incidence of 
all forms of SCID is variously reported to be between 1: 50,000 and 1: 75,000 and the 
incidence of ADA-SCID to be in the order of 1: 200,000 and 1: 1,000,000 live births (Hershfield 
and Mitchell 1995) although the prevalence may be higher within specific ethnic populations 
(Sanchez et al., 2007). 
3.1.1 Adenosine deaminase deficiency 
Adenosine deaminase is an enzyme that is expressed in all tissues of the body. During DNA 
breakdown, ADA catalyses the deamination of deoxydenosine (dAdo) and adenosine to 
deoxyinosine and inosine respectively (reviewed in (Hirschhorn, 1993). The lack of ADA 
results in the accumulation of dAdo in both intracellular and extracellular compartments. 
Intracellularly, 
dAdo 
is 
then 
converted 
by 
deoxycytidine 
kinase 
(dCydK) 
to 
deoxyadenosinetrisphosphate (dATP) which accumulates within the cell. The buildup of these 
two metabolites has profound effects on lymphocyte development and function and is the most 
likely cause of the immunological defects. dATP inhibits the enzyme ribonucleotide reductase 
which is necessary for DNA replication and repair (Takeda et al., 1991; Lee et al., 1984) and 
also induces apoptosis in immature thymocytes. dAdo inactivates the enzyme S-
adenosylhomocysteinehydrolase (SAHH) (Hershfield et al., 1979) (Benveniste et al., 1995): 
SAHH accumulation inhibits transmethylation reactions and the lack of SAHH activity as a 


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consequence of ADA deficiency may also contribute to the immunodeficiency. It has also been 
speculated that the effects of adenosine acting through G protein receptors on the surface of 
thymocytes may play a role in the pathogenesis of the disease (Benveniste and Cohen, 1995) 
(Apasov et al., 2001). 
Although ADA is expressed ubiquitously, the most profound effects are manifest in the immune 
system (non-immunological consequences are present however and are described in section 
3.1.4.2.). This may be explained by the expression pattern of ADA which is highest in the 
thymus as a result of high lymphocyte turnover (Adams and Harkness, 1976) (Van der 
Weyden and Kelley, 1976) and also by increased expression of dCydK in lymphocytes which 
serves to increase dATP accumulation in immune cells more than in other tissues (Carson et 
al., 1977). 
3.1.2 ADA gene: structure and function 
The ADA gene locus has been mapped to 20q13.11 (Tischfield et al., 1974) on the basis of 
evidence from studies of somatic cell hybrids containing translocations involving chromosome 
20 ((Mohandas et al., 1980), ADA activity in patients with chromosome 20 structural 
abnormalities (Philip et al., 1980) (Petersen et al., 1987) and in situ hybridisation with an ADA 
cDNA probe (Jhanwar et al., 1989). The gene spans 32kb and is organised into 12 exons 
separated by 11 introns (Valerio et al., 1983) (Wiginton et al., 1983). Promoter activity was 
localised to a 135bp region immediately 5’ of the major transcription start site  
(-95 relative to the AUG translation start site). The promoter consists of 82% G + C residues, 
lacks the TATA and CAAT sequences found in the promoter regions of many developmentally 
regulated genes and possesses six  GC boxes homologous to the consensus binding site for 
Spl, a eukaryotic ‘zinc finger’ transcriptional activator (Kadonaga et al., 1987) (Pugh and Tjian, 
1990). The murine ADA promoter has similar characteristics (Ingolia et al., 1986). 
cDNA sequences of normal human DNA were first characterized in 1983 by a number of 
different laboratories (Valerio et al., 1983) (Wiginton et al., 1983). The 1.5kb ADA mRNA 
consists of a 1089 nucleotide open reading frame together with 5’ and 3’ untranslated 
sequences. The resulting protein consists of 363 amino acids with a deduced molecular weight 
of 40.7Kd but in western blot studies the actual protein species  varies between 36-44Kd. The 
ADA enzyme is expressed in all tissues of the body but levels vary over a wide range (Adams 
and Harkness, 1976) (Van der Weyden and Kelley, 1976; Hirschhorn et al., 1978). In humans, 
the highest activity is in the thymus and in other lymphoid tissues with lowest expression in 
erythrocytes. Among non-lymphoid tissues, high levels are found in the villi of the duodenal 
epithelium and in other areas of the gastrointestinal tract (GIT) and also in the cerebrum. There 


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is a different pattern of expression in other species with mice having higher levels in the GIT 
than in the thymus (Lee, 1973) (Chinsky et al., 1990). 
ADA in human tissues exists in several different physical forms, which can be distinguished 
electrophoretically and by size. ADA is largely an intracellular enzyme although a low level of 
surface associated enzyme is detectable on a fraction of blood cells (SenGupta et al., 1985; 
Aran et al., 1991). The various forms of ADA represent a combination of genetic 
polymorphisms and isoenzymes generated by post-translational modifications and in some 
tissues, binding of the monomeric catalytic ADA gene product (Schrader et al., 1976) 
(Daddona and Kelley, 1977; Schrader and Stacy, 1977) to a non-catalytic (200kD) 
homodimeric glycoprotein termed ‘conversion factor’, ‘binding protein’ or ‘complexing protein’ 
(ADA-CP) (Daddona and Kelley, 1978; Schrader et al., 1976). It has been speculated that 
ADA-CP may regulate the activity of ADA  or contribute to extracellular Ado catabolism (Trotta, 
1982) or that it may mediate the renal clearance of monomeric ADA (Schrader and Bryer, 
1982; Schrader et al., 1990; Schrader et al., 1984). However, interaction with ADA-CP does 
not affect the activity of ADA and in some tissues the two proteins do not co-localise (Schrader 
et al., 1987; Dinjens et al., 1989). 
ADA-CP has been identified as a protein known both as CD26 and dipeptidyl peptidase IV 
(DPPIV) (Morrison et al., 1993; Kameoka et al., 1993). CD26 was first defined as an antigen 
on activated human T lymphocytes (Fox et al., 1984), and DPPIV as a widely distributed 
ectoenzyme that cleaves peptides including several hormones, neuropeptides, and cytokines 
(Hegen et al., 1990) (Ulmer et al., 1990). CD26 and DPPIV cDNAs from human T cells and 
intestine predict the same 766-residue, 88-kD polypeptide. This type II membrane protein has 
a large extracellular domain consisting of a glycosylated "stalk", a cysteine-rich segment 
(residues 290–552), and a COOH-terminal region that bears the serine protease (DPPIV) 
active site (residues 628–632). Residues 294 and 340–343 of the cysteine-rich segment are 
essential for binding ADA (Dong et al., 1997). Although it was initially suggested that binding 
of ADA to CD26 was important for protection of lymphocytes from the extracellular effects of 
Ado, recent studies on a healthy adult with defective ADA–CD26 binding suggests that 
interaction of these proteins is not essential for the development or maintenance of immune 
function in humans (Richard et al., 2000). 
3.1.3 Mutations in ADA-SCID patients 
Direct analysis of mutant ADA alleles from ADA deficient patients has revealed a wide variety 
of molecular defects. Most of the patients analysed are compound heterozygotes and several 
deletions and splicing defects have been found as have a larger number of point mutations in 
the coding region of the structural gene. Two large deletion mutations, one 3250bp deletion of 


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the promoter and exon 1 (Berkvens et al., 1987; Markert et al., 1988) and the other deleting 
exons 1-5 (Hirschhorn et al., 1992), led as might be expected to no mRNA production. A 
number of splice site mutations have been identified that result in skipping of specific exons 
or in activation of cryptic splice sites. Most of these mutations generate premature translation 
stop codons and result in a decreased level of mRNA. ADA missense mutations have been 
found throughout the coding region of the gene with no particular mutation ‘hotspots’ (reviewed 
in Hershfield and Mitchell 1995). Determination of the three-dimensional structure of the 
murine ADA has permitted modeling of the possible effects of the amino acid substitutions on 
human ADA function. Two mutations are directly involved in binding ADA substrates Glu217 
(Lys) hydrogen bonds to the N-1 atom of substrate through the side chain carboxyl and His15 
(Asp) coordinates with the zinc co-factor (Arrendondo-Vega et al., 1998). Several other 
mutated residues are close to the active site or to peptide segments that deploy active site 
residues. 
In vitro analysis of the residual ADA activity of mutant alleles has been determined by 
expression of the mutant in the ADA-deleted E.coli strain SO3834 (Arredondo-Vega et al., 
1998). Alleles associated with severe ADA SCID expressed 0.001%-0.6% of wild type activity 
whereas 3 alleles from partial ADA deficient patients expressed 5%-28% of normal. There was 
also a strong inverse correlation between mutant ADA activity and dATP level at time of patient 
diagnosis. These data suggest that the severity of the mutation and level of residual ADA 
activity may influence metabolic and clinical outcome. 
Although analysis of the effects of missense mutations by modeling of ADA structure or by in 
vitro assays may be informative, in most cases of ADA-SCID, mutant ADA protein is present 
at only very low levels or is undetectable by western blotting. Thus the primary effect of some 
point mutations in vivo may be to increase the rate of enzyme degradation by interfering with 
protein folding or by destabilizing the mature protein and predisposing it to proteolysis. 
3.1.4 The pattern of clinical disease 
3.1.4.1 Classical presentations 
ADA-SCID is estimated to affect between 1 in 200,000 and 1 in 1,000,000 births although the 
frequency may be greater in certain geographical areas (Hershfield and Mitchell 
1995)(Sanchez et al., 2007). Clinically it is characterised by severe and recurrent infections 
and a high frequency of opportunistic infections. The clinical presentation in ADA-SCID is 
similar to patients with autosomal forms of SCID and it is difficult to distinguish between the 
different forms of SCID on the basis of clinical presentation alone. The mean age at diagnosis 
for all types of SCID is 6.6 months (Buckley et al., 1997) and this most likely reflects the time 
when the protective effect of placentally transferred maternal immunoglobulin has diminished 


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and children have been exposed to a range of microorganisms. The most common infective 
problems are oral candidiasis, respiratory infection due to Pneumocystis jiroveci, respiratory 
syncitial virus and parainfluenza 3, adenoviral infection, persistent diarrhoea and failure to 
thrive. In countries, which administer anti-tuberculous vaccination to infants with bacillus 
Calmette-Guerin (BCG), disseminated infection with BCG has occurred. Live polio vaccine 
has also caused poliomyelitis and carditis but only rarely and this is probably due to the 
continued presence of maternal immunoglobulin at the time of initial vaccination. Physical 
findings are unremarkable except for evidence of infection and the absence of lymph nodes 
and pharyngeal lymphoid tissue. 
3.1.4.2 Non-immunological manifestations in ADA-SCID 
Unlike other forms of SCID, ADA deficient patients show a number of non-immunological 
abnormalities which may reflect the importance of ADA expression in other systems. 
Costochondral abnormalities and skeletal dysplasias are well documented (Cederbaum et al., 
1976) and ADA-SCID children have been noted to have a ‘rachitic rosary’ appearance. Other 
systemic abnormalities include neurological abnormalities involving motor function 
(Hirschhorn et al., 1980), bilateral sensorineuronal deafness (Tanaka et al., 1996), hepatic 
dysfunction (Bollinger et al., 1996) and renal mesangial sclerosis (Ratech et al., 1985). Non-
immunological manifestations are also found in ADA deficient mice, which die perinatally from 
hepatocyte degeneration but also show pulmonary and intestinal defects (Migchielson et al., 
1996; Mitani et al., 1993). More recently studies on ADA SCID patients post bone marrow 
transplantation show defects in cognitive and behavioural function despite correction of 
immunological abnormalities (Rogers et al., 2001; Titman et al., 2008) and again highlights 
the systemic nature of the disease. 
3.1.4.3 Delayed/late onset disease 
Approximately 10-15% of all cases of ADA deficient patients have a milder phenotype with 
less severe immunological abnormalities and clinical course. In these patients there is residual 
ADA activity as a result of the specific gene defect and consequently less profound metabolic 
derangement (Morgan et al., 1987). In these patients, recurrent infections may start to occur 
after 2-3 years of age and patients are eventually diagnosed following investigation for a 
combined immunodeficiency. At the far end of this spectrum are a handful of adults who have 
been diagnosed with ADA deficiency. Two sisters with a long-standing history of pulmonary 
insufficiency and warts were identified following investigation of their CD4 lymphopaenia 
(Shovlin et al., 1994). Other individuals with lymphopaenia and diagnosis in adulthood have 
been described (Ozsahin et al., 1997). In these patients there was only a mild metabolic 
abnormality in comparison with the levels of dATP and dAdo seen in patients with full blown 
ADA-SCID.  


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3.1.4.4 Partial ADA deficiency 
A number of individuals have now been described with partial ADA deficiency. In these 
individuals there is differential expression of ADA in different cell lineages with very low or 
undetectable levels in erythrocytes (<2%) but ~4-70% of normal activity in fibroblasts 
(Hirschhorn et al., 1979; Borkowsky et al., 1980). As a result of significant activity in nucleated 
cells, there is very little metabolic derangement in erythrocytes and there is normal immune 
function. In vitro expression studies of the mutant alleles from such individuals demonstrates 
residual activity and suggests that there is a genotype-phenotype correlation (Daddona et al., 
1983). 
3.1.4.5 Reversion mutations in ADA-SCID 
Patients have now been described in whom an in vivo reversion mutation to wild type 
sequence has resulted in restoration of wild type ADA activity to patient T cells. Detailed 
analysis of these patients showed that although B cells and other lineages showed the 
presence of two mutated alleles, T cell lines contained one mutant and one wild type allele. 
The wild type allele expressed a functional ADA protein resulting in half normal ADA activity 
in the cell lines. In one patient in vivo reversion resulted in progressive clinical improvement 
and unexpectedly mild biochemical and immunological abnormalities suggesting that T cells 
modified to wild type may have a powerful survival and growth advantage over ADA deficient 
cells (Hirschhorn et al., 1996). A similar phenomenon has been described in a patient with X-
SCID and was used as an important for model for subsequently successful clinical gene 
therapy trials (Stephan et al., 1996). In a second individual the use of PEG-ADA resulted in a 
decrease in ADA activity in peripheral blood mononuclear cells which could have resulted from 
PEG-ADA abolishing the selective advantage of revertant cells in vivo (Ariga et al., 2001).  
3.1.5 Diagnosis of ADA deficiency 
ADA deficiency unlike many of the other immunodeficiencies can be reliably diagnosed by 
enzymatic assays. Intracellular ADA activity can be measured by the ability of cells to convert 
the substrate adenosine to inosine and hypoxanthine using high performance liquid 
chromatography (HPLC). Further, dATP build-up in erythrocytes, accumulation of intracellular 
and extracellular dAdo and lack of SAHH activity are specific characteristics of the disease 
and can all be measured to give an accurate and unambiguous diagnosis. In carriers of the 
condition, intermediate levels of ADA activity have been found leading to reliable identification 
of carrier status. The availability of such precise enzymatic assays has to a certain extent 
obviated the need for genetic diagnosis, although this has been carried out in specialist 
laboratories. 


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Enzymatic assays have also been used in prenatal diagnosis of ADA deficiency. In the first 
trimester of pregnancy, cultured cells from chorionic villus sampling (CVS) can be assayed for 
ADA enzymatic activity (Dooley et al., 1987). This is a more reliable source than fresh material 
from the CVS. In the second trimester diagnosis has been performed on cultured amniotic 
cells. Direct analysis of enzyme and metabolite levels in amniotic fluid has not proved useful. 
Genetic diagnosis is also available in specialist laboratories and is useful for confirming the 
findings observed by metabolic analysis. In certain cases, the use of genetic analysis and 
mutant allele expression has been able to shed light on the variability in severity of 
presentation. 
3.1.6 Prognosis 
Classic ADA-SCID has an extremely poor prognosis without treatment. Death occurs in the 
first year of life from infectious complications. Although treatments based on physical isolation 
into a sterile environment can provide protection from infection and prolong life (for example, 
the case of the ‘bubble baby’), such cases are extremely rare. Upon diagnosis, patients are 
commenced on bacterial, viral and pneumocystis prophylaxis and immunoglobulin substitution 
therapy. In some cases fungal prophylaxis is also commenced. In a few atypical cases, 
patients have been maintained on this regime for a number of years. However, it is generally 
accepted that in the vast majority of cases, prophylactic therapy is only a means of protecting 
the child until more definitive treatment with either stem cell transplantation or PEG-ADA can 
be performed. 
3.2 
Management options for ADA-SCID 
3.2.1 Haematopoietic stem cell transplantation for ADA-SCID 
Haematopoietic stem cell transplantation (HCST) is the treatment choice that is most widely 
available to most physicians and transplant centres. Until recently, data on the outcome of 
ADA-SCID transplants has been limited as most SCID transplant papers have presented data 
on the outcome of all SCID types rather than by specific molecular defect (Antoine et al., 
2003). For these reasons, a multicenter retrospective study gathering data on outcome of 
haematopoietic stem cell transplantation for ADA-SCID has been conducted.  In this study, 
we analysed outcome of HSCT in 106 patients with ADA-SCID who received a total of 119 
transplants.(Hassan, 2012 ) 
Donor source had a major impact on the outcome of HSCT (fig 1). Overall survival was higher 
in Matched Sibling Donor (MSD) and Matched Family Donor(MFD) HSCTs (86% (42 patients) 
and 81% (12 patients) respectively). Comparison of survival outcomes in MisMatched 
Unrelated Donor (MMUD) and haploidentical transplants [29% (7 patients) and 43% (30 


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patients) respectively]to those of MSD HSCT showed a highly significant difference 
(p<0.0001). Comparison of overall survival in MSD to MUD HSCT also showed a significant 
difference (p<0.05). In haploidentical transplants, even though overall survival was 43%, 
stable engraftment was only noted in 11 (37%) patients. The majority of deaths in the Matched 
Unrelated Donor (MUD), MMUD and haploidentical transplants (HAPLO) were in the first 100 
days after HSCT, with relatively few late deaths. The causes of death were mainly related to 
pneumonitis/respiratory failure and sepsis which formed over 50% of all deaths. Other major 
causes included graft versus host disease (15%) and fungal infection (11%). 
 
Figure 1: Kaplan-Meier curve showing overall survival in relation to donor source. MSD–matched sibling donor; 
MFD–matched family donor; MUD-matched unrelated donor; MMUD-mismatched unrelated donor; HAPLO-
haploidentical donor (data from manuscript in preparation). 
The majority of MSD/MFD transplants were undertaken without any chemotherapy as were a 
smaller number of MUD and HAPLO transplants and this may have had a major effect on 
outcome. When conditioning itself was studied in a univariate analysis, there was a 
significantly improved overall survival in unconditioned transplants in comparison to 
myeloablative intensity conditioning transplants (79 % vs 55%; p=0.003). Reduced intensity 
conditioning transplants had an overall survival of 64% and this was not statistically different 
from outcomes following unconditioned procedures.  
The poor outcome in mismatched donor transplants may be attributable to the vulnerability of 
patients to the toxicities associated with cytoreductive chemotherapy and it has been argued 
that an alternative approach would be to infuse T cell depleted stem cells without any 
chemotherapy. Data available from a single North American centre suggests however, that 
this may not necessarily improve Event Free Survival (EFS) outcome. Of 19 patients 
undergoing such procedures, 14 survived (73%) but of these only 7 were successfully 
engrafted (DFS 37%) with the remainder rejecting the unconditioned transplant (Booth et al., 
0
5000
10000
15000
0
20
40
60
80
100
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DAYS
MSD
MFD
MUD
MMUD
HAPLO


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2007). Similarly in the HSCT data collected recently, of 4 fully matched unrelated donor 
transplants performed without conditioning, 2 patients did not engraft effectively suggesting 
that conditioning is necessary in all scenarios other than a fully matched sibling or family 
donor. 
The above data on HSCT outcome may help in guiding decision making with respect to the 
use of gene therapy. Certainly a fully matched sibling or family donor transplant is associated 
with a considerable degree of success both in terms of survival and sustained immune 
recovery and would be the definitive treatment of choice. By contrast, the current evidence 
suggests that mismatched transplants, both from unrelated and haploidentical donors, 
whether undertaken with or without conditioning have a poor chance of success and in the 
conditioned setting are associated with high mortality rate. The data from matched unrelated 
donor transplants (66% survival in 15 patients (fig 1)) also suggests that there is a significant 
risk of mortality associated with the procedure. 
3.2.2 ADA enzyme replacement therapy 
The toxic compounds that accumulate in ADA deficiency (dATP) cross the cell membrane 
poorly but are metabolised to ADA substrates (dAdo) that rapidly equilibrate with plasma via 
the nucleoside transporter. Thus maintaining sufficient levels of circulating ‘ectopic’ ADA either 
in the plasma or in a population of cells can normalise metabolite levels in enzyme deficient 
cells. This was initially demonstrated by the use of repeated red cell transfusions for the 
treatment of ADA deficiency (Polmar, 1978). Metabolic and immunological correction was 
seen but the effects were transient and outweighed by the risk of viral transmission and iron 
overload. 
PEG-ADA is a bovine form of ADA conjugated to polyethylene glycol. The covalently bound 
PEG is intended to prevent proteolysis and uptake by cells and to prolong circulating life and 
reduce immunogenicity. PEG-ADA has been used since 1987 for the treatment of ADA-SCID 
patients who lacked a genotypically identical donor (reviewed in (Hershfield, 1995a; 
Hershfield, 1995b). PEG-ADA avoids the risks of red cell transfusion and the amount of 
enzyme activity provided is equivalent on a ml to ml basis to ~1800 times the ADA activity of 
packed erythrocytes. Three mls of PEG-ADA contains the ADA equivalent of ~1012 T 
lymphocytes. The preparation is given as an intramuscular injection at ~30 u/kg on a weekly 
basis. 
Data on long term follow up of immunologic function and outcome in PEG-ADA treated 
patients has been limited since there has been no central point for data collection. However a 
recent review (Gaspar et al., 2009) has highlighted outcomes in over 185 patients treated with 


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PEG-ADA until September 2008 (about 90% of those ever treated). PEG-ADA has been used 
as initial therapy for patients who lacked a related HLA-identical donor, when assessment of 
risk and benefit by physicians and parents favoured Enzyme Replacement Therapy (ERT) 
over other options (Hershfield, 2004; Booth et al., 2007). 
Overall, 70% of patients treated with PEG-ADA began ERT at <1 year of age (50% were <6 
months old). Half of the remaining patients started treatment at 1-3 years of age, and half at 
3-34 years of age. Many of these latter “delayed” or “late’ onset patients had pulmonary 
disease or other consequences of chronic immune deficiency, which made them poor 
candidates for partially mismatched HSCT with conditioning. 
Up to September 2008, 98 patients were receiving PEG-ADA approximately half of the number 
that had begun ERT. About 20% of patients had died while on therapy; the remainder had 
discontinued ERT to undergo a potentially curative procedure. More than two thirds of the 
transplants were performed within a year of starting PEG-ADA, as soon as clinical condition 
was stable and a suitable donor had been identified. During the first decade after PEG-ADA 
received FDA approval in 1990, survival following these “elective” transplants was about 50% 
(Hershfield, 2004), similar to that for partially mismatched transplants in ADA-deficient SCID 
patients who had not received prior ERT.  
Figure 2 plots the estimated probability of survival vs. length of treatment with PEG-ADA. Half 
of the deaths on ERT occurred within the first 6 months (40% in the first month), due to 
conditions present at diagnosis. The overall probability of surviving 20 years on ERT is 
estimated to be 78%. A patient alive 6 months after starting ERT had about 90% probability of 
surviving the next 12 years. Conditions contributing significantly (3-5 patients each) to mortality 
beyond 6 months include refractory hemolytic anaemia at 1-3 years; chronic pulmonary 
insufficiency after 5 to 15 years; and lymphoproliferative disorders after 5 to 15 years of ERT 
(Kaufman et al., 2005; Husain et al., 2007). Hepatocellular carcinoma developed in two 
patients, one just starting ERT after failing an unconditioned haploidentical HSCT, and a 
second after 10 years of ERT. Another patient died of hepatoblastoma discovered after 2 years 
of ERT, but thought to be present at diagnosis of ADA deficiency. Late deaths due to acute 
infection appear to be uncommon, but a patient recently died of measles after 10 years of 
treatment.  
 
 
 


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Figure 2: Estimated probability of survival while receiving PEG-ADA (from Gaspar et al., 2009). 
Immune dysregulation has been seen in a few patients following commencement of PEG-
ADA. Thrombocytosis has been described although there were no adverse consequences 
with resolution after a few weeks of treatment (Marwaha et al., 2000). Two patients developed 
refractory immune haemolytic anaemia (Hershfield, 1995b), one of whom required prolonged 
immune suppression and died of candida sepsis and the other discontinued PEG-ADA and 
died of complications of a mismatched HSCT. In a significant number of patients (~50%), 
recovery of immune function also leads to development of antibodies against bovine epitopes 
of PEG-ADA (Chaffee et al., 1992). In a minority of these individuals an inhibitory antibody 
directed at the ADA active site develops and results in enhanced ADA clearance. In one case 
tolerance was induced and in another increased clearance was overcome by increasing the 
PEG-ADA dose. 
The other major limitation to PEG-ADA therapy is cost. As an orphan drug, the cost is high 
and treatment of a small child may cost between £150,000–£300,000 per year. With increase 
in age and size, these costs will only increase and since ADA therapy is palliative and not 
curative, treatment must be continued throughout the life of the patient. 
In summary, PEG-ADA provides an often life-saving therapy at the time of diagnosis, when 
other options may be unavailable or less predictably effective. If ERT is continued beyond 6 
months, there is a high probability that clinical benefit can be sustained for at least a decade. 
However, PEG-ADA may not be easily available in some countries and its high cost is a barrier 
to long-term ERT, and more uncertainty exists about how long term immunological and clinical 
benefit can be maintained beyond 8-10 years.  An additional concern with ERT beyond 5yrs 
is the emergence of serious complications, described above, including lymphoid and possibly 
hepatic malignancies, and progression of chronic pulmonary insufficiency. 
 


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3.2.2.1 PEG-ADA treatment and long-term outcome  
In the absence of a fully matched related or unrelated donor, a number of centres have chosen 
to maintain patients on long term PEG-ADA replacement therapy. Until very recently no formal 
data has been published on the immunological and clinical outcome in these patients but a 
number of reports now suggest that maintaining patients on long-term PEG-ADA may result 
in prolonged immunodeficiency and increased susceptibility to infection. 
Paper 1: (Chan et al., 2005)  
This paper evaluated the immune reconstitution of nine ADA SCID patients who have received 
PEG-ADA for 5-12 years (median 9 years). The T lymphocyte counts in these patients 
increased initially but as seen also in patients treated at GOSH, decreased thereafter to low 
levels. The graph below (Fig 3) shows that levels range between 12-500 T cells/mm3 after 5-
12 years and in all patients the absolute level is well below the lower limit of normal. Further 
impairment of cellular immunity was demonstrated by low levels of CD4+ cells and impaired 
T cell responses to both mitogens and specific antigens.  
 
 
 
 
 
 
 
 
 
 
 
Figure 3: Analysis of thymic output and TCR diversity. (A) Number of TREC and CD3+ lymphocytes in samples 
obtained from five PEG-ADA-treated patients (#1–#5) at 4.1 and 6.1 years after PEG-ADA therapy initiation. The 
mean (*) and standard deviation (vertical line) of values obtained in age-matched controls are shown for 
comparison. Figure from Chan B et al., 2005. 
 


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Paper 2: (Malacarne et al., 2005)  
In this study, five ADA-SCID patients who received PEG-ADA for 5-8 years (mean 6.7yrs) 
were assessed. These patients were also T lymphopaenic and importantly also showed 
decreased levels of TRECs (T cell receptor excision circles – an indicator of thymic activity) in 
comparison to age matched controls (Fig 4). These patients also showed a decreased 
response to mitogens and an increased tendency of T cells to apoptosis.  
 
Figure 4: Analysis of thymic output and TCR diversity. (A) Number of TREC and CD3+ lymphocytes in samples 
obtained from five PEG-ADA-treated patients (#1–#5) at 4.1 and 6.1 years after PEG-ADA therapy initiation. The 
mean (*) and standard deviation (vertical line) of values obtained in age-matched controls are shown for 
comparison. Figure from Malacarne F et al., 2005. 
Paper 3:(Serana et al., 2010)  
In a follow up to the Malacarne study above, a comparative analysis of patients receiving 
HSCT (n=5) and PEG-ADA (n=8) was undertaken. This study demonstrates that patients on 
ERT show decreased numbers of total lymphocytes/CD3+/CD19+ cells over time, all of which 
are below the normal range for age. Patients on PEG-ADA in comparison to HSCT recipients 
also show decreased thymic output and restricted T cell receptor repertoires (Fig 5). 
Papers 4 and 5: (Kaufman et al., 2005; Husain et al., 2007) 
These clinical reports document 2 children both of whom after 10 years of PEG-ADA therapy 
developed an EBV positive cerebral lymphoma and Burkitt’s lymphoma respectively. Despite 
chemotherapy the first patient died 5 months after diagnosis of the tumour and the second 
patient responded to chemotherapy. In both patients there was poor recovery of cellular 
immunity on PEG-ADA which may have contributed to development of lymphoma. Other 
patients have also been reported who have developed malignancies after long term PEG-ADA 
administration. 
 


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Figure 5: Analysis of thymic output and T cell numbers in 5 HSCT treated patients and 8 PEG-ADA-treated ADA 
patients over time. The shaded areas represent the age related normal values in healthy children. A) Number of 
TRECs B) Naïve T cell numbers as characterized by CD4+CD45RA+ expression C) Total T cell numbers. Figure 
from Serana et al., 2010. 
These reports suggest that continued PEG-ADA treatment leads to demonstrable defects of 
T cell function and impaired thymic activity that may lead eventually to significant clinical 
infectious complications. It is also very probable that attempts to treat patients by either HSCT 
or stem cell gene therapy at this late stage will be unsuccessful or of limited efficacy due to 
reduced thymic function and increased infective burden (Thrasher et al., 2005). 
Thus although PEG-ADA maybe an excellent short term measure to stabilise children with 
ADA-SCID, its prolonged use may be counter-productive. 
3.2.2.2 PEG-ADA and neurological outcome 


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PEG-ADA results in more systemic delivery of ADA enzyme. As a result it may be thought that 
PEG-ADA therapy may have a more beneficial effect on the non-immunological 
consequences of ADA deficiency. However, at present there is no conclusive data to suggest 
that PEG-ADA treatment improves neurocognitive outcome. In a preliminary study, we have 
compared patients treated with PEG-ADA alone with those treated by HSCT and although the 
numbers are small, there is no significant difference between the two groups. Anecdotal 
evidence from other physicians also suggests that PEG-ADA treated children continue to have 
behavioural problems. 
3.2.3 Alternative therapy for ADA-SCID based on somatic gene transfer 
Allogeneic bone marrow transplantation for ADA-SCID has a number of limitations especially 
when a genotypically matched donor is unavailable. PEG-ADA can be very beneficial but also 
has certain side-effects, poor long term immune recovery and is extremely expensive. An 
alternative therapy based on somatic gene transfer of the ADA gene into haematopoietic stem 
cells has been proposed by several investigators and clinical trials have been undertaken with 
variable results. For the reasons stated below, ADA-SCID been considered an attractive 
condition for treatment by somatic gene therapy. 
1. 
It is a monogenic disease well characterised at the molecular level. 
2. 
ADA is widely expressed in haematopoietic and non-haematopoietic cells and 
is not under complex regulatory control. 
3. 
Over-expression of the gene has not been associated with significant 
complications. 
4. 
Suboptimal levels of gene expression result in immunological correction. 
5. 
Introduction of the ADA gene into T cell precursors confers a survival and 
developmental advantage to their progeny. 
6. 
Introduction of the ADA gene into haematopoietic stem cells may restore 
functionality of B lymphoid and T lymphoid cells. 
Three recent clinical trials of somatic gene therapy for ADA-SCID have shown complete 
correction of the immunological and metabolic defects and thus verifies the feasibility and 
efficacy of this approach in human subjects. In all three studies, no adverse effects have been 
seen. 


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3.2.4 Clinical trials of gene therapy for ADA-SCID 
3.2.4.1 T lymphocyte gene therapy 
In 1990 the first clinical trial of gene therapy for ADA deficiency was started on two girls in the 
United States (Blaese et al., 1995). Both patients had had commenced PEG-ADA therapy but 
in each case there had been an initial good response to PEG-ADA with a subsequent 
deterioration in total lymphocyte numbers and in vitro and in vivo specific lymphocyte 
responses. The treatment protocol involved LASN retroviral vector supernatant transduction 
of peripheral T cells apheresed from each patient and stimulated with a combination of 
recombinant interleukin 2 (rIL-2) and OKT3 (an anti-CD3 complex antibody). The transduced 
expanded T cells were returned to the patients 9-12 days later by peripheral infusion. The 
procedure was repeated 11-12 times at regular intervals for each patient over a period of 18-
24 months. The efficiency of retroviral gene transfer varied considerably from one round of 
transduction to another with a range of 0.1%-10% transduced cells. The level of peripheral 
blood lymphocyte ADA was in keeping with the PCR findings with a significant rise in Patient 
1 but no change from pre-treatment levels in Patient 2. In both patients there was an initial 
rapid rise in the T cell count before stabilising in the normal range for Patient 1 and a slight 
increase from the pre-treatment levels for Patient 2. Cell mediated immunity was shown to 
improve in both patients as assessed by the development of DTH skin test reactivity to a 
variety of antigens and also by improvement in in vitro T cell immune responses. Humoral 
immune function improved with an increased antibody response to Hib and tetanus 
immunisation. One interesting observation arising from this study is the prolonged survival of 
the transduced T cells. Vector positive cells are detectable for over 10 years after the last 
infusion, a far longer survival time than originally predicted (Muul et al., 2003). 
3.2.4.2 Peripheral blood lymphocyte and T cell depleted bone marrow gene therapy 
A slightly different approach to gene therapy was taken by Bordignon and colleagues in 1992 
(Bordignon et al., 1995). Two structurally identical vectors expressing the human ADA cDNA 
and distinguishable by the presence of alternative restriction sites in a non-functional region 
of the viral LTR, were used to transduce peripheral blood lymphocytes (PBLs) and T cell 
depleted bone marrow independently. Thus using restriction digest analysis, it was possible 
to identify the origin of transduced cells. Efficiency of transduction varied from 2% to 40% of 
PBLs and from 30%-40% of bone marrow cells. 
Two patients were initially treated by this protocol (4 further patients have been treated 
following the initial report). Both had followed a similar clinical course with diagnosis at the age 
of two years and treatment with PEG-ADA. Both patients received infusions of gene modified 
PBLs and T cell depleted bone marrow regularly over a 1-2 year period. Analysis of vector 
transduced cells from peripheral blood indicated that initially all vector-positive cells were 


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derived from transduced PBLs. However, 3 years after initiation, and 1 year after 
discontinuation of gene therapy, peripheral lymphocytes showed the alternative restriction 
digest pattern indicating conversion of the circulating transduced lymphocytes from a PBL-
derived to a bone marrow derived population. Further analysis of T lymphocyte clones, 
granulocytes and erythrocytes taken at this time point also showed a predominantly bone 
marrow origin. These findings suggested that there had been stable transduction of early 
haemopoietic progenitors capable of generating multilineage progenies. Moreover, the 
continued presence of transduced bone marrow cells and their differentiation into mature 
circulating cells appears to confirm the assumption that a small number of ADA+ cells have a 
survival advantage in an ADA -ve environment. 
The frequency of transduced cells varied with 2-4.7% of T cells being vector positive and 17-
25% of clonable bone marrow progenitors. Unsurprisingly, given the low frequency of 
transduced cells in the circulation the level of total ADA activity in total circulating nucleated 
cells has remained low (5-18% of normal values). There was an increase in absolute 
lymphocyte and T cell numbers in both children into the normal range and there was an 
improvement in T cell proliferative responses to non-specific and specific stimuli. The T cell 
repertoire was analysed by V usage and demonstrated the development of a normal T cell 
repertoire. 
Although these results appeared promising, both children remained on PEG-ADA therapy, 
albeit with decreasing doses. With greater follow-up, reports from this study did not show any 
significant improvement in immunological or metabolic parameters and patients remained on 
PEG-ADA. 
3.2.4.3 CD34+ stem cell gene therapy 
Hoogerbrugge and colleagues performed retroviral mediated gene transfer into CD34+ bone 
marrow cells of 3 children with ADA deficiency in an attempt to effect a cure with a once only 
procedure (Hoogerbrugge et al., 1996). Two of the subjects were on enzyme replacement 
therapy at the start of the trial, and in the third child PEG-ADA was only started 3 months after 
the gene therapy procedure. Gene transfer resulted in a 5-12% transduction frequency in in 
vitro colony forming cells. Transduced granulocytes and mononuclear cells persisted in the 
circulation for 3 months as shown by PCR analysis. In one patient the transduced gene was 
detected in the bone marrow at 6 months after gene transfer. Subsequently, no evidence of 
the transduced gene was detectable in either the bone marrow or peripheral blood. In addition, 
ADA gene expression was not detected at any time point. 


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A further study also targeted the CD34+ population but on this occasion from cord blood 
derived cells (Kohn et al., 1995). Three prenatally diagnosed infants were treated by 
transplantation of transduced autologous CD34+ selected umbilical cord blood cells. All 3 
patients were started on PEG-ADA in the first few days of life. By progenitor colony assay, it 
was shown that 12-21% of CD34+ cells had been transduced in the three patients. The 
frequency of peripheral lymphocytes containing the LASN vector as shown by semiquantative 
PCR, was 1/3000 to 1/100000 18 months after transplantation. At this time point, ADA activity 
in unselected T cells was measured and found to be barely above the levels found in ADA 
SCID patients. The results suggested that retroviral mediated stable transduction of early 
progenitors in cord blood was possible and that engraftment of these cells without prior 
cytoablation was possible. However, the small numbers of transduced peripheral blood 
lymphocytes and the very low level of ADA expression in unselected cells suggested that 
significant immune reconstitution was unlikely and PEG-ADA administration was continued. 
3.2.4.4 PEG-ADA withdrawal leads to proliferation of gene transduced cells 
The use of PEG-ADA has always clouded the issue of whether these early trials were 
successful in correcting immune function and it has always been felt that only complete 
withdrawal of replacement therapy would answer this question. To date two patients from two 
different studies have undergone elective withdrawal of PEG-ADA. Kohn et al. at Children’s 
Hospital Los Angeles (UCLA) attempted withdrawal in one patient from their cord blood gene 
transfer program. Over two months of observation, there was a 30-100 fold increase in the 
proportion of gene transduced T cells. However, there was an overall 25% decrease in the 
total numbers of T cells and a decrease in the B and NK cell numbers. This prompted 
investigators to restart PEG-ADA and the patients remains on enzyme replacement to this day 
(Kohn et al., 1998). 
A patient from the Milan study also stopped PEG-ADA on the grounds that there was a plateau 
in immune function and the investigators reasoned that stopping PEG-ADA may provide an 
advantage to transduced cells (Aiuti et al., 2002b). The discontinuation of PEG-ADA led to a 
selective growth advantage of gene transduced T lymphocytes as assessed by PCR 
quantification, fluorescence in situ hybridisation and T cell clonal analysis. Transduced cells 
expressing the ADA vector increased during PEG-ADA withdrawal eventually replacing the 
non-transduced T cell population, reaching nearly 100% of circulating lymphocytes. Absolute 
T cell counts increased and stabilised to levels significantly higher than those observed during 
PEG-ADA treatment. Functional immunological parameters also improved with demonstration 
of vaccine specific responses and responses to the X174 neoantigen. Intracellular PBL ADA 
activity also increased by 1 log and persisted at approximately 50% of normal individuals 
(which is in the range for immunologically normal heterozygote parents) but there was a rise 


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in the red blood cell dATP levels suggesting that the total mass of gene corrected lymphocytes 
producing ADA was not sufficient for systemic detoxification. Nevertheless this patient has 
remained off PEG-ADA and is clinically well. It has not been reported whether prophylactic 
medication has been discontinued.  
There may be several reasons for the difference in outcome between the two patients 
described. However the most important factor is likely to be the total number of gene 
transduced cells at the time of PEG-ADA withdrawal. In the UCLA study this level was only 
0.01% whereas 1-3% of T cells in the Milan study were ADA gene positive prior to PEG-ADA 
discontinuation. Therefore the pool of cells capable of selective proliferation and immune 
system repopulation was initially much greater. However, both studies demonstrate that gene 
transduced cells do possess a proliferative advantage and this may have been blunted by the 
use of PEG-ADA. 
3.2.5 ADA-SCID gene therapy without PEG-ADA therapy and with the use of a mild 
conditioning regime 
A major progress in gene therapy for ADA-SCID was accomplished following the adoption of 
an improved gene transfer protocol into bone marrow CD34+ cells combined with the 
administration of a nonmyeloablative dose of busulfan chemotherapy prior cell reinfusion (Aiuti 
et al., 2002a). Since year 2000, 15 children lacking an HLA-identical sibling donor were 
enrolled in this experimental protocol (Aiuti et al., 2009)(and unpublished data). Most patients 
displayed an inadequate response to enzyme replacement therapy or had failed mismatched 
related transplant. To exploit the selective advantage for ADA-transduced cells in a toxic 
environment, no PEG-ADA was given after gene therapy. The reduced dose of busulfan 
induced a transient myelosuppression without organ toxicity, which was sufficient to achieve 
long-term engraftment of gene corrected hematopoietic stem cells (Aiuti et al., 2002a; Aiuti et 
al., 2009). The dose of CD34+ cells infused and the efficiency of gene transfer were shown to 
be critical factors in determining a higher proportion of gene corrected myeloid cells engrafting 
in patients. The large majority of lymphocytes were ADA-transduced, confirming that PEG-
ADA withdrawal favours the selective survival of gene corrected cells (Aiuti et al., 2002b; Kohn 
et al., 1998; Aiuti et al., 2009) (and unpublished data). Clonal analysis of long-term 
repopulating cells revealed the presence of shared vector integrations among multiple 
hematopoietic lineages, thus proving the engraftment of multipotent hematopoietic stem cells 
(Aiuti et al., 2007). All children are alive and the outcome of the first ten treated patients has 
been recently reported. Eight of the 10 patients remain currently off enzyme replacement 
therapy, with persisting ADA expression and systemic detoxification up to 8 years after 
treatment. Nine patients displayed recovery of polyclonal thymopoiesis, significant increase in 
T-cell counts (although 4 patients did not reach normal levels) and full correction of T-cell 


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function, including susceptibility to apoptosis and proliferative responses to mitogens and 
antigens (Cassani et al., 2008). Intravenous immunoglobulin treatment was discontinued in 5 
children with evidence of antigen-specific antibodies to vaccinal antigens and pathogens. The 
progressive restoration of immune and metabolic functions led to significant improvement of 
patients’ physical development and protection from severe infections, without adverse events 
related to gene therapy. 
In a similar trial performed in London at our centre (Great Ormond Street Hospital for Children 
NHS Trust), using an alternative retroviral construct and a single dose melphalan conditioning 
regime (and latterly busulfan iv 4mg/kg), 7 patients have been treated (Gaspar et al., 2006) 
(and unpublished data, manuscript submitted). Three patients have shown very good immune 
recovery and in a fourth, gene transduced cells contributed to immune recovery although PEG-
ADA was initially re-started and then stopped. Failure of gene therapy was seen in three 
patients, in one case as a result of a poor stem cell harvest and in two others due to low level 
stem cell transduction efficiency. In the two most successfully reconstituted patients, ADA 
expression was observed in different haematopoietic lineages, including red blood cells, 
leading to effective metabolic detoxification. The large majority of T cells and NK cells were 
gene corrected, while significant gene marking was also observed in granulocytes and 
monocytes. In these two individuals re-initiation of thymopoiesis after gene therapy was also 
noted. 
The clinical trials conducted at the National Institute of Health and Children’s Hospital of Los 
Angeles have provided important information on the role of myelosuppression in favouring 
engraftment of gene corrected HSC. In the first study, four patients undertook gene therapy 
without receiving conditioning, resulting in low levels of marking in two patients and no 
sustained immunological improvement (Sokolic et al., 2008). The second clinical trial included 
low-dose myelosuppression with busulfan and withdrawal of PEG-ADA and has enrolled six 
patients with a follow-up ranging between two months and two and a half years. This version 
of the trial led to superior immunological and metabolic outcome with two out of three patients 
with a follow-up longer than 1 year having derived clinical benefit as shown by normalisation 
of in vitro T function and improvement of immunoglobulin production that in one case allowed 
for normal responses to vaccinations. In all but one case, production of ADA by PBMCs is at 
levels between 30-100% of normal leading to sustained metabolic control similar to that 
observed after HSCT (K. Shaw, personal communication, ASGT 2009, San Diego, CA). One 
patient experienced a prolonged cytopenia following busulfan conditioning, as the 
consequence of a pre-existing cytogenetic abnormality, pointing out at a potential limitation for 
patients subject to autologous gene transfer (Engel et al., 2007). 


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Preliminary reports from a Japanese gene therapy trial under which two ADA-SCID patients 
were also treated with retroviral gene transfer into CD34+ bone marrow cells after withdrawal 
of PEG-ADA treatment, but in the absence of any myeloablative conditioning, have suggested 
that some immunological reconstitution can be observed in such a setting.  Pending the 
availability of further details, the extent of the improvement in comparison with the results 
obtained in patients subjected to prior conditioning remains unclear (Otsu et al., 2006). 
ADA-deficiency represents a paradigmatic approach for gene therapy approaches for 
inherited blood borne disorders. The results of clinical trials from 2000 onwards indicate that 
gene therapy with non-myeloablative conditioning is associated with significant clinical benefit 
and is now an option to be considered for all ADA-SCID patients lacking an HLA-identical 
sibling donor. The results from the trials in Milan, London and the US in which at least 30 
patients have been followed for longer than 1yr are impressive both in terms of toxicity and 
efficacy. All patients have survived therapy which compares favourably with HSCT data and 
~70% of patients have near full or partial recovery of immune function allowing complete 
cessation of PEG-ADA therapy (See Table 1). With respect to non-immunological 
manifestations, it remains to be determined whether gene therapy will be superior to allogeneic 
transplant in preventing the neuro-cognitive complications frequently observed in these 
children after transplant (Honig et al., 2007; Titman et al., 2008). 
 
 
 
 
 
 
 
 
Table 1: Results from 3 different trials of gene therapy for ADA-SCID (gathered from published data (Aiuti et al., 
NEJM 2009), GOSH experience in London and personal communication (D.B. Kohn/F. Candotti). 
3.2.6 Safety of ADA-SCID gene therapy using a gammaretroviral vector 
The use of gammaretroviral vector mediated gene therapy has been associated with 
insertional mutagenesis in 3 different gene therapy trials. This is includes 5 patients treated 
by gene therapy for SCID-X1 (Hacein-Bey-Abina et al., 2008; Howe et al., 2008), 2 patients 


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treated for X-CGD (Ott et al., 2006; Stein et al., 2010) and 1 patient treated for Wiskott-Aldrich 
syndrome (Christoph Klein, unpublished data). In both the X-CGD and the WAS trials, the 
vector backbone and promoter (SFFV) are very similar to the vector construct and promoter 
used in our current GTAC 073 gammaretroviral study. 
For ADA-SCID, analysis of gammaretroviral vector integrants has been analysed in a similar 
way to the studies for the other immunodeficiencies and shows a very similar insertional 
profile. Integrations are found predominantly in and around the transcriptional start site of 
genes and insertions have been found in genes associated with cell cycle control, cell 
signalling and near known oncogenes such as LMO2 (Aiuti et al., 2007). In our study (GTAC 
073) in which ADA transgene transcription is under the control of the SFFV LTR, we have 
seen insertions into LMO2 and particularly into the MDS-EVI1 locus which is a favoured 
insertion site for SFFV based vectors and was the insertional event that led to leukaemia 
formation in the X-CGD trial (Ott et al., 2006, Stein et al., 2010). 
Despite these worrying integration events, none of the patients treated (now over 30 patients) 
on the 3 trials have shown evidence of clonal proliferation or adverse events related to gene 
transfer. This may indicate that gene therapy for ADA-SCID has a favourable risk-benefit 
profile, as opposed to gene therapy for SCID-X1 (Hacein-Bey-Abina et al., 2003; Howe et al., 
2008) although further long term follow up is necessary. Nevertheless, safety monitoring 
should be continued to be strictly implemented long-term in all patients, according to 
guidelines of regulatory agencies. 
The safety of gene therapy for ADA-SCID is also highlighted by the long term follow-up 
(>10yrs) of patients treated in clinical trials in the 1990’s. Though these patients derived little 
benefit from the gene therapy procedure, a number of patients continue to have significant 
numbers of gene transduced cells (1-10%) without any evidence of clonal proliferation or 
insertional mutagenesis (Muul et al., 2003; Schmidt et al., 2003). 
From these studies it is clear that gene therapy is a very promising treatment strategy for the 
management of ADA-SCID. There is strong evidence for immunological and metabolic 
correction in the majority of patients treated. There is nevertheless, a potential risk associated 
with the continued use of conventional gammaretroviral vectors in which transcription of the 
ADA gene is under the control of the viral LTR. The use of alternative vector systems that can 
promote similar or improved immune recovery but with an enhanced safety profile would be 
of significant advantage in the furthering the use of gene therapy for ADA-SCID. 


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3.3 
Lentiviral vectors for gene therapy of ADA SCID 
3.3.1 Lentiviral vectors 
Lentiviral vectors were derived from components of the HIV-1 lentivirus and have been found 
to have several attributes that make them potentially more effective and safe than retroviral 
vectors (Naldini et al., 1996; Zufferey et al., 1998; Zufferey et al., 1997). Lentiviral vectors offer 
several potential advantages over conventional gammaretroviral vectors including higher 
efficiency, lack of dependence on cell division, and possibly a more favourable integration 
profile. This means that cell culture periods can be minimized, and that HSC activity can be 
more easily preserved. This may also mean that the risk of harmful mutagenesis during ex 
vivo cell culture is minimized. Recently, lentiviral vectors have been utilized in a clinical trial of 
HIV gene therapy in the US, and are currently in trial for adrenoleukodystrophy (Cartier et al., 
2009), -thalassemia in France (Cavazzana-Calvo et al., 2010) and Wiskott-Aldrich syndrome 
(GTAC 146) (where patients additionally receive full myeloablative alkylating agent 
conditioning). The lentiviral (LV) vector proposed for this clinical trial is a 3rd generation 
replication-defective hybrid viral particle made by core proteins derived from HIV and the 
envelope of the unrelated Vesicular Stomatitis Virus (VSV).  The system is based on 4 non-
overlapping expression constructs in order to maximize the segregation of cis and trans acting 
functions.  The lentivector is packaged using three separate plasmids, two expressing HIV 
proteins (gag/pol and rev), and the other expressing glycoprotein envelope protein of the VSV 
(VSV-G) combined with a self-inactivating (SIN) transfer vector carrying a major deletion in 
the 3’ long terminal repeat (LTR) region. The system is engineered in such a way that minimal 
homology regions are present between packaging and transfer vectors, thus minimizing the 
likelihood of homologous recombination events and the generation of replication competent 
lentiviruses (RCLs). The packaging construct is deleted of all HIV accessory proteins (vpu, 
vpr, nef, vif) and Tat. The packaging system segregates gag/pol and rev genes in two separate 
plasmids. The Rev responsive element (RRE) maintained in the gag/pol plasmid makes the 
gag/pol gene expression Rev dependent. As the transcripts of gag and pol genes contain cis 
acting repressor sequences, they are expressed only in the presence of Rev, expressed in 
trans on a separate plasmid, which promotes their nuclear export and expression by binding 
to RRE. The transfer vector encodes a codon optimised human ADA gene cDNA and 
sequences necessary for expression, incapsidation, reverse transcription and integration of 
the viral genome. In order to minimize the risk of RCL generation, the 3’ LTR of the transfer 
vector has been deleted in the U3 region. A mutated woodchuck post-transcriptional regulatory 
element (WPRE) sequence is incorporated in the vector to enhance transgene expression. 
 


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3.3.2 Rationale for using a lentiviral vector for gene delivery 
Lentiviral vectors were developed in the mid 1990’s with the potential advantage over retroviral 
vectors of being capable of gene transduction of non-dividing cells (Naldini et al. Science, 
1996).  Ideally, this will lead to more effective transduction of quiescent HSC with preservation 
of stem cell function.  Lentiviral vectors are more effective than retroviral vectors when 
compared using multiple surrogate assays for human HSC, including in vivo growth in 
NOD/SCID mice and LTCIC assays of quiescent CD34+/CD38- cells (Miyoshi et al., 1999; Bai 
et al., 2003; Case et al., 1999). Lentiviral vectors can perform effective transduction during 
just 1-2 days of culture; this shorter time of culture has been clearly proven to increase the 
survival of pluripotent stem cells (Mazurier et al., 2004). More effective gene transfer to long-
term reconstituting pluripotent hematopoietic stem cells may lead to more robust and rapid 
immune reconstitution than with using retroviral vectors.  Additionally, lentiviral vectors 
configured to lack strong the strong enhancer elements of retroviral long-terminal repeats, 
present in typical retroviral vectors, have been shown to have lower risks for causing 
insertional oncogenesis in a number of in vitro and in vivo models (Modlich et al., 2006; 
Modlich et al., 2009; Zychlinski et al., 2008; Montini et al., 2006). This may translate into a 
lower risk for causing lymphoproliferative disorders in gene therapy subjects. 
3.3.3 The EF1αS-ADA lentiviral vector 
Our laboratory has constructed an ADA cDNA lentiviral vector containing the 
enhancer/promoter from the human EF1-α gene (Figure 6). The EF1-α enhancer/promoter 
used is “shortened” by deletion of the first intron of EF-1α, which possesses strong enhancer 
activity and because of its potential to trans-activate adjacent cellular genes upon insertion. 
The “EF1αS” 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 retroviral LTR (Zychlinski et al., 2008). 
 
 
 
 
Figure 6: huADA cDNA = human ADA cDNA; EF1αS = elongation factor 1α short (see text for description); WPRE 
= woodchuck hepatitis post-transcriptional regulatory element. 
 
Hu ADA
EF1αS
Ψ
WPRE
The EF1αS-ADA Lentiviral Vector


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There have been no clinical trials to date utilising lentiviral vectors for gene-correction of ADA-
SCID.  We propose to use the EF1αS-ADA lentiviral vector with the human ADA cDNA to 
transduce autologous CD34+ cells from the bone marrow of ADA-deficient SCID patients.  We 
will evaluate transduction efficiency, gene marking, ADA levels, and immune function in ten 
(n=10) subjects.  We hypothesise that the EF1αS-ADA lentiviral vector will result in increased 
transduction efficiency of the CD34+ cells.  We anticipate that this increased transduction will 
result in better engraftment of gene-corrected stem cells, which will in turn result in increased 
gene marking, more rapid and robust production of gene-corrected lymphocytes that express 
ADA, and a reconstituted immune system. 
3.4 
Pre-clinical data 
3.4.1 Transduction and expression of ADA in cell lines 
In initial experiments, we compared the ability of the EF1αS-ADA lentiviral vector to express 
ADA in comparison to the current clinical grade gammaretroviral vector (Sfada/W) used in the 
present clinical trial GTAC 073. Following transduction of 3 different cell lines, ADA expression 
following transduction of cells with the EF1αS-ADA lentiviral vector (normalised for vector copy 
number) was either equivalent to or superior than the expression seen in cells transduced with 
the Sfada/W vector. This included an EBV transformed LCL from a patient with ADA deficiency 
(Fig 7). Total ADA activity is shown in Fig 7a and activity normalised for vector copy number 
is shown in Fig 7b. 
a 
 
 
 
b 
 
 
 
 
Figure 7: ADA expression/vector copy number following transduction of Jurkat cells, ADA deficient EBV-LCLs and 
normal T cells using a EF1αS-ADA lentiviral vector or the Sfada/W gammaretroviral vector - a) total ADA activity, 
b) activity normalised for vector copy number. 
ADA activity 
0
2
4
6
8
10
12
14
16
18
20
Jurkat
ADA -ve LCL
Normal T cells
Cell type
nmol/hr mg protein/vector copy
CCL-EFS-hADA
Sfada/W


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In further studies in our laboratory we compared the EF1αS-ADA lentiviral vector with a similar 
lentiviral vector in which the ADA gene was transcribed by an internal PGK promoter. A 
number of different cell lines were transduced including ADA-ve LCLs, primary T cells from a 
normal individual, Jurkat T cells, U937 cells and K562 cells. Viral supernatant of a similar titre 
and similar MOI were used for transduction purposes and in all cell lines other than the U937 
line, the EF1αS-ADA lentiviral vector showed a higher transduction efficiency when 
transduced cells were analysed by intracellular ADA analysis. Cells were then analysed for 
ADA activity above baseline levels (as a low level of ADA activity is found in all human cell 
lines) and activity normalised for vector copy number which was determined by vector specific 
qPCR. In all cell lines other than normal primary T cells, the EF1αS-ADA lentiviral vector 
showed markedly higher ADA activity/copy number than the PGK-ADA lentiviral vector (Figure 
8). Total ADA activity is shown in Fig 8a and activity normalised for vector copy number is 
shown in Fig 8b.  
a 
 
 
 
 
 
b 
 
 
 
 
 
Figure 8: ADA activity measured by HPLC and corrected for copy number in a variety of cell lines and in normal T 
cells - a) total ADA activity, b) activity normalised for vector copy number. 
 


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In similar experiments performed by collaborators at UCLA, transduction of HT29 (colon 
carcinoma), C.E.M. (T cell leukemia), and an HTLV-1-transformed T cell line from a patient 
with ADA-deficient SCID again showed increased ADA activity/vector copy in cells transduced 
with the EF1αS-ADA lentiviral vector. 
3.4.2 Transduction and expression of ADA in CD34+ cells  
To determine the ability of the EF1αS-ADA lentiviral vector to express ADA in human 
haematopoietic stem cells, CD34+ cells from cord blood or bone marrow of normal or ADA 
deficient patients respectively was obtained and transduced with the EF1αS-ADA lentiviral 
vector using the same cytokine cocktail to be used in the clinical transduction protocol. 
Following culture to allow cell number expansion, cells were stained and assessed for 
intracellular ADA expression by flow cytometry. Significant levels of ADA expression are seen 
in transduced cells from both ADA deficient and wild type CD34+ cells (Fig 9). 
 
 
 
 
 
 
 
 
 
Figure 9: Intracellular ADA expression following EF1αS-ADA lentiviral vector transduction of CD34+ cells from wild 
type and ADA deficient individuals (VCN=vector copy number). 
In further experiments, CD34+ cells bone marrow from an ADA deficient patient were 
transduced using either the EF1αS-ADA lentiviral vector, the PGK-ADA lentiviral vector or the 
gammaretroviral vector currently used in the GTAC 073 trial. Transduced cells were then 
grown on stromal cells in conditions conducive for development into T or NK cells (Schmitt 
and Zuniga-Pflucker, 2002). Viability and the extent of differentiation into the different lineages 
were similar for all 3 vectors. Following differentiation, the percentage of cells showing 
expression of ADA transgene was highest in those transduced with the gammaretroviral vector 


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and at slightly lower levels for those transduced with either the EF1αS-ADA lentiviral vector or 
the PGK-ADA lentiviral vector (Fig 10). 
 
Figure 10: CD34-ve cells from an ADA deficient patient were taken and transduced with the clinical grade 
gammaretroviral vector, pCCL EF1aS-ADA LLV vector or a pCCL PGK ADA LV vector and grown on a murine 
stromal layer expressing Notch-delta 1ligand. Following T cell differentiation the percentage of cells expressing 
ADA is shown. 
Studies of these lentiviral vectors were also performed using human CD34+ cells isolated from 
normal umbilical cord blood by collaborators at UCLA. The CD34+ cells were transduced using 
the culture conditions to be used for the proposed clinical trial, testing a range of vector 
concentrations (10
6, 10
7 and 10
8 TU/ml). The transduced CD34+ cells were grown in culture 
for two weeks after transduction, 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 
showed that transduction, as measured by vector copy per cell, is directly related to the 
concentration of vector during transduction, with 10
7 TU/ml leading to approximately 2 
copies/cell. Expression of the transferred ADA cDNA was at a similar level by a lentiviral vector 
with an internal viral LTR (pCSO-re-MCU3-hADA), the EF1αS-ADA lentiviral vector to be used 
in this clinical trial and a gammaretroviral vector.  
These in vitro studies demonstrate that in both cell lines and in primary progenitor cells, the 
EF1αS-ADA lentiviral vector is able to transduce cells at efficiencies comparable to 
gammaretroviral vectors used successfully in current gene therapy studies and is also able to 
express the ADA cDNA leading to ADA activity at similar levels to that shown by 
gammaretroviral constructs. 


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3.4.3 Transduction of CD34+ cells and expression of ADA in vivo  
In further experiments, we have transduced CD34+ cells selected from ADA-SCID patient 
bone marrow with either the EF1αS-ADA lentiviral vector (LV.SIN.EFS.hADA), the clinical 
SFFV LTR based clinical vector used in the current GTAC 073 trial (SFada) or a vector 
expressing GFP only (LV.SIN.EFS.eGFP) and engrafted transduced cells into 8-10 week old 
irradiated immunodeficient mice (NSG (NOD/SCID/gamma c-/-) mice) (Fig 11). Six mice were 
used in each group although one of the SFada cohort died. In both bone marrow (Fig 11a) 
and the spleen (Fig 11b) of recipient NSG mice, cells transduced with either the 
LV.SIN.EFS.hADA or SFada construct engrafted at similar levels whereas no evidence of 
CD45+ cells was seen in mice receiving cells transduced with a GFP containing vector. 
Development of CD19 B cells or CD3 T cells in the spleen was similar between the two 
constructs. B cell differentiation was the most abundant cell type seen in the bone marrow or 
spleen and formed the vast majority of engrafted human CD45+ cells. 
 
a 
 
 
 
 
 
 
b 
 
 
 
 
 
 
Figure 11: Transduction of ADA deficient CD34+ bone marrow with the LV.SIN.EFS.eGFP, SFada and 
LV.SIN.EFS.hADA vectors and engraftment into immunodeficient NOD/SCID/γc-/- murine recipients. a) levels of 
bone marrow engraftment of hCD45+ cells, CD34+ cells, CD19+ B and CD13+ myeloid cells, b) levels of splenic 
engraftment of hCD45+ cells, CD19+ B cells and CD3+ T cells and TCRα/β. 
These studies show that following transduction with the EF1αS-ADA lentiviral vector, ADA 
deficient CD34+ cells are able to engraft and differentiate in a human xenograft model at levels 
that are similar to that seen following transduction with the current SFada vector. 


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3.4.4 SIN lentiviral vector promotes immune recovery in a murine model of ADA 
deficiency 
Early models of ADA deficient mice were limited by embryonic or peri-natal lethality as a result 
of severe hepatocellular and pulmonary damage. A two stage transgenic strategy in which 
ADA expression is restricted to trophoblasts rescues ADA null mice from pre- and peri-natal 
lethality and allows post natal study of mice lacking ADA expression. In this model, many 
features of the human disease are accurately represented. Mice show abnormalities of thymic 
architecture with a significant block in thymocyte development at the CD4-CD8- double 
negative stage. In vitro analysis of thymocytes from these mice also demonstrates defects in 
later stages of thymocyte development due to the toxic effects of dATP and deoxyadenosine 
accumulation. In the periphery, there is a profound lymphopaenia with a severe T, B and NK 
cell deficiency that is seen in humans together with abnormalities in splenic lymphoid 
architecture and a paucity of splenic T cell populations. Murine ADA deficiency also manifests 
a number of non-immunological abnormalities including lung, renal, costochondral and 
neurological changes. Pulmonary problems are present in mice from ~postnatal day 12 and 
are associated with significant lung inflammatory changes, eventually leading to the death of 
mice by postnatal day 22 from pulmonary insufficiency, unless early treatment is initiated. Mice 
die by three weeks of age but can survive in response to enzyme replacement therapy with 
PEG-ADA (see below). The immunological and systemic defects found in this murine model 
resemble closely many features of the human disease and this model is therefore an ideal 
system in which to test the ability of new ADA vectors to correct the disease phenotype. 
The ADA-/- mouse colony was purchased and established at the ICH mouse facility. Murine 
lin-ve cells were either transduced using standard protocols with 1) the LV.SIN.EFS.hADA 
(n=3) or 2) SFada (n=2). Cells were returned to lethally irradiated recipients. Mice were 
analysed after 12 weeks and compared with ADA-/- mice, mice that had undergone wild type 
transplant experiments (WT) or mice that had been treated with enzyme replacement therapy 
alone. Mice reconstituted with the LV.SIN.EFS.hADA vector have increased numbers of total 
cells in the spleen in comparison to ADA-/- mice (Fig 12a). The recovery of total cell numbers 
and of specific lymphocyte subpopulations including CD3, CD4 and CD8 T cells, are similar 
to that seen in mice reconstituted with SFada vector transduced cells and mice treated with 
wild type cells or PEG-ADA replacement therapy. Similarly analysis of peripheral blood (Fig 
12b) shows very low % of T cells in the mononuclear fraction of ADA-/- mice whereas 
reconstituted mice with both vectors, WT cells or PEG-ADA treatment all show a marked 
increase in T cell recovery. B cell reconstitution is again similar between the different treatment 
groups. A representation of the equivalence in T cell reconstitution is shown (Fig 12c). 
Importantly, the vector copy number in the peripheral blood of mice transduced with either the 


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LV-EFS or SFada vector were similar suggesting that even with similar levels of virus 
transduction, equivalent levels of T cell reconstitution can be achieved (Fig 12d). ADA 
enzymatic activity was similar in both the marrow and spleen of mice transduced with LV-EFS 
vector in comparison to mice treated with SFada vector, WT transplants or PEG-ADA and 
were greatly increased from levels seen in ADA-/- mice. Similar results were also seen in a 
non-immune organ such as the lung (Fig 12e). 
Figure 12 
a 
 
 
 
 
 
b 
 
 
 
 
 
 
c 
 
 
 
 
 
d 
 
 
 
 
 
 
 
e 
 
 
 
 
 


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In the thymus, the major abnormalities in the ADA-/- mice are a block in thymocyte 
development with an accumulation of CD4-CD8- thymocytes. Reconstituted mice with either 
vector or with WT or PEG-ADA treatment show similar levels of reduction of this population 
(fig 13).  
Figure 13 
a 
 
 
 
 
 
b 
 
 
 
 
 
 
 
 
 
 
 
 
Importantly, all the reconstitution experiments in ADA-/- mice were undertaken with clinical 
grade LV.SIN.EFS.hADA vector demonstrating that the clinical grade vector has equivalent 
immune reconstitution potential to the current SFada clinical vector that has been used 
successfully in clinical trials. 
3.4.5 Genotoxicity of the EF1αS-ADA lentiviral vector  
It is clear from pre-clinical and clinical trial data that there is a need to move away from the 
use of gammaretroviral vectors which have the potential for insertional mutagenesis to the use 
of vector designs which have reduced transformation potential. The EF1αS-ADA lentiviral 
vector design is likely to be significantly less mutagenic than current gammaretroviral vectors 
for two major reasons: 
• 
The use of the self-inactivating (SIN) lentiviral vector backbone  
• 
The use of the internal EF1αS promoter for transgene transcription rather than the viral 
LTR 
3.4.5.1 SIN Lentiviral vector constructs are safer than LTR intact gammaretroviral 
constructs 
The risk of insertional mutagenesis from a SIN lentiviral vector design in general is low as 
determined in both in vitro and in vivo systems. One in vitro assay has now been developed 
and tested by a number of groups and is gaining recognition as the most reliable test of the 
transformation potential of different viral constructs. The in vitro immortalisation assay (IVIM) 


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(Modlich et al., 2006; Modlich et al., 2009) detects clonal dominance of insertional mutants 
which expand in initially polyclonal cultures of primary murine haematopoietic cells within two 
weeks after gene transfer and acquire serial replating ability, the extent of which provides a 
measure of clonal fitness. When different vector designs were tested in this assay the SIN 
lentiviral vector design shows significantly less transformation potential than current LTR intact 
gammaretroviral designs (Modlich et al., 2006; Modlich et al., 2009). 
To test the oncogenic capacity of SIN-lentiviral vectors in vivo, Montini et al., utilized the tumor-
prone Cdkn2a-/- mouse model (Montini et al., 2006). Haematopoietic stem cells from these 
mice were isolated and transduced in similar conditions with SIN LV or LTR intact 
gammaretroviral constructs expressing reporter genes. A vector with a similar backbone as 
the proposed ADA vector, but expressing a neutral eGFP reporter gene, was tested in this 
system and no genotoxicity was found even upon integration of ~6 copies of vector per cell. 
In contrast, LTR-active gammaretroviral vectors were measurably genotoxic, suggesting that 
the intrinsic genotoxicity of the pRRL LV (the backbone used in this study) is low. 
3.4.5.2 The EF1αS promoter is safer than the viral LTR 
Using the IVIM assay, the EF1αS promoter was compared under equivalent conditions to the 
SFFV LTR in the context of a SIN gammaretroviral vector (Zychlinski et al., 2008). In this assay 
a SIN vector with the internal EF1αS promoter was significantly (P<0.01) less mutagenic than 
a SIN vector with the internal SFFV promoter. Under conditions that resulted in death of mock-
transduced cells, the SIN vector containing an internal EF1αS promoter was unable to induce 
sustained growth of mutants, even when the average copy number was >40. In contrast, 
transformed clones were always observed with the SIN vector containing the internal SFFV 
promoter, and with this vector, an average copy number of ~2 was sufficient to induce 
transformation. This indicates that the transactivating potential of the SIN vector with the 
EF1αS promoter is at least substantially diminished. Of note, SIN vectors containing ‘insulator’ 
elements flanking the internal SFFV promoter-driven cassette showed a significantly greater 
degree of transforming activity that the SIN vector with the EF1αS promoter (4/4 experiments; 
P< 0.01). Together these studies indicate that the transactivating potential of gammaretroviral 
vectors are substantially reduced by SIN configurations (Modlich et al, 2006), and further 
reduced by the utilization of EF1αS internal regulatory sequences. 
Further in vivo studies have also established the low transformation capacity of SIN vectors 
containing the EF1αS promoter. These experiments were conducted as safety studies for a 
trial of gene therapy for SCID-X1 (GTAC 116) and are relevant here because of the use of the 
same EF1αS promoter. SIN gammaretroviral constructs containing the EF1αS promoter and 
driving expression of the IL2RG or GFP reporter transgene were evaluated in murine 


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transplant experiments in wild type C57/BL6 experiments. Briefly lin-ve cells from donor mice 
were isolated and transduced under standard conditions with vectors containing EF1αs-IL2RG 
or EF1αS-GFP or media alone (mock controls). Twenty mice were transplanted for each 
experimental group and were observed for a period of 10-12 months. There were 17 evaluable 
mice in the IL2RG group, 17 evaluable mice in the EGFP group and 18 evaluable mice in the 
mock group. On average, there was 60-80% engraftment in each group. The average vector 
copy number in peripheral blood mononuclear cells, determined by real-time PCR at 2 months 
post-transplant was 1.25 copies/cell for the IL2RG test group and 2.2 copies/cell for the EGFP 
control group. Two host cell derived malignancies were noted in the test IL2RG group. One 
vector-negative thymoma and an ovarian tumor were noted in the GFP vector group. Host cell 
derived malignancies are not unexpected in this mouse model, and the frequency is not higher 
than previously reported values by our group. There were no cases of vector positive donor 
cell derived leukemias in the IL2RG test group. There was one vector positive, donor cell 
derived T cell lymphoma noted in a C57BL/6J recipient mouse which received the EGFP 
control vector. These data suggest that in the context of a SIN configuration and coupled with 
the IL2RG gene, the EF1αS promoter has not been associated with demonstrable 
tumorigenesis in wild type murine transplant experiments.  
3.4.5.3 Data from other similar LV constructs 
Some surrogate information can also be gained from analysis of another SIN LV vector. The 
EF1αS-ADA lentiviral vector backbone for this study is the same as that approved in a trial of 
lentiviral gene therapy for Wiskott-Aldrich (WAS) syndrome (GTAC 146). The promoter and 
transgene for the WAS vector have been removed and replaced with the EF1αS-ADA cassette 
but the other structural elements of the virus remain the same. The WAS vector has been 
tested in vivo in wasnull recipients with a follow up of 12 months. wasnull mice transplanted with 
cells transduced with the WAS vector at high or low copy number had no difference in survival 
or tumour formation in comparison to wasnull mice transplanted with untransduced cells from 
WT or wasnull mice. Tumours seen were either of non-haematopoietic origin or were derived 
from host cells suggesting that tumour formation was not driven by vector integration. A similar 
frequency of tumours was seen in control cohorts (Marangoni et al., 2009). These data 
suggest that this WAS SIN LV vector construct has no significantly increased risk of tumour 
formation compared to standard transplant experiments. The EF1αS-ADA lentiviral vector is 
therefore likely to show similar safety to the above construct. 
3.4.5.4 IVIM assay to test genotoxic potential of the EF1αS-ADA lentiviral vector 
We have set up the IVIM assay at our laboratory and tested the transformation ability of SFFV 
LTR driven vectors expressing GFP (SF91.GFP) or dsRED (SF91,dsRED) reporter genes, 
the current SFFV LTR clinical gammaretroviral vector used in GTAC 073 (SFada/W) and the 


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EF1αS-ADA lentiviral vector. In consecutive experiments, cells transduced with the LTR driven 
gammaretroviral vectors give rise to transformed clones with replating ability whereas no 
transformed clones above background (or mock transduced) levels were detectable in cells 
transduced with the EF1αS-ADA lentiviral vector (Table 2) despite equivalent or higher vector 
copy numbers. In this assay, the genotoxic potential of the EF1αS-ADA lentiviral vector is 
considered to be low and significantly lower than the transformation potential of the vector 
currently used in clinical trials. 
 
Table 2: IVIM data 
 
 
 
 
 
 
 
In order to test objectively whether clones derived following transduction were viable and had 
significant cytokine independent proliferative capacity, clones were analysed using the WST-
1 assay. The assay is based on the reduction of WST-1 by viable cells which produces a 
soluble formazan salt. The procedure involves initial incubation of cells with the WST-1 
reagent, followed by spectrophotometric assay of coloured product. 
In two experiments independent experiments, any clones derived from cells transduced with 
the SFada/W, SF91.GFP, EF1αS-ADA LV vectors or mock transduced cells were studied 
further in the WST-1 assay. As seen in Fig 12, in both experiments, clones derived following 
SFada/W or SF91.GFP transduction showed levels of proliferation that were far higher than 
the baseline proliferation seen in mock transduced clones. In contrast any clones derived 
following EF1αS-ADA LV transduction showed negligible proliferative capacity and were no 
greater than levels of proliferation seen in mock transduced cells. 
 
 


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Figure 12: Proliferative capacity of clones generated following transduction were tested in a WST-1 assay. Clones 
from SFada/W and SF91.GFP transduced cells show high levels of proliferation whereas clones from EF1αS-ADA 
LV transduced cells have levels of proliferation no higher than that seen in mock transduced cell clones. 
3.4.6 Clinical grade vector is able to effectively transduce human ADA-/- CD34+ stem 
cells 
Clinical grade LV.SIN.EFS.hADA vector was characterised for its ability to transduce CD34+ 
cells from ADA-/- patients. Using clinical grade reagents and a clinical protocol (see section 
7.6.4) involving 18 hrs prestimulation and followed by 1 round of lentiviral transduction for 
24hrs, either 1 x 108 (~MOI 100) or  5 x 107 (~MOI 50) viral genomes were added to ~1 x 106 
CD34+ cells. As shown in table 3a, these clinically applicable transduction conditions resulted 
in effective CD34+ cell transduction with viral copy numbers of ~1–1.3 copies/cell. In addition 
there was preservation of CD34+ integrity with little change in the percentage of stem cells 
through the culture period form D0–D3 (table 3b). 
Table 3 
a 
 
 
 
b 
 
 
 


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These data show that using a clinically applicable protocol, the clinical grade vector generated 
for this study is able to show effective transduction of CD34+ cells from ADA deficient patients 
at levels that are acceptable for clinical use and maintain CD34+ cell integrity. 
3.5 
Continuation of PEG-ADA for 1 month after infusion of gene modified cells 
There has been considerable debate as whether PEG-ADA either blunts or improves the 
survival of gene modified cells. In previous clinical trials, PEG-ADA has been withdrawn prior 
to the infusion of gene-modified cells. 
Two lines of evidence now suggest that continuation of PEG-ADA may be of benefit: 
a) In a recently published murine study (Carbonaro et al., 2012) demonstrated that the 
continued use of PEG-ADA for 1 month after gene modified cell infusion did not 
diminish immune reconstitution but in fact allowed better thymic development possibly 
due to its protective effect on thymic epithelium. 
b) In two ADA-patients treated off trial on a compassionate basis, we also continued PEG-
ADA for 1 month after gene modified cell infusion. In both cases PEG-ADA was 
continued because of the clinical state of the patients and the concern that removal of 
enzyme may have adverse clinical consequences. In both cases we have seen 
engraftment of gene modified cells and emergence of naïve T cell cells at 6 and 7 
months post treatment. 
From this we would suggest that 1) continued PEG-ADA usage is not detrimental to gene 
modified cell engraftment, 2) it may promote improved thymic T cell development and 3) its 
continued use is clinically safer for patients. 
For these reasons we intend to continue PEG-ADA for 1 month after infusion of gene modified 
cells. 
3.6 
Busulfan dosage 
The busulfan dosage to be used in this study differs from previous studies. In this study, weight 
adjusted iv busulfan dosing will be given as detailed below: 


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This allows us to use the maximum daily dosage of iv busulfan and is based on European 
Society for Blood and Marrow Transplantation (EBMT) guidelines for Busulfan dosing in 
children and a recent publication (Bartelink et al., 2012). 
The reason for the change is to achieve the correct balance between level of conditioning and 
safety. A higher busulfan dose will allow a greater degree of myeloablation and therefore 
greater engraftment of gene modified cells. This dosing scheme has recently been used in 
two ADA-SCID patients treated off-trial on a compassionate basis and in both cases the dose 
(both received 5.1mg/kg) was well tolerated with no toxicities. 
3.7 
Summary and rationale for use of the EF1αS-ADA lentiviral vector for gene 
therapy 
• 
Gammaretroviral vector mediated gene therapy for ADA has shown impressive 
efficacy and safety long term in 3 international studies thus far. 
• 
Safety has been excellent with 100% survival and no serious adverse events but 
concerns regarding the insertional profile of gammaretroviral vectors need to be 
addressed. 
• 
Lentiviral vectors encoding the EF1αS internal promoter driving human ADA show 
efficacy of transduction and expression that is comparable to gammaretroviral 
constructs in vitro. 
• 
Lentiviral vectors have an insertional profile that differs from gammaretroviral 
constructs and show decreased potential for transformation in vitro and in in vivo 
tumour prone models. 
• 
The LV.SIN.EFS.hADA vector shows decreased transformation potential in an in vitro 
model of stem cell transformation and in in vivo transplantation experiments the 
EF1αS-promoter in a SIN configuration does not show transformation potential. 


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• 
The LV.SIN.EFS.hADA vector shows effective reconstitution of T cell numbers and 
ADA activity in a murine model of ADA-/- deficiency and is comparable to the current 
clinical grade SFada gammaretroviral vector. 
• 
The LV.SIN.EFS.hADA vector shows effective transduction of ADA-/- CD34+ cells and 
leads to successful engraftment in NSG xenograft experiments and is comparable to 
the current clinical grade SFada gammaretroviral vector. 
• 
A clinically applicable protocol for transduction of ADA-/- CD34+ cells leads to levels 
of stem cell transduction that are applicable for clinical use. 
• 
Lentiviral vector mediated gene therapy for ADA-SCID has potential for high levels of 
efficacy and improved safety. 
4 
Trial Design 
This is a historical controlled, non-randomised phase I/II clinical trial to assess the safety and 
efficacy of autologous transplantation of CD34+ cells from ADA-deficient SCID infants 
following human ADA cDNA transfer by the EF1αS-ADA lentiviral vector with comparison of 
outcomes to historical control group. Subjects will receive the infusion of their autologous, 
transduced cells following marrow cytoreduction with busulfan. 
4.1 
Patients treated with gene therapy (on study) 
• 
Patients will be selected on the basis of inclusion and exclusion criteria as detailed 
below 
• 
A back-up harvest will be retained in case of failure of reconstitution following 
conditioning and re-infusion of transduced cells 
• 
For the transduction protocol (see appendix 3 for schematics), CD34+ cells will be 
purified from G-CSF mobilised Peripheral Blood Stem Cell (PBSCs) recovered by 
leukapheresis (Plerixafor may be added to enhance mobilization), whenever possible 
or from bone marrow harvested under general anaesthetic. Back-up cells of 3x106 
CD34+/kg will be stored from a single procedure if sufficient cell counts available (and 
if not previously stored). The remaining CD34+ cells will be taken forward for 
transduction. If <3x106 CD34+ cells/kg are harvested from the back-up, the patient will 
be withdrawn from the study 


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• 
Patients in whom of <1 x 106 CD34+ cells/kg are available for transduction will not 
proceed to conditioning, will be withdrawn from the study and will be offered a repeat 
harvest in approximately. 3 months 
• 
Patient conditioning will be initiated immediately after bone marrow or PBSC collection 
(busulfan iv. weight adjusted dose) 
• 
After purification, CD34+ cells will be immediately transduced with the lentiviral vector 
as detailed below, then evaluated for sterility, cell count, and viability, and immediately 
infused into patients 
• 
Several other tests for safety including testing for RCLs will be performed on re-infused 
cells and results will be recorded retrospectively 
• 
If less than 0.5 x 106 viable transduced CD34+ cells/kg are returned to the patient, then 
back-up bone marrow/PBSCs will be returned and patients will be withdrawn from the 
study  
• 
PEG-ADA will be withdrawn one month (+/- 6 days) post-infusion  
• 
If there is no bone marrow recovery by 6 weeks (ANC<0.5 x 109/L or platelets < 20 x 
109/L) the back-up harvest will be re-infused 
• 
Patients will be followed up on study at 1 month, 6 weeks, 3, 6, 9, 12, 18, 24, 30 and 
36 months post gene therapy. Visit windows will be  +/- 5 days for visits at 1 month, 6 
weeks and +/- 2 weeks for visits at 3, 6, 9, 12, 18, 24, 30 and 36 months post gene 
therapy If after 180 days there is no evidence of the transgene in PBMCs by qPCR or 
there is no evidence of T cell recovery, then patients will re-start PEG-ADA and will be 
withdrawn from the study 
PEG-ADA may be restarted at PIs discretion prior to this time point on clinical grounds e.g 
infective problems or delayed immune reconstitution and at this stage will be withdrawn from 
the study.  
4.2 
Patients treated with gene therapy (off study): CUP group 
In addition to the patient treated on trial, another sub-group of patients treated on 
compassionate basis (CUP) with the same gene therapy under GOSH License, that followed 
the procedure of this protocol are included within this protocol and will be part of the analysis 
(see section 8.9). Details about this group of patient can be found in Appendix 9.  
4.3 
Historical control group: HSCT group  


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Historical HSCT data from a database of ADA-SCID patients treated with allogeneic HSCT 
from GOSH will be collected as comparator group and considered for the analyses (see 
section 8.9). Details about this group of patient can be found in Appendix 9.  


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Trial summary   
 
4.2 
Trial objectives 
4.2.1 Primary objectives 
The primary objective is to assess the safety and efficacy of EFS-ADA LV mediated gene 
therapy, the Investigational Medicine Product (IMP) for treatment of ADA-SCID patients. This 
will be achieved via the following specific objectives: 
Safety 
1. To assess clinical, haematological and immunological progress of patients 
2. To assess vector integration sites and clonal proliferation. This analysis will be 
performed at a specialised laboratory in Germany and the data will be reviewed and 
interpreted by the PI and the study team  
Efficacy 
1. To assess overall survival (OS) at 1 year for patients treated with IMP 
2. To assess event free survival (EFS, defined below in Section 5.2.2) at 1 year for 
patients treated with IMP 
o To compare OS and EFS at 1 year between patients treated with IMP and 
patients treated with allogeneic HSCT 
3. To assess at each visit engraftment success and resulting immunological and 
metabolic effects in IMP treated patients using:  


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a. Vector copy numbers in peripheral blood leukocytes 
b. Cellular and humoral immune system recovery 
c. ADA activity and reduction in dATP in peripheral blood cells 
4.2.2 Secondary objectives 
The secondary objectives are to: 
• 
Compare survival and event free survival at 2 and 3 years between patients treated 
with IMP and patients treated with allogeneic HSCT 
• 
Determine the percentage of IMP treated patients requiring immunoglobulin 
replacement therapy at all evaluation points from 18 months onwards  
• 
Compare frequency of infections, and growth of pathogenic microorganisms over 3 
years 
• 
Evaluate the longitudinal clinical effect in terms of improved immunity 
• 
Evaluate tolerability of conditioning regimen. These data will be reviewed and 
interpreted by the PI and the study team 
• 
Evaluate feasibility of the transduction procedure. These data will be reviewed and 
interpreted by the PI and the study team 
4.3 
Study endpoints 
4.3.1 Primary endpoints 
• 
To assess survival at 1 year for patients treated with IMP. 
• 
To assess event free survival at 1 year for patients treated with IMP. 
o To compare survival and event free survival at 1 year for patients treated with 
IMP compared to patients treated with allogeneic HSCT. 
• 
Success of engraftment of transduced cells as assessed by vector copy number in the 
cells at each study visit. 
• 
Reconstitution of cell mediated and humoral immunity as assessed by evidence of 
changes in T cell number, function and circulating immunoglobulin levels at each study 
visit. 
• 
Correction of metabolic abnormalities as assessed by dATP and evidence of ADA 
activity in erythrocytes at each study visit compared to published data from HSCT 
treated patients 


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The following will be analysed at a specialist laboratory in Germany and the results reviewed 
and interpreted by the PI and the study team.  
• 
Analysis of frequency of vector integration into known proto oncogenes  
• 
Analysis of frequency of clonal expansion associated with vector integration near proto 
oncogene 
4.3.2 Secondary endpoints 
1) 
Survival and Event free survival at 2 years and 3 years 
2) 
Reduction in frequency of infections (evaluated at 1, 2 and 3 years after 
treatment based on clinical history and examination)  
3) 
Long term immune reconstitution as assessed by sustained improvement in 
thymic function assessed by TRECS after 2 years post-gene therapy 
4) 
Serum immunoglobulin levels for IgA, IgG and IgM and patients requiring 
immunoglobulin replacement therapy (recorded dichotomously).  
5) 
Feasibility of the transduction procedure assessed by availability of greater 
than 0.5 x 106 CD34+ cells/kg after transduction; undetectable RCL (determined 
retrospectively); and CD45+ cell viability after transduction equal to or greater than 
50%, in accordance with the final product release criteria. These data will be reviewed 
and interpreted by the PI and the study team. 
4.3.3 Additional Endpoints 
1) 
T Cell V Beta Panel 
2) 
Phytohaemagglutinin (PHA) stimulation Result 
3) 
CD3 Stimulation 
4) 
Integration Analysis Result 
5) 
Bone Marrow Aspirate (if performed) 
Morphology (Normal/ Abnormal). 
Integration (Normal/ Abnormal). 
Vector copy number in BM-MNC. 
6) 
Tetanus Vaccination Response 


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5  
Patient selection and recruitment  
5.1 Patients treated with gene therapy (on trial) 
Up to 10 treated patients will be recruited from the Immunology Unit at Great Ormond Street 
Hospital. Referrals for enrolment in the study will be accepted from centres worldwide. 
Individuals will be selected for inclusion on the basis of the following defined criteria: 
5.1.1 Inclusion criteria  
1. Diagnosis of ADA-SCID confirmed by DNA sequencing OR by confirmed absence of 
<3% of ADA enzymatic activity in peripheral blood or (for neonates) in umbilical cord 
blood erythrocytes and/or leucocytes or in cultured foetal cells derived from either 
chorionic villus biopsy or amniocentesis, prior to institution of PEG-ADA replacement 
therapy 
2. Patients who lack a fully HLA-matched family donor 
3. Patients (male or female) < 5years of age OR 
Patients (male or female) ≥ 5years to 15rs of age who have preserved thymic function 
as evidenced by presence of >10 % naïve T cells (CD4+45RA+27+ cells) 
4. Parental/guardian signed informed consent 
5.1.2 Exclusion criteria  
1. Cytogenetic abnormalities on peripheral blood 
2. Evidence of active malignant disease 
3. Known sensitivity to busulfan 
4. If applicable, confirmed pregnancy (to be tested in patients above 12 years old) 
5.1.3 Justification for enrolment criteria 
In this study gene therapy will be offered in preference to a matched unrelated donor HSCT. 
To date over 30 patients have been treated with a gene therapy protocol involving cessation 
of PEG-ADA and non-myeloablative conditioning. All patients have survived the treatment and 
~70% have been able to stop PEG-ADA. Furthermore, patients who have failed gene therapy 
have been able to restart PEG-ADA without any compromise to clinical or immunological well-
being. In contrast, MUD transplants have a 67% survival (n=15). For these reasons, gene 
therapy offers a safer initial alternative and carries a high rate of success. If gene therapy fails, 


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patients can restart PEG-ADA or proceed to a transplant without compromising the success 
of the latter procedure. 
Older patients have been shown to have a poorer response to gene therapy for ADA-SCID 
and also for SCID-X1 (Thrasher et al., 2005; Aiuti et al., 2009). This most probably relates to 
the low level of residual thymic function seen in older SCID patients as a result of diminished 
population of thymic tissue and also in the case of ADA-SCID ongoing thymic toxicity. 
This also suggests that in the case of older patients (>5yrs of age), gene therapy will be of 
greatest benefit in those who have preserved thymic function as evidenced by the presence 
of > 10 %  naïve t cells (CD4+45RA+27+ cells) and TRECs.  
5.2 Patients treated with gene therapy (off trial): CUP group 
A group of patients were treated under the special compassionate group program (GOSH 
special license) either because the study was not yet open and patients needed urgent 
treatment, or because they were outside of the inclusion/exclusion criteria or received followed 
a different process (ie, received the Imp in two infusions). Patients followed however the same 
protocol steps and study visits. For this reason, CUP patients are included in the analysis, as 
described in the Statistical Analysis Plan (SAP) 
5.3 Historical Control group: HSCT group  
A group of historical retrospective data has been collected from GOSH ADA-SCID patients 
treated with HSCT from year 2000. This group will be use as comparator data vs patients 
treated with gene therapy, to evaluate survival and event free survival, as described in the 
SAP. Below are the inclusion criteria for this population 
Inclusion criteria 
1. Diagnosis of ADA-SCID confirmed by DNA sequencing OR by confirmed absence of 
<3% of ADA enzymatic activity in peripheral blood or (for neonates) in umbilical cord 
blood erythrocytes and/or leucocytes or in cultured foetal cells derived from either 
chorionic villus biopsy or amniocentesis, prior to institution of PEG-ADA replacement 
therapy 
2. Patients (male or female) between 0-18 years at time of treatment 
3. Patient treated with allogeneic haematopoietic stem cell transplantation since 2000 
5.4 
Concomitant medication 
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 


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withdrawn from cotrimoxazole prophylaxis at least day -1 prior to IMP infusion. Cotrimoxazole 
will be restarted when ANC > 1 x 109/l on 3 consecutive days after reinfusion of transduced 
cells. Aerosolised or intravenous pentamidine will be used as prophylaxis for Pneumocystis 
jirovecii in the intervening time period.  
PEG-ADA will be continued until one month (+/- 6 days) post-infusion of gene-modified cells. 
PEG-ADA will be restarted if i) the child is withdrawn from the study ii) if after 180 days there 
is no evidence of gene modified cell engraftment and/or failure of T cell recovery. 
PEG-ADA may be restarted at PIs discretion prior to this time point on clinical grounds e.g 
infective problems or delayed T cell reconstitution. 
6 
Enrolment of subjects 
Patients will be enrolled following diagnosis and referral to GOSH. ADA is an inherited disease 
and infants normally present in the first year of life. Patients less than 5 years of age will be 
enrolled and also those >5 years who show evidence of thymic function will also be eligible 
for enrolment. Informed consent will be obtained from parents/guardians and assent where 
appropriate.  
The Chief/Principal investigator will discuss the study at length with the parent/guardian of a 
potential new subject. A printed information leaflet will be supplied and the parent/guardian 
given ample time to consider his/her decision. The parent/guardian will be encouraged to ask 
further questions about the study to the Investigator or the clinical nurse specialist. Interpreters 
will be used if English is not the native tongue. Patient Information Leaflet (PILs) will be 
translated into native language. 
Should a parent/guardian decide that the patient will participate they will be invited to sign the 
study consent form. 
Consent will take place prior to any study specific procedures. 
Once a patient has been enrolled onto the study, the ADA-SCID pre-gene therapy patient 
protocol (see Appendix 4 for details) will be completed, reviewed, signed off by the investigator 
and distributed to all concerned parties. 


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7 
Treatment protocol 
7.1 
Informed consent procedure 
ADA deficiency is an inherited disease and therefore most patients with this inherited disease 
are diagnosed while they are still infants and are therefore incapable of giving informed 
consent.  Therefore, it is expected that the majority of the informed consent will be obtained 
from parents/guardians by the Chief investigator or co-investigator. 
The Chief investigator/co-investigator will discuss the study at length with the parent/guardian 
of a potential new subject, including an explanation of the aims, methods, anticipated benefits 
and potential risks of the study.  The Chief investigator/co-investigator will explain that the 
patients are under no obligation to enter the trial and that they can withdraw at any time during 
the trial, without having to give a reason. 
A patient information leaflet will be supplied and the parent/guardian will be given ample time 
to consider his/her decision.  The parent/guardian will be encouraged to ask further questions 
about the study to the Investigator or the clinical nurse specialist. 
Should a parent/guardian decide that the patient will participate they will be invited to sign the 
study consent form.  A copy of the patient information leaflet and signed consent form will be 
provided to the patient to take home.  A further copy will be retained in the patient’s medical 
notes and the original will be filed in the Trial Master File.  
Informed consent will be obtained before any trial-related procedures are performed. 
7.2 
Randomisation procedures 
This section is not applicable as the trial is non-randomised. 
7.3 
Emergency unblinding  
This section is not applicable as this is an open-label trial. 
7.4 
Screening assessments  
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 contraindication which would exclude 
them from the trial.  


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7.5 
Treatment procedures 
7.5.1 Harvest of a back-up autologous graft 
If a back-up has not previously been stored, haematopoietic stem cells will be collected from 
the patient as a back-up at the time of leukapheresis (on day -3) or bone marrow harvest (on 
day -2) (see section 7.5.2). A back-up of 3 x 10 6 CD34+/kg will be stored.  
The total cell count required for both back-up (3 x 106 CD34+ cells/kg) and for transduction (1 
x 106  CD34+ cells/kg) is 4 x 106 CD34+ cells/kg.  
If insufficient cells are collected then the total collection will be stored as back-up, the patient 
will be withdrawn from the trial and a further collection will be attempted 2-3 months later in 
order to meet the criteria specified above. 
The back-up cells serve as a salvage procedure, should there be a lack of haematopoietic 
recovery observed following the injection of genetically-manipulated cells (back-up graft).  
The bone marrow or leukapheresis cells will be frozen and stored un-manipulated in liquid 
nitrogen vapours (-1620C and -1800C) to constitute the back-up graft. The back-up will be used 
in case the final cell dose is very low as specified or if no haematopoietic recovery is observed 
after 6 weeks, following the gene therapy treatment. 
7.5.2 CD34+ cells for gene therapy 
CD34+ cells for the gene therapy will be obtained preferably by leukapheresis (possibly 
augmented with Plerixafor) of G-CSF mobilised peripheral blood stem cells (PBSCs). If PBSC 
leukapheresis is not clinically appropriate or is contraindicated or where mobilisation fails, a 
bone marrow harvest will be performed. PBSCs are the preferred choice since we will obtain 
far higher numbers of CD34+ cells which in turn will allow gene modification of larger stem cell 
numbers prior to reinfusion into the patient. Autologous cells will be stored if cell count is >4 x 
106 CD34+ cells/kg (back-up: 3 x 106 CD34+ cells/kg and cells for transduction: 1x106 CD34+ 
cells/kg).  
For PBSC collection, recombinant human granulocyte colony stimulating factor (rhu G-CSF) 
will be administered by subcutaneous injection (usually in the abdominal region) for 5 to 6 
successive days (dose 5-16 mg/kg per day) (possibly augmented with Plerixafor) prior to 
harvest. CD34+ cell counts will be monitored from day 5, and leukapheresis performed on 
days 5 and/or 6 providing that the CD34+ cell count is greater than 1 x 104/ml. If a bone marrow 
harvest is to be used as the source of CD34+ cells, bone marrow will be harvested from the 
patient under general anaesthesia from the posterior iliac crests on both sites by multiple 
punctures. The amount of marrow collected will be equivalent to 20 ml/kg of body weight. This 


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will give a total nucleated cell count of greater than 108cells/kg. This in turn should yield a 
CD34+ cell dose of greater than 106cells/kg after CD34+ cell selection.  
Patients from whom <1 x 106 CD34+cells/kg are available, will be withdrawn from the study 
prior to conditioning. A repeat harvest will be considered after a period of 2-3 months and the 
patient will be re-enrolled onto the study. 
Patients withdrawn from the study prior to administration of transduced CD34+ cells will 
resume normal clinical care. Efficacy and safety assessments will not be carried out from the 
point of withdrawal and data will not be recorded in the Case Report Forms (CRFs).  
7.5.3 CD34+ cell purification 
CD34+ cells will be separated by standard CliniMACs cell purification protocols which are in 
routine use as part of the bone marrow transplantation programme. Operating procedures are 
available on request. Purified cells will be immediately transferred to the gene therapy 
laboratory for transduction. 
7.5.4 CD34+ cell culture and transduction 
CD34+ cells will be purified, cultured and transduced in a dedicated clinical gene therapy 
laboratory at Great Ormond Street Hospital. CD34+ cells will be purified using standard 
protocols for stem cell transplantation (CliniMACs), and seeded into fresh closed cell culture 
bags or flasks in serum free medium (X-VIVO-20, Lonza) supplemented with 1% human serum 
albumin (HSA, Baxter), and cytokines IL-3 (20ng/ml), SCF (300ng/ml), Flt-3 ligand (300ng/ml), 
TPO (100ng/ml), at a density of 0.5 - 1 x 106/ml. All cytokines (Peprotech) and culture reagents 
are approved for ex vivo clinical use. A small culture sample will be removed for sterility testing. 
After ~18 hours total pre-stimulation, cells will be cultured with EF1αS-ADA lentiviral vector 
supernatant for 24 hours in the same complete media at a cell concentration of 0.5 - 1 x 106/ml. 
Following transduction, a portion of cells will be removed for quality control (analysis of 
transduction efficiency, viability, sterility, RCL). The remaining cells will be resuspended in 
Human Serum Albumin (HSA) 2% in order to be administered to patient. 
7.5.5 Testing prior to patient re-infusion  
Samples are collected during and at the end of the procedure for cell viability (trypan blue 
stain, 7AAD staining) and sterility (for bacteria, fungi and mycoplasma). Transduction 
efficiency (transgene PCR), and RCL testing will be performed after infusion of cells and 
recorded retrospectively. 
7.5.6 Patient conditioning regimen  


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During transduction of CD34+ cells, patient will commence conditioning. Patients will receive 
non-myeloablative conditioning with intravenous busulfan.  Busulfan dose will be weight-
dependent, in accordance with the recommendation below (table taken from (Bartelink et al., 
2012)). 
 
A wash-out period of at least 24 hours will be maintained prior to reinfusion of the transduced 
cells. Pharmacokinetic monitoring of busulfan levels will be performed after administration of 
the first dose.  
7.5.7 Infusion of transduced cells  
Cells will be washed and infused in a volume up to 20mls/kg intravenously over 30-45 minutes 
A baseline set of observations will be carried out before the infusion begins (temperature, 
pulse, respirations, oxygen saturations). These will then be repeated 15 minutes into the 
infusion and then again at the end of infusion.  Regular monitoring (frequency and duration 
dependent on reaction/occurrence) will be commenced if anything abnormal (e.g. allergic 
reaction) occurs during the infusion or if antihistamines are administered. 
7.5.8 Discontinuation of PEG-ADA one month after infusion of gene modified cells 
PEG-ADA will be continued until one month (+/- 6 days) post-infusion of gene-modified cells. 
PEG-ADA will be restarted if i) the child is withdrawn from the study ii) if after 180 days there 
is no evidence of gene modified cell engraftment and/or failure of T cell recovery. 
PEG-ADA may be restarted at PI’s discretion prior to this time point on clinical grounds e.g. 
infective problems or delayed T cell reconstitution. In instances when PEG-ADA has been re-
started, PEG-ADA may be withdrawn again, if the patient remains well and meets withdrawal 
criteria (i.e. has detectable PBSC ADA activity and/or detection of gene modified cells above 
baseline levels). 


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8 
Evaluation and Follow-up  
8.1 
Assessment of safety and efficacy 
8.1.1 Immunological reconstitution 
Detailed analysis of immune recovery following gene therapy for primary immune deficiency 
is essential to evaluate the effectiveness of the procedure, and is largely beyond the scope of 
our routine clinical immunology resources. We have therefore established dedicated 
resources and methodologies that enable us to 1) measure the diversity and complexity of T 
and B reconstitution 2) analyse thymic education and output following gene modification of 
lymphocyte precursors and 3) measure cellular antigen specific responses. 
The analyses will be carried out on pre- and post-treatment samples as shown in Appendix 5. 
The results will be interpreted as a change in value over time through a longitudinal analysis, 
for each patient. 
• 
The lymphocyte subset counts (LSC) immunophenotyping panel will be carried out to 
show the distribution of cells and is used to detect an increase in naïve CD3+ T 
lymphocyte cell numbers and assess the development of normal distribution of cell 
populations.  This will include the evaluation of CD3+, CD19+, CD16+, CD56+, CD3+, 
CD4+, CD8+ and naïve CD4+CD45RA+CD27+ cell numbers. 
• 
B cell memory tests will be performed at months 12, 24, and 36.  This parameter should 
be recorded as “not done” in the CRF for subjects that have already completed any of 
these three visits at the time of amendment (Version) implementation. 
• 
TCR excision circles (TRECs) may be enumerated as a surrogate marker for new 
thymic emigrants following gene therapy. 
• 
Whole blood lymphocyte proliferation assays will be carried out to test function of T 
cells and will include responses to mitogens (PHA stimulation and CD3 stimulation).  
• 
Representation of TCR families by flow cytometric analysis (Vβ phenotyping), also 
forms an important part of monitoring for both physiological and potentially pathological 
clonal expansions. 
• 
Restoration of antibody production (IgA, IgM, IgG), and serological responses to 
tetanus vaccinations will be assessed. 
8.1.2 Metabolic correction 


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Metabolic assays will be carried out at the Purine Research Laboratory and will include 
analysis of red blood cell ADA activity, dATP and dAdo levels. These will indicate how 
effectively gene therapy has corrected the metabolic phenotype of ADA-SCID. 
8.1.3 Molecular characterisation of gene transfer 
Molecular characterisation of gene transfer in patient cells is also an important parameter for 
assessment of efficiency, and potentially for assessment of safety: 
• 
Quantification of transgene copy numbers is determined on sorted cell populations by 
real-time PCR methodology. This will be performed on peripheral blood samples and 
on bone marrow samples (if available). Detailed integration analysis maybe used to 
investigate specific clonal expansions. 
• 
Analysis of lentiviral vector integration sites will be performed by specialised PCR 
based techniques and by high throughput sequencing of lineage specific populations. 
This will be performed on peripheral blood samples and on bone marrow samples (if 
available). These assays will be performed at the times specified in appendix 5. 
8.2 
Investigations and monitoring schedule  
See appendix 5 for tables of investigations. 
8.2.1 Pre-treatment investigations  
Pregene therapy visit will be carried out prior to gene therapy. This visit will include pre-
treatment monitoring investigations, which should be carried out within 3 months of the start 
of gene therapy treatment according to Appendix 5 
8.2.2 Post-infusion monitoring  
Patients will be monitored on protocol at 1 month, 6 weeks, 3, 6, 9, 12, 18, 24, 30 and 36 
months post-infusion. Window to carry out these visits is +/- 5 days for visits at 1 month, 6 
weeks and  +/- 2 weeks for visits at  3, 6, 9, 12, 18, 24, 30 and 36 months. Follow-up as part 
of the routine clinical care of post-transplant patients will be annual after this. 
Monitoring, testing being performed and information to be collected on CRF are fully detailed 
in Appendix 5. Modifications to this protocol will be adopted as necessary to improve sample-
processing capability, and a degree of flexibility regarding the actual dates of assessment will 
be maintained. Additional tests may be carried out in the event of a significant adverse effect 
to ensure optimal clinical care.  
8.2.3 Safety monitoring 


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The safety of treatment will be summarised using AEs, laboratory assessments and clinical 
examination assessments.  These will be analysed continuously from pre-gene therapy to 
study completion, depending on the data available.  Further details are given in the SAP 
8.2.4. Neurological and behavioural monitoring of patients 
Cognitive and behavioural defects are well documented in ADA-SCID patients following HSCT 
(Rogers et al., 2001; Titman et al., 2008). It is thought that patients undergoing gene therapy 
will also suffer similar consequences.  
We have recently set up a study protocol (funded by the Department of Health) to assess all 
immunodeficiency patients (both ADA-SCID and non ADA-SCID) for cognitive, educational 
and behavioural outcomes following HSCT. This assessment will therefore also be applied to 
all ADA-SCID patients undergoing gene therapy. In addition to this, we will administer a 
standard health related quality of life questionnaire to assess the general impact of the 
treatment on the child functions. 
8.3 
Treatment failure/lack of response  
• 
Failure of immune reconstitution is determined by poor recovery of T lymphocyte 
numbers and function by day 180 
OR  
• 
by absence of gene corrected cell engraftment (<0.1 %) tested by quantitative PCR in 
peripheral blood and/or BM samples 6 months after the procedure. 
• 
If there is no immune recovery/evidence of gene modified cell engraftment seen 180 
days after gene therapy, PEG-ADA will be commenced OR the patient will undergo 
stem cell transplant from a matched unrelated donor depending on the clinical state of 
the child. 
• 
If there is no matched unrelated donor available, the patient will be commenced on 
PEG-ADA OR if the referring health agency is unable to provide funding, the patient 
will undergo stem cell transplant from an unrelated or haploidentical parental donor. 
• 
In the absence of a matched family or unrelated donor a second administration of the 
gene therapy treatment could be considered following specific approval from 
GTAC/MHRA on a case by case basis. 
8.4 
Withdrawal of individual subjects 


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A patient can withdraw or be withdrawn from protocol treatment in the study at any time from 
enrolment until the transduced CD34+ cells have been administered. Once a patient has been 
administered the transduced CD34+ cells study treatment is complete. 
• 
Patients from whom <3x106 CD34+ cells/kg are harvested for the back-up, will be 
withdrawn from the study prior to conditioning and offered a repeat harvest 
• 
Patients from whom <1x106 CD34+ cells/kg are harvested for transduction, will be 
withdrawn from the study prior to conditioning and offered a repeat harvest 
• 
If less than 0.5 x 106 viable transduced CD34 + cells/kg are returned to the patient, 
then back-up bone marrow/PBSCs will be returned and patients will be withdrawn from 
the study  
• 
If after 180 days there is no evidence of the transgene in PBMCs by qPCR or there is 
no evidence of T cell recovery, then patients will re-start PEG-ADA and will be 
withdrawn from the study 
PEG-ADA may be restarted at PIs discretion prior to this time point on clinical grounds e.g 
infective problems or delayed immune reconstitution and at this stage will be withdrawn from 
the study. The decision for patients to remain off PEG-ADA is dependent on a combination of 
factors including: 
1) metabolic detoxification as assessed by sustained red blood cell dATP levels <250 
umol/l 
2) T cell recovery as assessed by absolute T cell numbers  (CD3+ cells) >300cells/mm3 
3) engraftment of gene modified cells as assessed by vector copy number in CD3+ cells 
>0.05 (>5% of total T cells 
 
Subjects who wish to discontinue from the study at any time are free to do so. However the 
reasons for discontinuation should be documented by the investigator if possible.  
Patients who withdraw from the study after administration of the transduced CD34+ cells for 
reasons other than those given above will be encouraged to have follow-up investigations so 
that the consequences of the administration can be documented and analysed.  
Any patient withdrawn prior to administration of transduced CD34+ cells will be replaced in the 
study.  
8.5 
Off study criteria 


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A patient will be considered off study under the following circumstances:  
1. The patient is withdrawn from the study prior to administration of transduced CD34+ 
cells 
2. The patients/parent/guardian withdraws consent for study procedures and data 
collection 
3. The patient is lost to follow-up 
8.6 
Follow-up of withdrawn subjects 
Patients withdrawn from the study prior to administration of transduced CD34+ cells and 
patients for whom consent has been withdrawn for the study, will resume normal clinical care. 
Efficacy and safety assessments will not be carried out from the point of withdrawal and data 
will not be recorded in the Case Report Forms (CRFs). 
8.7 
Definition of end of trial 
• 
The sponsor will notify GTAC and the MHRA of the end of the study within a period of 
90 days. 
• 
The end of the study is defined as the last patient’s last scheduled visit according to 
the protocol, which will be the 3-year follow-up visit of the last patient entered into the 
trial.  
• 
In case the study is ended prematurely, the sponsor will notify GTAC and the MHRA 
within 15 days, including the reasons for the premature termination. 
• 
Within one year after the end of the study, the investigator/sponsor will submit a final 
study report with the results of the study, including any publications/abstracts of the 
study, to GTAC and the MHRA. 
8.8 
Trial stopping criteria 
The trial will be stopped prematurely under the following circumstances:     
1. Evidence of oncogenesis in any trial patient.  The trial will only be re-started following 
agreement from both the MHRA and GTAC. 
The events below will result in halting of the trial and will require a formal evaluation of the 
reasons for non-efficacy. The trial will only be re-started after discussion with and approval 
from the MHRA and GTAC: 


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1. Unsuccessful transduction of 3 consecutive patient CD34+ cells to specified criteria  
2. No evidence of vector integration in 3 consecutive patients at 6 months post-treatment  
3. 3 consecutive patients needing to re-start enzyme replacement therapy prior to 1 year 
post-gene therapy 
(From our experience of these studies, 3 consecutive patients would be seen as a number whereby this 
could not have happened by chance and is most likely related to processes or procedures) 
4. Emergence of new therapies for ADA-SCID, believed by the CI to be of greater benefit 
to this population of trial patients.  The CI is in regular contact with other experts in the 
field of ADA-deficiency therefore is aware of new developments in the field.  Regular 
reviews of the currently available treatment options for the subject population will be 
conducted by the PI throughout the trial. 
Complete cessation of the trial would be made in consultation with the MHRA and GTAC. 
8.9 
Statistical analysis 
Timing of analyses 
One interim analysis, one primary analysis, one longer term follow-up and one end-of-study 
analysis are planned for this study. 
An interim look will be done when all on-study patients (not including CUP) have reached at 
least 6 months’ follow-up. 
The primary analysis for the study will be done when all on-study patients have reached at 
least 12 months’ follow-up. This interim analysis will be including CUP and HSCT groups. 
The third analysis will be done when all IMP on-study patients have reached at least 24 
months’ follow-up. This interim analysis will be including CUP and HSCT groups. 
The final analysis will be done at the end of the study. i.e. after on study patients, have 
completed the planned follow up of 3 years. This analysis will also include HSCT group.  
 
8.9.1 Analysis populations 
There will be 3 populations analysed in this study: The on-study patients treated with IMP 
(gene therapy), patients who were treated under compassionate use (CUP patients) and the 
historical comparator group of patients who received HSCT. 


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The safety analysis population will include all patients who received the IMP infusion, including 
both on-study patients and patients who were treated on a compassionate use basis.  In 
addition, all the allogeneic HSCT controls patients (with any type of donor) will be included. 
Further efficacy populations will be detailed in the statistical analysis plan (SAP). 
8.9.2 Data analysis/description 
Descriptive statistics will be employed for the socio-demographic characteristics of the study 
population.  
The primary efficacy endpoints, overall survival (OS) and event-free survival (EFS) will be 
compared descriptively to a group of ADA-SCID patients treated by allogeneic HSCT 
(historical control group). The main secondary endpoint, will be summarised descriptively. 
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 as appropriate.  
All statistical analyses will be performed and data appendices will be created using SAS® 
version 9.4 or higher 
Safety 
Safety will be analysed descriptively by arm and study phase using the safety analysis 
population. Coding will be done using MedDRA version 18.1 or higher.  The safety of 
treatment will be summarised using AEs, laboratory assessments and clinical examination 
assessments.  These will be analysed continuously from pre-gene therapy to study 
completion, depending on the data available.  Further details are given in the SAP. 
Efficacy 
Primary Endpoints 
Statistical methodologies for comparing gene therapy to historical controls at all stages will be 
descriptive in nature and will utilize the following approaches: 
1. OS and EFS will be described using Kaplan-Meier curves for patients treated with 
investigational gene therapy product and HSCT, respectively, and statistically 
compared by the log-rank test and proportional hazards models (where applicable).  
2. OS and EFS rates post treatment will be described by group and their differences 
between groups presented along with associated exact binomial confidence intervals.  


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Main Secondary Endpoint 
Percentage of subjects requiring immunoglobulin replacement therapy will be provided for 
each cohort (See Overview Section above) along with associated, two-sided, exact binomial, 
95% confidence intervals. 
The detailed statistical procedures will be provided in the statistical analysis plan (SAP). 
9 
Safety issues related to the gene therapy protocol 
9.1 
Risks 
9.1.1 Venepuncture 
Hazards associated with blood drawing are those associated with venepuncture and, those 
associated with the loss of red blood cells as a result of removal of blood from the procedures 
described in this protocol. Risks of venepuncture are pain, the potential for formation of a 
haematoma at the site of the needle stick and a small potential for infection at the site of needle 
stick. Occasionally, individuals having venepuncture may faint as a result of a vasovagal 
reaction to the procedure. These risks will be minimised by carefully preparing the site of 
needle puncture with alcohol swab, by using the smallest size needle required for the 
procedure, and by having the patient lie down or sit as appropriate for 5 or 10 minutes after 
venepuncture.  The major risk of repeated blood drawing is anaemia from removal of red blood 
cells.  To minimise this risk the amount of blood drawn for all purposes will be adjusted so as 
not to exceed the guidelines of 450 ml or 7.5 ml/kg (whichever is less) over any six-week 
period. 
9.1.2 Infection 
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 a severe infection 
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 therapy may have to be resumed. 
It is also possible that the cells for infusion 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 be returned to 
the subject with appropriate antibiotic cover. If the infection significantly affects viability (to a 
total cell dose of <0.5 x 106/kg) then transduced cells will not be returned and the back-up 


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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).  
9.1.3 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 (4mg/kg iv. total dose) in a paediatric 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). Our own experience (of 3 patients) 
has shown no significant toxicity. The complications of high dose busulfan in combination with 
other chemotherapy drugs have not been seen in any of the more than 25 ADA-deficient 
patients treated using low dose busulfan. Busulfan is mutagenic (a clastogen, inducing 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. 
The dosage schedule in this study is based on the differential clearance of busulfan in children 
of different weights. There is no evidence for any greater toxicity as a result of this weight 
adjusted dosing schedule. In two patients treated off trial on a compassionate basis and who 
received 5.1mg/kg of iv busulfan, no toxicity was seen.  
9.1.4 Bone marrow harvest 
Bone marrow will be harvested from the patient under general anaesthesia from the posterior 
iliac crests on both sites by multiple punctures. Marrow will be collected into sterile bags 
containing ACD (acid citrate dextrose) (Baxter). The amount of marrow collected will be 
equivalent to 20 ml/kg of body weight. The risks of the procedure include the standard risks of 
general anaesthesia and complications associated with the bone marrow harvest procedure. 
The latter include pain at the site of puncture following the procedure, excessive blood loss 
resulting in anaemia and the potential for infection at the puncture site. The anaesthetic risks 
will be minimised by the anaesthetic being administered by a paediatric anaesthetist at a major 
paediatric tertiary referral centre. The patient will be given paracetamol pain relief after the 


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procedure and will be monitored carefully to check haemodynamic status. It is likely that the 
patient will need a blood transfusion after the bone marrow harvest. Infection risk will be 
minimised by careful cleansing of the site prior to needle insertion and strict adherence to 
sterile technique during the procedure. 
9.1.5 Peripheral blood stem cell harvest 
Treatment of patients or donors with granulocyte colony stimulating factor (G-CSF) and 
Plerixafor results in the mobilisation of bone marrow stem and progenitor cells into the 
peripheral blood, from where they can be harvested. The quality of cells collected in terms of 
engrafting potential has been such that many centres have virtually replaced transplantation 
of bone marrow with that of peripheral blood stem cells (PBSCs) particularly in the autologous 
setting. The advantages of this procedure (over bone marrow harvest) are that a larger number 
of stem cells can be harvested, and no anaesthetic is required. G-CSF is administered by 
subcutaneous injection for 6 days. On day 5, Plerixafor may also be administered by 
subcutaneous injection. The commonest side effect is of bone and muscle pain, which is easily 
controlled with mild analgesics. Rare reports (<1 in 1000 patients) exist of allergic-type 
reactions (including anaphylaxis, skin rash, urticaria, angioedema, dyspnoea and 
hypotension), usually when the drug has been injected intravenously. Temporary 
abnormalities of liver function and skin rashes have occurred in a very small number of 
patients, usually on long term therapy for neutropenia. Apart from extremely rare reports of 
spontaneous splenic rupture, the safety records of both G-CSF and Plerixafor are very good. 
If patients develop unanticipated inflammatory reactions, which in the opinion of the 
investigator are due to G-CSF and/or to Plerixafor toxicity, then administration will be 
terminated and the patient would then be proposed a bone marrow harvest. 
After mobilisation, the cells are harvested by leukapheresis on two consecutive days from 
centrally placed cannulae. Each leukapheresis lasts from three to four hours and has been 
shown to be extremely safe both in adults and in children. Adverse reactions to apheresis 
procedures are rare, although vasovagal episodes related to needle insertions or transient 
volume loss can occur. The former reaction is prevented by lying down and if necessary, fluid 
administration. Paraesthesiae or tingling can also sometimes occur due to lowering of calcium 
levels by the citrate anticoagulant. This is readily relieved by slowing the rate or temporarily 
interrupting the anticoagulant infusion. The precautions taken to minimise pain, haematoma 
formation, risk of infection at needle site and vasovagal fainting are outlined above. 
Leukapheresis can be associated with some loss of red blood cells and plasma equivalent to 
100 to 200 ml of whole blood though every effort will be made to keep this volume as low as 
possible. Insertion of the apheresis line can lead to occlusion of blood flow and therefore can 
result in thrombus formation.  This is treated with anti-coagulant and regular screening to 


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monitor reduction of the size of the thrombus. Leukapheresis is a routine procedure with 
minimum side effects or complications. Potential complications include formation of 
haematoma at the site and a small risk of infection as with any intravenous catheter. 
9.1.6 CD34+ cell infusion 
Based on results from other gene transfer studies and our own experience, the infusion of 
cultured and gene altered autologous blood progenitors does not appear to be associated with 
any significant reactions.  In this study, the medium used to culture the CD34+ cells is free of 
animal serum. However, infusion of any type of blood cell product can be associated with 
reactions resulting from clumping of these cells or other immediate reactions related to sticking 
of these cells to blood vessels in the lungs.  Patients will be monitored as per national and 
hospital Blood product administration policy.  Reactions are treated by stopping the infusion 
and providing oxygen, antihistamines, steroids and medications or fluids to increase blood 
pressure. Because infused cells in this study are autologous CD34+ cells, the possibility that 
this type of agglutination or vascular reaction will occur is very low.  It is theoretically possible 
for the CD34+ cell cultures to become contaminated with microorganisms. One of the 
important safety features of this gene therapy protocol is that CD34+ cells will be cultured in 
a sealed bag culture system or in flasks in an isolator unit. Routine cultures will be performed 
on cell samples before infusion. Only interim sterility results will be available before infusion 
of cells. If significant growth of pathogenic microorganisms is detected, the cells will be 
returned with appropriate antibiotic cover, or if the infection significantly affects cell viability, 
the cells will not be returned to the patient. 
9.2 
Risks associated with the use of lentiviral vector 
9.2.1 Insertional mutagenesis 
Currently, there have been no reported cases of insertional mutagenesis in clinical trials using 
a lentiviral vector for gene therapy.  A number of studies both in vitro and in in vivo (Modlich 
et al., 2009; Modlich et al., 2006; Montini et al., 2006) showed that the risk for insertional 
mutagenesis is less genotoxic with lentiviral vectors that lack strong retroviral LTR and may 
be further reduced with improved vector designs. 
Risks from insertional mutagenesis have occurred in clinical trials of gene transfer using 
retroviral 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 leukaemia ≥ 2½ years 
after receiving the gene-transduced CD34+ cells (Hacein-Bey-Abina et al., 2003; Hacein-Bey-
Abina et al., 2008). In a similar trial of gene therapy for X-SCID performed in London, England, 
one of 10 subjects also developed T cell leukaemia (Howe et al., 2008).  One of these five 
patients who developed T cell leukaemia has died and the other four are in leukaemia 


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remission and continue to have restored immunity from the gene therapy.  For three of the 
subjects with leukaemia, the proviral insertion sites have been mapped within or near (3 kb) 
of the LMO-2 gene, a human proto-oncogene associated with T-cell leukaemia.  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 leukaemia developing 
in subjects participating in a clinical study of retroviral-mediated gene transfer to CD34+ cells.  
The probability of leukaemia developing in other participants of the French trial, or in 
participants of other trials, is not known. Similarly in a trial of gene therapy for X-CGD, 2 
patients developed a myelodysplasia and an acute myeloid leukaemia as result of vector 
mediated integration events disrupting expression of the MDS-EVI1 oncogene ((Ott et al., 
2006; Stein et al., 2010). 
As of April 2011, 17 ADA-deficient SCID patients have undergone these protocols in Milan 
and 8 in London and 11 in the CHLA/NIH and UCLA/NIH trials (by personal communication 
from site PIs).  No cases of leukaemia have occurred in any of the ADA subjects, in sharp 
contrast to the X-SCID or X-CGD results.  
9.2.2 Replication-competent lentivirus (RCL) exposure 
Generation of RCL is highly unlikely due to the multiply deleted nature of the vector and SIN 
configuration, and has not been noted in any preclinical study with similar vectors. Lentiviral 
supernatant and patient samples will however be tested for the presence of RCL by cell culture 
and PCR methodology. Patient samples will be tested at time points 0 (pre-treatment), 3 
months, 6 months and one year after treatment. If all tests are negative during the first year, 
further samples will be taken annually through-out the patient’s care at GOSH and archived 
for at least 15 years. If in the unlikely event that post-treatment samples are positive, further 
analysis will be undertaken to characterize the RCL. 
9.2.3 Germ-line transmission of vector sequences 
Issues of contraception and pregnancy are not applicable in infants and children before 
puberty.  Because we will only be putting the EF1α-ADA vector into the CD34+ cells from bone 
marrow ex vivo, we do not anticipate that they can enter the germ cells. 
9.2.4 Allergic/immunological responses to cell processing excipients 
Stem Cell Factor (SCF) and 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 


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may remain, it is possible that the presence of these, and other cytokines used during the 
transduction process, 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.  
9.2.5 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 "immunisation" 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 with any new form of therapy, there may be risks which are unknown or not anticipated.  
10 
Name description of the IMP and critical raw materials 
10.1 
Name and description of each IMP 
• 
EF1αS-ADA lentiviral vector transduced patient CD34+ cells is designated as an IMP 
• 
The IMP is patient-specific and corresponds to autologous CD34+ cells transduced ex 
vivo with the EF1αS-ADA lentiviral vector containing the human ADA gene in final 
formulation and container closure system, ready for intended medical use 
• 
In this study EF1αS-ADA lentiviral vector is designated as a critical raw material 
• 
The starting materials used for the production of the IMP consist of the viral vector and 
the patient’s CD34+ cells 
10.2 
Production, supply and release of the IMP 


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• 
Transduction of autologous CD34+ cells with the lentiviral vector will be carried out in 
the Gene Therapy Laboratory, Camelia Botnar laboratories, Great Ormond Street 
Hospital for Children NHS Trust. 
• 
The transduced cells will be packaged in sealed transfer bags prior to being 
transported to the ward at Great Ormond Street Hospital (GOSH) for administration. 
• 
An independent Qualified Person (QP) will be responsible for importation, sign off and 
release of the vector for use in the study 
10.3 
IMP accountability 
IMP accountability is ultimately the responsibility of the Chief Investigator. This responsibility 
however will be delegated to the Clinical Scientist responsible for the transduction procedure.  
Detailed records will be kept to allow for accurate accountability of the vector and transduced 
CD34+ cells. These records will include details of shipping, receipt, storage, use and 
destruction of the vector. Transfer of CD34+ cells from the gene therapy laboratory to the ward 
at GOSH and administration of the transduced CD34+ cells to patients will be recorded. 
10.4 
Description and justification of the trial treatment and dosage 
The EF1αS-ADA lentiviral vector is used to transduce autologous CD34+ cells ex vivo. These 
transduced cells are then infused into the patient. The cell/product dose will consist of at least 
0.5 x 106 CD34+ cells per kg of body weight transduced ex vivo with 1 x 108 i.g./ml of lentiviral 
vector to achieve an average of 0.3-2 integrated copies per cell.  A higher dose would be 
expected but given that there is a survival advantage to gene corrected cells, this lower level 
has been designated as final release threshold in this study.  This is supported by evidence 
gained from our first retroviral ADA trial here at GOSH, where two patients reconstituted 
effectively, having received less than 0.5x106 transduced CD34+ cells/kg (Gaspar et al., 2011). 
10.5 
Dose modifications 
A single dose of cells will be administered to the patient and the dose is individual to the patient 
since the gene-modified cells are the IMP. 


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10.6 
Assessment of compliance 
There is no compliance issue with IMP administration since gene modified cells are infused 
through a Hickman line and the infusion is monitored by a trained nurse. 
10.7 
Post-trial IMP arrangements 
There is no provision to provide patients with IMP post-trial participation. 
10.8 
Name of each NIMP 
• 
Cotrimoxazole 
• 
Itraconazole 
• 
Penicillin 
• 
Aciclovir 
• 
Immunoglobulin 
• 
Calcium Folinate 
• 
Busulfan 
NIMP suspected Adverse Drug Reactions (ADR) or side effects will be reported through the 
yellow card system.  
11 
Recording and reporting of adverse events and reactions 
11.1 
Definitions 
Adverse event  
This is defined as any untoward medical occurrence in the patient administered an IMP that 
does not necessarily have a causal relationship with this treatment.  
Adverse reaction  
This is defined as any untoward and unintended response in a subject to an IMP, which is 
related to any dose administered to that subject.  
Serious adverse event 


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A serious adverse event is defined as any untoward medical occurrence in the patient 
administered an IMP, which does not necessarily have a causal relationship with this 
treatment, and that at any dose: 
• 
Results in death 
• 
Is life threatening 
• 
Results in persistent or significant or disability/incapacity 
• 
Requires in-patient hospitalisation or prolongs existing hospitalisation 
• 
Results in a congenital abnormality or birth defect 
Medically significant (i.e. any event which the investigator considers significant but which is 
not covered by the above) 
Suspected Serious Adverse Reaction (SSAR) 
This is defined as an adverse reaction that is classed in nature as serious and which is 
consistent with the information about the IMP in question as set out in the Investigator’s 
Brochure. 
Suspected Unexpected Serious Adverse Reaction (SUSAR)  
The definition of a SUSAR is a serious adverse drug reaction, the nature or severity of which 
is not consistent with the applicable product information (as set out in the Investigator’s 
Brochure) or which adds significant information on the specificity or severity of an expected 
event. 
11.2 
Recording adverse events 
All adverse events (related to the IMP, unrelated to the IMP, serious or not) will be recorded 
in the hospital notes and in the electronic Case Report Form (eCRF). An adverse events log 
may be adopted for the hospital notes.   
If the investigator suspects that the disease has progressed faster due to the administration 
of the IMP, then he will report this as an unexpected adverse event to the sponsor.  
Clinically significant abnormalities in the results of objective tests may also be recorded as 
adverse events.   
The record of adverse events will include the following: 


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• 
Clinical symptoms: A simple, brief description 
• 
Severity:  we will categorise according to WHO classification (see appendix 7) 
• 
Causality assessment (relationship to treatment):  The assessment of relationship 
of adverse events to the administration of IMP is a clinical decision based on all 
available information at the time of the completion of the case report form.  The 
following categories will be used: 
Definitely: There is clear evidence to suggest a causal relationship, and other possible 
contributing factors can be ruled out. 
Probably: There is evidence to suggest a causal relationship, and the influence of other 
factors is unlikely. 
Possibly: 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 patient’s clinical 
condition, other concomitant events). 
Unlikely:  There is little evidence to suggest there is a causal relationship (e.g. the event 
did not occur within a reasonable time after administration of the trial medication). There 
is another reasonable explanation for the event (e.g. the patient’s clinical condition, other 
concomitant treatments). 
Not related: There is no evidence of any causal relationship. 
Not Assessable 
Note: events assessed as possibly, probably or definitely related to the IMP are considered 
adverse reactions (ARs) 
• 
Expectedness evaluation: The following categories will be used: 
Expected: 
An adverse event which is consistent with the information about the IMP listed 
in the Investigator Brochure or clearly defined in this protocol 
Unexpected: An adverse event which is not consistent with the information about the IMP 
listed in the Investigator Brochure  
• 
Seriousness: as defined for an SAE in section 11.2.1.  


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Collection, recording and reporting of adverse events (including serious and non-serious 
events and reactions) to the sponsor will be done according to the sponsor’s SOP. 
11.3 
Expected disease adverse events 
The following SAEs are considered disease related:  
• 
Death from complications of the underlying disease process (SAE) 
• 
Hospitalisation from complications of the disease (SAE) 
• 
Prolonged hospitalisation from complications of the disease (SAE) 
• 
Bone marrow aplasia for greater than 1 month (SAE/SSAR) 
The following SAEs are considered related to the side effects of chemotherapy:  
• 
Renal/liver/gastrointestinal/pulmonary/metabolic problems (SAE) 
• 
Viral re-activation following chemotherapy and prior to immune recovery (SAE) 
• 
Bone marrow aplasia for greater than 1 month (SAE/SSAR) 
A full list of AEs considered related to the side effects of the chemotherapy are detailed in the 
undesirable effects section of the Summary of Product Characteristics (see Appendix 7). 
11.3 
IMP expected adverse events 
The following ARs are considered expected reactions that could occur following administration 
of the IMP: 
1. Lympho- or myelo-proliferation due to insertional mutagenesis (SSAR) 
2. Chills/fevers/rigors at time of infusion (AR)  
3. Rash (AR) 
4. ADA overexpression leading to haemolysis (AR) 
5. Immune response to transgene (AR) 
6. Bone marrow aplasia for greater than 1 month (SAE/SAR) 
11.4 
Procedures for recording and reporting Serious Adverse Events 
All SAEs will first be clearly documented in the patient’s notes. 


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Events defined as serious will be reported immediately (within 24 hours of the any study team 
member observing or learning of the event), in as much detail as possible to the sponsor (Joint 
R&D office). 
This event will be recorded in the sponsor’s SAE form (F44) and emailed to 
 or 
 within the 
reporting timelines. 
The Investigator may be asked by the sponsor to provide follow-up information, which should 
be reported to sponsor by completing SAE from (F44). 
All SUSARs will be notified to the sponsor immediately (within 24 hours of any study team 
member observing or learning of the event) according to the sponsor’s written SOP. 
Elective and/or planned procedures do not require sponsor’s SAE reporting, unless of course 
there is any complication arise that fulfils the definition as listed section 11.1 
Definitions. 
In any case, these elective and/or planned procedures will be documented in the patient’s 
notes and CRF. 
 
11.4.1 Notification of deaths 
 Death will be reported to the sponsor irrespective of whether it is related to disease 
progression, the IMP, or an unrelated event.  
11.4.2 Reporting SUSARs 
SAE reported to the sponsor that is evaluated by the Investigator as related to the IMP and 
unexpected will be reported to the regulatory authorities (MHRA) and the ethics committee 
(GTAC) by the sponsor within the expedited reporting timelines: 
Fatal or life threatening SUSARS: not later than 7 days after the sponsor has information that 
the case fulfils the criteria for a fatal or life threatening SUSAR, and any follow up information 
within a further 8 days.  
All other SUSARs: not later than 15 days after the sponsor for pharmacovigilance has 
information that the case fulfils the criteria for a SUSAR. 
11.4.3 Medically important events considered critical to the safety evaluation of the 
study 
Abnormal transgene related lympho- or myelo-proliferation (i.e. in the absence of viral 
reactivation) is considered critical to the safety evaluation of the study and will be reported 
immediately (within 24-48hrs) to the sponsor using the sponsors reports of other safety issues 


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(F20) form. Such events will be reported to the MHRA and the GTAC by the sponsor within 
the expedited reporting timelines. 
11.4.4 Long term follow-up 
All patients are followed up lifelong and once these studies are officially closed the PI will 
continue to report SARs and SUSARS to the MHRA and GTAC in the same way as during the 
study. 
11.4.5 Annual safety reports 
Within 60 days of the anniversary of the clinical trial authorisation (CTA) date, an annual safety 
report will be sent to the MHRA and GTAC. 
11.4.6 Annual progress reports 
An annual progress report (APR) will be submitted to GTAC within 30 days of the anniversary 
date on which the favourable opinion was given, and annually until the trial is declared ended. 
11.4.7 Pregnancy  
Not applicable for this study. 
11.4.8 Reporting urgent safety measures 
The sponsor and the investigator may take appropriate urgent safety measures in order to 
protect the subjects of a clinical trial against any immediate hazard to their health or safety. If 
measures are taken, the sponsor shall immediately and in any event no later than 3 days from 
the date the measures are taken, give written notice, in the form of a substantial amendment, 
to the MHRA and GTAC of the measures taken and the circumstances giving rise to those 
measures. 
In order to prevent any delays in the reporting timelines the sponsor has delegated this 
responsibility to the PI. Therefore the PI must report any urgent safety measures to the MHRA 
directly and in parallel to the sponsor.   
11.4.9 Notification of serious breaches to GCP and/or the protocol 
Any deviations, violations, potential serious breaches and urgent safety measures will be 
recorded. 
The sponsor will be notified immediately of any case where the above definition applies during 
the trial conduct phase.  The sponsor’s SOP will be followed and all these events will be 
notified to GTAC and MHRA by sponsor.  


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12 
Administrative aspects 
12.1 Data handling, record keeping and sample storage 
12.1.1 eCRF completion 
All study data will be recorded on study specific eCRF. The eCRF will be completed by 
personnel authorised to enter or change data on the eCRF. The eCRF will track all 
modifications done by the relevant authorised person    
12.1.2 Sample storage  
A record of retained body fluids / tissue samples will be completed every time sample is stored. 
This includes the patient trial identification number, initials, date sample was stored, and 
storage location as well as the date the sample was moved or destroyed.  
Samples (Cells, DNA & RNA) will be stored pre cell therapy and at regular intervals post cell 
therapy, and kept indefinitely at the bone marrow laboratory GOSH (or alternative approved 
location). Should it be required, samples will be disposed of in the appropriate manner 
according to local GOSH procedures, this will be detailed in the parent/guardian information 
sheet and consent obtained. Samples for RCL testing, integration analysis are sent outside 
GOSH for analysis only.  
12.1.3 Record retention  
Essential documents will be retained for a minimum of 25 years after completion of the trial. 
These documents will be retained for longer if required by the applicable regulatory 
requirements or the sponsor. 
Records related to traceability of the IMP at site along with the patient identification code list 
will be retained at site for at least 30 years from the expiry date of the transduced cells.  
The sponsor (or PI if delegated) will retain a copy of the final clinical trial protocol and 
Investigator Brochure along with the traceability records for the trial for at least 30 years from 
the expiry date of the transduced cells. 
12.2 Patient confidentiality 
In order to maintain patient privacy, all eCRFs, IMP accountability records, study reports and 
communications will identify the patient by initials, date of birth and the assigned unique patient 
trial number. 
Direct access to the patient’s original medical records for verification of data gathered on the 
eCRFs and to audit the data collection process will be permitted for trial-related monitoring & 
audits by the sponsor, Research ethics committee review, and regulatory inspection(s).  


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12.3 Monitoring and audit 
The trial has been initially monitored and audited by GOSH, this activity is now delegated by 
GOSH to Voisin Consulting Life Science. 
12.4 Direct access to source data/documents 
The investigator(s)/ institution(s) will permit trial-related monitoring, audits, GTAC review and 
regulatory inspection(s), providing direct access to source data/documents. Trial participants 
are informed of this during the informed consent discussion.  Participants will consent to 
provide access to their medical notes. 
13 
Ethics and regulatory requirements 
The sponsor will ensure that the trial protocol, patient information sheet, consent form, GP 
letter and submitted supporting documents have been approved by the MHRA and GTAC, 
prior to any patient recruitment. The protocol and all agreed substantial protocol amendments, 
will be documented and submitted for ethical and regulatory approval prior to implementation. 
Before the site can enrol patients into the trial, the Chief Investigator or designee must apply 
for Site Specific Assessment from Trust Research & Development (R&D) and be granted 
written NHS R&D approval.  It is the responsibility of the Chief Investigator or designee at each 
site to ensure that all subsequent amendments gain the necessary approval.  This does not 
affect the individual clinician’s responsibility to take immediate action if thought necessary to 
protect the health and interest of individual patients (see section 11.4.8 for reporting urgent 
safety measures). 
Within 90 days after the end of the trial, the CI and sponsor will ensure that GTAC and the 
MHRA are notified that the trial has finished.  If the trial is terminated prematurely, those 
reports will be made within 15 days after the end of the trial. 
The CI will submit a summary report of the clinical trial to the MHRA and GTAC within 1 year 
after the end of the trial.  
14 
Finance 
Finance for the study provided by 
• 
Department of Health – vector costs 
• 
Internal funds – to cover clinical costs 


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• 
Orchard Therapeutics 
15 
Insurance 
Cover for negligent harm will be provided by the Great Ormond Street Hospital NHS Trust 
through the Clinical Negligent Scheme for Trusts (CNST). No-fault compensation insurance 
cover for any non-negligent harm will be provided by University College London (UCL). 
16 
Publication policy 
Results of this study will be disseminated by publication, oral presentation at scientific 
meetings, and by direct communication with regulatory agencies. 
17 
Statement of compliance 
The trial will be conducted in compliance with the protocol, GCP and the applicable regulatory 
requirement(s). 


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18 
References 
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human tissues. Clin. Exp. Immunol. 26, 647-649. 
Aiuti,A., Cassani,B., Andolfi,G., Mirolo,M., Biasco,L., Recchia,A., Urbinati,F., Valacca,C., 
Scaramuzza,S., 
Aker,M., 
Slavin,S., 
Cazzola,M., 
Sartori,D., 
Ambrosi,A., 
Di,S.C., 
Roncarolo,M.G., 
Mavilio,F., 
and 
Bordignon,C. 
(2007). 
Multilineage 
hematopoietic 
reconstitution without clonal selection in ADA-SCID patients treated with stem cell gene 
therapy. J. Clin. Invest 117, 2233-2240. 
Aiuti,A., Cattaneo,F., Galimberti,S., Benninghoff,U., Cassani,B., Callegaro,L., Scaramuzza,S., 
Andolfi,G., Mirolo,M., Brigida,I., Tabucchi,A., Carlucci,F., Eibl,M., Aker,M., Slavin,S., Al-
Mousa,H., Al,G.A., Ferster,A., Duppenthaler,A., Notarangelo,L., Wintergerst,U., Buckley,R.H., 
Bregni,M., Marktel,S., Valsecchi,M.G., Rossi,P., Ciceri,F., Miniero,R., Bordignon,C., and 
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Ref Type: Abstract 


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in 
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Ref Type: Abstract 


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Appendix 1: 
Construction of EF1αS-ADA lentiviral vector 
 
ADA Vector 
 
 
 
 
 
EF1αS: short version of the EF1α promoter 
Hu ADA: human ADA cDNA (codon optimised) 
WPRE: mutated form of Woodchuck Hepatitis Virus post transcriptional regulatory element 
 
Hu ADA
EF1αS
Ψ
WPRE
The EF1αS-ADA Lentiviral Vector


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Appendix 3: 
Transduction protocol schematic 
 
 
 
 
 
 
 
 
 














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Appendix 5: ADA-SCID patient monitoring protocol 


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Timepoint (months) ^ 
 
Visit window 
 
 
+/-
5d 
+/-
5d 
+/- 
2w 
+/- 
2w 
+/-
2w 
+/-
2w 
+/-
2w 
+/- 
2w 
+/-
2w 
+/-
2w 
Assessment 
Parameter to be reported on the eCRF 
PreGT 
Treat
ment 
1 
1.5 
3 
6 
9 
12 
18 
24 
30 
36 
Inclusion / Exclusion criteria 
- 
X 
 
 
 
 
 
 
 
 
 
 
 
Pregnancy test for patient >12 years 
old 
-  
X* 
 
 
 
 
 
 
 
 
 
 
 
Diagnosis 
DNA sequencing (mutation, exon, polymorphism) OR 
Confirmed absence of <3% enzymatic activity 
X 
 
 
 
 
 
 
 
 
 
 
 
Medical history  
Infection/hospitalisation history 
Transplantation history 
X 
 
 
 
 
 
 
 
 
 
 
 
Coagulation testing 
PTT, APT, Thrombin Time, Fibrinogen 
X 
 
 
 
 
 
 
 
 
 
 
 
Blood ABO testing 
Determination, Rhesus 
X 
 
 
 
 
 
 
 
 
 
 
 
Extended B cell memory  
CD19 
Naïve B cells CD19+IgD+CD27- 
Non Switched memory B cells CD19+IgD+CD27+ 
Class Switched memory B cells  
CD19+IgD-CD27+ 
Transitional B cells CD19+IgM++CD38++ 
Plasmablasts CD19+CD38++IgMwk 
CD21 low B cells CD19+CD21wkCD38wk 
X 
 
 
 
 
 
 
X 
 
X 
 
X 
Virology 
CMV, EBV, Adenovirus, HIV1, HIV2, VZV, Hep A, Hep B, 
HSV, Hep C, Syphilis, HTLV1, HTLV2, Toxoplasma 
X 
 
 
 
 
 
 
 
 
 
 
 
Biochemistry  
Sodium, Potassium, Urea, Creatinine, Calcium, 
Magnesium, Phosphate, Albumin, Alkaline Phosphatase 
(ALP), Alanine Transaminase(ALT), gamma-
glutamyltranferase(GGT), Bilirubin 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Glomerular Filtration  
Weight, Creatinine,Distribution volume,Half-life 
Glomerular Filtration, corrected GFR 
X 
 
 
 
 
 
 
 
 
 
 
 
Chest x-ray 
Normal/Abnormal 
X 
 
 
 
 
 
 
 
 
 
 
 
ECG 
Normal/Abnormal 
X 
 
 
 
 
 
 
 
 
 
 
 
Echocardiogram 
Normal/Abnormal 
X 
 
 
 
 
 
 
 
 
 
 
 
Lung function test if applicable\\ 
Normal/Abnormal 
X 
 
 
 
 
 
 
 
 
 
 
 
Dental 
Normal/Abnormal 
X 
 
 
 
 
 
 
 
 
 
 
 
Audiology 
Normal/Abnormal 
X 
 
 
 
 
 
 
 
 
 
 
 
Maternal engraftment studies 
Normal/Abnormal 
X 
 
 
 
 
 
 
 
 
 
 
 


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Assessment 
Result to be reported on the eCRF 
PreGT 
Treat
ment 
1 
1.5 
3 
6 
9 
12 
18 
24 
30 
36 
Back-up harvest 
Harvest type BM or leukapheresis, CD34+cell count 
 
X 
 
 
 
 
 
 
 
 
 
 
Harvest for IMP preparation 
If leukapheresis : Mobilisation: G-CSF (date dose) +/- 
Plerixafor (date, dose), Leukapheresis harvest: patient’s 
weight, date, CD34+cell dose for transduction 
If BM: patient’s weight, date, CD34+cell dose for 
transduction) 
 
X 
 
 
 
 
 
 
 
 
 
 
Conditioning 
Patient weight, Busulfan dose, Busulfan AUC 
 
X 
 
 
 
 
 
 
 
 
 
 
IMP administration 
Infusion date, IMP expiry, IMP start date, IMP end date, 
IMP cell dose, IMP viability, IMP volume 
Observation before, +15min,  after infusion 
 
X 
 
 
 
 
 
 
 
 
 
 
Vital Signs 
Patient’s weight 
Temperature 
Heart rate 
Respiratory rate 
Oxygen saturation 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Physical examination 
Cardiovascular system 
Respiratory system 
Ear, Nose, Throat, Neck and Eyes (only at PreGT, 36 m) 
Neurological System (only at PreGT, 36 months) 
Abdominal 
Skin 
Other 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Infection status 
Evaluate any active infection at time of visit (respiratory 
tract, meningitis, sepsis, skin and mucosa, ear, nose and 
throat, abscess, persistent fever, other) 
Evaluate if there is any infection resulting in 
hospitalisation since last visit 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
PEG-ADA ERT status 
Confirmation that if PEG-ADA has been administered or 
not since last visit 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Blood Count 
Haemoglobin, 
Platelet count 
White cell count 
Neutrophil count 
Lymphocyte count 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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Assessment 
Result to be reported on the CRF 
PreGT 
Treat
ment 
1 
1.5 
3 
6 
9 
12 
18 
24 
30 
36 
Lymphocyte subset counts 
 
CD 3 
CD19 
CD16+ CD 56+ 
CD3+ CD4+ 
CD3+ CD8+ 
Naïve: CD4+CD45RA+CD27+ 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
T cell function PHA stimulation** 
Normal/Abnormal 
X 
 
 
 
X 
X 
 
X 
X 
X 
 
 
Lymphocyte function CD3 
stimulation** 
Normal/Abnormal 
 
 
 
 
 
 
 
X 
 
 
X 
 
GAM *** 
Ig G level 
Ig A level 
Ig M level 
Immunoglobin status since last visit 
X 
 
 
 
 
X 
X 
X 
X 
X 
X 
X 
TRECS 
TRECS on T cells 
TRECS on MNCs 
X 
 
 
 
 
X 
 
X 
 
X 
 
 
Vβ Spectratyping 
Reported as Normal/Abnormal 
 
 
 
 
 
X 
 
X 
X 
X 
X 
X 
Vector copy number in cells (qPCR) 
vector copy number  in PBMC 
vector copy number  in CD34 
vector copy number  in CD3 
vector copy number  in CD19 
vector copy number  in myeloid 
 
 
 
 
X 
X 
 
X 
 
X 
 
X 
Integration analysis  
Normal/Abnormal 
 
 
 
 
X**** 
X**** 
 
X 
 
X**** 
 
X 
Tetanus vaccination responses  
Normal/Abnormal 
 
 
 
 
 
 
 
 
 
X 
 
X 
ADA metabolite analysis (dATP) and 
ADA activity 
Plasma deoxyadenosine level 
ADA (rbc) level 
dATP level 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
RCL (blood) 
Reported as detectable / not detectable 
X 
 
 
 
X**** 
X**** 
 
X 
 
X**** 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Assessment 
Result to be reported on the CRF 
PreGT 
Treat
ment 
1 
1.5 
3 
6 
9 
12 
18 
24 
30 
36 


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Bone marrow aspirate for vector copy 
number qPCR and integration analysis 
(optional) 
Morphology (Normal/ Abnormal) 
Vector copy number in BM-MNC 
Integration analysis (Normal / Abnormal) 
 
 
 
 
 
 
 
X 
 
X 
 
X 
Immunology save serum & SCLN 
(stored cells) 
Serum collection date 
SCLN s collection date 
X 
 
 
 
 
X 
 
X 
X 
X 
X 
X 
Reporting of Adverse Event 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Reporting of Concomitant 
Treatment^^ 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
^Lab results to be entered into eCRF for these time points should be the ones closer to the consent date/ visit date 
^^ Concomitant reporting: In case of any change in administering a concomitant medication i.e. dose, frequency etc. the previous medication should be stopped 
where the new medication has started 
* Assay to be performed only if patient > 12 years of age 
** Assay can only be performed if the lymphocyte count > 0.5 x 109/L  
# Assay to be carried out until evidence of T cell recovery 
*** Pre-gene therapy assay should be carried out only if not already done at diagnosis 
****Sample are stored but analysed retrospectively only in case sample at 1 year visit or 3 year visit are positive 
 
PHA - phytohaemagglutinin stimulation, Ig – immunoglobulin, TCR - T cell receptor, RCL - replication competent lentivirus, TREC - TCR excision 
circles, PCR - polymerase chain reaction, SCLN - save cells in liquid nitrogen 


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Appendix 6: Undesirable effects of Busilvex (Busulfan)  
(taken from the summary of product characteristics (Pierre Fabre Limited)) 
4.8 Undesirable effects 
Undesirable effects in adults: Adverse events information is derived from two clinical trials 
(n=103) of Busilvex.  
Serious toxicities involving the hematologic, hepatic and respiratory systems were considered 
as expected consequences of the conditioning regimen and transplant process. These include 
infection and Graft-versus host disease (GVHD) which although not directly related, were the 
major causes of morbidity and mortality, especially in allogeneic HPCT.  
Blood and the lymphatic system disorders:  
Myelo-suppression and immuno-suppression were the desired therapeutic effects of the 
conditioning regimen. Therefore all patients experienced profound cytopenia: leukpoenia 96%, 
thrombocytopenia 94% and anemia 88%. The median time to neutropenia was 4 days for both 
autologous and allogeneic patients. The median duration of neutropenia was 6 days and 9 
days for autologous and allogeneic patients.  
Immune system disorders:  
The incidence of acute graft versus host disease (a-GVHD) data was collected in OMC-BUS-
4 study (allogeneic) (n=61). A total of 11 patients (18%) experienced a-GVHD. The incidence 
of a-GVHD grades I-II was 13% (8/61), while the incidence of grade III-IV was 5% (3/61). 
Acute GVHD was rated as serious in 3 patients. Chronic GVHD (c-GVHD) was reported if 
serious or the cause of death, and was reported as the cause of death in 3 patients.  
Infections and infestations:  
39% of patients (40/103) experienced one or more episodes of infection, of which 83% (33/40) 
were rated as mild or moderate. Pneumonia was fatal in 1% (1/103) and life-threatening in 3% 
of patients. Other infections were considered severe in 3% of patients. Fever was reported in 
87% of patients and graded as mild/moderate in 84% and severe in 3%. 47% of patients 
experienced chills which were mild/moderate in 46% and severe in 1%.  
 
 


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Hepato-biliary disorders:  
15% of SAEs involved liver toxicity. HVOD is a recognised potential complication of 
conditioning therapy post-transplant. Six of 103 patients (6%) experienced HVOD. HVOD 
occurred in: 8.2% (5/61) allogeneic patients (fatal in 2 patients) and 2.5% (1/42) of autologous 
patients. Elevated bilirubin (n=3) and elevated AST (n=1) were also observed. Two of the 
above four patients with serious serum hepatotoxicity were among patients with diagnosed 
HVOD.  
Respiratory, thoracic and mediastinal disorders:  
One patient experienced a fatal case of acute respiratory distress syndrome with subsequent 
respiratory failure associated with interstitial pulmonary fibrosis in the Busilvex studies.  
In addition, the literature review reports alterations of cornea and lens of the eye with oral 
busulfan.  
Adverse events in newborn infants, children and adolescents:  
Adverse events information are derived from the clinical study in paediatrics (n=55). Serious 
toxicities involving the hepatic and respiratory systems were considered as expected 
consequences of the conditioning regimen and transplant process.  
Immune system disorders: The incidence of acute graft versus host disease (a-GVHD) data 
was collected in allogeneic patients (n=28). A total of 14 patients (50%) experienced a-GVHD. 
The incidence of a-GVHD grades I-II was 46.4% (13/28), while the incidence of grade III-IV 
was 3.6% (1/28). Chronic GVHD was reported only if it is the cause of death: one patient died 
13 months post-transplant.  
Infections and infestations:  
Infections (documented and non-documented febrile neutropenia) were experienced in 89% 
of patients (49/55). Mild/moderate fever was reported in 76% of patients.  
Hepato-biliary disorders:  
Grade 3 elevated transaminases were reported in 24% of patients. VOD was reported in 15% 
(4/27) and 7% (2/28) of the autologous and allogenic transplant respectively. VOD observed 
were neither fatal nor severe and resolved in all cases.  


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Adverse reactions reported both in adults and paediatric patients as more than an isolated 
case are listed below, by system organ class and by frequency. Frequencies are defined as: 
very common (>1/10), common (>1/100, <1/10), uncommon (>1/1,000, <1/100). 
System organ class 
Very common 
Common 
Uncommon 
Blood and lymphatic system disorders 
Neutropenia 
Thrombocytopenia 
Anaemia 
Pancytopenia 
Febrile neutropenia  
 
 
Nervous system disorders 
Insomnia 
Dizziness 
Depression 
Confusion 
Delirium Nervousness 
Hallucination 
Agitation 
Encephalopathy 
Cerebral haemorrhage 
Seizure 
Metabolism And nutrition 
disorders 
Anorexia 
Hyperglycaemia 
Hypomagnesaemia 
Hypokalaemia 
Hypocalcaemia 
Hypophosphatemia 
Oedema 
Hyponatraemia 
 
Psychiatric disorders 
Anxiety  
 
 
Cardiac disorders 
Tachycardia 
Hypertension 
Hypotension 
Vasodilatation 
Thrombosis 
Arrhythmia 
Atrial fibrillation 
Cardiomegaly 
Pericardial effusion 
Pericarditis 
Decrease ejection 
fraction 
Femoral artery 
thrombosis 
Ventricular 
extrasystoles 
Bradycardia 
Capillary leak 
syndrome 
Respiratory thoracic and 
mediastinal disorders 
Dyspnoea 
Rhinitis 
Pharyngitis 
Cough 
Hiccup 
Epistaxis 
Abnormal breath 
sounds 
Hyperventilation 
Respiratory failure 
Alveolar 
haemorrhages 
Asthma 
Atelectasis 
Pleural effusion 
Hypoxia 
Gastrointestinal disorders 
Nausea 
Stomatitis 
Vomiting 
Diarrhoea 
Constipation 
Dyspepsia 
Anus discomfort 
Abdominal pain 
Ascites 
Oesophagitis 
Ileus 
Haematemesis 
Gastrointestinal 
haemorrhage 
Hepato-biliary disorders 
Hepatomegaly 
Jaundice  
 
 
Skin and subcutaneous tissue 
disorders 
Rash 
Pruritis 
Alopecia 
Erythema 
Pigmentation disorder 
Skin desquamation 
 
Musculoskeletal connective 
tissue disorders 
Back pain 
Myalgia 
Arthralgia  
 
 
Renal and urinary disorders 
Creatinine elevated 
Dysuria 
Oligurea 
Bun increase 
Haematuria 
Moderate renal 
insufficiency 
 
General disorders and 
administration site conditions 
Fever 
Headache 
Asthenia 
Chills 
Pain 
Allergic reaction 
Oedema general 
Pain or inflammation at 
injection site 
Chest pain 
Mucositis  
 
 


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Appendix 7: 
WHO classification of acute/subacute toxic effects 
ITEM  
Grade 1 Toxicity 
Grade 2 Toxicity 
Grade 3 Toxicity 
Grade 4 Toxicity 
HEMATOLOGY  
Hemoglobin  
9.5 - 10.5 gm/Dl  
8.0 - 9.4 gm/Dl  
6.5 - 7.9 gm/Dl  
< 6.5 gm/Dl  
Absolute Neutrophil 
Count  
1000-1500/mm
3 
 
750-999/mm
3 
 
500-749/mm
3 
 
<500/mm
3 
 
Platelets  
75000-99000/mm
3 
 
50000-74999/mm
3 
 
20000-49000/mm
3 
 
<20000/mm
3 
 
Prothrombin  
Time (PT)  
1.01 - 1.25 x ULN  
1.26-1.5 x ULN  
1.51 -3.0 x ULN  
>3 x ULN  
Activated Partial 
Thromboplastin (APPT)  
1.01 -1.66 x ULN  
1.67 - 2.33 x ULN  
2.34 - 3 x ULN  
> 3 x ULN  
Fibrinogen  
0.75 - 0.99 X LLN  
0.50 - 0.74 x LLN  
0.25 - 0.49 x LLN  
< 0.25 x LLN  
Fibrin Split Product  
20-40 mcg/ml  
41-50 mcg/ml  
51-60 mcg/ml  
> 60 mcg/ml  
Methemoglobin  
5 - 9.9 %  
10.0 - 14.9 %  
15.0 - 19.9%  
> 20 %  
LIVER ENZYMES  
AST (SGOT)  
1.25 - 2.5 x ULN  
2.6 - 5 x ULN  
5.1 - 10 x ULN  
> 10 x ULN  
ALT (SGPT)  
1.25 - 2.5 x ULN  
2.6 - 5 x ULN  
5.1 - 10 x ULN  
> 10 x ULN  
GGT  
1.25 -2.5 x ULN  
1.6 - 5 x ULN  
5.1 - 10 x ULN  
> 10 x ULN  
Alkaline Phosphatase  
1.25 - 2. 5 x ULN  
1.6 - 5 x ULN  
5.1 - 10 x ULN  
> 10 x ULN  
Amylase  
1.1 - 1.5 x ULN  
1.6 - 2.0 x ULN  
2.1 - 5.0 x ULN  
> 5.1 x ULN  
CHEMISTRIES  
Hyponatremia  
130-135 mEq/L  
123-129 mEq/L  
116-122 mEq/L  
< 116 or mental 
status changes or 
seizures  
Hypernatremia  
146-150 mEq/L  
151-157 mEq/L  
158-165 mEq/L  
> 165 mEq/L or 
mental status 
changes or seizures  
Hypokalemia  
3.0 - 3.4 mEq/L  
2.5 - 2.9 mEq/L  
2.0 - 2.4 mEq/L  
or intensive 
replacement Rx 
required or 
hospitalization 
required.  
< 2.0 mEq/L  
or paresis or ileus or 
life-threatening 
arrhythmia  
Hyperkalemia  
5.6 - 6.0 mEq/L  
6.1 - 6.5 mEq/L  
6.6 - 7.0 mEq/l  
> 7.0 mEq/L  
or life-threatening 
arrhythmia  
Hypoglycemia  
55-64 mg/dL  
40-54 mg/dL  
30-39 mg/dL  
<30 mg/dL or 
mental status 
changes or coma  
Hyperglycemia  
(note if fasting)  
116 - 160 mg/dL  
161- 250 mg/dL  
251 - 500 mg/dL  
> 500 mg/dL  
or ketoacidosis  
or seizures  
Hypocalcemia  
(corrected for albumin)  
8.4 - 7.8 mg/dL  
7.7 - 7.0 mg/dL  
6.9 - 6.1 mg/dL  
< 6.1 mg/dL  
or life threatening 
arrhythmia or tetany  
Hypercalcemia  
(correct for albumin)  
10.6 - 11.5 mg/dL  
11.6 - 12.5 mg/dL  
12.6 - 13.5 mg/dL  
> 13.5 mg/dL  
life-threatening  
arrhythmia  


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Hypomagnesemia  
1.4 - 1.2 mEq/L  
1.1 - 0.9 mEq/L  
0.8 - 0.6 mEq/L  
< 0.6 mEq/L or life-
threatening 
arrhythmia  
Hypophosphatemia  
2.0 - 2.4 mg/dL  
1.5 -1.9 mg/dL or  
replacement Rx 
required  
1.0 -1.4 mg/dL  
intensive Rx or  
hospitalization required  
< 1.0 mg/dL or life-
threatening 
arrhythmia  
Hyperbilirubinemia  
1.1 - 1.5 x ULN  
1.6 - 2.5 x ULN  
2.6 - 5 x ULN  
> 5 x ULN  
BUN  
1.25 - 2.5 x ULN  
2.6 - 5 x ULN  
5.1 - 10 x ULN  
> 10 x ULN  
Creatinine  
1.1 x 1.5 x ULN  
1.6 - 3.0 x ULN  
3.1 - 6 x ULN  
> 6 x ULN or  
required dialysis  
URINALYSIS  
Proteinuria  
1+ or < 0.3% or 
<3g/L or 200 mg - 1 
gm loss/day  
2 -3 + or 0.3 - 1.0% or 
3-10 g/L  
1- 2 gm loss/day  
4+ or > 1.0% or > 10 
g/L  
2-3.5 gm loss/day  
nephrotic syndrome 
or > 3.5 gm loss/day  
Hematuria  
microscopic only  
gross, no clots  
gross+ clots  
obstructive or 
required transfusion  
CARDIAC DYSFUNCTION  
Cardiac Rhythm  
  
asymptomatic, 
transient signs, no  
Rx required 
recurrent/persistent;  
No Rx required  
requires treatment  
Hypertension  
transient inc. > 20  
mm; no Rx  
recurrent, chronic, > 
20 mm, Rx required  
requires acute Rx; No 
hospitalization  
requires 
hospitalization  
Hypotension  
transient orthostatic  
hypotension, No Rx  
symptoms correctable 
with oral fluids Rx  
requires IV fluids; no 
hospitalization required  
requires 
hospitalization  
Pericarditis  
minimal effusion  
mild/moderate 
asymptomatic 
effusion, no Rx  
symptomatic effusion; 
pain; EKG changes  
tamponade; 
pericardiocentesis 
or surgery required  
Hemorrhage, Blood Loss  
microscopic/occult  
mild, no transfusion  
gross blood loss;  
1-2 units transfused  
massive blood loss; 
> 3 units transfused  
RESPIRATORY  
Cough  
transient- no Rx  
treatment associated  
cough  
local Rx  
uncontrolled  
 
Bronchospasm, Acute  
transient; no Rx  
< 80% - 70% FEV1  
(or peak flow)  
requires Rx 
normalizes with 
bronchodilator;  
FEV1 50% - 70%  
(or peak Flow)  
no normalization with 
bronchodilator;  
FEV1 25% - 50%  
(or peak flow 
retractions)  
cyanosis: FEV1  
< 25%  
(or peak flow) or 
intubated  
GASTROINTESTINAL  
Stomatitis  
mild discomfort; no 
limits on activity  
some limits on 
eating/drinking  
eating/talking very 
limited  
requires IV fluids  
Nausea  
mild discomfort; 
maintains 
reasonable intake  
moderate discomfort; 
intake decreased 
significantly; some 
activity limited  
severe discomfort; no 
significant intake; 
activities limited  
minimal fluid intake  
Vomiting  
transient emesis  
occasional/moderate 
vomiting  
orthostatic hypotension 
or IV  
fluids required  
hypotensive shock 
or hospitalization 
required for IV fluid 
therapy  
Constipation  
mild  
moderate  
severe  
distensions 
w/vomiting  


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Diarrhea  
transient 3-4 loose 
stools/day  
5-7 loose stools/day  
orthostatic hypotension 
or  
> 7 loose stools/day or  
required IV fluids  
hypotensive shock 
or hospitalization for 
IV fluid therapy 
required  
NEURO & NEUROMUSCULAR  
Neuro-Cerebellar  
slight incoordination 
dysdiadochokinesis  
intention tremor, 
dysmetria, slurred 
speech; nystagmus  
locomotor ataxia  
incapacitated  
Mood  
mild anxiety or 
depression  
moderate anxiety or 
depression and 
therapy required  
severe anxiety or 
depression or mania; 
needs assistance  
acute psychosis; 
incapacitated, 
requires 
hospitalization  
Neuro Control  
(ADL = activities of daily 
living)  
mild difficulty 
concentrating; no 
Rx; mild 
confusion/agitation; 
ADL unaffected  
moderate 
confusion/agitation 
some limitation of 
ADL; minimal Rx  
severe 
confusion/agitation  
needs assistance for 
ADL; therapy required  
toxic psychosis; 
hospitalization  
Muscle Strength  
subjective 
weakness  
no objective 
symptoms/ signs  
mild objective  
signs/symptoms  
no decrease in 
function  
objective weakness 
function limited  
paralysis  
OTHER PARAMETERS  
Fever: oral, > 12 hours  
37.7 - 38.5 C or  
100.0 - 101.5 F  
38.6 - 39.5 C or  
101.6 - 102.9 F  
39.6 - 40.5 C or  
103 - 105 F  
> 40 C or  
> 105 F  
Headache  
mild, no Rx therapy  transient, moderate; Rx 
required  
severe; responds to 
initial narcotic therapy  
intractable; required 
repeated narcotic 
therapy  
Fatigue  
no decrease in 
ADL  
normal activity 
decreased 25- 50%  
normal activity 
decreased > 50% can’t 
work  
unable to care for 
self  
Allergic Reaction  
pruritus without 
rash  
localized urticaria  
generalized urticaria; 
angioedema  
anaphylaxis  
Local Reaction  
tenderness or 
erythema  
induration < 10 cm or 
phlebitis or 
inflammation  
induration > 10 cm or 
ulceration  
necrosis  
Mucocutaneous  
erythema; pruritus  
diffuse, maculo papular 
rash, dry desquamation  
vesiculation, moist 
desquamation, or 
ulceration  
exfoliative 
dermatitis, mucous 
membrane 
involvement or 
erythema, 
multiforme or 
suspected Stevens-
Johnson or necrosis 
requiring surgery  
  
 
 


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Appendix 8: Details of the historical HSCT control group 
1. Objective 
Historical data from ADA-SCID patients who were treated with Hematopoietic Stem Cell 
Transplantation (HSCT) at GOSH during the period of 2000-2016 will be collected. HSCT is 
currently the standard of care for ADA-SCID patients. These data will be used as historical 
control group in a current on-going gene therapy study “Phase I/II, historical 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 efficacy and safety of gene therapy versus HSCT to 
determine the optimal treatment. 
2. Study population 
The control group will be patients from 0-18 years of age with a confirmed diagnosis of ADA 
deficiency transplanted at Great Ormond Street Hospital (GOSH) since 2000. See inclusion 
criteria page 64. 
3. Data to be collected 
GOSH holds a registry / clinical database (European Society for Immunodeficiencies (ESID) 
and European Society for Blood and  Marrow Transplant (EBMT) Registry, GOSH BMT clinical 
database) displaying information on these types of patients. Patients treated with HSCT have 
been identified who received a transplant at GOSH between 2000-2016. . ESID and EBMT 
will allow us to collect the below information: 
• 
Demographics (gender, date of birth, date of death if any), 
• 
Method of ADA-SCID diagnosis, 
• 
HSCT treatment(s) including: date of treatment, transplant cells, donor type and 
outcome 
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 history and immunological response to vaccination 


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• 
Auto-immune disease 
• 
Malignant disease / leukaemia 
• 
Graft versus host disease 
• 
ADA activity and ADA metabolites 
• 
AEs and SAEs 
This information is only available from the patient’s records; we therefore plan to re-consent 
these patients to access their full medical records. 
4. Data collection process 
This is a retrospective, non-interventional data collection activity; the patients follow their 
routine medical practice. We will contact these patients or their authorised representative and 
seek their consent to access their medical notes and share the information with regulatory 
agencies and pharmaceutical companies involved with the licensing process. HSCT historical 
control group data team will work on the collection of data from GOSH registries and patients’ 
records, 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. 
5. Data Analysis 
The comparison analysis between gene therapy and HSCT will be performed evaluating data 
related to: 
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 infections post treatments. Infections 
are defined as infections requiring hospitalisation or prolonging hospitalization and/or 
documented infections by opportunistic pathogens (interstitial pneumonia, intractable 
diarrhoea) 
 
 


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Appendix 9: Details of the CUP group 
Great Ormond Street Hospital holds a Specials License from the MHRA for the manufacture 
of gene modified products for compassionate use. This license allows a physician to treat a 
patient with an unlicensed product in case of a special need. These ADA-SCID patients treated 
under this Special License could not be enrolled into the clinical trial for various reasons: the 
trial was not yet opened and patients were in clinical need of treatment; the patient could not 
meet the inclusion/exclusion criteria; the final formulation of the product was different 
(cryopreserved instead of fresh transduced cells); the product could not meet the release 
specification, the trial recruitment was already completed and patients were in clinical need of 
treatment.  
However, in the treatment of these patients we have applied similar standards to clinical trial 
i.e. the patient gives their consent to collect data, the same trial monitoring schedule/trial 
procedure is applied, and pharmacovigilance (SAE) is documented in the same way. These 
patients will be included part of the analysis for the safety and secondary efficacy objectives 
only as mentioned in section 8.9.  
 
 

SAP_001.pdf: 
COVER PAGE
Official Study Title: Phase I/II, Historical 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
Statistical Analysis Plan v12.0, 10-Jan-2018
NCT01380990


Statistical Analysis Plan
Study Name: 
Phase I/II, historical 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 
Protocol Number: 
10-MI-29
Protocol Version: 
Version 17.0 
Protocol Date:  
28 June 2017 
SAP Version 12.0: 
10 January 2018 


Study: 10-MI-29 
 
 
 
 
 
 
Statistical Analysis Plan 
Final Version 12.0 
Page 2 of 31 


Study: 10-MI-29 
 
 
 
 
 
 
Statistical Analysis Plan 
Final Version 12.0 
Page 3 of 31 
Table of Contents 
1 
Introduction ...................................................................................................................... 8 
2 
Study Design .................................................................................................................... 8 
3 
Study Schedule ................................................................................................................. 9 
4 
Study Objectives ............................................................................................................. 14 
4.1 
Primary Objectives ......................................................................................................... 14 
4.2 
Secondary Objectives ..................................................................................................... 14 
5 
Endpoints ........................................................................................................................ 14 
5.1 
Primary Endpoints .......................................................................................................... 14 
5.2 
Secondary Endpoints ...................................................................................................... 15 
5.3 
Additional Endpoints ...................................................................................................... 15 
6 
Timing of Analyses ........................................................................................................ 15 
7 
Analysis Populations ...................................................................................................... 16 
7.1 
Efficacy Population ........................................................................................................ 16 
7.2 
Safety Population............................................................................................................ 17 
8 
Statistical Methods ......................................................................................................... 17 
8.1 
Subject Disposition ......................................................................................................... 17 
8.1.1 
Summary of Populations ............................................................................. 17 
8.1.2 
Summary of Withdrawals ............................................................................ 17 
8.2 
Demography and Baseline Characteristics ..................................................................... 18 
8.2.1 
Demography ................................................................................................ 18 
8.2.2 
ADA Deficiency Diagnosis ......................................................................... 18 
8.2.3 
Disease Treatment History .......................................................................... 18 
8.2.4 
Medical History ........................................................................................... 19 
8.2.5 
Infection at Screening .................................................................................. 19 
8.2.6 
Coagulation Tests ........................................................................................ 19 
8.2.7 
Glomerular Filtration Rate (GFR) at screening ........................................... 19 
8.2.8 
Virology Tests ............................................................................................. 19 
8.2.9 
Cardiopulmonary Tests at Screening ........................................................... 19 
8.2.10 
General Tests at Screening .......................................................................... 19 
8.2.11 
Harvest for Gene Therapy ........................................................................... 20 
8.2.12 
Back-up Harvest .......................................................................................... 20 
8.2.13 
Myeloablative Conditioning ........................................................................ 20 


Study: 10-MI-29 
 
 
 
 
 
 
Statistical Analysis Plan 
Final Version 12.0 
Page 4 of 31 
8.2.14 
IMP Administration ..................................................................................... 20 
8.2.15 
Transplant Details for the HSCT Controls .................................................. 20 
8.2.16 
Exposure ...................................................................................................... 21 
8.3 
Efficacy Analyses ........................................................................................................... 21 
8.3.1 
Primary Efficacy Endpoints ........................................................................ 21 
8.3.1.1 
Overall Survival at 12 Months .................................................................... 21 
8.3.1.2 
Event-free Survival at 12 Months ................................................................ 22 
8.3.1.3 
Engraftment ................................................................................................. 22 
8.3.1.4 
Cellular and Humoral Immune System Recovery ....................................... 23 
8.3.1.5 
RBC ADA Enzyme Activity and Reduction in dATP ................................ 23 
8.3.2 
Secondary Efficacy Endpoints..................................................................... 23 
8.3.2.1 
Overall and Event-free Survival at 2 and 3 Years ....................................... 23 
8.3.2.2 
Use of Immunoglobulin Replacement Therapy........................................... 24 
8.3.2.3 
Rate of Severe Infections ............................................................................. 25 
8.3.2.4 
T Cell Receptor Excision Circles (TREC) in the Peripheral Blood ............ 25 
8.3.3 
Additional Efficacy Endpoints .................................................................... 25 
8.3.3.1 
Use of PEG-ADA Enzyme Replacement Therapy ...................................... 25 
8.3.3.2 
T Cell Vβ Panel ........................................................................................... 25 
8.3.3.3 
PHA Stimulation ......................................................................................... 26 
8.3.3.4 
CD3 Stimulation .......................................................................................... 26 
8.3.3.5 
Vector Integration Analysis ......................................................................... 26 
8.3.3.6 
Bone Marrow Aspirate ................................................................................ 26 
8.3.3.7 
Response to Tetanus Vaccination ................................................................ 26 
8.3.4 
Sensitivity Analyses .................................................................................... 26 
8.4 
Safety Analyses .............................................................................................................. 27 
8.4.1 
Adverse Events ............................................................................................ 27 
8.4.1.1 
Definition of Treatment-emergent Adverse Events .................................... 27 
8.4.1.2 
Adverse Events ............................................................................................ 27 
8.4.1.3 
Serious Adverse Events and Death .............................................................. 28 
8.4.2 
Concomitant Medications ............................................................................ 28 
8.4.3 
RCL Testing ................................................................................................ 28 
8.4.4 
Laboratory Evaluations................................................................................ 28 
8.4.4.1 
Haematology ................................................................................................ 28 


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8.4.4.2 
Biochemistry ................................................................................................ 29 
8.4.5 
Vital Signs and Weight ................................................................................ 29 
8.4.6 
Physical Examination .................................................................................. 30 
9 
Analysis Software ........................................................................................................... 30 
10 
Changes in Statistical Analysis Plan .............................................................................. 30 
10.1 
Changes in Version 10.0 .............................................................................. 30 
10.2 
Changes in Version 11.0 .............................................................................. 31 
 
 
 
 


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List of Abbreviations 
 
 
ADA 
Adenosine deaminase 
ADA-SCID 
Adenosine deaminase severe combined immunodeficiency 
AE 
Adverse event 
ALT 
Alanine aminotransferase 
APT 
Activated partial thromboplastin 
AST 
Aspartate aminotransferase 
AUC 
Area under the curve 
BM 
Bone marrow 
BSA 
Body surface area 
CI 
Confidence interval 
CMV 
Cytomegalovirus 
CUP 
Compassionate use programme 
dATP 
Deoxyadenosine triphosphate 
DNA 
Deoxyribonucleic acid 
EBV 
Epstein-Barr virus 
ECG 
Electrocardiogram 
eCRF 
Electronic case report form 
EFS 
Elongation Factor 1α Short form 
ERT 
Enzyme replacement therapy 
G-CSF 
Granulocyte-colony stimulating factor 
GFR 
Glomerular filtration rate 
GGT 
Gamma glutamyl transferase 
GOSH 
Great Ormond Street Hospital 
HIV 
Human immunodeficiency virus 
HLA 
Human leukocyte antigen 
HSC 
Hematopoietic stem cell 
HSCT 
Hematopoietic stem cell transplant 
HSV 
Herpes simplex virus 
HTLV 
Human T-lymphotropic virus 
Ig 
Immunoglobulin 
IgA 
Immunoglobulin A 
IgG 
Immunoglobulin G 
IgM 
Immunoglobulin M 
IgRT 
Immunoglobulin replacement therapy 
IMP 
Investigational medicinal product 
LV 
Lentiviral vector 
MCH 
Mean corpuscular haemoglobin 
MCHC 
Mean corpuscular haemoglobin concentration 
MCV 
Mean corpuscular volume 


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MedDRA 
Medical Dictionary for Regulatory Activities 
MMRM 
Mixed model for repeated measures 
MNC 
Mononuclear cells 
NK 
Natural Killer 
PBMC 
Peripheral blood mononuclear cells 
PBSC 
Peripheral blood stem cells 
PCR 
Polymerase chain reaction 
PEG 
Polyethylene-glycol 
PHA 
Phytohemagglutinin 
PI 
Principal Investigator 
PT 
Preferred Term 
qPCR 
Quantitative polymerase chain reaction 
RBC 
Red blood cell(s) 
RCL 
Replication competent lentivirus 
SAE 
Serious Adverse Event 
SAP 
Statistical analysis plan 
SCID 
Severe combined immunodeficiency 
SCLN 
Save cells in liquid nitrogen 
SOC 
System Organ Class 
TCR 
T-cell receptor 
TREC 
T-cell receptor excision circle 
UK 
United Kingdom 
VZV 
Varicella zoster virus 
WBC 
White blood cell(s) 
 
 
 


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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 medicinal product (IMP) includes autologous CD34+ HSC of 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).  The IMP is used as fresh 
cell dispersion for infusion after non-myeloablative conditioning. 
2 Study Design 
This is a Phase I/II, historical controlled, open-label, non-randomised, single-centre trial to 
assess the safety and efficacy of the IMP, i.e. autologous CD34+ HSC transduced ex vivo with 
EFS-ADA LV in ADA-SCID patients. 
 
The IMP will be compared with an historical control group treated with allogeneic 
Hematopoietic Stem Cell Transplantation (HSCT), representative of standard of care. 
  
The study is conducted in a single centre at Great Ormond Street Hospital (GOSH) for 
Children, London, UK. 
 
 
 
 


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3 Study Schedule 


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Timepoint (months) ^ 
 
Visit window 
 
 
+/-
5d 
+/-
5d 
+/- 
2w 
+/- 
2w 
+/-
2w 
+/-
2w 
+/-
2w 
+/- 
2w 
+/-
2w 
+/-
2w 
Assessment 
Parameter to be reported on the eCRF 
PreGT 
Treat
ment 
1 
1.5 
3 
6 
9 
12 
18 
24 
30 
36 
Inclusion / Exclusion criteria 
- 
X 
 
 
 
 
 
 
 
 
 
 
 
Pregnancy test for patient >12 years 
old 
-  
X* 
 
 
 
 
 
 
 
 
 
 
 
Diagnosis 
DNA sequencing (mutation, exon, polymorphism) OR 
Confirmed absence of <3% enzymatic activity 
X 
 
 
 
 
 
 
 
 
 
 
 
Medical history  
Infection/hospitalisation history 
Transplantation history 
X 
 
 
 
 
 
 
 
 
 
 
 
Coagulation testing 
PTT, APT, Thrombin Time, Fibrinogen 
X 
 
 
 
 
 
 
 
 
 
 
 
Blood ABO testing 
Determination, Rhesus 
X 
 
 
 
 
 
 
 
 
 
 
 
Extended B cell memory  
CD19 
Naïve B cells CD19+IgD+CD27- 
Non Switched memory B cells CD19+IgD+CD27+ 
Class Switched memory B cells  
CD19+IgD-CD27+ 
Transitional B cells CD19+IgM++CD38++ 
Plasmablasts CD19+CD38++IgMwk 
CD21 low B cells CD19+CD21wkCD38wk 
X 
 
 
 
 
 
 
X 
 
X 
 
X 
Virology 
CMV, EBV, Adenovirus, HIV1, HIV2, VZV, Hep A, Hep B, 
HSV, Hep C, Syphilis, HTLV1, HTLV2, Toxoplasma 
X 
 
 
 
 
 
 
 
 
 
 
 
Biochemistry  
Sodium, Potassium, Urea, Creatinine, Calcium, 
Magnesium, Phosphate, Albumin, Alkaline Phosphatase 
(ALP), Alanine Transaminase(ALT), gamma-
glutamyltranferase(GGT), Bilirubin 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Glomerular Filtration  
Weight, Creatinine,Distribution volume,Half-life 
Glomerular Filtration, corrected GFR 
X 
 
 
 
 
 
 
 
 
 
 
 
Chest x-ray 
Normal/Abnormal 
X 
 
 
 
 
 
 
 
 
 
 
 
ECG 
Normal/Abnormal 
X 
 
 
 
 
 
 
 
 
 
 
 
Echocardiogram 
Normal/Abnormal 
X 
 
 
 
 
 
 
 
 
 
 
 
Lung function test if applicable\\ 
Normal/Abnormal 
X 
 
 
 
 
 
 
 
 
 
 
 
Dental 
Normal/Abnormal 
X 
 
 
 
 
 
 
 
 
 
 
 
Audiology 
Normal/Abnormal 
X 
 
 
 
 
 
 
 
 
 
 
 
Maternal engraftment studies 
Normal/Abnormal 
X 
 
 
 
 
 
 
 
 
 
 
 


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Assessment 
Result to be reported on the eCRF 
PreGT 
Treat
ment 
1 
1.5 
3 
6 
9 
12 
18 
24 
30 
36 
Back-up harvest 
Harvest type BM or leukapheresis, CD34+cell count 
 
X 
 
 
 
 
 
 
 
 
 
 
Harvest for IMP preparation 
If leukapheresis : Mobilisation: G-CSF (date dose) +/- 
Plerixafor (date, dose), Leukapheresis harvest: patient’s 
weight, date, CD34+cell dose for transduction 
If BM: patient’s weight, date, CD34+cell dose for 
transduction) 
 
X 
 
 
 
 
 
 
 
 
 
 
Conditioning 
Patient weight, Busulfan dose, Busulfan AUC 
 
X 
 
 
 
 
 
 
 
 
 
 
IMP administration 
Infusion date, IMP expiry, IMP start date, IMP end date, 
IMP cell dose, IMP viability, IMP volume 
Observation before, +15min,  after infusion 
 
X 
 
 
 
 
 
 
 
 
 
 
Vital Signs 
Patient’s weight 
Temperature 
Heart rate 
Respiratory rate 
Oxygen saturation 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Physical examination 
Cardiovascular system 
Respiratory system 
Ear, Nose, Throat, Neck and Eyes (only at PreGT, 36 m) 
Neurological System (only at PreGT, 36 months) 
Abdominal 
Skin 
Other 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Infection status 
Evaluate any active infection at time of visit (respiratory 
tract, meningitis, sepsis, skin and mucosa, ear, nose and 
throat, abscess, persistent fever, other) 
Evaluate if there is any infection resulting in 
hospitalisation since last visit 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
PEG-ADA ERT status 
Confirmation that if PEG-ADA has been administered or 
not since last visit 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Blood Count 
Haemoglobin, 
Platelet count 
White cell count 
Neutrophil count 
Lymphocyte count 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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Assessment 
Result to be reported on the CRF 
PreGT 
Treat
ment 
1 
1.5 
3 
6 
9 
12 
18 
24 
30 
36 
Lymphocyte subset counts 
 
CD 3 
CD19 
CD16+ CD 56+ 
CD3+ CD4+ 
CD3+ CD8+ 
Naïve: CD4+CD45RA+CD27+ 
 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
T cell function PHA stimulation** 
Normal/Abnormal 
X 
 
 
 
X 
X 
 
X 
X 
X 
 
 
Lymphocyte function CD3 
stimulation** 
Normal/Abnormal 
 
 
 
 
 
 
 
X 
 
 
X 
 
GAM *** 
Ig G level 
Ig A level 
Ig M level 
Immunoglobin status since last visit 
 
X 
 
 
 
 
X 
X 
X 
X 
X 
X 
X 
TRECS 
TRECS on T cells 
TRECS on MNCs 
 
X 
 
 
 
 
X 
 
X 
 
X 
 
 
Vβ Spectratyping 
Reported as Normal/Abnormal 
 
 
 
 
 
X 
 
X 
X 
X 
X 
X 
Vector copy number in cells (qPCR) 
vector copy number  in PBMC 
vector copy number  in CD34 
vector copy number  in CD3 
vector copy number  in CD19 
vector copy number  in myeloid 
 
 
 
 
 
X 
X 
 
X 
 
X 
 
X 
Integration analysis  
Normal/Abnormal 
 
 
 
 
X**** 
X**** 
 
X 
 
X**** 
 
X 
Tetanus vaccination responses  
Normal/Abnormal 
 
 
 
 
 
 
 
 
 
X 
 
X 
ADA metabolite analysis (dATP) and 
ADA activity 
Plasma deoxyadenosine level 
ADA (rbc) level 
dATP level 
 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
RCL (blood) 
Reported as detectable / not detectable 
X 
 
 
 
X**** 
X**** 
 
X 
 
X**** 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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Assessment 
Result to be reported on the CRF 
PreGT 
Treat
ment 
1 
1.5 
3 
6 
9 
12 
18 
24 
30 
36 
Bone marrow aspirate for vector copy 
number qPCR and integration analysis 
(optional) 
Morphology (Normal/ Abnormal) 
Vector copy number in BM-MNC 
Integration analysis (Normal / Abnormal) 
 
 
 
 
 
 
 
X 
 
X 
 
X 
Immunology save serum & SCLN 
(stored cells) 
Serum collection date 
SCLN s collection date 
X 
 
 
 
 
X 
 
X 
X 
X 
X 
X 
Reporting of Adverse Event 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Reporting of Concomitant Treatment^^ 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
^Lab results to be entered into eCRF for these time points should be the ones closer to the consent date/ visit date 
^^ Concomitant reporting: In case of any change in administering a concomitant medication i.e. dose, frequency etc. the previous medication should be stopped where the new 
medication has started 
* Assay to be performed only if patient > 12 years of age 
** Assay can only be performed if the lymphocyte count > 0.5 x 109/L  
# Assay to be carried out until evidence of T cell recovery 
*** Pre-gene therapy assay should be carried out only if not already done at diagnosis 
****Sample are stored but analysed retrospectively only in case sample at 1 year visit or 3 year visit are positive 
 
PHA - phytohaemagglutinin stimulation, Ig – immunoglobulin, TCR - T cell receptor, RCL - replication competent lentivirus, TREC - TCR excision circles, PCR - polymerase 
chain reaction, SCLN - save cells in liquid nitrogen  
 


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4 Study Objectives 
4.1 Primary Objectives 
The primary objective is to assess the safety and efficacy of EFS-ADA LV mediated gene 
therapy, the Investigational Medicinal Product (IMP) for treatment of ADA-SCID patients.  
This will be achieved via the following specific objectives: 
 
Efficacy 
• 
To assess overall survival and event-free survival at 1 year for subjects treated with IMP 
and compare with patients treated with allogeneic HSCT 
• 
To assess at each visit engraftment success and resulting immunological and metabolic 
effects in IMP treated subjects using:  
o 
Vector copy numbers in peripheral blood leukocytes 
o 
Cellular and humoral immune system recovery 
o 
ADA enzyme activity and reduction in dATP in peripheral blood cells 
 
Safety 
• 
To assess clinical, haematological and immunological progress of subjects 
• 
To assess vector integration sites and clonal proliferation.  This analysis will be performed 
at a specialised laboratory in Germany and the data will be reviewed and interpreted by 
the Principal Investigator (PI) and the study team 
4.2 Secondary Objectives 
The secondary objectives are to: 
• 
Compare overall survival and event-free survival at 2 and 3 years between patients treated 
with IMP and patients treated with allogeneic HSCT 
• 
Determine the percentage of IMP treated patients requiring immunoglobulin replacement 
therapy at all evaluation points from 18 months onwards  
• 
Compare frequency of infections, and growth of pathogenic microorganisms over 3 years 
• 
Evaluate the longitudinal clinical effect in terms of improved immunity 
• 
Evaluate tolerability of conditioning regimen. These data will be reviewed and interpreted 
by the PI and the study team 
• 
Evaluate feasibility of the transduction procedure. These data will be reviewed and 
interpreted by the PI and the study team. 
5 Endpoints 
5.1 Primary Endpoints 
Primary endpoints for this study are: 
• 
Overall survival at 1 year for patients treated with IMP. 
• 
Event-free survival at 1 year for patients treated with IMP. 


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• 
Vector copy number in the cells at each study visit. 
• 
Changes in T cell number, function and circulating immunoglobulin levels at each 
study visit. 
• 
dATP and ADA enzyme activity in erythrocytes at each study visit. 
The following endpoints will be analysed at a specialist laboratory in Germany and the results 
will be reviewed and interpreted by the PI and the study team, so these will not be included in 
this Statistical Analysis Plan (SAP). 
• 
Frequency of vector integration into known proto oncogenes.  
• 
Frequency of clonal expansion associated with vector integration near proto 
oncogene. 
5.2 Secondary Endpoints 
• 
Overall survival and event-free survival at 2 years and 3 years 
• 
Frequency of infections (evaluated at 1, 2 and 3 years after treatment)  
• 
TRECS after 2 years post-gene therapy 
• 
Requirement for immunoglobulin replacement therapy (IgRT) and serum 
immunoglobulin levels for IgA, IgG and IgM.  
• 
Feasibility of the transduction procedure assessed by availability of greater than 0.5 x 
106 
CD34+ 
cells/kg 
after 
transduction; 
undetectable 
RCL 
(determined 
retrospectively); and CD45+ cell viability after transduction equal to or greater than 
50%, in accordance with the final product release criteria. These data will be reviewed 
and interpreted by the PI and the study team and so will not be included in this SAP. 
5.3 Additional Endpoints 
Additional endpoints for this study are: 
• 
T cell Vβ panel 
• 
Phytohaemagglutinin (PHA) stimulation result 
• 
CD3 stimulation 
• 
Vector integration analysis result 
• 
Bone marrow aspirate (if performed) 
o Morphology (Normal/ Abnormal). 
o Integration (Normal/ Abnormal). 
o Vector copy number in BM-MNC. 
• 
Tetanus vaccination response 
6 Timing of Analyses 
The statistical analyses of data from this study will be performed in four stages: 
 
Interim look: 
• Performed after all on-study subjects have completed 6 months of follow-up post 
infusion 
• Descriptive statistics for all available data 


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• Comparisons between IMP treated subjects and HSCT control cohorts of 
demographics and overall survival 
 
Primary analysis: 
• Performed after all on-study 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 at 12 months 
(primary efficacy endpoints), use of immunoglobulin replacement therapy (IgRT) and 
infection rate (key secondary endpoints). 
 
Long-term follow-up analysis: 
• Performed after all on-study subjects have completed 2 years of follow-up post 
infusion  
• Descriptive statistics for all available data 
• Comparison to HSCT control cohorts on overall and event-free survival at 2 years, use 
of immunoglobulin replacement therapy (IgRT) and infection rate (key secondary 
endpoints).  
 
End of study analysis: 
• Performed after all on-study subjects have completed the study (3 years of follow-up 
post infusion)  
• Descriptive statistics for the complete study cohort 
• Comparison to HSCT control cohorts on overall and event-free survival at 3 years, use 
of immunoglobulin replacement therapy (IgRT) and infection rate (key secondary 
endpoints).  
7 Analysis Populations 
7.1 Efficacy Population 
IMP Treated Subjects 
The study protocol recruited 10 subjects with a diagnosis of ADA-SCID to be treated with IMP 
at GOSH.  These subjects will be known as the “on-study” subjects. 
 
In addition, a group of subjects were treated under a Compassionate Use Programme (CUP 
under GOSH Special License) either because the study was not yet open and urgent treatment 
was needed, because they were outside of the inclusion/exclusion criteria or because they 
followed a different process (i.e., received the IMP in two infusions).  These subjects followed 
the same study schedule as the on-study subjects. 
 
The primary efficacy population for analysis will consist of the on-study IMP treated subjects.  
A secondary efficacy population will consist of all IMP treated subjects (on-study and CUP) 
 


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HSCT Control Patients 
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 Great Ormond Street Hospital (GOSH) from the year 2000 onwards. 
 
Secondary efficacy populations will comprise: 
• ADA-SCID patients with matched related donors treated with HSCT at GOSH 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 GOSH from the year 2000 onwards. 
7.2 Safety Population 
The safety population will consist of all IMP treated subjects (on-study and CUP) and the 
complete HSCT historical control cohort consisting of ADA-SCID patients with any type of 
donor treated with HSCT at GOSH 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 IMP treated subjects will be summarised in the groups of on-study only 
and on-study plus CUP subjects.  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 IMP 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, the primary analysis and the long-term follow-up analysis, the number (%) 
completing the designated follow-up period, and ongoing in the study will also be summarized. 


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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. 
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 IMP treated on-study subjects will be calculated as:  
12*(date informed consent signed – date of birth)/365.25. 
 
Age in months for the IMP treated CUP subjects and the HSCT controls will be captured on 
the eCRF. 
 
Age in months will be summarized for the IMP treated and HSCT control groups.  Sex, 
ethnicity, location, weight and height at screening will be summarised for the IMP treated on-
study group. 
8.2.2 ADA Deficiency Diagnosis 
Age at diagnosis in months will be calculated for the IMP treated subjects (on-study and CUP) 
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 and time since diagnosis will be summarized using descriptive statistics for 
the IMP treated subjects and HSCT control groups.  In addition, the number (%) of subjects 
with each method of diagnosis (DNA sequencing/ confirmed absence of <3% enzymatic 
activity/ unknown) will be summarized.  For DNA sequencing, the details of mutations, exons 
and polymorphisms will be listed. 
 
8.2.3 Disease Treatment History 
Number (%) of IMP treated subjects who previously received PEG-ADA treatment, receiving 
immunoglobulin replacement therapy, receiving infection prophylaxis, any previous BM or 
PBSC transplant (with the outcome) will be summarized. 


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8.2.4 Medical History 
The number (%) of subjects reporting medical history in each category will be summarised for 
the IMP treated and the HSCT control groups. 
8.2.5 Infection at Screening 
The number (%) of IMP treated subjects reporting active infections at screening will be 
summarised overall and by localisation/event type. 
8.2.6 Coagulation Tests 
Coagulation tests (partial thromboplastin time, APT, thrombin time and fibrinogen) will be 
evaluated at screening for the IMP treated subjects and summarised using descriptive statistics. 
8.2.7 Glomerular Filtration Rate (GFR) at screening 
Distribution volume, half-life, glomerular filtration rate and corrected glomerular filtration rate 
will be summarised for the IMP treated subjects using descriptive statistics.  In addition, the 
number (%) of subjects with a distribution volume outside the normal range that was 
considered to be clinically non-significant or clinically significant will be summarized. 
8.2.8 Virology Tests 
The number (%) of IMP treated subjects with positive results for CMV, EBV, Adenovirus, 
HIV1, HIV2, VZV, Hep A, Hep B, HSV, Hep C, Syphilis, HTLV1, HTLV2, Toxoplasma will 
be summarised. 
8.2.9 Cardiopulmonary Tests at Screening 
A chest x-ray, ECG, Echocardiogram and lung function test will be carried out at screening.  
The results of each test will be categorized as normal/ abnormal, not clinically significant/ 
abnormal, clinically significant.  The number (%) of IMP treated subjects with evaluations 
falling into each category will be summarised for each test.  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, details of any clinically significant findings will be listed.  
8.2.10 
General Tests at Screening 
Dental, audiology and maternal engraftment studies tests will be carried out at screening.  The 
results of each test will be categorized as normal/ abnormal, not clinically significant/ 
abnormal, clinically significant.  The number (%) of IMP treated subjects with evaluations 
falling into each category will be summarised for each test.  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. 
 


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In addition, details of any clinically significant findings will be listed.  
8.2.11 
Harvest for Gene Therapy 
The number (%) of IMP treated subjects with each blood type and with stem cells from each 
source (leukapheresis/bone marrow) will be summarised. 
 
For subjects with a leukapheresis harvest, the dose of G-CSF administered on Days 1-6, the 
details of any plerixafor administration and the CD34+ cell counts in blood will be listed. 
 
For subjects with a leukapheresis harvest on multiple days, the total number of cells will be the 
sum across the days.  The number of cells stored for the gene therapy will be calculated as the 
total cell count – number of back up cells.  Body weight at harvest, total number of nucleated 
cells, total CD34+ cell count and CD34+ cell count excluding back-up cells will be summarised 
using descriptive statistics. 
8.2.12 
Back-up Harvest 
The number (%) of IMP treated subjects for whom ≥3x106 CD34+ cells/kg have been stored as 
back-up will be summarised along with the CD34+ count.  For those that have a back-up 
harvest, the number (%) with stem cells from each source (leukapheresis/bone marrow) will be 
summarized using the number with a back-up harvest as denominator for the percentage.  For 
those without a back-up harvest, the number (%) with each reason for not performing the 
harvest will be summarized using the number without a back-up harvest as denominator for the 
percentage. 
8.2.13 
Myeloablative Conditioning 
The number (%) of IMP treated subjects for whom conditioning was done will be summarised 
along with the body weight, height, BSA, busulfan dose and busulfan AUC. 
8.2.14 
IMP Administration 
Age at transplant in months will be calculated for the IMP treated subjects (on-study and CUP) 
as:  
12*(date of transplant – date of birth)/365.25. 
 
The duration of IMP infusion (in minutes) will be calculated as (end time – start time)/60. 
 
The number (%) of subjects who received the IMP infusion and for whom the entire content of 
the bag was infused will be summarised along with the age at transplant, the duration of 
infusion, CD34+ cell dose, cell viability and volume.  
8.2.15 
Transplant Details for the HSCT Controls 
For the HSCT control groups, the age in months at transplant will be recorded on the eCRF. 
 


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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. 
 
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. 
8.2.16 
Exposure 
Duration of follow-up in months will be calculated for the IMP 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 IMP 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 Analyses 
The primary comparison for all efficacy analyses will be between the IMP treated on-study 
subjects and the allogeneic HSCT controls treated at GOSH without a matched related donor.   
Secondary comparisons will be between: 
• the IMP treated on-study subjects and the HSCT controls treated at GOSH with a matched 
related donor; 
• the IMP treated on-study subjects and the total group of HSCT controls; 
• all IMP treated subjects (on-study and CUP) and the HSCT controls treated at GOSH 
without a matched related donor; 
• all IMP treated subjects (on-study and CUP) and the HSCT controls treated at GOSH with 
a matched related donor; 
• all IMP treated subjects (on-study and CUP) 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 


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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 each of the IMP treated and HSCT control groups along 
with the difference in proportions (with 95% CI) between the IMP treated and control groups, 
excluding those with unknown status.   
 
Overall survival in each treatment group will also be represented 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% CI 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 IMP treated and HSCT control groups.  
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. 
 
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. 
8.3.1.3 
Engraftment 
Engraftment of transduced cells will be assessed using vector gene marking in granulocytes,  
PBMC, CD3, CD34, CD19 and myeloid progenitor cells.  Any assessments in which the result 
indicates that cells were not detected, will be assigned a value of 0. 
 
The vector copy numbers in each type of cell will be summarised for IMP treated subjects by 
visit using descriptive statistics.  In addition, for each cell type, plots of the median and range 
for copy numbers over time on a logarithmic scale will be produced along with individual 
subject plots. 


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8.3.1.4 
Cellular and Humoral Immune System Recovery  
Immune reconstitution will be assessed using lymphocyte subsets.  Absolute numbers and 
percentages of CD3+, CD4+, CD8+ CD4+CD45RA+CD27+ T-lymphocytes, CD19+ B-
lymphocytes and CD16+CD56+ NK cells will be measured. 
 
For each of the lymphocyte subsets, the actual values and change from baseline will be 
summarized by visit for each of the IMP treated groups using descriptive statistics.  The median 
value and range will be plotted by treatment group over time on a logarithmic scale and 
individual counts will be plotted along with the age-dependent normal ranges provided by the 
study site. 
 
In addition, statistical testing of the change from baseline to 6 and 12 months will be performed 
for the IMP treated subjects.  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 in the IMP treated subjects 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. 
Otherwise, if 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. 
 
8.3.1.5 
RBC ADA Enzyme Activity and Reduction in dATP  
RBC ADA enzyme activity, dATP and plasma deoxyadenosine will be summarized by visit 
for each of the IMP treated groups using descriptive statistics.  The median value and range 
will be plotted by treatment group over time on a logarithmic scale.  In addition, individual 
subject plots will be created. 
 
Changes from baseline in each of the parameters will be calculated with baseline being the pre-
treatment evaluation.  The change from baseline will be summarized by visit for each of the 
IMP treated and HSCT control groups (data permitting) using descriptive statistics and 95% 
confidence intervals. 
 
8.3.2 Secondary Efficacy Endpoints 
8.3.2.1 
Overall and Event-free Survival at 2 and 3 Years 
Overall and event-free survival at 2 and 3 years 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. 


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8.3.2.2 
Use of Immunoglobulin Replacement Therapy 
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 each of the IMP 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, 24, 30 and 36 
months without restarting at any subsequent time point will be summarized for each of the IMP 
treated and HSCT control groups (data permitting) along with exact 95% CIs. 
 
In addition, serum immunoglobulin levels (IgG, IgA and IgM) will be measured at each visit 
for the IMP 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 site. 
 
 
 


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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 each of the IMP treated and 
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 
second and third years. 
8.3.2.4 
T Cell Receptor Excision Circles (TREC) in the 
Peripheral Blood 
TREC levels on T cells and mononuclear cells will be summarised for each group of IMP 
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. 
8.3.3 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 each of the IMP 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. 
8.3.3.2 
T Cell Vβ Panel 
Results of the T cell Vβ panel spectratyping will be reported as normal or abnormal.  The 
number (%) of subjects with normal/abnormal results will be summarized by visit for each of 
the IMP treated groups along with the number (%) with an abnormal result at any post-infusion 
visit.  


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8.3.3.3 
PHA Stimulation 
PHA stimulation results will be categorised as normal proliferation, impaired proliferation, 
absence of proliferation or other.  The number (%) of subjects falling into each category will 
be summarized by visit for each of the IMP treated groups. 
  
The PHA stimulation index will be calculated as the stimulated PHA/unstimulated PHA and 
will be summarized at each visit for each of the IMP treated groups.  In addition, the median 
and range will be plotted over time on a logarithmic scale.   
8.3.3.4 
CD3 Stimulation 
CD3 stimulation results will be reported as normal or abnormal.  The number (%) of subjects 
with normal/abnormal results will be summarized by visit for each of the IMP treated groups 
along with the number (%) with an abnormal result at any post-infusion visit.  
8.3.3.5 
Vector Integration Analysis 
The number (%) of subjects with one integration site representing > 30% of total integration 
will be summarized by visit for each of the IMP treated groups.  The number (%) of subjects 
for whom one integration site contributed > 30% of total integration at 2 or more time points 
will be summarized for each of the IMP treated groups.  In addition, the total number of 
integration sites will be summarized by visit for each of the IMP treated groups. 
8.3.3.6 
Bone Marrow Aspirate 
A bone marrow aspirate may be performed for vector copy number, qPCR and integration 
analysis.  Morphology and integration analysis results will be reported as normal or abnormal.  
The number (%) of subjects with normal/abnormal results for each of the tests will be 
summarized by visit for each of the IMP treated groups.  In addition, the vector copy number 
in BM-MNC will be summarised using descriptive statistics. 
8.3.3.7 
Response to Tetanus Vaccination 
The response to the tetanus vaccination will be reported as normal or abnormal.  The number 
(%) of subjects with normal/abnormal results will be summarised for each of the IMP treated 
groups. 
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) 


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• Measures of engraftment (descriptive statistics and plots) 
8.4 Safety Analyses 
8.4.1 Adverse Events 
All adverse events will be coded using MedDRA version 20.0 or higher 
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 IMP 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, 
• the entire date is missing and the end date is missing or is not before the date of infusion. 
8.4.1.2 
Adverse Events 
Treatment emergent adverse events will be summarised for each of the IMP treated groups in 
an overall summary presenting the number (%) of subjects with: 
• any AE,  
• moderate or severe AE,  
• severe AE,  
• treatment related AE (possibly/probably/definitely/not assessable/missing),  
• 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 for each of the IMP 
treated groups by system organ class (SOC) and preferred term (PT): 
• overall, 
• by maximum severity, 
• by maximum relationship to treatment, 
• treatment related AEs, 
• 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. 


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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. 
8.4.1.3 
Serious Adverse Events and Death 
Incidence and frequency of treatment emergent SAEs will be summarised for each of the IMP 
treated and HSCT control groups (data permitting) 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 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 SAEs that are not treatment emergent (occurred prior to the infusion) will be listed. 
 
If there are any deaths, the details will be listed. 
8.4.2 Concomitant Medications 
Concomitant medications taken during the study will be listed. 
8.4.3 RCL Testing 
Number (%) of subjects testing positive for RCL will be summarised for each of the IMP 
treated groups 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, 


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monocytes, eosinophils, basophils and platelet count) will be evaluated over time for the IMP 
treated subjects.  The baseline evaluation for each parameter will be the final evaluation prior 
to the IMP infusion. 
 
 For each parameter, the actual value and change from baseline will be summarised at each 
visit using descriptive statistics for each of the IMP treated groups. 
 
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-infusion.  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 parameter at each visit and at any time post-infusion. 
 
8.4.4.2 
Biochemistry 
Biochemistry parameters (sodium, glucose, chloride, potassium, urea, creatinine, calcium, 
magnesium, phosphates, albumin, alkaline phosphatase, alanine transaminase (ALT), aspartate 
transaminase (AST), gamma glutamyl transferase (GGT), bilirubin, total protein) will be 
evaluated over time for the IMP treated subjects.  The baseline evaluation for each parameter 
will be the final evaluation prior to the IMP infusion. 
 
For each parameter, the actual value and change from baseline will be summarised for each 
IMP treated group 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 parameter at each visit and at any time post-infusion. 
8.4.5 Vital Signs and Weight 
Vital signs (temperature, pulse rate, respiratory rate, oxygen saturation) and weight are 
measured over time for the IMP treated subjects. 
 
The baseline evaluation for each of the vital signs and weight will be the final evaluation prior 
to the IMP infusion. 
 
For each parameter, the actual value and change from baseline will be summarised for each of 
the IMP treated groups at each visit using descriptive statistics. 


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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 for each parameter by visit and at any time post-
infusion.  In addition, the number (%) of subjects requiring additional O2 will be summarised 
by visit. 
8.4.6 Physical Examination 
Physical examinations will be performed over time for the IMP treated subjects.  The number 
(%) of subjects with abnormal evaluations will be summarized by body system for each of the 
IMP treated groups at each visit.  An overall summary will also be produced for each body 
system showing the number (%) of subjects with normal evaluations at screening who had one 
or more abnormal evaluations post-treatment. 
9 Analysis Software 
All summaries and analyses will be carried out using SAS version 9.4 or higher. 
10 Changes in Statistical Analysis Plan 
10.1 Changes in Version 10.0 
Since Statistical Analysis Plan version 9.0 was signed off on 14th March, 2017, Version 17.0 
of the protocol has been signed off and changes have been made to the eCRF, so a number of 
modifications were needed to this SAP in order to account for the changes in the protocol and 
eCRF.   
 
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 17.0. 
• 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. 
• 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. 


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• The eCRFs for the vector integration and PHA stimulation have been changed, so the 
summaries have been modified to take account of the change in the data being collected. 
• The infection summaries have been reworded to match the definition of severe 
infections from the protocol.  
• Description of possible sensitivity analyses has been added. 
• Additional adverse event summaries have been added and it has been explicitly stated 
that all summaries will include only 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. 
10.2   Changes in Version 11.0 
Since version 10.0 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 site to be used in the analyses.  
• The summary of coagulation data has been removed from the safety section as these 
data are only collected at screening. 
• The MedDRA version to be used has been updated to 20.0.   
10.3   Changes in Version 12.0 
Since version 11.0 of the Statistical Analysis Plan was signed off, the sponsor has made the 
decision to restrict the HSCT control data to be only those from subjects treated at GOSH, so 
this SAP has been updated to take account of this change.