Multi-site Phase I/II Trial Evaluating the Treatment of SCID-X1 Patients With Retrovirus-mediated Gene Transfer
Phase: PHASE1
Conditions: Severe Combined Immunodeficiency
Sponsor: David Williams

1 
Version 12.0 
January 4, 2022 
 
Gene transfer for SCID-X1 using a self-inactivating (SIN) gammaretroviral vector 
 
Protocol short title: Gene transfer for SCID-X1 
 
IND 14067 
Boston Children's Hospital IRB # 09-05-0254 
NCT01129544 
 
IMP: pSRS11.EFS.IL2RG.pre* retroviral vector 
 
A multi-institutional phase I/II trial evaluating the treatment of SCID-X1 patients with retrovirus-mediated 
gene transfer 
 
 
Version 12.0 – January 4, 2022 
 
 
Dr. David A. Williams, IND Sponsor & Grant PI/PD, US sites 
Division of Hematology/Oncology 
Children’s Hospital Boston,  
Harvard Medical School 
Boston, MA 
 
Primary Institution: Children’s Hospital 
Boston 
Children’s Hospital Boston,   
Site PI: Dr. Christine Duncan 
Division of Hematology/Oncology 
Children’s Hospital Boston,  
Harvard Medical School 
Boston, MA 
 
 
Dr. Wendy B. London, PhD, 
Co-Investigator, Division of 
Hematology/Oncology 
Children’s Hospital Boston,  
Harvard Medical School 
Boston, MA 
 
 
 
 
 
 
Collaborating sites:  
Mattel Children’s Hospital UCLA 
 
Dr. Donald Kohn, Site PI 
Departments of MIMG and Pediatrics 
Mattel Children’s Hospital 
University of California, Los Angeles 
Los Angeles, CA 90095 
 
 
Dr. Satiro De Oliveira, Co-Investigator 
Pediatric Hematology/Oncology 
Mattel Children’s Hospital 
University of California, Los Angeles 
Los Angeles, CA 90095 
 
Cincinnati Children’s Hospital Medical 
Center. 
 
Dr. Rebecca Marsh, Site PI 
Division of Hematology/Oncology 
Cincinnati Children’s Hospital Medical Center 
Cincinnati, Ohio 
 
Dr. Punam Malik, Co-investigator 
Division of Experimental Hematology 
Cincinnati Children’s Hospital Medical Center 
Cincinnati, OH 45229 

2 
Version 12.0 
January 4, 2022 
Funding Sponsor for Cooperative 
Agreement:  
National Institutes of Health, National Institute 
of Allergy and Infectious Diseases/Division of 
Allergy, Immunology and Transplantation 
 
Dr. Linda M. Griffith 
Medical Officer / Medical Monitor 
Clinical Immunology Branch 
Division of Allergy, Immunology and 
Transplantation 
NIAID / NIH 
6610 Rockledge Drive, Room 6716, MSC 
6601 
Bethesda, MD 20892-6601 
(20817 for express delivery) 
Phone: (301) 496-7104 
Fax: (301) 480-1450 
Email: LGriffith@niaid.nih.gov 
 
 
Steering Committee 
 
Dr. David Williams 
Dr. Luigi Notarangelo 
Dr. Donald Kohn 
Dr. Rebecca Marsh 
Dr. Linda M. Griffith 
Dr. Sung-Yun Pai 
Dr. Frederic Bushman 
Dr. Sarah Nikforow 
Ms. Colleen H. Dansereau 
 
 
 
International Collaborators 
(These sites are conducting a parallel trial using 
the same vector. They do not fall under Dr. 
Williams’ Sponsorship or under the US sites 
IND) 
 
Overall Chief Investigator for European 
sites: 
Dr. Adrian Thrasher 
Molecular Immunology Unit 
Institute of Child Health 
London 
 
 
 
 
 
 
Co-Investigators, European sites: 
 
Prof Alain Fischer 
Unité d’immunologie et d’hematologie 
pediatriques 
Inserm U 768 
Hôpital Necker - Enfants Malades 
Paris France 
 
Prof Marina Cavazzana-Calvo 
Department Biotherapie 
Hôpital  Necker - Enfants Malades 
Paris France 
 
Salima Hacein-Bey-Abina 
Department de Biotherapies 
Hôpital  Necker 
Paris France 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

3 
Version 12.0 
January 4, 2022 
INVESTIGATOR SIGNATURE PAGE 
 
Protocol Title: A multi-institutional phase I/II trial evaluating the treatment of SCID-X1 patients with 
retrovirus-mediated gene transfer 
Protocol Number:  
Protocol Version: Version12.0  
Funding Agency:  
Division of Allergy, Immunology and Transplantation (DAIT) 
National Institute of Allergy and Infectious Diseases 
6610 Rockledge Drive, Room 6716 
Bethesda, MD 20892 
 
The Principal Investigator at each collaborating clinical site should print, sign, and date at the indicated 
location below. A copy should be kept for your records and the original signature page sent to the Data 
Management Coordinating Center. 
 
I confirm that I have read the above protocol and the corresponding consent form in the latest version. I 
understand them, and I will work according to the principles of Good Clinical Practice (GCP) as 
described in the United States Code of Federal Regulations (CFR) – 21 CFR Parts 45, 50, 56, and 312, 
and the International Conference on Harmonization (ICH) document “Guidance for Industry – E6 Good 
Clinical Practice: Consolidated Guidance”, dated April 1996. Further, I will conduct the study in keeping 
with local, legal, and regulatory requirements. 
 
As the Principal Investigator, I agree to conduct “A multi-institutional phase I/II trial evaluating the 
treatment of SCID-X1 patients with retrovirus-mediated gene transfer”. I agree to carry out the study by 
the criteria written in the protocol and understand that no changes can be made to this protocol without 
written permission of the NIAID. 
 
 
 
Principal Investigator (Print): -------------------------------------------- 
 
Principal Investigator Signature: --------------------------------------- 
 
Date: ------------------ 
 

4 
Version 12.0 
January 4, 2022 
ABBREVIATIONS 
 
BCG  
Bacillus Calmette-Guerin  
BMT   
Bone Marrow Transplant 
CBC 
 
Complete Blood Count  
CBER  
Center for Biologics Evaluation and Research 
CDR3  
Complementarity Determining Region-3  
CFCs  
Colony Forming Cells  
CFR 
 
Code of Federal Regulations  
CFU 
 
Colony-Forming Unit 
CGD  
Chronic Granulomatous Disease  
CIBMTR 
The Center for International Blood and Marrow Transplant Research  
CMCF  
Cell Manipulation Core Facility  
CNS   
Central Nervous System 
CpG 
 
Cytosine Phosphate Guanine 
CRF   
Case Report Form 
CSM  
Committee for Safety of Medicines  
CTC 
 
Common Toxicity Criteria  
CTIP  
Clinical and Translational Investigator Program  
DAIT   
Division of Allergy, Immunology and Transplantation  
DFCI  
Dana-Farber Cancer Institute  
DMSO  
Dimethyl Sulfoxide 
DNA   
Deoxyribonucleic Acid 
DSMB  
Data and Safety Monitoring Board 
EBMT  
European group for Blood and Marrow Transplantation  
EBV 
 
Epstein Barr Virus 
EF-1α  
Elongation factor-1α  
EGFP   
Enhanced Green Fluorescent Protein 
ESID  
European Society for Immune Deficiency 
FACT  
Foundation for the Accreditation of Cell Therapy 
FDA 
 
Food and Drug Administration 
GALV  
Gibbon Ape Leukemia Virus  
GCP 
 
Good Clinical Practice  
GeMCRIS  
Genetic Modification Clinical Research Information System  
GMP  
Good Manufacture ring Practice 
GOSH  
Great Ormond Street Hospital  
GTAC  
UK Gene Therapy Advisory Committee  
GvHD  
Graft-versus-host disease  
HEK 
 
Human Embryonic Kidney 
HLA 
 
Human leukocyte antigen 
HSCs  
Hematopoietic Stem Cells  
HSCT  
Hematopoietic Stem Cell transplantation 
ICH 
 
International Conference on Harmonization  
IDE  
 
Investigational Device Exception 
IL-2RG 
Interleukin-2 Receptor Gamma 
IMP 
 
Investigational Medicinal Product  
IRB  
 
Institutional Review Board 
JAK   
Janus Kinase 
LAM   
Linear Amplification Mediated  
LSS 
 
Lymphocyte Subsets  
LTC-ICs 
Long Term Culture-Initiating cells  
 
 

5 
Version 12.0 
January 4, 2022 
 
ABBREVIATIONS 
 
LTR 
 
Long Terminal Repeat  
MMRD  
Mismatched Related Donor 
MOI 
 
Multiplicity of Infection 
MUD  
Matched Unrelated  Donor 
NCI 
 
National Cancer Institute 
NIAID   
National Institute of Allergy and Infectious Diseases 
NIH/OBA  
National Institute of Health - Office of Biotechnology Activities 
NK 
 
Natural Killer  
PCR   
Polymerase Chain Reaction 
PHA   
Phytohemagglutinin  
PI 
 
Principal Investigator 
PID 
 
Primary Immune Deficiency  
PTC 
 
Points To Consider 
pUC 
 
Plasmid University of California 
QACT  
Quality Assurance for Clinical Trials Office  
RCR 
 
Replication Competent Retrovirus  
SAE 
 
Serious Adverse Event 
SAS   
Statistical Analysis Software 
SCETIDE  
Stem Cell Transplantation for Immunodeficiencies 
SCID  
Severe Combined Immunodeficiencies  
SCID-X1  
X-linked Severe Combined Immunodeficiency  
SFFV  
Spleen Focus Forming Virus  
SIN 
 
Self-inactivating  
SOPs  
Standard Operating Procedures 
SRC 
 
SCID-repopulating cells  
SRCs  
SCID-repopulating cells  
STAT   
Signal Tranducers and Activators of Transcription 
T-ALL  
T acute lymphoblastic leukemia 
TCR 
 
T-cell Receptor  
TRECs 
TCR Excision Circles  
UCLA   
University of California at Los Angeles 
USP 
 
United States Pharmacopeia 
VPF 
 
Vector Production Facility  
WBC's 
White Blood Cells  
WPRE  
Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element 
 
 

6 
Version 12.0 
January 4, 2022 
Contents 
Front cover 
Contact details 
Investigator Signature Page 
Abbreviations 
Contents 
 
 
1. Abstract ................................................................................................................................................... 9 
2. Lay summary of proposal ....................................................................................................................... 9 
3. Overall objectives ................................................................................................................................. 10 
4.  Background: X-linked severe combined immunodeficiency (SCID-X1) ............................................ 10 
4.1 Molecular Pathology of SCID-X1 .................................................................................................. 11 
4.2 The pattern of clinical disease......................................................................................................... 12 
4.2.1 Classical presentations ............................................................................................................. 12 
4.2.2 Atypical presentation ............................................................................................................... 13 
4.2.3 Spontaneous reversion mutations ............................................................................................ 13 
4.3 Diagnosis of SCID-X1 .................................................................................................................... 13 
4.4 Prognosis and conventional treatment ............................................................................................ 14 
4.4.1 Survival after hematopoietic stem cell transplantation ............................................................ 14 
4.4.2 Quality of immunological reconstitution after HSCT ............................................................. 17 
5. Alternative therapy for SCID-X1 based on somatic gene transfer ....................................................... 18 
5.1 Target cell populations for somatic gene transfer of SCID-X1 ...................................................... 18 
5.2 Studies in γc-deficient mice generated by gene targeting ............................................................... 19 
5.3 Clinical trials of gene transfer for SCID-X1 ................................................................................... 20 
5.3.1 Successful restoration of immunological function .................................................................. 20 
5.3.2 Insertional mutagenesis and lymphoproliferative disease ....................................................... 22 
5.3.3 Mechanisms of leukemogenesis in SCID-X1 gene transfer .................................................... 22 
5.3.4 Strategies to overcome insertional mutagenesis ...................................................................... 23 
6. Preliminary studies relevant to the proposed clinical trial .................................................................... 23 
6.1 Development of a novel vector to enhance safety of gene transfer protocols for SCID-X1 .......... 23 
6.1.1 Self-inactivating (SIN) gammaretroviral vectors for expression of IL2RG ............................ 23 
6.1.2 SIN vector development .......................................................................................................... 24 
6.1.3 Platform studies addressing the genotoxic impact of gammaretroviral SIN vectors ............... 25 
6.1.4 Toxicological studies in murine models .................................................................................. 26 
6.1.5 Evaluation of potency in murine model systems in vitro and in vivo ...................................... 29 
6.1.6 Evaluation in human T cell lines and CD34+ cells.................................................................. 31 
6.1.7 Details of final vector construction and derivation (Appendix 1) ........................................... 32 
6.1.8 Clinical grade vector production .............................................................................................. 32 
6.2 General Information related to the Investigational Medicinal Product (IMP) ................................ 32 
6.2.1 Name description of the IMP: .................................................................................................. 32 
6.2.2 Production, Supply and Release of the IMP: ........................................................................... 33 
6.2.3 Drug accountability .................................................................................................................. 33 
6.2.4 Description of and justification of the trial treatment & dosage .............................................. 33 
7. Study Design ......................................................................................................................................... 33 
7.1 Protocol summary ........................................................................................................................... 34 
7.2 Study objectives .............................................................................................................................. 34 
7.3 Study endpoints ............................................................................................................................... 35 
8.  Patient selection and recruitment ......................................................................................................... 35 

7 
Version 12.0 
January 4, 2022 
8.1 Inclusion and exclusion criteria ...................................................................................................... 35 
8.2 Enrollment of subjects .................................................................................................................... 36 
9. Treatment protocol ................................................................................................................................ 37 
9.1 Harvest of CD34+ cells ................................................................................................................... 37 
9.1.1 Bone marrow harvest or mobilized peripheral blood leukapheresis ........................................ 37 
9.1.2 Back-up product collection in patients receiving busulfan ...................................................... 37 
9.2 Low dose busulfan conditioning for selected subjects ................................................................... 37 
9.3 CD34+ cell culture, transduction and infusion ............................................................................... 37 
9.3.1 CD34+ cell culture and transduction ....................................................................................... 37 
9.3.2 Testing of product prior to patient re-infusion ......................................................................... 38 
9.3.3 Infusion of transduced cells ..................................................................................................... 39 
9.3.4 Criteria for co-infusion of back-up product in patients receiving busulfan ............................. 39 
10.  Evaluation and Follow-up .................................................................................................................. 39 
10.1 Assessment of safety and efficacy (Appendix 4) .......................................................................... 39 
10.1.1 Immunological reconstitution ................................................................................................ 39 
10.1.2 Molecular characterization of gene transfer .......................................................................... 40 
10.1.3 Safety assessments ................................................................................................................. 41 
10.2 Investigations & Monitoring schedule .......................................................................................... 41 
10.2.1 Pre-treatment Investigations .................................................................................................. 42 
10.2.2 Post-infusion monitoring ....................................................................................................... 42 
10.2.3 Early failure of immune reconstitution .................................................................................. 43 
10.3 Procedure for failure of hematologic reconstitution in patients receiving busulfan ..................... 43 
10.3 Withdrawal of individual subjects ................................................................................................ 43 
10.3.1 Off study criteria .................................................................................................................... 44 
10.3.2 Follow-up of withdrawn subjects........................................................................................... 44 
10.4 Premature termination of the study ............................................................................................... 44 
10.5 Statistical considerations ............................................................................................................... 44 
10.5.1 Stopping rule for lack of efficacy .............................................................................................. 45 
10.5.2 Data analysis and reporting .................................................................................................... 47 
11. Safety issues related to gene transfer protocol .................................................................................... 47 
11.1 Venipuncture ................................................................................................................................. 47 
11.2 Bone marrow harvest .................................................................................................................... 47 
11.3 Retrovirus mediated gene transfer ................................................................................................ 47 
11.3.1 Insertional mutagenesis .......................................................................................................... 47 
11.3.2 Germline transmission of vector sequences ........................................................................... 48 
11.3.3 Quality control of harvest and transduction process .............................................................. 48 
11.3.4 Infusion of transduced cells ................................................................................................... 48 
12. Reporting Adverse Events .................................................................................................................. 49 
13. Public Health Considerations .............................................................................................................. 56 
14. Ethical considerations ......................................................................................................................... 56 
15. Administrative aspects ........................................................................................................................ 57 
15.1 Data Handling, Record Keeping, Sample Storage ........................................................................ 57 
15.1.1 CRF completion ..................................................................................................................... 57 
15.1.2 Sample storage ....................................................................................................................... 57 
15.1.3 Record retention ..................................................................................................................... 57 
15.2 Patient confidentiality ................................................................................................................... 57 
15.5 Definition of end of study & end of study report.......................................................................... 58 
15.6 Insurance & indemnity .................................................................................................................. 58 

8 
Version 12.0 
January 4, 2022 
15.7. Dissemination of results ............................................................................................................... 58 
16. Clinical facilities and arrangements .................................................................................................... 58 
17. Qualifications of responsible clinicians and investigators .................................................................. 59 
18. References cited .................................................................................................................................. 59 
Appendix 1: Construction and sequence of SIN gammaretroviral vector pSRS11.EFS.IL2RG.pre* ...... 68 
Appendix 2:  Outline of proposed criteria for enrollment ........................................................................ 69 
Appendix 3: Transduction......................................................................................................................... 70 
Appendix 4: Patient  Monitoring  Plan ..................................................................................................... 71 
Appendix 5: Busulfan dosing guidelines .................................................................................................. 73 
Appendix 6: Changes to protocol ............................................................................................................. 74 
 
  
 
 

9 
Version 12.0 
January 4, 2022 
1. Abstract 
Severe combined immunodeficiencies (SCID) are a heterogeneous group of inherited disorders 
characterized by a profound reduction or absence of T lymphocyte function. They arise from a variety of 
molecular defects which affect lymphocyte development and function.  The most common form of SCID 
is an X-linked form (SCID-X1) which accounts for 40-50% of all cases. SCID-X1 is caused by defects in 
the common cytokine receptor γ chain (γc), which was originally identified as a component of the high 
affinity interleukin-2 receptor (IL-2RG), but is now known to be an essential component of the IL-4, -7, -
9 -15, and -21 cytokine receptor complexes. Classic SCID-X1 has an extremely poor prognosis without 
treatment. Death usually occurs in the first year of life from infectious complications unless definitive 
treatment can be administered. Until the recent advent of somatic gene therapy, hematopoietic stem 
cell transplantation (HSCT) offered the only curative option for patients with any form of SCID. If a 
genotypically matched sibling donor is available, HSCT is a highly successful procedure.  However a 
genotypically matched family donor is only available for approximately 30% of patients. For the 
remaining individuals, alternative donor transplants, principally from matched unrelated (MUD) or 
haploidentical parental donors have been performed. These approaches are still problematic with 
toxicity from ablative therapy, graft-versus-host disease and incomplete lymphoid reconstitution. Recent 
gene transfer trials have documented the efficacy of gene transfer in this disease, albeit with toxicity 
related to insertional mutagenesis. A new generation of SIN vectors has been developed which lack all 
enhancer-promoter elements of the LTR U3 region and are also devoid of all gammaretroviral coding 
regions. A SIN vector expressing the IL-2RG gene, pSRS11.EFS.IL2RG.pre* has been developed and 
has shown a reduction in mutagenic potential compared to LTR configuration in non-clinical studies. 
The current study is a phase I/II trial of somatic gene therapy for patients with SCID-X1. Inclusion 
criteria include patients with a definitive diagnosis of SCIDX1 in whom HLA-matched family donors are 
unavailable and lack an HLA identical (A,B,C,DR,DQ) unrelated donor OR patients of any age with an 
active, therapy-resistant infection or other medical conditions that significantly increase the risk of 
allogeneic transplant. Primary endpoints include immunological reconstitution defined as absolute CD3 
cells of >300/μl and PHA stimulation index >15 at 6 months post infusion and the incidence of life-
threatening adverse reactions related to the gene transfer procedure. 
 
2. Lay summary of proposal 
X-linked severe combined immunodeficiency (SCID-X1) is a disease that runs in families, such that 
babies are born missing key parts of the immune system. While there are different types of SCID, 
SCID-X1 only affects boys because the genetic mistake is on the X-chromosome. In normal healthy 
people special cells in the blood called lymphocytes protect against many viruses and other infectious 
agents. In SCID-X1, the bone marrow cells which develop into lymphocytes do not grow, and as a 
result vital lymphocytes are either virtually absent from the blood (T lymphocytes and natural killer cells) 
or do not work (B lymphocytes). Therefore, affected boys are extremely susceptible to infection. 
Common viruses which have little effect on normal individuals can cause life-threatening illness. 
Although drugs such as antibiotics can offer partial protection, untreated boys typically die in the first 
year of life. 
 
Bone marrow transplantation can cure SCID-X1. When an exact donor match from a brother or sister is 
available virtually all patients are cured. However, less than a third of affected boys have a fully 
matched brother or sister, and the chances of success from other donor sources, such as a parent or 
unrelated people, are not as good. In these situations 20-30% of patients will not survive. When a 
parent is the donor, about half of patients who do survive are not fully cured because the T lymphocytes 
are replaced but the B lymphocytes are still broken. When an unrelated person is the donor, the patient 
typically gets high dose chemotherapy, which kills off the bone marrow and allows full replacement of T 
and B cells. But these patients often have long term problems related to the chemotherapy. Both 
transplants from parents and especially from unrelated people can cause graft versus host disease 
(GvHD), a condition where donor lymphocytes in the transplant recognize the patient’s own organs, 
such as skin and gut, as foreign and cause severe damage. Simply put, transplantation can cure SCID-

10 
Version 12.0 
January 4, 2022 
X1 much of the time but current therapy is not 100% effective and has short-term and long-term 
toxicities. 
 
Over the past decade a new treatment has been developed based on our knowledge of the defective 
gene causing SCID-X1. We can now use genes as a type of medicine that corrects the problem in the 
patient’s own bone marrow cells and allows the development of a new immune system, with or without 
chemotherapy. The use of gene transfer on the patient’s own bone marrow cells results in normal 
numbers of functional T and B lymphocytes, and avoids the risk of GvHD entirely. Although still 
experimental in nature, this procedure has been carried out effectively in around 40 patients with 
different forms of SCID including 20 children with SCID-X1, and most of these children are living 
healthy lives. Unfortunately, in a few patients the gene therapy vector (the vehicle that carries the new 
gene into the bone marrow cells) has caused leukemia a few years after treatment because it has 
accidentally altered the way in which the growth of lymphocytes is normally controlled. Due to scientific 
advances, the technology now exists to reduce this risk by changing the design of the vector. In this 
trial we aim to evaluate the treatment of patients with SCID-X1 with a new and safer gene transfer 
vector. 
 
3. Overall objectives 
The objectives of this proposal are to initiate a trial of somatic gene transfer for patients with SCID-X1 in 
whom HLA-matched family or unrelated donors are unavailable in a timely fashion or for patients 
deemed not suitable for allogeneic stem cell transplantation due to infection. For this study, CD34+ 
cells will be purified from harvested patient bone marrow or mobilized peripheral blood, and transduced 
ex vivo using a novel gibbon ape leukaemia (GALV)-virus pseudotyped gammaretroviral vector 
encoding the human common cytokine receptor gamma chain (γc). The vector has been designed with 
a self inactivating (SIN) configuration in which expression of the therapeutic gene is regulated by an 
internal housekeeping gene promoter derived from human elongation factor-1α (EF-1α) gene. These 
technological features are anticipated to substantially reduce risks of insertional mutagenesis and to 
retain efficacy. 
 
4.  Background: X-linked severe combined immunodeficiency (SCID-X1) 
Severe combined immunodeficiencies (SCID) are a heterogeneous group of inherited disorders 
characterized by a profound reduction or absence of T lymphocyte function. They arise from a variety of 
molecular defects which affect lymphocyte development and function(1).  The most common form of SCID 
is an X-linked form (SCID-X1) which accounts for 40-50% of all cases. SCID-X1 is caused by defects in 
the common cytokine receptor γ chain (γc), which was originally identified as a component of the high 
affinity interleukin-2 receptor (IL-2RG(2), but is now known to be an essential component of the IL-4, -7, -9 
-15, and -21 cytokine receptor complexes(3)  (Figure 1).  

11 
Version 12.0 
January 4, 2022 
 
 
Figure 1. Cartoon depicting participation of γc in multiple cytokine receptor complexes. 
 
The molecular defect in SCID-X1 results in the complete absence of T cell and natural killer (NK) cell 
development and an as yet uncharacterized defect of B cell function (T-B+NK- SCID). These profound 
abnormalities in cellular and humoral immune function leave patients extremely susceptible to recurrent 
and opportunistic infection. The prognosis without treatment is uniformly fatal and affected boys normally 
die in the first year of life.  
 
4.1 Molecular Pathology of SCID-X1 
The SCID-X1 gene locus was first mapped by linkage analysis to Xq12-13(4). In 1993, it was shown by 
two groups that the IL2RG gene was located precisely in the critical region where the SCID-X1 locus 
had been placed(2, 5, 6).  Subsequent analysis of a number of unrelated patients with T-B+NK- SCID 
demonstrated disease-causing mutations in the IL2RG gene(2)IL2RG is organized into eight exons and 
spans 4.5kb of genomic DNA in Xq13.1(2).  The coding sequence of 1,124 nucleotides gives rise to a 
232 amino acid transmembrane glycoprotein which is expressed constitutively in all lymphoid cells(7, 
8).  The protein has a number of structural motifs characteristic of cytokine receptor superfamily 
members. Four conserved cysteine residues are found at the extracellular amino-terminal end; a 
juxtamembrane conserved extracellular motif, the WSXSW box, found in all cytokine receptors is 
encoded in exon 5; the highly hydrophobic transmembrane domain of 29 amino acids occupies most of 
exon 6 and the proximal intracellular domain in exon 7 has a signaling sequence called Box1/Box 2 
which has homology to the SH2 domains of Src family tyrosine kinases. 
 
Genetic analysis of boys with T-B+NK- SCID has now identified hundreds of patients with mutations in 
the IL2RG gene. Although mutations have been found in all the exons, they are not evenly distributed. 
Exon 5 is the site of over 25% of all mutations followed by exons 3 and 4 with 15% each. Only a 
relatively small number of mutations have been found in exon 8. A number of hotspots for mutation 
have also been noted within CpG dinucleotides, the most prominent being in exon 5 where 16 
independent missense mutations have been reported(9). The most frequently encountered defects are 
missense mutations, resulting in non-conservative amino acid substitutions, followed by nonsense 
mutations and together these account for approximately two thirds of all mutations. 
 

12 
Version 12.0 
January 4, 2022 
The consequences of the different mutations on γc protein expression and function are variable. 
Missense mutations in the extracellular domain of the protein can result in normal, trace or absent 
expression of γc but missense defects in the intracellular domains appear to result in intact cell surface 
protein expression(10), although the intensity of staining by flow cytometry is reduced compared to 
expression in normal lymphocytes (Filipovich et al, unpublished observations). γc was initially thought 
only to be a component of the IL-2R complex and thus did not offer a satisfactory explanation for the T-
B+NK- immunophenotype of SCID-X1. Subsequent realization that it was also an essential component 
of the IL-4,-7,-9 –15 and -21 receptor complexes has since allowed a greater understanding of the 
lineage specific abnormalities. Murine ‘knockout’ models and in vitro cellular studies have shown that 
functional IL-7/IL-7R signaling is essential for normal T cell development at an early stage in 
thymopoiesis(11). NK cell development is unaffected in these mice but B cell development is 
significantly disrupted in contrast to the human phenotype(12). The essential and lineage specific role 
of IL-7/IL-7R function in human T cell development is further illustrated by the finding of mutations in the 
IL-7Rα gene in some patients with T-B+NK+ SCID(13). Similar in vitro and murine studies have 
identified the dominant role of IL-15 in NK cell development and survival. IL-15 efficiently promotes NK 
cell differentiation from bone marrow precursors in humans and mice and maintains the survival of 
mature NK cells even at low concentrations(14, 15). Mice deficient in IL-15Rα or IL-15 completely lack 
mature NK cells but show normal B and T cell development(16, 17). 
 
B lymphopoiesis appears to proceed normally in most cases of SCID-X1, but a number of studies 
suggest that there are intrinsic functional B cell abnormalities. IL-2 and IL-15 fail to induce B cell 
responses in vitro and signaling molecules downstream of the IL-4R receptor (JAK3 and STAT6) are 
not activated after ligand binding(18, 19). Molecular detail of VDJ recombination in B cells from patients 
by analysis of complementarity determining region-3 (CDR3) sequences showed that the diversity of 
CDR3 lengths was characteristic of a normal primary repertoire, indicating that the ability to generate 
junction diversity is retained although there was little evidence for somatic hypermutation(20). It has 
also been shown using B cell spectratyping analysis that γc-deficient B cells are unable to class switch 
and undergo somatic hypermutation even in the presence of adequate T cell help after bone marrow 
transplantation(21). 
 
The downstream consequences of γc activation have been defined (reviewed in(22, 23). Stimulation of 
the receptor complex by cytokine results in the heterodimerization of the receptor subunits and tyrosine 
phosphorylation of JAK3, a cytoplasmic tyrosine kinase which binds specifically to the γc subunit. 
Tyrosine phosphorylated JAK3 in turn phosphorylates one of the STAT (Signal Tranducers and 
Activators of Transcription) family of transcription factors which then dimerizes and translocates to the 
nucleus where it binds to specific sites to initiate transcriptional events. The specificity of γc binding to 
JAK3 rather than to other JAK molecules is demonstrated by the lack of JAK3 tyrosine phosphorylation 
in patients with SCID-X1 and also by the identification of mutations in JAK3 in patients with an 
autosomal recessive form of T-B+NK- SCID(24, 25). This latter finding implies that any disruption to the 
specific interaction between these two molecules can lead to the T-B+NK- immunophenotype. Further 
downstream, all γc cytokine stimulation pathways (except IL-4) activate the same members of the STAT 
family of molecules, STAT 3 and STAT 5, whereas IL-4 activates STAT6(26). 
 
4.2 The pattern of clinical disease 
4.2.1 Classical presentations 
SCID-X1 is estimated to affect around 1 in 100,000 births although the frequency may be greater in 
certain geographical areas or ethnic groups. Clinically it is characterized by severe and recurrent 
infections and a high frequency of opportunistic infections. The clinical presentation in SCID-X1 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 around 6 months(27) and this most likely reflects the time when the protective effect of 

13 
Version 12.0 
January 4, 2022 
placentally transferred maternal immunoglobulin has diminished and children have been exposed to a 
range of microorganisms. The most common infective problems are oral candidiasis, respiratory 
infection due to Pneumocytis  jiroveci, respiratory syncytial virus and parainfluenza 3, adenoviral 
infection, persistent diarrhea and failure to thrive. In countries which administer antituberculous 
vaccination to infants with bacillus Calmette-Guerin (BCG), disseminated infection with BCG has 
occurred. Live polio vaccine has also caused poliomyelitis and myocarditis but only rarely and this may 
be due to the continued presence of maternal immunoglobulin at the time of initial vaccination. 
 
4.2.2 Atypical presentation  
Atypical forms in terms of both immunological and clinical phenotype have been described. B cell 
numbers are normal in most, but pedigrees have been described in which B cell development is also 
affected thus presenting as a T-B- form of SCID(28). In the reported cases the clinical presentation was 
no different from the typical phenotypes. Certain individuals have also been reported who, despite a 
mutation in the γc gene, have some residual T cell function and thus present with a less severe clinical 
phenotype. In these cases, patients present later, have less severe infections and a prolonged clinical 
course with progressive loss of T and B cell function. In one pedigree, this was associated with a splice 
site mutation that generated two transcripts: one truncated and one normal sized, which accounted for 
80% and 20% of the total γc mRNA respectively. IL-2 binding to high affinity receptor complexes was 
severely reduced and T cells from affected individuals showed impaired in vitro stimulation and a 
restricted TCR repertoire(29).  A missense mutation (R222C) in the extracellular region of the γc was 
shown to result in preserved expression of the γc, and normal development of T cells, that however 
failed to respond to IL-2(30). Finally, a few patients have preserved NK cell development(31). 
 
4.2.3 Spontaneous reversion mutations 
SCID-X1 patients in whom somatic reversions have occurred provide important information for a gene 
transfer approach to the treatment of the condition  One patient with a normal number of poorly 
functional T cells, high B cell count, hypogammaglobulinemia, absent NK cells and an X-linked family 
history has been described in detail(32). B cells from the patient showed absent γc expression and 
analysis of genomic DNA derived from a B cell line demonstrated a missense mutation in exon 3 of IL-
2RG. Analysis of monocyte and neutrophil populations also showed the presence of the mutation. 
However, T cells displayed normal γc surface expression and sequencing revealed wild-type sequence. 
These results are explained by a reversion of the mutation in a committed T cell precursor giving rise to 
a pool of mature T cells. Similar findings have been observed in other patients (A. Thrasher, 
unpublished observations). These cases confirm previous experimental data that γc expression and 
signaling are essential for T lineage development and suggests that a significant growth advantage was 
conferred to the reverted T cell precursors(33). Such data also suggests that introduction of γc into 
lymphoid precursors by gene transfer even at relatively low frequency, could have significant 
therapeutic benefit. 
 
4.3 Diagnosis of SCID-X1 
Prior to the identification of the genetic defects, diagnosis was based on family history and clinical and 
immunological profile. Linkage analysis and examination of X-inactivation pattern in T cells of female 
relatives (a unilateral pattern is seen in T and B lymphocytes from female carriers) was used to guide 
diagnosis and carrier status but could only offer a degree of probability(34).  These techniques have 
largely been replaced by direct analysis of the IL2RG gene(35). If a mutation is identified an 
unambiguous molecular diagnosis can be made. In addition carrier assessment for female relatives can 
be made with absolute certainty and accurate prenatal diagnosis can be offered. More rapid tests 
based on the expression patterns and function of the mutant γc are also now available for diagnosis of 
affected infants. Approximately 65–90% of children have abnormal expression of  γc on the surface of 

14 
Version 12.0 
January 4, 2022 
mononuclear cells, allowing confirmation of the molecular diagnosis by flow cytometric analysis of 
peripheral blood mononuclear cells(10). In infants affected by T-B+NK- SCID who have normal γc 
expression, further dissection of the signalling pathway can now be undertaken. IL-2 stimulation of 
mononuclear cells results in tyrosine phosphorylation of JAK3 at specific tyrosine based motifs. A 
monoclonal antibody directed against phosphotyrosine residues can be used to demonstrate JAK3 
activation, so abnormalities in this signalling pathway can be detected at a protein level prior to genetic 
analysis(36).  Similarly, STAT-5 phosphorylation can now also be determined by flow cytometric 
methodology. The variability in clinical presentation again underlines the need to identify the molecular 
defect so that earlier referral for definitive therapy by bone marrow transplantation or gene transfer can 
be made. 
 
4.4 Prognosis and conventional treatment 
Classic SCID-X1 has an extremely poor prognosis without treatment. Death usually occurs in the first 
year of life from infectious complications unless definitive treatment can be administered. Upon 
diagnosis, patients are started on bacterial, fungal and pneumocystis prophylaxis and immunoglobulin 
substitution therapy. In some cases fungal prophylaxis is also initiated. In a few atypical cases (see 
above), 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 stem cell transplantation or gene transfer can be performed.  
 
Due to the severity of the condition and the risks associated with infection in the untreated child , 
recommendations from the European group for Blood and Marrow Transplantation (EBMT)/European 
Society for Immune Deficiency (ESID) inborn errors working party state that a definitive procedure 
should be undertaken as soon as possible, and within 3 months of diagnosis (including donor search 
time). 
 
4.4.1 Survival after hematopoietic stem cell transplantation  
Until the recent advent of somatic gene transfer, hematopoietic stem cell transplantation (HSCT) 
offered the only curative option for patients with any form of SCID. If a genotypically matched sibling 
donor is available, HSCT is a highly successful procedure.  Based on data accumulated from the 
European SCETIDE (Stem Cell Transplantation for Immunodeficiencies) database of 566 transplants 
(in 475 patients with all forms of SCID), the 3 year survival rate for HLA-matched related (matched 
sibling donor, MSD) procedures was over 80% (n=104) for patients treated from 1968 until 1999, and 
over 90% for patients treated since 1996(1, 37). The high survival rates are partly due to the fact that 
the absence of T and sometimes NK cells allows infusion of an HLA-identical donor graft without the 
need for prior myeloablative conditioning. However a genotypically matched family donor is only 
available for approximately 30% of patients. For the remaining individuals, alternative donor 
transplants, principally from matched unrelated (MUD) or haploidentical parental donors have been 
performed. In the same cohort noted above, 3 year survival rates following MUD transplants (n=28) 
were not significantly different from genotypically HLA-identical related grafts (n=104). The survival 
rates for patients treated by a haploidentical T-cell depleted HSCT were less good overall, although 
they have improved over time due to better GvHD prophylaxis and treatment of infective complications 
(from 35% 3 year survival before 1985, to 75% for those treated between 1996 and 1999)(38). For B+ 
SCID (which predominantly includes SCID-X1 patients), outcome has also improved over time and the 
1 year survival for patients treated between 1996-1999 was 84%. While a significant improvement in 5-
year survival rate had been observed between T-cell depleted mismatched related transplants 
performed prior to 1995 (survival rate: 47%) and the period 1995-1999 (65%), no further improvement 
has been observed for transplants performed in the period 2000-2005 (66% survival rate), as shown 
below for all forms of SCID (Figure 2). 

15 
Version 12.0 
January 4, 2022 
 
 
 
 
 
Figure 2. Data from SCETIDE 
Registry for hematopoietic cell 
transplantation for SCID 
 
 
 
 
 
 
 
 
Moreover, the 10-year survival rate 
for 223 patients with B+ SCID (most 
of whom have SCIDX1) after T-cell 
depleted mismatched related 
transplantation is 63% (Figure 3), with no significant improvement in recent years. 
 
 
 
 
 
 
 
 
Figure 3. 10-year 
survival rate after MUD 
or T-cell depleted 
mismatched related 
transplantation for B+ 
and B- SCID. Data 
from the SCETIDE 
European Registry 
 
 
 
 
 
A recent analysis of the outcome of HLA-mismatched related (MMRD) HSCT for SCIDX1 and JAK3, 
performed in Paris (France) and Brescia (Italy) after 1990, indicates that 30 such patients received 
MMRD-HSCT. Of these, 16 had severe infections at the time of transplantation. Only 8 of these 16 
patients (50%) are currently alive. This contrasts with the fact that 12 out of 14 patients (85.7%) who 
received MMRD HCT and who did not have significant infections at the time of HSCT, are currently 
alive. Furthermore, among the 20 patients in this combined series who are currently alive after MMRD 
HCT for SCIDX1 and JAK3 deficiency, 7 have significant long-term problems (chronic/persistent 
infections, colitis requiring nutritional support). In addition, 8 patients have not attained humoral immune 
reconstitution and remain dependent on IVIG.  Similar data on inadequate immune reconstitution after 
MMRD HCT for patients with SCID (and specifically for patients with SCIDX1) have been reported by 
other groups. In particular, 57% of the patients who have received unconditioned HSCT at Duke 

16 
Version 12.0 
January 4, 2022 
University (NC) have inadequate B cell function and require intravenous immune globulin (IVIG) 
infusions monthly. The percentage requiring IVIG is highest in patients with γc, Jak 3 and RAG 
deficiencies (73%), and those with γc and Jak3 deficiencies also usually remain NK cell deficient 
despite adequate T cell reconstitution (39). 
 
Finally, the Primary Immune Deficiency Treatment Consortium (PIDTC) has conducted a detailed 
retrospective study of 240 patients with SCID undergoing HSCT between 2000-2009 (100). This study 
confirms the inferior survival after alternative donor HSCT including MMRD, whether without 
conditioning (none) or with immunosuppression, reduced intensity or myeloablative conditioning 
(IS/RIC/MAC), adult volunteer unrelated donor or phenotypically matched related donors (OD) or 
unrelated umbilical cord blood (UCB). Additionally, the poor survival with these donor types is 
accentuated in patients with active infection at the time of HSCT (Figure 4). These data were published 
in 2014 (100). 
 
Figure 4. 5-year overall survival after matched sibling donor 
(MSD), mismatched unrelated donor (MMRD), other donor (OD), 
umbilical cord blood donor (UCB) in 2000-2009 at PIDTC centers 
of 240 SCID patients (left) or of 107 patients with active infection at 
time of HSCT (right). 
 
 
 
Overall, these data reinforce the notion that MMRD HSCT may not 
provide rapid and sufficient immune reconstitution in SCIDX1 infants 
with severe infections. Also, while difficult to summarize the data from 
many centers, overall the survival rate after matched unrelated donor 
transplantation appears to be around 70-80%. Gene modification of autologous hematopoietic progenitor 
cells can be expected to provide a more rapid and robust immune reconstitution in these infants, and 
hence improve survival.  
 
Information is also available from large individual centers experienced in the treatment of SCID by HSCT.  
 
1. At Great Ormond Street Hospital (GOSH) in London, 8 haploidentical transplants were performed 
for SCID-X1 from1982 (6 with conditioning chemotherapy), of which 1 patient died. Of 7 
survivors, the levels of immune reconstitution were similarly variable in the long term, and one 
received gene transfer as an adult in an attempt to alleviate severe lung and gut disease. 7 
patients at GOSH received MUD transplants, of which 1 patient died and 1 (treated in 2005) 
suffered from chronic GvHD with persisting poor immune reconstitution. All MUD transplants 
received chemotherapy as pre-conditioning.  
2. At Necker Hospital in Paris, 33 haploidentical transplants were performed specifically for SCID-
X1 from 1971, of which 18 patients survived. 8 patients of these were treated after 1998, of which 
6 are alive with variable levels of T cell reconstitution. Interestingly, long term morbidity and 
mortality in this cohort was also significant. Of those surviving past 2 years, 3 subsequently died 
from autoimmune and immunopathological disease. More than half of the others suffered from 
recurrent respiratory tract infections despite immunoglobulin prophylaxis. Analysis of patients 
treated after 1997 suggests that the clinical status of the patients at the time of transplantation 
has a significant influence on outcome. Five out of five patients who had no active infection at 
time of HSCT are alive (follow up 1 to 8 years, median 4). All have normal T cell counts and 
function, and 3 continue immunoglobulin substitution. In contrast, among 6 patients who had 
active infection or inflammation at time of HSCT (including disseminated  BCG infection in 2, B 
cell lymphoproliferation in 1, protracted diarrhea requiring parenteral nutrition in 4, viral infection 

17 
Version 12.0 
January 4, 2022 
of the lung in 2), only 3 are alive (1,2 years and 4 years follow up respectively), one with 
persistent BCG infection despite the presence of functional donor T cells.  
3. In a joint Italian/Canadian study, 19 SCID-X1 patients treated between 1990-2004 were reported. 
The survival rates for HSCT from the different donor sources were: MSD 100% (3/3), MUD 89% 
(8/9), HLA-haploidentical 29% (2/7). 
4. In a cohort of patients treated at Duke University Medical Centre (which does not use pre-
conditioning), the long term survival of 132 SCID patients (of which 70 had B+ SCID) was 77% 
(40, 41). This included 117 HLA-haploidentical and 15 HLA-identical related transplants most of 
which were T cell depleted. More than a quarter of these patients required up to 3 additional 
transplants, mostly unconditioned, and many had residual immunological deficits (see below). 
5. The Center for International Blood and Marrow Transplant Research (CIBMTR) data collected 
between 1990 and 2005 reports 248 cases of BMT for XSCID, the majority performed with 
haploidentical parental BM donors. Overall 5 year survival is 70%; however survival of the 35 
boys treated under 6 months without conditioning was 95%. 
6. Six patients with XSCID have received transplants from unrelated donors at Cincinnati Children’s 
Hospital Medical Center during the past 6 years following conditioning with rituxan and 
thymoglobulin – all are alive and well, but none have fully recovered B and NK cell function.  
 
4.4.2 Quality of immunological reconstitution after HSCT 
The strategies for transplantation from alternative donors are variable. In the case of transplantation from 
HLA-mismatched related donors, T cell depletion of the graft is used, thus minimizing the risk of GvHD. In 
certain centers, unconditioned transplants are undertaken thus decreasing the short term and long term 
side effects associated with the cytotoxicity of conditioning agents used in young children (including 
infertility, endocrine abnormalities, and dental abnormalities). Alternatively a conditioning regimen may be 
used to facilitate engraftment of donor cells, which may have important implications for long term efficacy 
(42). Although an unconditioned transplant would appear to be a more favorable approach for both 
genotypically matched and alternative donor transplants, there are certain disadvantages. Under these 
circumstances, donor derived T cell function becomes established in patients with SCID-X1, rapidly in the 
case of full grafts from matched family donors and more slowly following T cell depleted grafts, and co-
exists with the host derived γc-defective B cells. Following myeloablative conditioning regimens, the 
pattern of engraftment is difficult to predict but most commonly results in complete donor-derived T and B 
cell lineages presumably due to enhanced engraftment of donor stem cells. Residual humoral deficits 
have been described following both types of transplantation(43-45)  but are more commonly seen 
following unconditioned transplants. In a single center report of 102 unconditioned transplants from both 
HLA and non-HLA identical donors, 62% of patients remained on immunoglobulin substitution(40) 
suggesting that the continued presence of host γc defective B cells might result in residual humoral 
defects. Studies of SCID-X1 patients following HSCT showed that there are intrinsic defects of isotype 
switching and somatic mutation in host γc-defective B cells and effective humoral function is presumably 
derived almost entirely from the engrafted donor B cell pool (21). Since donor B cells are derived from the 
engraftment of donor stem cells, this suggests that a degree of donor stem cell engraftment (or correction) 
is necessary for functional cellular and humoral reconstitution.  
 

18 
Version 12.0 
January 4, 2022 
These findings of improved humoral immune reconstitution 
have been confirmed in a retrospective study of 240 SCID 
patients transplanted from 2000-2009 at PIDTC centers 
(100). In multivariate analysis of 149 2-5 year survivors, 
receipt of busulfan-based conditioning was associated with 
a higher likelihood of humoral immune reconstitution 
defined as freedom from immunoglobulin substitution 
(84% versus 41% without busulfan, OR 8.87, p<0.001). Additionally, busulfan based conditioning was 
associated with higher T cell counts in 2-year survivors in multivariate analysis, defined as CD3+ T cell 
count >1000 (89% versus 62% without busulfan, OR 8.84, p=0.007). The improvement in total T cell 
numbers may be due to improved thymic function, as shown by a dramatic increase in CD4+ T cells with a 
naïve, CD45RA+ phenotype (Figure 5). These data form the basis for inclusion of low dose busulfan-
based chemotherapy in patients without active infection, to increase the likelihood of complete immune 
reconstitution in survivors of SCID-X1 patients following transplant. 
 
 
5. Alternative therapy for SCID-X1 based on somatic gene transfer  
As outlined above, allogeneic bone marrow transplantation for SCID-X1 remains problematic when HLA-
matched donors are unavailable. A number of features make SCID-X1 an attractive candidate for 
treatment by somatic gene transfer. 
1. It is a monogenic disease well characterized at the molecular level. 
2. Introduction of γc into deficient T lymphoid precursor cells confers a powerful survival and 
developmental advantage to their progeny.  
3. Introduction of γc into hematopoietic stem cells can restore the long term functionality of B 
lymphoid, T lymphoid, and NK cells. 
4. The survival advantage of T cells obviates the absolute requirement for myeloablative conditioning. 
5. Two recent clinical trials of somatic gene transfer for SCID-X1 have demonstrated good correction 
of the immunological defects in 17 patients, thus verifying the feasibility and efficacy of this approach in 
human subjects. 
 
5.1 Target cell populations for somatic gene transfer of SCID-X1 
Hematopoietic stem cells (HSCs) are capable of self-renewal and differentiation to all blood lineages. In 
mice, this activity is highly enriched in the Thy-1.1lolineage (Lin)-/loSca-1+ population which represents 
about 0.05% of bone marrow cells(46, 47). This heterogeneous population of cells has been further 
sub-divided based on surface immunophenotype and the ability to self-renew, but all retain 
multipotency(48). The question of whether HSCs commit to specific lineages, and at that point lose 
their characteristic high proliferative potential or whether they give rise to lineage-restricted progenitors 
with significant proliferative and repopulating activity has been more difficult to answer. The study of 
radiation-induced chromosomal aberrations in the progeny of stem cells injected into mice at limiting 
dilution originally suggested that in addition to the multipotent population there existed myeloid and 
lymphoid restricted stem cells(49). These findings were later supported by retroviral marking 
studies(50).  However, it has been difficult to define a clear phenotypic identity for these lineage-
Figure 5. Naïve CD4+ CD45RA+ T cell 
counts are higher in SCID patients receiving 
busulfan based reduced intensity or 
myeloablative conditioning (RIC/MAC, 
median 1094 cells/ul) compared to those 
receiving no conditioning or 
immunosuppression. without busulfan 
(none/IS, median 192 cells/ul, p<0.001). 

19 
Version 12.0 
January 4, 2022 
restricted cells, in part because of contamination of sorted populations with multipotent HSCs, and their 
existence has therefore been questioned. For example, Thy-1loMac-1+B220- and Thy-1loMac-1-B220+ 
populations which were originally identified as highly proliferative myeloid and B lymphoid-restricted 
progenitors respectively, were later shown to be unable to significantly repopulate lethally-irradiated 
recipients(51). Despite these reservations, good evidence has emerged for the existence of murine 
clonogenic common lymphoid stem cells which possess lymphoid-restricted (T,B and natural killer 
(NK)) repopulating activity, and which are defined phenotypically as a Lin–IL-7R+Thy-1–Sca-1loc-kitlo 
population(52). An equivalent population of cells with restricted myeloid potential has also been 
isolated(53). 
 
Less is known about the human HSC compartment predominantly because of the failure of in vitro 
assay systems to measure defining activities. However, studies have recently been facilitated by the 
development of xenogeneic transplantation assays for the ability of human hematopoietic cells to 
competitively repopulate immunodeficient mice. SCID-repopulating cells (SRCs) are a population of 
human hematopoietic cells that have been conventionally defined by their capacity for bone marrow 
engraftment, extensive proliferation and multilineage (lymphoid and myeloid) differentiation in SCID, 
NOD/SCID and most recently NOD/SCID/IL-2RG-deficient mice. This activity is highly enriched in 
CD34+CD38– fractions, and based on the kinetics of engraftment, defines a more primitive cell 
population than most long term culture-initiating cells (LTC-ICs) and colony forming cells (CFCs)(54-
56).  A small fraction of CD34–CD38–lin– cells have been shown to possess multilineage repopulating 
activity in vivo (CD34–SRC), and to have the capacity to mature into CD34+ cells both in vitro and in 
vivo (54).  These studies are consistent with other experiments which have identified stem cell activity 
in CD34– populations of mice, primates, and humans, and together indicate that as in mice, the human 
repopulating stem cell compartment is phenotypically heterogeneous(57-60). The phenotypic definition 
of murine clonogenic common lymphoid and myeloid progenitor populations goes some way to support 
these ideas, and may be paralleled by similar populations of human committed cells(52, 53, 61-63).  
 
For SCID-X1, gene transfer to pluripotential hematopoietic stem cells in vivo would provide a continuing 
source of cells expressing γc, and is therefore predicted to result in complete reconstitution of the 
immunological deficit including B cell function. For reasons discussed previously, it is also likely that 
gene transfer to more committed populations, for example common lymphoid progenitors, will be 
sufficient to reconstitute complex T cell, B cell and NK cell immunity. Furthermore, conditioning with 
busulfan would promote engraftment of gene marked HSC that can give rise to multi-lineage gene 
marked cells, including B cells. Therefore, both HSCs and lymphoid precursor cells are the target for 
gene transfer.  
 
5.2 Studies in γc-deficient mice generated by gene targeting  
The existence of murine and canine models of γc deficiency offers the possibility to test gene transfer 
as an alternative treatment for SCID-X1. In contrast to the human disease, γc deficient mice are 
characterized by severe reductions in B cells, NK cells and gut associated intraepithelial cells(64, 65). 
Mature activated T cells do develop but are not able to proliferate in response to mitogens or in a mixed 
lymphocyte culture and lack the capacity to reject tumors. Thus, this murine model functionally 
represents many of the features of the human disease, but is not a phenocopy of the human disease. 
Several laboratories have reported the correction of SCID-X1 in mice by gammaretrovirus-mediated 
gene transfer(66, 67). In one study using a vector subsequently used for clinical study (see below), 
transduced γc- cells were transferred into an alymphoid recipient mice (a γc-/RAG- double knockout 
strain, which is deficient in both B and T cells and therefore a clearer indicator of the effect of IL-2RG 
gene transfer on B and T cell development)(68). Circulating B and T lymphocytes developed after 4 
weeks, showing integration of the transgene and cell surface expression of γc. Humoral immunity was 
reconstituted as evidenced by normal production of all immunoglobulin subclasses and by normal 
antibody responses following vaccination. Cellular function was also restored with normal T cell 
numbers, CD4/CD8 ratio and proliferative responses to IL-2, -4 and –7. However, in more recent and 

20 
Version 12.0 
January 4, 2022 
upublished studies studies in our own laboratories using the γc-/RAG- double knockout strain of mice, 
we have noted a high incidence of ‘spontaneous’ leukemia/lymphoma development when cells are 
‘mock’ transduced, calling into question the validity of this model for safety studies. 
 
5.3 Clinical trials of gene transfer for SCID-X1 
5.3.1 Successful restoration of immunological function 
Many incremental advances in gene transfer technology, several by investigators of this protocol,  have 
contributed to the successful application of gene transfer for SCID-X1 (including the optimization of cell 
culture and gene transfer conditions ex vivo), which complement the intrinsic profound selective growth 
advantage imparted to successfully transduced cells. Two studies carried out in Hospital Necker, Paris 
and ICH/GOSH, London by Thrasher and colleagues and Fisher and colleagues in a total of 20 patients 
with classical presentation have demonstrated that gene transfer is highly effective in SCID-X1(69-71).  
Both studies utilized an MFG-based gammaretroviral vector encoding a IL2RG cDNA (regulated by 
intact Moloney murine leukemia virus long terminal repeat (LTR) sequences), to transduce autologous 
CD34+ cells ex vivo which were re-infused into the patients in the absence of pre-conditioning. In nearly 
all patients NK cells appeared between 2 and 4 weeks after infusion of cells, followed by new thymic T 
lymphocyte emigrants at 10-12 weeks (see Figure 4 below showing Paris patients in black and London 
patients in red, normal range for age matched controls in blue, yellow stars represent lymphocyte 
recovery in 3 patients who developed abnormal lymphoproliferation).  
 
Figure 6. T cell recovery in previous SCID-X1 trials. 
 
With some variation which may relate to the age of the patients treated, dosage of transduced cells, 
and clinical status, the number and distribution of these T cells increased rapidly (often more rapidly 
than observed following haploidentical transplantation), usually achieving normal numbers compared to 
age-matched control values. These T cells also functioned normally in terms of proliferative response to 
mitogens, T-cell receptor (TCR), and specific antigen stimulation, and were shown to have a complex 
phenotypic and molecular diversity of TCR. Functionality of the humoral system was also restored to a 

21 
Version 12.0 
January 4, 2022 
sufficient degree that discontinuation of immunoglobulin therapy was possible in most patients with 
sufficient follow-up. In Paris, 8/10 patients recovered good levels of immunity up to 8 years after 
treatment, 1/10 failed to develop T cells after receiving a low dose of transduced CD34+ cells (31), 4/8 
patients developed T-ALL like complications (2 of whom were successfully treated and recovered good 
immunity, 1 patient died of refractory lymphoproliferation, and one of which is currently under treatment 
(8.2.07) see below). In London, 9/10 patients recovered good levels of immunity up to 5 years after 
treatment, 1 patient developed T-ALL complications and was successfully treated and recovered good 
immunity. In both studies, evidence exists for long term engraftment of transduced HSCs at low levels 
(estimated at 0.1-2% based on marking in myeloid lineages), which may have significant implications 
for maintenance of thymopoiesis long term and for persisting B cell functionality. This is further 
supported by the observed recovery of thymopoiesis after chemotherapy in 4 patients (see below). 
Interestingly, NK cell reconstitution has been partial in all patients, but without obvious clinical 
sequelae, and follows an identical pattern to that observed after allogeneic transplantation. All 10 
patients treated in London were able to resume normal social interactions, with good quality of life and 
normal psychosocial development. Most patients were treated with only minimal hospitalization at the 
time of bone marrow harvest and cell infusion. This is in marked contrast to patients receiving 
conditioning chemotherapy in the context of mismatched allogeneic transplantation in whom there are 
significant risks associated with GvHD and prolonged immunosuppression. In 2 older patients, 
immunological reconstitution failed despite effective gene transfer to bone marrow CD34+ cells(72). It is 
therefore likely that there are host-related restrictions to efficacy, primarily due to the inability to 
reinitiate an exhausted or failed program of thymopoiesis. 
 
Long term follow up of patients also suggests that non-myeloablative regimens can have detrimental 
effects on T cell functionality many years after transplant, probably because there is insufficient HSC 
engraftment to sustain prolonged effective thymopoiesis(1).  One probable explanation is that the initial 
unconditioned transplant only allowed the engraftment of committed T cell precursors or expanded 
populations of mature T cells. Therefore, the complexity of the T cell repertoire may diminish over time, 
and the engraftment of NK cells may not be durable. It therefore seems desirable to design protocols that 
augment long term engraftment of thymocyte precursor cells.  
 
Potentially beneficial effects of busulfan based conditioning regimens on T cell number and thymic 
output are suggested by recent studies of allogeneic transplant outcome (Section 4.4.2). The likelihood 
of engrafting pluripotential hematopoietic stem cells is increased by complete or partial ablation of the 
recipient hematopoietic stem cell compartment with agents such as busulfan. This has been clearly 
demonstrated in gene transfer for adenosine deaminase deficient (ADA) SCID, where trials conducted 
using a total of 4 mg/kg busulfan dosed either every 8 hours over 2 days (101) or dosed daily once 
(102) resulted in multilineage engraftment with gene marked cells in T, B and myeloid compartments. 
The range of busulfan exposures measured as area-under-the-curve in these patients is 2377-7482 
uM*minute or 9.7-30.7 mg*hour/liter, clearly submyeloablative and were well tolerated. As a reference, 
full myeloablative exposure is 20,740-19200 uM*minute or 85-95 mg*hour/liter. Metabolism of busulfan 
is highly variable according to age (103, 104). While the exposure required for significant hematopoietic 
stem cell engraftment in SCID-X1 is not known, exposures in higher end of the range effective in ADA 
SCID are likely to be effective. 
 
Moreover, results of gene transfer for SCID-X1 using a self-inactivating lentiviral vector in 5 older 
patients who have failed allogeneic HSCT (NCT 01306019, 2011) have been recently reported (105). 
These patients received a total of 6 mg/kg busulfan given over 2 days resulting in exposure of 2519.6-
4528.9 uM*min, a cumulative exposure of 5039.2-9057.8 uM*min or 20.7-37.1 mg*hour/liter (105). 
Preliminary data of immune reconstitution show evidence of gene marking in B and myeloid cells, and 
ability to maintain stable IgG trough levels without immunoglobulin replacement therapy (105). Thus a 
similar target exposure could be expected to enhance gene marking and function of B cells in SCID-X1 
patients undergoing gene transfer in this protocol.  
 

22 
Version 12.0 
January 4, 2022 
5.3.2 Insertional mutagenesis and lymphoproliferative disease 
The dependence of retroviruses on chromosomal integration for stability of transduction brings with it 
the risk of insertional mutagenesis. Reproducible leukemogenesis and oncogenesis has now clearly 
been demonstrated in pre-clinical models, and may be directly associated with vector dose or cell copy 
number. Co-operating effects from expression of the transgene, or from other elements within the 
vector backbone may also be important, and are likely to be disease or context dependent(73). In 
human clinical trials, 5 patients with SCID-X1 having initially achieved successful immunological 
reconstitution, developed T cell lymphoproliferative disease 3-5 years after the gene transfer 
procedure(74, 75). Of 20 patients with SCIDX1 treated by gene transfer in these trials, 18 are currently 
alive and with good immune reconstitution but 5 have experienced a serious side effect. These 5 
children developed leukemia related to the gene transfer itself. Of these children, 4 of the 5 have 
received chemotherapy are currently in clinical and molecular remission, whereas one died of therapy-
related leukemia. One additional patient, who did not develop leukemia, died of complications of 
subsequent bone marrow transplant that was performed as a result of the gene transfer not working. 
This child had received a low dose of transduced CD34+ cells during the gene therapy protocol. In at 
least four of these patients, retroviral vector insertion into or near the LMO-2 proto-oncogene resulted in 
high level expression of LMO-2 in the clones, as a result of retroviral enhancer-mediated activation of 
transcription (Figure 5). In another trial of gene transfer for Chronic Granulomatous Disease (CGD), 
non-malignant amplification of myeloid clones leading to acquisition of monosomy 7 and 
myelodysplasia occurred in 3 patients and contributed to the initial efficacy of the therapy, but occurred 
due to similar Spleen Focus Forming Virus (SFFV) LTR-mediated activation of MDS1-EVI1, PRDM16 
or SETBP1 genes(76). All three CGD patients demonstrated signficant improvement in infections 
resistant to state-of-the-art medical management following gene therapeutic approach.   
 
 
Figure 7. Insertional activation of LMO-2 from retrovirus insertion in SCID-X1 trial. 
 
 
5.3.3 Mechanisms of leukemogenesis in SCID-X1 gene transfer 
Activation of LMO-2 is known to participate in human leukemogenesis by chromosomal translocation, 
and results in the development of T cell lymphoproliferation and leukemia in mice, albeit with a long 
latency. It is therefore likely that other contributing factors are required for leukemia to evolve. Cells with 
high proliferative potential such as HSC and thymocytes are also likely to be more susceptible to 

23 
Version 12.0 
January 4, 2022 
transformation following an insertional event than quiescent cells if they acquire additional adverse 
mutations unrelated to the gene transfer itself. One consideration is a contribution from the activity of 
the IL-2RG transgene. At least one tumor derived in susceptible mice following infection with replication 
gammaretroviruses has been shown to harbor separate but coincident integrations at the Il2rg and lmo-
2 gene loci, suggesting that there may be a significant synergistic interaction(77).  However, this clone 
also contained insertions at least 2 other known oncogenes including Bmi1 and Rap1gds1. Another 
study using forced high level lentiviral-vector-mediated expression of γc in mice claimed to demonstrate 
a pro-oncogenic effect of the transgene(78).   However, the influence of insertional mutagenesis was 
not evaluated (which may be important given the high copy numbers of transgene), and other 
investigators using in vitro and in vivo murine models either in classical transgenic or retroviral gene 
transfer experiments have not observed this effect(79-81).  In treated patients there is currently no 
evidence of dysregulated expression or signaling in lymphoid cells. The levels of expression of the IL-
2RG transgene are generally normal or less than normal in primary lymphocytes (including the 
leukemic clones from patients). Furthermore, there has been no selection over time for more highly 
expressing cells. The accumulated evidence from murine studies (gene therapy and transgenic models) 
and expression data for human leukemias from the previous X-SCID trials have not shown constitutive 
activation of JAK/STAT pathways in presence of transgenic γc expression. In addition, in primary 
human cells, it has been shown that over-expression of γc does not alter T cell development in vitro 
(82).  Therefore there is no good evidence at this time to support the suggestion that transgenic IL-2RG 
is potently oncogenic in its own right (see (80).  
 
5.3.4 Strategies to overcome insertional mutagenesis 
It is now apparent from animal studies that mutagenesis can be reproducibly induced by 
gammaretrovirus-mediated gene transfer, at least using vectors with intact LTR sequences. Strategies 
for improvement of safety are therefore of general importance. According to updated recommendations 
from the UK Gene Therapy Advisory Committee (GTAC) and Committee for safety of Medicines (CSM), 
a number of advances in vector design should be pursued that may be advantageous in the longer term 
and for future generations of retroviral vectors:  
 
Recommendation 6 states: ‘Additional safety features should be considered for retroviral 
protocols, including the use of self-inactivating vectors, and non viral promoters to drive 
therapeutic genes. Ideally, new vectors should be selected on the basis of improved safety in 
pre-clinical testing models (in vitro and/or in vivo). However the current lack of validated 
systems remains a constraint to application of this principle.’ 
(http://www.advisorybodies.doh.gov.uk/genetics/gtac/). 
 
6. Preliminary studies relevant to the proposed clinical trial 
 
6.1 Development of a novel vector to enhance safety of gene transfer protocols for SCID-X1  
 
The existing gene transfer study for SCID-X1 at ICH/GOSH (GTAC: 045) has been recently closed after 
successful treatment of 10 patients due to exhaustion of vector stocks. It was felt that significant 
advances in vector design should be incorporated into future studies to enhance the safety profile. 
 
6.1.1 Self-inactivating (SIN) gammaretroviral vectors for expression of IL2RG  
Several strategies have been suggested to improve efficiency and safety of current protocols. Patterns 
of integration into host chromosomes are to some degree vector dependent and could thereby 
contribute to the likelihood of inadvertent gene activation. However, both gammaretroviral vectors and 
lentiviral vectors have been shown to integrate preferentially within genes, and both are likely therefore 
to be susceptible to induction of mutagenic side effects. The overall design of vectors used for gene 

24 
Version 12.0 
January 4, 2022 
delivery is probably  most important and modifications may be possible that will limit the risks of 
mutagenesis, for example by the use of self-inactivating (SIN) configurations in which the powerful 
duplicated viral LTR enhancer sequences are deleted, and by the incorporation of regulatory domains 
with reduced transactivation potential.  
 
6.1.2 SIN vector development  
Dr. Christopher Baum and colleagues at Hannover Medical School have refined retroviral vectors for 
application to human gene transfer, and have been at the forefront of SIN gammaretroviral vector 
development. A new generation of SIN vectors has been developed which lack all enhancer-promoter 
elements of the LTR U3 region and are also devoid of all gammaretroviral coding regions, as previously 
described(83). In particular, modifications of the 5’ promoter of the SIN vector plasmid (which is not 
present in the integrated provirus) have contributed to high titers after transient transfection in 293T-
based packaging cells, thereby obviating some of the difficulties previously encountered with SIN vector 
production(84).  On the basis of these studies, a series of gammaretroviral vectors have been 
constructed containing different internal promoters to express the IL2RGcDNA (Figure 6).  All vectors 
contained the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), as this 
increases titers after transient transfection of 293T-based packaging cells (at present efficient stable 
packaging systems for production of SIN gammaretroviral vectors are not available). The WPRE 
sequence is devoid of the hepadnaviral X-protein open reading frame and contains a point mutation 
that destroys the largest residual open reading frame of this element (pre*)(84).  In retrovirally 
transduced cells, SIN vectors carry an internal promoter immediately upstream of the IL2RG cDNA, 
without an additional element in the 5’UTR. After preliminary testing for expression in cell lines the 
following two internal promoters were selected for further evaluation: murine spleen focus-forming virus 
(SF promoter vector designation pSRS11.SF.IL2RG.pre* SF), human polypeptide chain elongation 
factor-1α (EFS promoter, vector designation pSRS11.EFS.IL2RG.pre*) promoter short version lacking 
the first intron. A gammaretroviral MFG-γc vector used in the French phaseI/II clinical SCID-X1 trial 
(kindly provided by Professor Marina Cavazzana-Calvo, Paris, France) was used as a positive control. 
Schematics of vectors are also shown below. 
 
 
 
Figure 8. IL2RG vector design. 
 
In mammalian cells, EF-1α is present in most tissues, and the associated CpG-rich gene regulatory 
domains have therefore been attractive for utilization in expression vectors, including those under 
development for gene transfer as an alternative to viral sequences(85-87). To address whether the 
internal EFS has a reduced potential to transactivate neighbouring genes, two reporter systems were 
designed and evaluated in vitro. (1) In a plasmid-based assay the enhancer interactions of different 
promoter/enhancer configurations on a minimal promoter driving a luciferase expression cassette were 
tested following transient transfection in human fibrosarcoma cells (293T) and murine hematopoietic 
progenitor cells (32D).  (2) To test potential transactivating effects in retrovirally transduced cells, a 
novel design of a SIN vector was constructed with a minigene cassette placed in the residual U3 of the 
long terminal repeat (which would only be active if transactivated by the internal enhancer of the vector 
or a neighbouring cellular enhancer). Expression of the minigene cassette was examined in polyclonal 
cultures of murine SC1 fibroblasts and murine primary hematopoietic progenitor cells several days after 

25 
Version 12.0 
January 4, 2022 
retroviral transduction, without previous selection.  In both experimental settings, the cell-derived 
promoter EFS was a significantly weaker transactivator than viral enhancer-promoters such as the 
SFFV LTR. In the case of the minigene cassette, only the internal retroviral enhancer-promoter of SFFV 
was able to increase its expression above the background level observed in primary hematopoietic 
cells(88).  
 
6.1.3 Platform studies addressing the genotoxic impact of gammaretroviral SIN vectors 
In vivo murine studies in γc-deficient mice (and low dose transduction of normal bone marrow) have not 
previously been predictive of leukemogenesis as observed in human studies, and this remains a 
problem for pre-clinical evaluation as acknowledged in a recent GTAC/CSM report (see above).  Thus, 
there is currently no uniformly accepted assay to predict mutagenic potential in humans. Surrogate 
assays are available. One of the more advanced assays is the cell based transformation assays 
established in the Baum laboratory (89, 90)In this assay, we have shown that gammaretroviral SIN 
vectors containing the SFFV promoter have a residual propensity to immortalize murine hematopoietic 
progenitor cells when examined in serial replating assay, though this was significantly reduced 
compared to vectors with intact SFFV LTRs(91). Recently, we further increased the sensitivity of the 
assay so that we can now introduce more than 20 vector copies per cell. Despite this high efficiency of 
retroviral transduction and very high mean copy number/cell (Figure 7), cell viability is maintained, so 
that the assay is sufficiently sensitive and reproducible to discriminate the genotoxic impact of different 
internal promoters in SIN vectors expressing a neutral reporter transgene (eGFP). Under equivalent 
conditions, a SIN vector with the internal EFS promoter (pSRS11.EFS.eGFP.pre* or ‘Sin.EFS’ in Figure 
7) was significantly (P<0.01) less mutagenic than a SIN vector with the internal SF promoter 
(pSRS11.SF.eGFP.pre* or ‘SinSF’ in Figure 7).  In these assays to date, the backbone containing EFS 
as the internal promoter to drive EGFP expression in the SIN configuration has not been associated 
with immortalization in MOI dose escalation leading to >30 copies/cell.  Sin.EFS is the gammaretroviral 
SIN vector backbone identical to  pSRS11.EFS.IL2RG.pre*, but with the cDNA of IL2RG replaced by 
EGFP. No induction of replating activity was observed with this backbone, although the copy number 
was escalated from 11 up to 95 copies per cell (Table 1 in (90). Sin.SF is the same vector with the 
internal SFFV promoter instead of EFS. Replating activity was observed in all 6 assays with doses 
ranging from 16 to 27 copies per cell.  Sin.SF.1xHS4 is Sin.SF carrying an insulator element in the Ur 
region of the SIN LTR. Of note, SIN vectors containing ‘insulator’ elements flanking the  
 
internal SF promoter-
driven cassette showed a 
significantly greater 
degree of transforming 
activity that the SIN vector 
with the EFS promoter 
(4/4 experiments; P< 
0.01).The mean copy 
number of the vectors was 
determined by real-time 
PCR 4 days after 
transduction. Clones were 
counted under conditions 
of limiting dilution (10 cells 
plated per well and 
observed for 14 days), 
starting from polyclonal  
 
 

26 
Version 12.0 
January 4, 2022 
Figure 9. Immortalization of primary mouse bone marrow after transduction.  
 
cultures that were expanded for 14 days after transduction prior to replating in limiting dilution. 
Transformed clones were always observed with the SIN vector containing the internal SFFV promoter 
(3/3 cases), and with this vector, an average copy number of 1.7 was sufficient to induce 
transformation. This indicates that the transactivating potential of the SIN vector with the EFS promoter 
is at least substantially diminished(88).  In summary,  it has not been possible to induce transformation 
in vitro with a SIN vector harboring an internal EFS promoter(88). Finally, we have directly compared 
the potential for the SRS11.EFS.IL2RG.pre* vector to activate LMO2 in a model system developed by 
Nienhuis and colleagues that utilizes the Jurkat T cell line (92) Jurkat T cells express barely detectable 
LMO2 transcripts compared with K562 cells (3590-fold expression relative to Jurkat) (Figure 8). In this 
assay, a targeted insertion of the proviral genome is accomplished at the precise location of vector 
integration in one of the patients with X-SCID and LMO-2 mediated leukaemogenesis. Preliminary 
studies obtained from 2 clones indicate that SRS11.EFS.IL2RG.pre* (EFS#19,21) results in marked 
reduction in gene activation compared with a single LTR (G25-1-26), a double LTR provirus (656 #16), 
and an MFG-based LTR with a deletion of one enhancer repeat (MFG#43,5.2,54,92) (vectors used for 
both Paris and London clinical studies contained conventional dual enhancer LTRs). Furthermore the 
relative expression of LMO-2 was very similar in these two clones to an insulated SIN lentiviral vector in 
the same context. All of these values are derived by mRNA analysis using qRT-PCR, and are shown on 
a log scale relative to unmodified Jurkat cells.  
 
 
Figure 10. Expression of LMO2 in Jurkat Cells after Integration of Retrovirus Vectors (note log scale). 
Together these studies indicate that the transactivating potential of gammaretroviral vectors are 
substantially reduced by SIN configurations and further reduced by the utilization of EFS 
internal regulatory sequences. 
 
6.1.4 Toxicological studies in murine models 
Gammaretroviral vectors with intact LTRs (in the wild-type configuration) encoding reporter genes, or 
alternate transgenes induce leukemogenesis during experimental dose escalation through a process of 
combinatorial mutagenesis(93). This model is therefore being used to test for mutagenesis using the 
selected vectors to transduce lin- bone marrow cells from mice at variable multiplicities of infection(93).  

27 
Version 12.0 
January 4, 2022 
Safety-enhanced gammaretroviral vectors have since emerged, that incorporate the self-inactivating 
(SIN) LTRs. 
 
The potential of SIN gammaretroviral vectors encoding IL2RG (γc) driven by an internal promoter from 
the spleen focus forming virus (SF) to induce leukemia has been tested in a serial BMT model using 
C57BL/6J mice. Eight groups of 6 mice each received lin- bone marrow cells transduced with MFG-γc, 
a SIN gammaretroviral vector expressing IL2RG under control of an internal SF promoter 
(SRS11.SF.IL2RG.pre*), an equivalent SIN lentiviral vector (RRL.SF.IL2RG.pre*), and a control SIN 
gammaretroviral vector expressing the dsRedexpress fluorescent protein under control of an internal 
SF promoter (SRS11.SF.DsRedexpress.pre*).  No vector-associated leukemias were observed in 
primary transplant recipients observed for 11 months. In these mice, average transgene copy numbers 
(defined by real-time PCR in peripheral blood leukocytes) reached up to 0.6 copies/cell for 
SRS11.SF.DsRedexpress.pre*, 1.1 copies/cell for SRS11.SF.IL2RG.pre*, 2.6 for RRL.SF.IL2RG.pre*, 
and <0.2 for MFG-γc (this vector had a low infectious titer). Thus, in contrast to Woods et al.(78) 
gammaretroviral or lentiviral over-expression of IL2RG was not sufficient to induce leukemia in mice, 
despite a long observation time(80).   
 
After termination of this study and detailed necropsy confirming the absence of leukemia, serial or 
secondary BMT was performed with bone marrow cells from the  SRS11.SF.DsRedexpress.pre* or 
SRS11.SF.IL2RG.pre* groups of mice. The four donors of each group that showed the highest level of 
gene marking were chosen for these studies. Two recipients were transplanted from each donor, 
resulting in a total of 16 mice that were observed for another 7.5 months (18.5 months clone 
observation time since gene transfer). Two secondary recipients of SRS11.SF.DsRedexpress.pre* 
transduced cells showed signs of disease and were humanely sacrificed. Diseases were unrelated to 
vector insertion (one mouse suffering from leukopenia, another from a large renal cyst). No leukemia 
occurred in this group. However, two distinct leukemic clones were observed in secondary recipients of 
cells transduced with SRS11.SF.IL2RG.pre*. One clone showed a B cell precursor phenotype (latency 
3-5 months in secondary recipients), the other a myeloid phenotype (latency 6 .5 months). The single 
vector insertion site of the B cell leukemia was found to map to the Evi1 gene, encoding a transcription 
factor whose up-regulation by retroviruses may induce leukemia in mice(91, 94). Preliminary data 
showed that the leukemic cells did not require interleukins for growth in vitro and lost the B cell marker 
(B220) after culture in the presence of the cytokines mSCF and Flt3L, therefore questioning a 
significant role of the transgenic IL2RG in leukemia induction. The myeloid leukemia observed in the 
same transplant group had four vector insertion sites, and again one of these was found in Evi1. This 
study has been published by Baum’s group(95). 
 
These initial data suggest that leukemogenesis may be induced by random insertion of 
SRS11.SF.IL2RG.pre* (ie a viral LTR at an internal position) in the vicinity of a potent cellular proto-
oncogene, in line with the predictions of the in vitro immortalization assay(91). These data raise caution 
regarding the therapeutic use of SIN vectors with the very strong internal viral promoters and 
enhancers such as the SF promoter, and supports for clinical use a vector that relies on an internal 
cellular gene promoter which may have reduced transactivation potential, such as the promoter of 
elongation factor-1α gene (EFS).  
 
Therefore, in additional studies the SRS11.EFS.IL2RG.pre* vector was tested in two different mouse 
models. In studies using γc/Rag2 double knockout (gc-/-Rag-/-) mice we found high frequencies of both 
host tumors and donor-derived leukemia and lymphoma formation even in the absence of vector 
insertions (including ‘mock transduced’ cells). Host cell derived malignancies are not unexpected in this 
mouse model, and the frequency is not higher than previously reported values by our group(96). More 
importantly, given that even donor derived cells without any vector insertions develop 
leukemia/lymphoma in these recipient mice, these data strongly suggest that gc-/-Rag-/- mice are not 
satisfactory for analysis of vector-induced leukemogenesis.  Evaluation of vector-mediated toxicity was 

28 
Version 12.0 
January 4, 2022 
therefore conducted in a standardinbred mouse strain. Safety studies were conducted using C57/BL6 
Ly5.1/Ly5.2 congenic donors and recipients were undertaken in two experimental groups. To 
summarize data detailed below, in a total of 31 mice transplanted with the clinical vector and followed 
for a minimum of 10 months, there were no donor derived tumors noted.  An additional 8 animals 
received secondary transplants from a cohort of these primary mice and were observed for up to one 
additional year. No donor derived tumors were noted.  Details of these cohorts are provided below.  
 
Experimental group 1: In one set of studies, lineage negative (Lin-) cells from three experimental 
groups of Ly5.2 (CD45.1+) mice were concurrently transduced with 1) SRS11.EFS.IL2RG.pre* (IL2RG 
group), 2) control EGFP vector SERS11.EFS.EGFP.pre* (EGFP group), or 3) media alone (mock 
controls). Briefly, lineage negative bone marrow derived stem cells were isolated and pre-stimulated for 
48 hours in Stemspan with 1% (v/v) Penicillin/Streptomycin, murine recombinant (m) SCF (100 ng/ml), 
mFlt3 (100 ng/ml), mIL-3 (20 ng/ml) and human recombinant IL-11 (100 ng/ml). The cells were then 
harvested and transduced on fibronectin (FN) CH296 fragment on two consecutive days with a 
multiplicity of infection (MOI) which was demonstrated in preliminary studies to yield approximately 40% 
transduced bone marrow clonogenic progenitors. Cells were harvested after the second transduction, 
washed and injected into recipient mice at doses of at least 4x10e5 cells per mouse. Twenty mice were 
transplanted for each experimental group, in 2 cohorts of 10 mice per cohort. The mice were observed 
for a period of 10-12 months. In this set of studies, evaluable mice were those that had >25% 
engraftment at 5 weeks and survived >10 months following transplant. Any mouse that died after 1 
month of transplant was also autopsied to determine the cause of death, presence of donor/host 
derived tumor/s and vector status of the tumor. Peripheral blood was taken from the tail vein at 5 
weeks, 2 months, 4 months, 6 months and 9 months post transplant. Complete blood counts were 
measured at each time point, and additional analysis by flow cytometry and real-time PCR was 
alternated. At sacrifice, all tissues were weighed and examined grossly. Liver, spleen, thymus, bone 
marrow, kidney, lung as well as any masses or enlarged organs were evaluated by a pathologist. 
Additionally, bone marrow, spleen, and thymus were examined by flow cytometry.  
 
Details of the individual mice in all three cohorts (and data from the second experimental group) are 
summarized in Table 1.   
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 
Table 1:  SRS11.EFS.IL2RG.pre* Animal Data Analysis 
Recipient 
Donor 
Vector 
Evaluable 
Mice^ 
Donor 
Derived 
Lymphomas Percent 
C57 (CD45.2) BoyJ (CD45.1) SRS11.EFS.IL2RG.pre*  
31++ 
0 
0 
C57 (CD45.2) BoyJ (CD45.1) SERS11.EFS.GFP.pre*  
17 
0^^ 
0 
C57 (CD45.2) BoyJ (CD45.1) Mock 
18 
0# 
0 
 
^ Evaluable mice in the C57 recipients were all mice followed for at least 10 months and 
with more than 30% engraftment at 5 weeks. On average, there was 60-80% 
engraftment in this group of mice. ++ includes 14 mice followed for 14 months in primary 
recipients.  Four animals from this cohort with peripheral blood and spleen engraftment of 
30-60% and 15-60% respectively (copy number 0.04-0.74) were sacrificed at 14 months 
and 1x10e6 cells transplanted into secondary recipient mice. Eight secondary mice were 
followed for 1 year. 
^^in one mouse tumor (thymoma), the origin could not be determined; however, the 
thymoma mass was vector positive. 
# one mouse found dead with enlarged thymus 

29 
Version 12.0 
January 4, 2022 
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 (96).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 (mouse ID H.8). Of note, there was also an animal found dead with enlarged 
thymus in the mock control group.  
 
Experimental group 2: In a second set of studies, the transduction protocol was identical to above 
except that the MOI was equal to 2. Cells were harvested after the second transduction and injected 
into recipient mice at doses of 5x10e5 cells per mouse. In this second group of mice, 13 out of 14 
animals were followed for 14-16 months after reconstitution. The copy number in peripheral blood 
mononuclear cells determined by QT-PCR after 14 months was 0.008-0.74. These animals had a 
CD45.1 engraftment level of 4-60% in the peripheral blood and 4-60% in the spleen (at sacrifice). One 
host derived B220 leukemia was noted at 264 days. One mouse had a host derived liver mass at final 
sacrifice. Four healthy animals from this primary cohort with peripheral blood and spleen engraftment of 
30-60% and 15-60% respectively (copy number 0.04-0.74) were sacrificed at 14 months and 1x10e6 
cells transplanted into secondary recipient mice. Eight mice were followed for up to 1 year. Engraftment 
in the spleen was 4-33% and peripheral blood was 4-21% at sacrifice of these animals. The peripheral 
blood mononuclear cell copy number per CD45.1 cell 4 months post injection was 0.04-0.1. In the 
secondary animals, one host-derived B220 leukemia (10 weeks after engraftment) and one host-
derived thymoma (7 months after engraftment) were noted. One animal was lost to evaluation. Five 
animals survived to one year and were normal at sacrifice. No donor derived tumors were noted in any 
primary or secondary mice.  
 
In summary, the basic configuration of pSRS11.EFS.IL2RG.pre* has a clearly proven reduction 
in mutagenic potential compared to LTR configuration in in vitro assays and has not been 
associated with vector-positive tumors in a large number of mice followed over an extensive 
period, including after serial transplantation stress.  
 
6.1.5 Evaluation of potency in murine model systems in vitro and in vivo  
To investigate γc expression in hematopoietic progenitors, lin- cells from young adult donor C57Bl6 
mice were transduced cells as described(97). Both SIN configuration vectors (incorporating SF or EFS 
promoters) mediated good surface expression of γc, as detected by flow cytometry, although at a 
somewhat lower level than that obtained with MFG-γc. More importantly, both were able to efficiently 
rescue the in vitro B and T cell differentiation capacity of lin- cells derived from Il2Rγ -/- mice in a 
surrogate lymphocyte differentiation system, and at equivalent levels to that achieved using the MFG-
based vector. These results reveal that both SIN vectors are sufficiently potent to compensate the 
genetic deficiency in primary murine progenitor cells when examined in cell culture. 
pSRS11.EFS.IL2RG.pre* was further evaluated in vivo in preference to pSRS11.SF.IL2RG.pre to avoid 
use of potentially mutagenic viral LTR sequences (see above). 
 
The γc-deficient mouse provides a convenient, though not perfect, model with which to compare gene 
transfer strategies for SCID-X1 in vivo. pSRS11.EFS.IL2RG.pre* was therefore compared to the vector 
currently used in the clinical trial, MFG γc, for efficacy of immune reconstitution following gene transfer 
in vivo. Briefly, lineage negative (lin-) bone marrow cells from γc-/- mice were transduced ex vivo using a 
low MOI with either the SIN vector, the LTR-regulated clinical vector or with a gammaretroviral vector 
encoding an eGFP transgene (SFFV-eGFP) under serum-free conditions in the presence of cytokines. 
Transduction levels of between 55-80% were achieved using this method. Transduced cells were 
subsequently injected into sublethally-irradiated alymphoid γc-/rag2-/c5- recipient mice. Transplanted 

30 
Version 12.0 
January 4, 2022 
animals were maintained in pathogen-free environments for at least three months to allow engraftment 
and repopulation. 
 
Fifteen to eighteen weeks post-transplant circulating T and B lymphocytes were detectable in the 
peripheral blood of all seven mice transplanted with pSRS11.EFS.IL2RG.pre* transduced cells and all 
six MFG γc mice. Four out of four mice repopulated with SFFV-eGFP transduced cells remained 
alymphoid. Experimental animals were sacrificed approximately five months post-transplant for analysis 
of immune reconstitution. Flow cytometric analysis of the spleens and bone marrow revealed 
restoration of mature B220+IgM+ B cells and NK cell populations in all pSRS11.EFS.IL2RG.pre and 
MFG γc transplanted mice. CD4+ and CD8+ T cells were also detected in both tissues and in thymi 
recovered from transplanted animals indicating restoration of thymopoiesis (Figure 9). The restored T 
lymphocyte populations in these mice appeared functional with splenocytes able to proliferate in 
response to mitogenic stimuli and at increased levels in the presence of a γc-dependent cytokine. 
Immunoglobulin subclasses IgG1 and IgG2a detected in the plasma from pSRS11.EFS.IL2RG.pre* 
reconstituted mice also indicate restored B cell function in these animals. Proviral copy number in 
splenocytes and bone marrow cells was detected by real-time PCR and found to be low (less than one 
copy per cell in all but one of the reconstituted mice).  All control mice repopulated with cells transduced 
with the SFFV-eGFP vector remained athymic and had no detectable T, NK or mature IgM+ B cells in 
the spleens and bone marrow at the time of analysis. Further engrafted animals will be evaluated at 
later time points. 
 
Figure 11.   T and B cell compartments following gene therapy in a murine model. 
EF1αrepresents pSRS11.EFS.IL2RG.pre*, and SF represents pSRS11.SF.IL2RG.pre*. 
 

31 
Version 12.0 
January 4, 2022 
6.1.6 Evaluation in human T cell lines and CD34+ cells 
The ability of pSRS11.EFS.IL2RG.pre* to reconstitute signaling activity in human cells has been tested 
on a human γc-deficient T cell leukemia cell subline (ED7R) which retains expression of the IL2R α and 
β chains(98). Transduction of these cells in vitro using GLP-produced GALV-pseudotyped vector 
results in normal reconstitution of STAT5 phosphorylation following stimulation with γc-dependent 
cytokines such as IL-2 or IL-7 (Figure 10). 
 
 
  
 
 
 
 
 
 
 
 
 
 
Figure 12. Restoration of Stat5 activation following gene transfer. 
 
Preparations of GLP-produced GALV-pseudotyped vector stocks have also been tested on human 
CD34+ cells using standard transduction conditions (as used previously). The titer of the viral 
preparations was approximately 1x106 transducing units per ml measured by expression of human γc in 
transduced HT1080 cells. In two independent experiments normal peripheral blood derived CD34+ 
cells were used as targets (Figure 11). In a further experiment, a limited number of CD34+ bone 
marrow cells were purified from a patient with classical SCID-X1. Overall transduction efficiency of 
CD34+ using pSRS11.EFS.IL2RG.pre* was between 20 and 60%. In one experiment, clinical grade 
MFG-γc vector was used to transduce cells in parallel. Transduction efficiency was in this case similar, 
although as expected the levels of expression were lower in cells transduced with 
pSRS11.EFS.IL2RG.pre*.  
 
Figure 13. Expression of γc in transduced CD34+ cells. 
 
Transduced cells derived from a SCID-X1 patient were also cultured under conditions favoring NK cell 
development in vitro (in the presence of IL-2 and IL-15 for 21 days). Due to the low number of cells 

32 
Version 12.0 
January 4, 2022 
available for analysis, detailed confirmation of NK cell differentiation and function was not possible. 
However, untransduced cells cultured under the same conditions all died, whereas transduced SCID-
X1 cells remained viable after 21 days suggesting that cell signaling had been successfully restored. 
 
6.1.7 Details of final vector construction and derivation (Appendix 1) 
The SIN gammaretroviral vector backbone used in this study has been described in an earlier section. 
A schematic map of the plasmid (backbone based on pUC) and detailed sequence data shown in 
Appendix 1.  
6.1.8 Clinical grade vector production 
This has been undertaken by the vector production facility (VPF) at Cincinnati Children’s Research 
Foundation. Previously, production of SIN gammaretroviral vectors has been compromised by 
difficulties in achieving usable titers. However, advances in the design and configuration of these 
vectors, has resulted in greatly improved yields, albeit by transient production techniques in clinically 
validated cell lines such as 293 HEK, or following virus concentration. The vectors proposed for this 
study have been shown to yield effective titers of up to 107 transducing units (γc expression in cell lines) 
per ml of cell culture supernatant, which is readily applicable to existing clinical protocols. Consistent 
with the pre-existing vector, viral particles will be GALV-pseudotyped, as this may confer advantages 
with respect to transduction of early progenitor cells and HSC. Certification of clinical grade vector 
supernatant with a titer of >1 X 106 transducing units/ml is currently underway.  
 
All clinical viral vector production open manipulations are performed in an ISO Class 5 Biosafety 
Cabinet located in an ISO Class 7 segregated tissue culture suite with ISO Class 8 support areas.  All 
practices and documentation are consistent with GMP recommendations for early phase US FDA 
retroviral vector products to be administered under an IND.  The retroviral vector is produced by 
transient transfection in a closed system disposable bioreactor. Cells from a 293T certified Master Cell 
Bank are expanded using standard tissue culture flasks and roller bottles in a fetal bovine 
supplemented cell culture medium. Cells are harvested, transfected with plasmids 
SRS11.EFS.IL2Rg.pre*, pCDNA3.MLV.g/p, and K83-GALV using Calcium Phosphate transfection 
reagents and seeded into Corning Cell Bind 10 Stacks. The 10 Stacks are placed in a temperature and 
CO2 controlled incubator.  Following overnight incubation tissue culture medium is replaced and virus is 
harvested with media re-feeds at three 12 hours intervals.  The harvested virus is aliquoted into closed 
system cryobags and frozen at <-70o C., with appropriate samples aliquoted for certification and 
evaluation.  Following the final harvest, end of production cells are removed for certification assays and 
appropriate reserves are cryopreserved in DMSO in vapor phase liquid nitrogen.  
 
Vector and end of production cells are submitted for certification for purity, potency, and identity.  These 
assays include sterility/bacteriostasis and fungistasis (21CFR610.12), mycoplasma (PTC), endotoxin 
(USP), in-vitro adventitious virus (3 cell lines), RCR (5% of supernatant and 108 post production cells by 
S+L- assay (using 293 HEK cells for amplification) performed at IU NGVL certification laboratory), 
residual DNA (PCR), identity (non-GMP LTR to LTR sequencing of proviral insert in permissive target 
cell), and potency (sponsor defined).  All results and records are Quality Assurance reviewed prior to 
product release. 
 
6.2 General Information related to the Investigational Medicinal Product (IMP)  
6.2.1 Name description of the IMP: 
 
pSRS11.EFS.IL2RG.pre* gammaretroviral vector (used to transduce autologous CD34+ cells, ex vivo), 
see Appendix 1.  This vector is based on a Moloney murine leukemia virus packaged in a gibbon ape 
leukemia (GALV) pseudotype. 

33 
Version 12.0 
January 4, 2022 
 
pSRS11.EFS.IL2RG.pre* is an unlicensed product (not applicable for licensing). 
 
6.2.2 Production, Supply and Release of the IMP: 
 
The Vector was produced, certified and will be supplied by the vector production facility of Cincinnati 
Children’s Research Foundation, Division of Experimental Hematology, Cincinnati Children’s Research 
Foundation, Cincinnati Children’s Hospital Medical Center, Cincinnati OH, 45229, USA. 
 
Transduction of autologous CD34+ cells with the retroviral vector pSRS11.EFS.IL2RG.pre* will be 
carried out in the GMP cell manipulation facilities at each participating institution.  
 
The transduced cells will be packaged in sealed transfer bags prior to being transported according to 
local practice for cellular product distribution at room temperature to the clinical site of administration. 
Cells released from the GMP facility will be infused according to local SOPs and practice, with minimum 
requirements specified in the Manual of Operations. 
 
6.2.3 Drug accountability  
 
Drug accountability is ultimately the responsibility of the Study Sponsor. 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 & transduced CD34+ 
cells. These records will include details of shipping, receipt, storage, use & destruction of the vector. 
Transfer of CD34+ cells from the cell manipulation laboratory at each institution to the site of 
administration. Administration of the transduced CD34+ cells to patients will be recorded. 
6.2.4 Description of and justification of the trial treatment & dosage 
 
The retroviral vector pSRS11.EFS.IL2RG.pre* is used to transduce autologous CD34+ cells. These 
transduced cells are then infused into the patient. The autologous CD34+ cells undergo 2 or 3 rounds 
of retroviral transduction. The transduced cells are administered intravenously into the patient as one 
infusion over 15-30 minutes.The transduction protocol has been optimized to achieve a gene transfer 
efficiency of 30-40% in starting cells, which will produce a mean copy number of ~1 for engrafted T-
cells. 
 
7. Study Design 
 
An open labeled, non-randomized, multi-center, phase I/II, cohort study involving a single infusion of 
autologous CD34+ cells transduced with the self-inactivating (SIN) gammaretroviral vector 
pSRS11.EFS.IL2RG.pre* in up to 9 patients with SCID-X1 at Children’s Hospital Boston, Mattel 
Children’s Hospital UCLA, Cincinnati Children’s Hospital Medical Center. These three sites are funded 
by NIAID/DAIT and fall under the IND 14067. 
 
Adequate vector has been produced by Cincinnati Children’s Medical Center to treat a minimum of 20 
patients. In addition to the 9 patients in this trial, 11 patients will be enrolled in parallel independent 
trials under separate protocols at two European Centers (Great Ormond Street Hospital and Hopital 
Necker). We will collect data only on the 9 patients enrolled on this trial in the United States. We will 
review limited safety and efficacy data provided to us by the European sites for statistical purposes and 
reporting to the DSMB as detailed in section 10.5. 

34 
Version 12.0 
January 4, 2022 
 
We detailed evidence in section 5.3.1 regarding improved humoral immune reconstitution in older 
SCID-X1 patients undergoing gene transfer after low dose busulfan.Thus, this protocol envisages that 
of 20 patients total, 5 who meet criteria for receiving chemotherapy conditioning will undergo treatment 
with low dose busulfan prior to receiving transduced cells. The remaining 15 will receive transduced 
cells without conditioning. The primary endpoint for all patients remains the same, T cell reconstitution 
and safety.  
 
7.1 Protocol summary 
 
 
 
7.2 Study objectives  
The objectives of this proposal are to initiate a trial of somatic gene transfer for patients with SCID-X1 in 
whom HLA-matched family donors are unavailable or in whom underlying clinical problems would 
exclude chemotherapy conditioning. For this study, autologous CD34+ cells will be transduced ex vivo 
using a novel gibbon ape leukemia (GALV)-virus pseudotyped self-inactivating (SIN) gammaretroviral 
vector (pSRS11.EFS.IL2RG.pre*). The specific objectives are as follows: 
 
1. Treatment of SCID-X1 patients by somatic gene transfer when HLA-matched family donors are 
unavailable. 
2. Successful ex vivo transduction of CD34+ hematopoietic cells from SCID-X1 patients by ex vivo 
gammaretrovirus-mediated gene transfer. 
3. Evaluation of immunological and functional reconstitution in progeny of engrafted cells. 
4. Longitudinal evaluation of clinical effect in terms of augmented immunity.  
5. Evaluation of the functional performance of novel SIN gammaretroviral configuration. 
6. Evaluation of the molecular characteristics of vector integration. 
7. Evaluation of safety. 
8. In a descriptive graphical fashion, to present immune reconstitution data for the subgroup of 
patients treated with a low-dose conditioning regimen including busulfan. 

35 
Version 12.0 
January 4, 2022 
 
7.3 Study endpoints 
 
Primary endpoint  
1) 
Immunological reconstitution defined as absolute CD3 cells of >300/μl and PHA stimulation 
index >15 at 6 months post infusion. 
2) 
Incidence of life-threatening adverse reactions related to the gene therapy procedure. 
 
Secondary Endpoints 
1) 
Molecular characterization of gene transfer. 
2) 
Ability to mount antibody responses to vaccination. 
3) 
Normalization of nutritional status, growth, and development. 
 
8.  Patient selection and recruitment 
 
Up to 9 patients will be recruited at Children’s Hospital Boston, UCLA Mattel Children’s Hospital and 
Cincinnati Children’s Hospital Medical Center and will be selected for inclusion on the basis of the 
following defined criteria (see Appendix 2 for decision tree): 
 
8.1 Inclusion and exclusion criteria 
 
Inclusion criteria 
 
1. 
Diagnosis of SCID-X1 based on immunophenotype (<200 CD3+ autologous T cells,, and 
confirmed by DNA sequencing) 
AND  
 
2. 
Lack an HLA identical (A, B, C, DR, DQ) related donor 
 
AND either one of the following 
 
3a. Patients in good clinical condition who do not have a readily available HLA identical (A,B,C,DR,DQ) 
unrelated donor (readily available defined as: a donor confirmed within 6 weeks of searching, with 
ability to transplant within 3 months of diagnosis). Such a patient would be eligible to receive busulfan 
conditioning. 
3b. Patients with an active, therapy-resistant infection or other medical conditions that significantly 
increase the risk of allogeneic transplant. Such a patient is NOT eligible to receive busulfan 
conditioning. Examples of “therapy-resistant infections that significantly increase the risk of allogeneic 
transplant” include but are not limited to:  
 
a) interstitial pneumonia due to adenovirus or parainfluenzae virus 3 
b) protracted diarrhea requiring total parenteral nutrition 
c) disseminated BCG infection  
d) virus-induced lymphoproliferative disease 
e) any active opportunistic infection (eg, due to Pneumocystis jiroveci, cytomegalovirus, 
                  cryptosporidium) that does not improve on medical management 
f) active and progressive pulmonary disease requiring mechanic ventilation. 
 
Inclusion of patients with disease-related organ dysfunction is justified by the known poor outcome for 
patients with active infection undergoing standard treatment and the potential life-saving nature of the 

36 
Version 12.0 
January 4, 2022 
treatment proposed. Patients who are on high-dose steroids or other immunosuppressive agents will 
also be considered eligible, because use of these drugs is common in patients with SCID and maternal 
T cell engraftment or who present with severe interstitial lung disease. Use of immunosuppressive 
drugs does not affect efficacy of hematopoietic cell transplantation, and therefore should not affect 
efficacy of gene transfer.  
 
Exclusion criteria   
1. No available molecular diagnosis confirming SCID-X1. 
2. Patients who have an available HLA-identical related donor. 
3. Diagnosis of active malignant disease other than EBV-associated lymphoproliferative disease 
4. Patients with evidence of infection with HIV-1 
5. Major (life-threatening) congenital anomalies. Examples of “major (life-threatening) congenital 
anomalies” include, but are not limited to: unrepaired cyanotic heart disease, hypoplastic lungs, 
anencephaly or other major CNS malformations, other severe non-repairable malformations of the 
gastrointestinal or genitourinary tracts that significantly impair organ function. 
6. Other conditions which in the opinion of the P.I. or co-investigators, contra-indicate collection and/or 
infusion of transduced cells or indicate patient’s inability to follow the protocol. These may include 
for example clinical ineligibility to receive anesthesia, severe deterioriation of clinical condition of the 
patient after collection of bone marrow but before infusion of transduced cells, or documented 
refusal or inability of the family to return for scheduled visits. There may be other unforeseen rare 
circumstances that would result in exclusion of the patient, such as sudden loss of legal 
guardianship. 
 
Although the presentation of the disease may be variable in type, the severity of the immunodeficiency 
is uniform. The gene transfer protocol will be instituted in the place of haploidentical transplant for those 
patients who do not have a matched family donor or in whom an unrelated donor transplant is not 
indicated for the reasons specified above.  Apart from the gene transfer protocol, the patients will not 
undergo additional procedures that would not form part of an equivalent haploidentical transplantation 
regimen.  
 
8.2 Enrollment of subjects 
Patients will be enrolled following diagnosis and referral to the Immunology/BMT Services at each 
institution. SCID-X1 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. Informed 
consent will be obtained from parents/guardians.  
 
The Principal Investigator at each site will discuss the study at length with the parent/guardian of a 
potential new subject, and provide as much time as possible for their review and consideration. The 
parent/guardian will be encouraged to ask further questions about the study to the Investigator or 
designees.  Consent procedures at each site will be in accordance with the site’s IRB requirements for 
obtaining informed consent, and reviewed by the sponsor prior to site initiation. 
 
After consent is obtained, the eligibility of each patient will be discussed by the site PIs, the study 
sponsor/grant PI, and grant co-PI by conference call. Study procedures will only commence if all PIs 
agree that the patient is eligible. 
 
Because of the life-threatening nature of the underlying disease, and the potential life-saving benefits of 
the procedure proposed, no staggering of patients is considered necessary. However, because of the 
rarity of the disorder and the planned recruitment of 3 patients per U.S. site in 5 years, we anticipate 
simultaneous enrollment of two or more patients to be very unlikely. 
 
 
 

37 
Version 12.0 
January 4, 2022 
9. Treatment protocol  
 
9.1 Harvest of CD34+ cells  
9.1.1 Bone marrow harvest or mobilized peripheral blood leukapheresis  
For patients weighing less than 10 kg, bone marrow harvest will generally be used as the source of 
CD34+ cells. For patients weighing 10 kg or more, mobilized peripheral blood cells will generally be 
used as the source of CD34+ cells. In situations where one method fails or is not preferred, the other 
method will be considered as an alternative. Cryopreserved umbilical cord blood cells may also be used 
if available.  
 
Bone marrow will be harvested from the patient under general anesthesia from the posterior iliac crests 
on both sides by multiple punctures. If necessary the anterior iliac crests may also be used. The 
amount of marrow collected will be up to 20 ml/kg of body weight.  
 
For mobilized peripheral blood cell collection, filgrastim will be administered by subcutaneous injection 
for 4 days at 10 micrograms per kilogram of body weight per day. The morning after the 4th dose, 
leukapheresis will be performed according to standard clinical procedures. Plerixafor (Mobozil), 
administered by subcutaneous injection at 0.24 milligrams per kilogram of body weight 10 hours prior to 
the day of collection, can be given daily for up to 4 days in consultation with the clinical apheresis team. 
The number of CD34+ cells from 2 consecutive days of apheresis may be added together for 
consideration. 
9.1.2 Back-up product collection in patients receiving busulfan 
A minimum of bone marrow or mobilized peripheral blood cells containing 2 x 106 CD34+ cells/kg and 
optimally 4 x 106 CD34+ cells/kg will be cryopreserved unmanipulated, according to standard 
institutional procedure, for infusion in situations described in Sectios 9.3.4 and 10.3. 
 
9.2 Low dose busulfan conditioning for selected subjects 
Patients who meet inclusion criteria 1, 2, and 3a will receive low dose busulfan conditioning on days -3 
and -2 with an optional dose on day -1. Conditioning may also be given starting day -4 to adjust for 
individual institutional or patient circumstances, and doses may be administered on non-consecutive days 
(e.g., -4, -2). Busulfan will be administered only after verifying an adequate estimated CD34+ cell dose of 
at least 4 x 106 CD34+ cells for transduction (pre-manipulation). The starting dose of busulfan will be 3 
mg/kg (see Appendix 5). Busulfan will be infused over 3 hours, and levels will be measured according to 
institutional norms starting at the end of infusion until 6-8 hours post infusion to calculate the area under 
the curve (AUC). The AUC will be calculated after each dose, and the dose will be adapted to reach a 
cumulative exposure of 30 mg*h/L. AUC exposure that falls +/- 5 mg*h/L will be accepted. If the total 
exposure falls short of the target after 2 doses, a 3rd dose may be given to achieve the goal. For further 
details see Appendix C. If the total exposure is achieved, subsequent doses should be omitted. At least 24 
hours must elapse between the end of infusion of the last dose of busulfan and infusion of transduced 
cells.  
 
Prophylaxis for seizures with levetiracetam or lorazepam should be given per institutional standards. 
 
9.3 CD34+ cell culture, transduction and infusion  
9.3.1 CD34+ cell culture and transduction 
CD34+ cells will be purified, cultured and transduced in a dedicated cell manipulation facility at each 
institution. CD34+ cells will be purified using standard protocols for stem cell transplantation 
(CliniMACs, see 9.1.2). Purified CD34+ cells will be seeded into gas permeable flexible plastic 

38 
Version 12.0 
January 4, 2022 
containers in serum free medium, and cytokines; IL-3 (20ng/ml), SCF (300ng/ml), FL (300ng/ml), TPO 
(100ng/ml).   After pre-stimulation and 2 repeats of bag transduction in serum free medium using vector 
preloaded on clinical grade RetroNectin (Takara Bio) and treated with Pulmozyme (Genentech) cells 
will be formulated and samples collected as described in section 9.2.2.  
The transduced cells that meet release criteria will be transported immediately to the patient for re-
infusion.  Infusion will occur less than or up to 4 days following initial vector exposure as described in 
section 9.3.  
9.3.2 Testing of product prior to patient re-infusion  
Samples are collected at approximately 0, 40 hours and at the end of incubation for cell viability (trypan 
blue stain, PI-annexin) and at 0 hr  and end of incubation for proliferation and sterility (for bacteria, 
endotoxin and mycoplasma). Samples are taken at 0, 40 hours and at the end of incubation for 
immunophenotyping (CD45+ & CD34+), in addition to samples taken at 0 hours and the end of 
incubation to monitor the gene transduction procedure (PCR for transgene, flow cytometric 
determination of γc expression). Similarly, at completion of the transduction process, a sample will be 
analyzed for Gram stain (the result of which will be known prior to re-infusion) and endotoxin 
measurement. The endotoxin result in EU/kg/hour will be calculated based on the final infusion volume 
and patient weight (EU per ml x infusion volume / patient weight), this will be available prior to infusion.  
A further minimum of 5% of the used culture medium, and 1% of the total cell number (or 108 cells, 
whichever is less) will also be archived to test for replication-competent retrovirus (RCR). The result of 
this investigation will not be known prior to reinfusion, although the viral stocks will have been 
previously tested, and the chances that RCR will be generated during the transduction process are 
extremely small. Some cells will also be frozen for later analysis if required. A minimum of >1 x 106/kg 
CD34+ cells after transduction are required for infusion into the patient. Transduced cells must be 
endotoxin negative and Gram stain negative for release.  
 
Failure to meet release criteria 
 
If transduced cells fail to meet release criteria for viability, sterility or endotoxin testing, they will not be 
infused. Therapeutic options, including a second attempt to perform gene transfer or allogeneic 
hematopoietic stem cell transplantation, will be discussed with the subject or legal guardian. If consent 
is given to perform a second attempt of gene transfer, the bone marrow harvest will be performed not 
earlier than 4 and not later than 8 weeks after the first harvest. If the product from the second harvest 
fails to meet release criteria, the subject will come off study and proceed to alternative treatment, i.e. 
allogeneic hematopoietic stem cell transplantation. 
 
Pre-infusion Sample Archive and testing. 
 
A pre infusion sample of recipient blood will be sent to for mononuclear cell preparation to be 
cryopreserved and stored within a designated repository.  
 
Pre-infusion RCR Screening: 
 
Pre-infusion samples of the transduced product and patient peripheral blood will be archived.  to test 
for replication competent retrovirus and  S+L- assays.     
 
Post-Infusion Patient Positive PCR Screening Results 
 
PCR for GALV-env (Gibbon Ape Leukemia Virus envelope) will be performed on patient peripheral 
blood samples. In the event of a positive GALV-env PCR testing on patient peripheral blood samples, a 
repeat PCR and S+L- assays will be performed and the archived sample of pre-infusion peripheral 

39 
Version 12.0 
January 4, 2022 
blood will be thawed, tested by PCR and S+L-assays.   
A subject with a persistently positive PCR assay but negative S+L- assay may continue on the study 
but only after consultation with the DSMB and the monitoring plan post infusion will require RCR 
monitoring by S+L- assay. 
9.3.3 Infusion of transduced cells  
Cells will be washed and infused in a volume of 20-50 mls intravenously over 15-30 minutes on day 0. 
Routine observations will be performed throughout the infusion period, and hourly thereafter for 6 hours. 
9.3.4 Criteria for co-infusion of back-up product in patients receiving busulfan  
CD34+ cells ≥1 x 106/kg after transduction is required to infuse transduced cells. If CD34+ cell count 
after transduction is <1 x 106/kg, transduced cells will not infused and back-up product will be thawed 
and infused. If CD34+ cell count after transduction is ≥1 x 106/kg but <2 x 106/kg, transduced cells will 
be co-infused with back-up product. If CD34+ cell count after transduction is ≥2 x 106/kg, transduced 
cells will be infused alone. 
 
 
10.  Evaluation and Follow-up  
 
10.1 Assessment of safety and efficacy (Appendix 4) 
10.1.1 Immunological reconstitution 
Detailed analysis of immune recovery following gene transfer for primary immune deficiency (PID) 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) analyze thymic 
education and output following gene modification of lymphocyte precursors and 3) measure cellular 
antigen specific responses: 
 
- The lymphocyte subsets (LSS) 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 CD4,CD8, TCRαβ, TCRγδ, CD16+CD56+ 
NK & γc surface expression. CD45RA, CD45RO, and CD31 expression is used to monitor naive 
and activated/memory T cells. TCR excision circles (TRECs) may be enumerated as a surrogate 
marker for new thymic emigrants following gene transfer. 
- Whole blood lymphocyte proliferation assays will be carried out to test function of T cells. 
- Representation of TCR families by flow cytometric analysis (Vβ phenotyping), combined with 
CDR3 PCR spectratyping (Vβ spectratyping) 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 vaccinations and 
natural infections (such as varicella) will be assessed. 
 
For the purpose of assessing success of gene transfer, we define successful reconstitution as a 
peripheral blood CD3 count of >300/μl and in vitro PHA stimulation index of >15 at 6 months post-
infusion of gene modified cells.  
 
Failure will be defined as patients who do not meet these reconstitution criteria at 6 months, OR are 
described as an early failure by 10.2.3. 
 
Weight and linear growth will be followed and recorded during each evaluation.  

40 
Version 12.0 
January 4, 2022 
10.1.2 Molecular characterization of gene transfer 
Molecular characterization of gene transfer in patient cells is also an important parameter for 
assessment of efficiency, and potentially for assessment of safety: 
 
-Percentage of vector positive T cells 
 
- Quantification of transgene copy numbers is determined on sorted cell populations by real-time 
PCR methodology. Detailed integration analysis maybe used to investigate specific clonal 
expansions. 
 
For the purposes of this protocol, subjects who have lack of gene marking (<0.1% of vector positive 
T cells detected by quantitative PCR i.e. <0.001 copies per cell) on two consecutive determinations 
will be considered to have failure of gene transfer. If insufficient T cells are available for gene 
marking, peripheral blood mononuclear cells will be tested instead by the same criteria. Such 
subjects will generally be taken off study (see section 10.3.1). If the subject is eligible for and 
proceeds to a 2nd gene transfer procedure (see Section 10.2.4), he will remain on study. 
 
Peripheral Blood Clonal Analysis 
 
Peripheral blood with detectable proviral sequences of > 0.01 copies per cell by 6 months post infusion 
of gene modified cells will be further analyzed for clonality by LAM PCR. A copy number of 0.01 copies 
per cell is presumed equal to 1% of circulating white blood cells (WBC's) containing the proviral 
sequence, based on a copy number of one proviral insertion site per transduced cell. Clonal analysis 
will be monitored at a minimum of every 6 months for the first 5 years and then yearly for up to 15 years 
post infusion if > 1% of the WBC's shows the presence of the corrective gene. Peripheral blood with < 
1% of the WBC's showing detectable proviral sequences during the first year post infusion will be 
analyzed for clonality by LAM-PCR at 6 months and 1 year. Peripheral blood with no detectable proviral 
sequences will not undergo clonal analysis.  
 
Clinical laboratory abnormalities may trigger further clinical work-up and insertional analysis, and 
conversely, insertional analysis suspicious for clonal expansion may trigger further clinical work-up. 
Laboratory abnormalities that may trigger clinical evaluation will include WBC >30,000/microliter, 
absolute lymphocyte count >10,000/microliter, or expansion of Vβ or Vδ TCR families suggesting 
proliferation of a homogenous population on two consecutive evaluations that are 1 week apart. If 
clonal analysis of peripheral blood with gene marking in > 1% of the WBC's reveals that greater than 
1/5th (>20%) of the gene modified population is derived from a single clone in conjunction with 
leukocytosis, the site investigator, Sponsor and Project Manager will be notified. This will also trigger 
additional clinical and laboratory investigation.  
 
Work-up will include detailed physical examination, complete blood count and differential, lymphocyte 
subsets, infectious studies of blood, stool, sputum, urine and/or tissue cultures, and imaging studies if 
deemed appropriate. If an infectious cause is found or is considered likely, monitoring of clinical lab 
abnormalities at regular intervals is recommended, every 1-4 weeks at the discretion of the site 
investigator, with regular updates to the Sponsor and Project Manager. If imaging studies are 
suggestive of a possible proliferative disorder, biopsies and appropriate infectious, pathological and 
molecular tests will be performed. If laboratory abnormalities resolve or improve significantly, the 
patient will be monitored at the next scheduled study point.  
 
If more than 20% of the gene modified population is derived from a single clone, and yet no other 
clinical or laboratory abnormalities are identified, close clinical monitoring (with visits at least every 2 
weeks) and careful repeat of peripheral blood clonal analysis (at least once a month) will be performed 
to document possible further expansion of the clonal population.  

41 
Version 12.0 
January 4, 2022 
 
If laboratory or pathology studies are suggestive of hematologic malignancy, a bone marrow aspirate 
will be performed to assess the possibility of myelodysplasia or leukemia, including morphology and 
cytogenetics and other clinically indicated molecular diagnostics. Insertion site analysis and 
immunophenotypic analysis will be performed on both blood and bone marrow. If a subject undergoes 
this evaluation, the DSMB, NIH and FDA will be informed for consideration of a temporary suspension 
of inclusion of new subjects in the study until diagnostic evaluation is complete. 
 
If more than 20% of the gene modified population is confirmed to be derived from a single clone for 3 
consecutive times, in the absence of clinical or laboratory signs indicative of infections or autoimmunity,  
a bone marrow aspirate and appropriate morphology, cytogenetics and molecular tests will be 
performed as indicated above. Insertion site analysis and immunophenotypic analysis will be performed 
on both blood and bone marrow. If a subject undergoes this evaluation, the DSMB will be informed. 
 
If there is no evidence of myelodysplasia, leukemia or any other tumors that might be caused by clonal 
expansion of gene modified cells, proviral copy number, clonal analysis, immunophenotyping, and TCR 
repertoire from peripheral blood will be performed a minimum of every 3 months as long as a 
predominant clone is detected and > 1% gene marking of circulating WBC's persists. If the predominant 
clone falls consistently below 20% of the gene modified population, the patient will be monitored at the 
next scheduled time point. If the predominant clone is expanding further or if other lab abnormalities are 
detected, bone marrow aspirate will be repeated.  
 
If the copy number decreases to < 1% of the circulating WBC's, the frequency of monitoring after the 
first year will be decreased to every 6 months for up to 5 years post infusion. If proviral sequences 
become undetectable, clonal analysis will not be performed. Proviral copy number will continue to be 
evaluated on a yearly basis for up to 15 years post infusion. Insertion site specific PCR may be utilized 
if available for early screening for re-emergence of specific clones. 
10.1.3 Safety assessments  
Efficacy and safety of the gene transfer procedure will be further assessed though clinical 
examinations, clinical laboratory assessments. Adverse reactions observed by the investigator or 
reported by the patient/parent/guardian during the study period will be recorded for evaluation (see 
section 11.0 Recording and Reporting of Adverse Events). Patient samples will be analysed for 
replication competent retrovirus (RCR), an essential safety test to detect the potentially pathogenic 
wildtype strains of the virus at 3 months, 6 months, 12 months and then yearly for 15 years. Discarded 
samples at any time point when blood is scheduled to be drawn will be archived for further studies 
relevant to this protocol. 
 
10.1.4 Additional assessments in patients receiving busulfan 
Complete blood counts will be monitored per institutional norm, at minimum twice weekly from date of 
infusion until documentation of neutrophil recovery, defined as the first day of absolute neutrophil count 
>500/microliter for 3 consecutive days after neutrophil nadir. Need for platelet transfusions and packed 
red blood cell transfusions will be recorded. Monitoring for veno-occlusive disease will be performed per 
institutional norm, including weight, liver function tests and physical examination, at minimum twice 
weekly from date of infusion until 21 days after infusion. 
 
10.2 Investigations & Monitoring schedule  
 
Trial Coordination and Monitoring Structure   
 
The Clinical and Translational Investigator Program (CTIP), Quality Assurance for Clinical Trials Office 
(QACT, Dana-Farber Cancer Institute), Project Manager will provide important services necessary for 

42 
Version 12.0 
January 4, 2022 
this multi-institutional clinical trial.  CTIP will provide the methodological and technical support needed 
to ensure excellence and quality in the proposed studies. More specifically CTIP will support 1) 
development of study forms and training for data entry; 2) statistical analysis and reporting of results to 
promote the rapid dissemination of the findings; 3) preparation of study data and adverse events to 
provide to the DSMB. QACT will be responsible for coordination of enrollment, with documentation of 
agreement of enrollment by the PI’s of participating institutions and the study sponsor. The Project 
Manager will be responsible for 1) development and maintenance of manuals of operation; 2) study 
coordination and standard operating procedures and monitoring; 3) coordination of the DSMP across all 
institutions; 4) coordination and dissemination of adverse event reports including expedited reports C.R. 
Matthew Hodgson will be responsible for trial monitoring at Children’s Hospital Boston and coordination 
of monitoring of other participating sites. 
10.2.1 Pre-treatment Investigations  
For all investigations requiring phlebotomy (pre-treatment and post-treatment) associated with this 
protocol, the volume of blood draws will be limited to 5ml/kg at one time. If maximum volume is 
reached, not all studies may be possible at each time point and a prioritization of tests would be 
utilized. The priority for testing would be 1) safety endpoints (CBC, chemistries, RCR, integration sites; 
2) clinical immunology end-points, gene marking; and 3) more research related analysis such as TCR 
and TREC.  
 
Routine gene transfer pre-treatment investigations will be carried out according to Appendix 4.  
 
Pre-treatment monitoring investigations will be carried out within 3 months of the start of gene transfer 
treatment according to Appendix 4. In case a second bone marrow harvest is planned, results of pre-
treatment investigations will be considered valid if performed within 3 months of the date of the repeat 
harvest. 
10.2.2 Post-infusion monitoring  
Monitoring of patients will follow the patient monitoring protocol (see Appendix 4). 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.  
 
CBC and blood chemistries will be performed prior to infusion and then daily for three consecutive days to 
detect post-infusion acute adverse events. For patients with evidence of pre-infusion organ dysfunction, 
CBC and blood chemistry data will be made available to the DSMB to help discriminate between disease-
related and procedure-related adverse events. In case of serious acute adverse events that are possibly- 
or probably related to the procedure, enrollment of new patients will be temporarily suspended until full 
review by the DSMB and the FDA. 
 
Samples for RCR analysis after the first year will be archived for future testing should it be required (See 
section 10.3.5). Serum samples and cells stored in liquid nitrogen will be kept indefinitely for future 
analysis should it be required.  
 
Bone marrow samples will be obtained if required as part of the clinical care of an unwell patient or in a 
situation where the patient is being administered a general anesthetic (a bone marrow sample would then 
be requested at the same time) or if clonal hematopoiesis is documented by LAM-PCR analysis.  Routine 
morphological and cytogenetic analysis, analysis of transgene copies in CD34+ cell populations, and also 
if sufficient cells are available progenitor cell (CFU), and xenograft repopulating cell assays (SRC) will be 
carried out on the bone marrow. Common integration sequences will be sought between myeloid cells, 
CFU and T cell populations.  

43 
Version 12.0 
January 4, 2022 
10.2.3 Early failure of immune reconstitution  
 
Early failure of immune reconstitution is determined by:  
 
1) lack of an increase in autologous T cell counts over baseline in at least two of three 
assessments at days 60, 90, and 120 
 
 
AND 
 
2.) lack of gene marking 
 
Patients who are deemed to be an early failure have the option of retreatment as described in 10.2.4 or  
of referral for allogeneic hematopoietic stem cell transplantation. 
 
Patients that undergo a allogeneic transplant will continue on with the monitoring protocol and data 
collection according to standard post BMT procedures. Subjects that give consent for retreatment with 
gene transfer will be monitored on study according to the monitoring protocol and data collection 
according to this protocol, however will be considered a failure under the criteria outlined in 10.1.1 with 
regards to analysis of the primary endpoint. 
 
10.2.4 Retreatment Option for failure of immune reconstitution 
Patients who are deemed to be a failure of immune reconstitution as detailed in Protocol section 10.1.1 
or 10.2.3, will be offered the option of a repeat gene transfer procedure if the following criteria are met: 
 
1.) Subject continues to meet the original protocol eligibility criteria per 8.1 including do not 
have a readily available HLA identical (A,B,C,DR,DQ) unrelated donor. Lack of a readily 
available HLA identical unrelated donor at the time that consent is obtained for the repeat 
procedure will be sufficient to meet this criterion. 
2.) Agreement and consensus of all study PIs of the eligiblitiy of the patient for a 2nd gene 
transfer procedure.  
 
The repeat gene transfer procedure will  follow the study procedures outlined in Section 9 – Treatment 
Procedures and Section 10 – Evaluation and Follow – up for the 1st Gene transfer procedures.  For 
purposes of study evaluation, patients would follow the evaluation and follow – up procedures with Day 
0 being the day of the 2nd Gene transfer infusion.  
 
10.3 Procedure for failure of hematologic reconstitution in patients receiving busulfan 
 
If absolute neutrophil count >500/microliter for 3 consecutive days after neutrophil nadir is not achieved 
by day +35, clinical evaluation which may include bone marrow aspirate and peripheral blood analysis 
for gene marking will be performed in consultation with the protocol PI/co-I. Persistent failure of 
hematopoietic recovery will be treated with infusion of thawed back-up cells and/or by proceeding 
immediately to allogeneic transplantation with the most suitable donor available. 
 
10.3 Withdrawal of individual subjects 
A patient can withdraw or be withdrawn from protocol treatment in the study at any time from enrollment 
until the transduced CD34+ cells have been administered. Once a patient has been administered the 
transduced CD34+ cells study treatment is complete.  
 
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.  
 

44 
Version 12.0 
January 4, 2022 
Patients who withdraw from the study after administration of the transduced CD34+ cells will be 
encouraged to have follow-up investigations so that the consequences of the administration can be 
documented and analyzed.  
 
Any patient withdrawn prior to administration of transduced CD34+ cells will be replaced in the study.  
10.3.1 Off study criteria 
 
A patient will be considered off study under the following circumstances:  
 
a) 
The patient is withdrawn from the study prior to administration of transduced CD34+ cells. 
 
b) 
The patients/parent/guardian withdraws consent for study procedures and data collection. 
 
c) 
The patient is lost to follow-up. 
 
d) 
The patient who has failure of gene transfer (defined as <0.1% of vector positive T cells or 
peripheral blood mononuclear cells on two consecutive determinations, see 10.1.2). 
 
e) 
Completion of protocol specified therapy and observation. 
 
Study procedures and assessments will not be performed once the patient is taken off study. 
Patient who meet the definition of early failure of immune reconstitution at day 120, however, will have 
assessment of gene marking performed according to the study schedule until the definition of failure of 
gene transfer above has been met. 
10.3.2 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). 
 
10.4 Premature termination of the study 
Enrollment of the study will be suspended under the following circumstances: 
a) 
Expiration or exhaustion of vector stock may prevent further recruitment of patients. 
b) 
Any leukemia potentially related to provirus insertion or treatment-related death. Planned 
infusion of a gene manipulated product in any subject already enrolled will be suspended; those 
who have already received a gene modified product will continue to be evaluated per protocol.  
The events will be reviewed as quickly as possible by the DSMB, IRB and FDA if one of these 
serious adverse events is observed. 
c) 
Overall stopping of trial would be made in consultation with the DSMB due to other serious and 
related adverse events occurring on trial. We will notify the FDA and each site Institutional 
Review Board if the study stopping rules are triggered.  
d) 
Lack of efficacy (see 10.5.1). 
 
10.5 Statistical considerations  
A maximum of 9 patients on this trial and an additional 11 patients in independent trials in Europe (total 
20) will undergo gene transfer with this vector. A formal stopping rule for lack of efficacy will be applied 
to all 20 patients (see below), and additional data analyses will be mostly descriptive in nature.  
 

45 
Version 12.0 
January 4, 2022 
“Success” in each individual patient will be determined by evidence of immune reconstitution defined by 
a peripheral blood CD3 lymphocyte count >300/μl and a PHA Stimulation Index >15 by 6 months post-
infusion of gene modified cells.  
 
“Failure” will be defined as patients who do not meet these reconstitution criteria at 6 months, OR are 
described as an early failure by 10.2.3. 
 
Evaluability 
All patients who receive an infusion of the transduced CD34+ cells are evaluable for inclusion in the 
stopping rule analysis.  Patients for whom the bone marrow harvest is unsatisfactory after a maximum 
of two attempts will go off study, and will be inevaluable for (excluded from) the stopping rule analysis. 
See Figure 10.5. 
 
Figure 10.5  Application of protocol definitions of evaluability, successes, failure, and off study 
 
10.5.1 Stopping rule for lack of efficacy 
This rule serves dual purposes of monitoring for safety and the determination of efficacy (assessment 
of primary objective 3, section 7.2). We designed a rule-based inferential procedure for this trial with the 
guidance of East clinical trial software (version 5, Cytel Inc., Cambridge, MA).  The null hypothesis (H0) 
is that the probability of success is at least P=0.65, a benchmark based on the historical success of 
haploidentical transplantation (37, 39, 99).  Our design specifications included a maximum of 20 
evaluable patients; decision points after 5, 10, and 20 evaluable patients; and a Type I error-spending 
budget proportional to the cumulative number of enrolled evaluable patients.  We rounded the 
asymptotic software-generated stopping criteria to the next lower integral number of failures and 
obtained the following rule for the 3 decision points: 
 
• 
If 4 of the first 5 evaluable patients fail immune reconstitution, then stop the trial and 
reject H0; otherwise continue. 
 
• 
If 7 of the first 10 evaluable patients fail, then stop the trial and reject H0; otherwise 
continue. 

46 
Version 12.0 
January 4, 2022 
 
• 
If 11 of the 20 evaluable patients fail, then reject H0.  Otherwise ‘accept’ H0, which is to 
say, the results are consistent with H0. 
 
Each of the above criteria is to be interpreted as specifying that the trial will stop as soon as the 
criterion is reached; e.g., if the first 4 evaluable patients are failures, then trial stops immediately without 
going on to a fifth patient.  Similarly, up to the point where 10 evaluable patients are enrolled and 
evaluated, the trial halts as soon as the seventh failure occurs.  Finally, beyond the tenth patient, the 
trial halts as soon as 11 failures occur, only proceeding through to the full sample of 20 if the number of 
failures has never exceeded 10. If there are 12 or more patients who are a success, then we will 
conclude that there is sufficient evidence of benefit from gene transfer therapy. We will provide a 
descriptive analysis of the data if fewer than 20 evaluable patients are enrolled and the stopping rule 
was not triggered, including placement of a 95% exact confidence interval on the success rate.  
 
 
We used SAS software (version 9, SAS Institute, Cary, NC) to simulate the performance of this rule 
under a range of assumed values for P.  We conducted enough repetitions to determine the likelihood 
of rejecting or ‘accepting’ H0 within 0.001.  Results are shown in the accompanying table. 
 
P (true 
success rate) 
Power (probability of 
rejecting H0: P ≥ 0.65) 
Expected 
evaluable 
patients 
0.75 
0.029 
20 
0.65 
0.099 
19 
0.60 
0.186 
18 
0.50 
0.474 
16 
0.40 
0.789 
13 
0.30 
0.960 
9 
 
These figures indicate that our stopping rule is a conservative strategy, in that ‘acceptance’ of H0 is 
virtually guaranteed only if the true rate somewhat higher than the hypothetical minimum for success 
(P=0.65).  For example (first line), if the true rate is P=0.75 then the trial has a 98% chance of running 
through the full 20 evaluable patients and H0 being ‘accepted.’  If the true rate is exactly P=0.65, the 
lower boundary of hypothesized success (second line), then the trial has only 90% probability of 
completing 20 evaluable patients and ‘accepting’ H0; a 10% chance remains that the trial will be 
stopped early and H0 rejected.  The expectation in this case, because of occasional early stopping, is 
an average of 19 rather than 20 evaluable patients. 
 
The table shows that smaller success rates carry higher probabilities of correctly rejecting H0, with 
smaller expected patient numbers due to early stopping.  At the extreme of the rates tabulated above, 
where the true value is only P=0.30 (bottom line), the trial has a 96% chance of stopping after an 
average of only 9 evaluable patients.  
 
The Type I error rate for this inferential procedure may be defined as the average power for all values 
of P greater than 0.65, weighted according to their a priori likelihood.  A conservative estimate is 
obtained by assuming equal likelihood for all values between 0.65 and 0.75.  We conducted simulations 
for this range, using close spacing (not shown) and obtained average power of 0.051.  Thus the chance 
of rejecting H0 under a conservative version of the null hypothesis, which is to say concluding that the 
success rate is lower than P=0.65 when in fact it is somewhere between 0.65 and 0.75, is 5.1%, 
virtually identical to the convention Type I error rate used in routine statistical inference. 

47 
Version 12.0 
January 4, 2022 
10.5.2 Data analysis and reporting 
 
Data to be reported, in addition to the outcome of the inferential procedure, will include detailed 
analysis of the immunological reconstitution of each patient, (detailed in Appendix 3), presence and 
copy number of the vector in specific myeloid and lymphoid cell populations with time after infusion of 
gene modified CD34+ cells, expression levels of the transgene by flow analysis in lymphoid cell 
populations and frequency of side effects.  Analysis of data for the subgroup of patients undergoing 
busulfan conditioning, beyond data contributing to the primary endpoint and safety stopping rules, will 
be performed separately and limited to descriptive graphical presentation. 
 
11. Safety issues related to gene transfer protocol 
 
11.1 Venipuncture 
Hazards associated with blood drawing are those associated with venipuncture 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 venipuncture are pain, the potential for formation of a hematoma at the site of the 
needle stick and a small potential for infection at the site of needle stick. Occasionally, individuals 
having venipuncture may faint as a result of a vasovagal reaction to the procedure. These risks will be 
minimized 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 venipuncture.  The major risk of repeated blood drawing is anemia from removal of red 
blood cells.  To minimize this risk the amount of blood being drawn for all purposes will be tracked to 
ensure that minimal volumes are collected, and to anticipate the need for blood transfusion.  
 
11.2 Bone marrow harvest 
Bone marrow will be harvested from the patient under general anesthesia from the posterior iliac crests on 
both sides by multiple punctures. The amount of marrow collected will be equivalent to 15% of total blood 
volume. The risks of the procedure include the standard risks of general anesthesia 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 anemia and the potential for infection at the 
puncture site. The anesthetic risks will be minimized by the anesthetic being administered by a pediatric 
anesthetist. The patient will be treated with appropriate pain medication after the procedure and will be 
monitored carefully to check hemodynamic status. The amount of marrow to be collected has been 
calculated such that the desired cell count will be obtained without the need for further transfusion. 
However, transfusion of red blood cells may be required. A CBC should be drawn no later than one day 
prior to marrow harvest. If anemia (Hb <9 g/dl) is observed prior to bone marrow harvest, the use of red 
blood cell transfusion will be recommended before the harvest is performed. At all other times, institutional 
guidelines should be followed to prevent and/or treat symptomatic anemia.  
 
Infection risk will be minimized by careful cleansing of the site prior to needle insertion and strict 
adherence to sterile technique during the procedure. A second bone marrow harvest may be performed if 
the product fails to meet release criteria. The second harvest would be performed no sooner than 4 weeks 
and no later than 8 weeks from the first harvest. The risks of the second harvest remain the same as for 
the first harvest. 
 
11.3 Retrovirus mediated gene transfer 
11.3.1 Insertional mutagenesis 
Insertional mutagenesis remains a finite risk of gene transfer using integrating vectors. However, 
modifications to the vector design including removal of LTR enhancer sequences, and utilization of a 
less potent promoter sequence for transgene expression, have been shown to significantly diminish 

48 
Version 12.0 
January 4, 2022 
mutagenic risk in experimental systems. It is therefore probable that the risk of clinically significant 
mutagenesis will be substantially reduced. 
11.3.2 Germline transmission of vector sequences 
Hematopoietic cells are manipulated and transduced ex vivo, and extensively washed prior to re-
infusion. In addition, retroviral particles are inactivated by human complement in vivo. Therefore the risk 
of gene transfer to other tissues, including gonads, is extremely small. 
11.3.3 Quality control of harvest and transduction process 
Manipulation of cells ex vivo is potentially associated with contamination. However, all manipulations 
will be undertaken in a dedicated cell manipulation laboratory at each site, following clinically 
acceptable guidelines and to GMP standards. To minimize contamination, all procedures are conducted 
within closed culture bag systems. In addition, cells will be tested for microbial contamination and 
endotoxin prior to re-infusion. A minimum of 1 x 106/kg CD34+ cells after transduction are required for 
infusion into the patient.  
11.3.4 Infusion of transduced cells 
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. The virus 
producer cell cultures must be maintained in medium with fetal calf serum and therefore the virus 
supernatant will contain animal serum. 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.  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 
stud 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 
microrganisms. One of the important safety features of this gene transfer protocol is that CD34+ cells will 
be cultured in a sealed bag culture system. Gram stains and routine cultures will be performed on cell 
samples the day before infusion and on the day of infusion. If significant growth of pathogenic 
microrganisms is detected, the cells will not be returned to the patient. Endotoxin levels will be measured 
prior to infusion and cells will not be infused if levels exceed 5 EU/kg per hour of infusion.  
 
Excess cells remaining after testing required for the protocol will be frozen in DMSO and archived at each 
participating site. Serum samples and cells stored in liquid nitrogen will be kept indefinitely for future 
analysis should it be required. 
 
11.4 Conditioning Regimen (Busulfan) 
Immediate side effects relating to the use of alkylating agent chemotherapy are mainly limited to the 
gastrointestinal tract with transient nausea and vomiting followed by stomatitis, esophagitis and diarrhea, 
which resolve with neutrophil recovery. All subjects will receive standard antimicrobial prophylaxis 
according to Children’s Hospital Boston Hematopoietic Stem Cell Transplantation Unit standard 
guidelines. With these measures, the immediate risk to the subject as a direct result of the conditioning 
and period of neutropenia will be no more than that associated with standard allogeneic HSCT. If there 
is a failure of bone marrow recovery after infusion of transduced cells, back up bone marrow harvested 
prior to therapy will be administered. The risk of secondary malignancy associated with the use of this 
regimen for HSCT in non-malignant disease is very low, however fertility may be compromised. As is 
standard prior to HSCT, sperm or testicular tissue preservation when appropriate and available will be 
offered prior to the procedure. 
 

49 
Version 12.0 
January 4, 2022 
 
12. Reporting Adverse Events 
 
Adverse Event Reporting  
 
A. Definitions of adverse events, serious events, and unexpected AEs 
 
For purposes of this study, an adverse event will be defined as any unfavorable and unintended 
diagnosis, symptom, sign (including any abnormal laboratory finding deemed by the PI to be clinically 
significant), syndrome or disease which either occurs during the study, having been absent at baseline, 
or, if present at baseline, appears to worsen. 
 
 
Unexpected adverse event will be defined as any adverse event that is not listed as a risk in the current 
version of the Protocol or Investigator Brochure. 
 
 
Adverse reaction will be defined as any adverse event caused by the Investigational Product. 
Suspected adverse reaction will be defined as any adverse event for which there is a reasonable 
possibility that the Investigational Product caused the event. 
 
 
Adverse Event recording and reporting begins once the subject starts the bone marrow harvest, which 
is the first protocol procedure.  
 
 
All adverse events will be graded by the site investigator according to the criteria specified by the 
National Cancer Institute’s (NCI) Common Toxicity Criteria (CTC) V 3.0.  
 
 
It is the responsibility of the treating physician to document all adverse experiences in the patient chart.  
At each assessment, adverse experiences will be evaluated and a detailed description of the event will 
be documented on an adverse event case report form that will include:  
 
• 
Description of event 
 
• 
Onset date/Start date 
 
• 
Resolution date/Stop date 
 
• 
Severity of event (Grade I-IV based on NCI common toxicity criteria) 
 
• 
Relationship to study: 
 
Unrelated 
 
Unlikely 
 
Possible  
 
Probable  
 
Definite 
 
• 
Classification of event: 
 
Expected 
 
Unexpected 
 
 
• 
Action taken: 
 
None 
 
 Medication Therapy  
 
Procedure 
 
Hospitalization  
 
Other 

50 
Version 12.0 
January 4, 2022 
 
• 
Patient outcome to date: 
 
Recovered 
 
Under Treatment/Observation 
 
Alive with sequelae 
 
Death 
 
Unknown 
 
All adverse events will be entered into the InForm database in the manner and timeframe specified in 
the Manual of Procedures. 
 
 
Serious Adverse Events: 
 
For purposes of this study, a serious adverse event will be defined as any untoward medical 
occurrence that: 
• 
Results in death: this is defined as death from any cause while the patient is on study.   
• 
Is life-threatening. This is defined as any Grade IV event that is possibly, probably or definitely 
related to the investigational agent, gene modified autologous CD34+ cells 
• 
Results in persistent or significant disability/incapacity 
• 
Is a new malignancy 
• 
Any clonal changes found on gene transfer safety testing analysis or routine bone marrow 
(defined as greater than 1/5 or 20% of clones derived from a single clone, as discussed in 
section 10.1.2) 
• Requires unanticipated in-patient hospitalization or prolongation of existing hospitalization 
• Leads to a congenital anomaly or birth defect 
• Is an event that required intervention to prevent permanent impairment or damage 
 
 
All serious adverse events that occur during the time the patient is on this study, whether or not related 
to the study, must be reported by the Principal Investigator to the Study Sponsor and  Project Manager 
within 24 business hours of knowledge of the occurrence.  
 
 
Investigator Reporting Requirements to Sponsor: 
 
 
 
Expedited Reporting: 
 
The following adverse events must be reported by the Principal Investigator to the Study Sponsor and 
Project Manager, within 24 business hours of knowledge of the occurrence. These events will be 
captured for up to 5 years post infusion. Sites will be provided with a Serious Adverse Event Form for 
expedited reporting purposes.  
 
• 
Any Adverse Event that meets the criteria for a Serious Adverse Event. 
• 
Grade IV events that are considered directly attributed to this study (related events: possible, 
probable or definite) and that are not listed as an expected event in the current version of the 
Investigator Brochure or Protocol. 
 
Adverse events: 
 
All adverse events that do not meet criteria for expedited reporting to the Sponsor will be reported at a 
minimum, monthly (first of the month) for the first year post infusion, on a quarterly basis (March 1st, 
June 1st, September 1st, and December 1st)  up to 5 years post infusion. After 5 years post infusion, 
grade 3 and higher adverse events that do not meet criteria for expedited reporting to the Sponsor will 
be reported at a minimum annually (March 1st) to 15 years post infusion.  Each site will be provided with 

51 
Version 12.0 
January 4, 2022 
an Adverse Event Log which should be reviewed, signed and dated by the Principal Investigator and 
submitted to the Study Sponsor and the Project Manager at the following email addresses noted below: 
 
 1. Sponsor: David Williams, MD 
Chief, Hematology/Oncology 
Children’s Hospital Boston 
Karp 8 
300 Longwood Ave.  
Boston, MA 02115 
Phone 617-919-2697 
FAX: 617-730-4697 
E-mail : DAWilliams@childrens.harvard.edu 
 
2. Project Manager:  
Colleen H. Dansereau,  MSN,RN, CPN 
Program Manager – Gene Therapy Program, Division of Hematology/Oncology 
Boston Children’s Hospital 
Boston, MA 02115 
Phone: 617-919-7008 
Email: colleen.dansereau@childrens.harvard.edu 
 
 
 
The CTIP will compile and report safety data information to the DSMB.  The Study Sponsor verifies 
grading and determines relatedness. The DSMB will verify these classifications at the next regularly 
scheduled meeting.  
 
 
Sponsor Reporting Requirements to Federal Regulatory Bodies 
 
Expedited reporting requirements: 
 
Serious adverse events, life threatening events or deaths are to be reported to the FDA and the Data 
Safety Monitoring Board by the Project Manager by telephone or fax as soon as possible but no later 
than 7 days after the IND’s sponsor’s receipt of the information. Report to the NIH/OBA (via GeMCRIS 
reporting mechanism) will occur concurrently.  
 
 
Reports of serious, unexpected adverse events associated with the use of the gene transfer agent, will 
be reported within 15 days after the sponsor’s receipt of information.  
 
 
A copy of expedited reports to the FDA will also be concurrently forwarded to all site PIs, IRBs and 
IBCs, and NIAID Medical Monitor. 
 
 
Non–expedited reporting requirements:  
 
Other adverse events occurring under this IND not meeting the criteria for expedited reporting will be 
documented during the course of the trial and will be included in the report to the FDA and OBA/RAC 
annually.   
 
 
Contact Information for Reporting Purposes: 
 
CBER, Office of Therapeutic Research and Review 
Food and Drug Administration 
Document Mail Center HFM-99, Room 200N 
1401 Rockville Pike 
Rockville, MD  201052 
301-1027-5101, phone 
301-1027-5397, fax 
 

52 
Version 12.0 
January 4, 2022 
Office of Biotechnology Activities                        
National Institutes of Health, 
6705 Rockledge Drive,  
Suite 750, MSC 7985                             
Bethesda, MD  20892-7985 
 (All non-USPS mail should use zip code 20817) 
 
301-496-9838, phone 
301-496-9839, fax 
oba@nih.gov 
 
Data Safety Monitoring Board (DSMB): 
 
The DSMB has been assembled, and has been approved by the National Institutes of Health (NIH), 
NIAID/DAIT. The Medical Monitor from NIAID/DAIT is invited to participate in the open sessions of the 
DSMB meetings. Arrangements will be made for DSMB members to meet with the Study Sponsor and 
other members of the study to review any interim SAEs and otherwise on no less than a biannual basis.   
The DSMB findings will be provided to the Sponsor, Dr. Williams. The Project Manager will ensure that 
the FDA, NIH/OBA, NIAID/DAIT Medical Monitor, and Site Investigators all receive copies of the report. 
 
European Data Consideration: 
 
Data from patients enrolled in the trials in Paris and London will be collected by the investigators at 
those sites. Serious adverse events occurring in Paris or London will be made available to the Sponsor 
(Dr. David Williams) for distribution to the DSMB, the FDA and NIH/OBA for evaluation and possible 
actions. While data collection forms will be harmonized for the purpose of data analysis and ultimate 
publication, United States sites will not monitor European sites with respect to data collection. 
NIAID/DAIT funds will be used solely for data collection at US sites, not at European sites.  
 

53 
Version 12.0 
January 4, 2022 
 
 
Table 2a: Years 0 – 5 Post Infusion (Subjects not receiving busulfan)~   
Relation to Gene Transfer 
 
Grade 1 
(Mild) 
Grade 2 
(Moderate) 
Grade 3 
(Severe) 
Grade 4 
(Life-threatening) 
Grade 5 
(Fatal) 
Unrelated 
Unlikely 
Required 
Report on the 1st of every month (-/+ 7 days) until 12 months 
post-infusion then quarterly* up to 5 years post infusion 
Required 
Reported to the 
Sponsor on the 1st of 
every month (-/+ 7 days) 
until 12 months post-
infusion then quarterly* 
up to 5 years post 
infusion 
Required 
Report within 24 
business hours to 
Sponsor 
Possible 
Probable 
Definite 
Required 
Report within 24 business hours to Sponsor 
Required 
Report within 24 
business hours to 
Sponsor 
Required 
Report within 24 
business hours to 
Sponsor 
Serious Adverse 
Event 
Required 
Report within 24 
business hours to 
Sponsor 
Required 
Report within 24 
business hours to 
Sponsor 
Required 
Report within 24 
business hours to 
Sponsor 
Required 
Report within 24 
business hours to 
Sponsor 
Required 
Report within 24 
business hours to 
Sponsor 
 
* Adverse events not meeting criteria for expedited reporting should be submitted to the sponsor on the first of the month in year 1, then on a 
quarterly basis year 2 -5 (March 1st, June 1st, September 1st, and December 1st).  
~ Events required to be reported within 24 hours to the Sponsor that meet criteria after Sponsor review will be reported as soon as possible 
but no later than 7 days after Sponsor receipt to DSMB, FDA and NIH/OBA. Concurrent copies of the FDA report will be reported to NIAID, 
to sites (PI, IRB, IBC). DSMB recommendations will be forwarded to sites (PI, IRB, IBC).  
 
 
 
 
 
 
 
 
 
 
 

54 
Version 12.0 
January 4, 2022 
Table 2b: Years 0-5 Post Infusion (Subjects receiving busulfan)~  
Relation to Gene Transfer 
 
Grade 1 
(Mild) 
Grade 2 
(Moderate) 
Grade 3 
(Severe) 
Grade 4 
(Life-threatening) 
Grade 5 
(Fatal) 
Unrelated 
Unlikely 
Not required to be collected 
Required – excluding 
abnormal hematologic 
laboratory results related to 
chemotherapy conditioning  
Reported to the Sponsor 
on the 1st of every month (-
/+ 7 days) until 12 months 
post-infusion then 
quarterly* up to 5 years 
post infusion 
Required - excluding 
abnormal hematologic 
laboratory results related 
to chemotherapy 
conditioning 
Report within 24 hours to 
Sponsor 
Required 
Report within 24 
hours to Sponsor 
Possible 
Probable 
Definite 
Required 
Report within 24 
business hours 
to Sponsor 
Required 
Report within 24 
business hours to 
Sponsor 
Required 
Report within 24 business 
hours to Sponsor 
Required 
Report within 24 business 
hours to Sponsor 
Required 
Report within 24 
business hours to 
Sponsor 
 
Serious 
Adverse 
Event 
Required 
Report within 24 
business hours 
to Sponsor 
Required 
Report within 24 
business hours to 
Sponsor 
Required 
Report within 24 business 
hours to Sponsor 
Required 
Report within 24 business 
hours to Sponsor 
Required 
Report within 24 
business hours to 
Sponsor 
 
* Adverse events not meeting criteria for expedited reporting should be submitted to the sponsor on the first of the month in year 1, then on a 
quarterly basis year 2 -5 (March 1st, June 1st, September 1st, and December 1st).  
~ Events required to be reported within 24 hours to the Sponsor that meet criteria after Sponsor review will be reported as soon as possible 
but no later than 7 days after Sponsor receipt to DSMB, FDA and NIH/OBA. Concurrent copies of the FDA report will be reported to NIAID, 
to sites (PI, IRB, IBC). DSMB recommendations will be forwarded to sites (PI, IRB, IBC).  
 
 
 
 
 
 
 
 
 
 

55 
Version 12.0 
January 4, 2022 
Table 2c: Years 6-15 Post Infusion (all subjects)~  
 
Relation to Gene 
Transfer 
 
Grade 1 
(Mild) 
Grade 2 
(Moderate) 
Grade 3 
(Severe) 
Grade 4 
(Life-threatening) 
Grade 5 
(Fatal) 
Unrelated 
Unlikely 
Not required to be collected 
Report annually 
Report annually 
Required 
Report within 24 
business hours to 
Sponsor 
Possible 
Probable 
Definite 
Required 
Report within 24 business hours to Sponsor 
Required 
Report within 24 
business hours to 
Sponsor 
Required 
Report within 24 
business hours to 
Sponsor 
Serious Adverse 
Event 
Required 
Report within 24 
business hours to 
Sponsor 
Required 
Report within 24 
business hours to 
Sponsor 
Required 
Report within 24 
business hours to 
Sponsor 
Required 
Report within 24 
business hours to 
Sponsor 
Required 
Report within 24 
business hours to 
Sponsor 
 
~ Events required to be reported within 24 hours to the Sponsor that meet criteria after Sponsor review will be reported as soon as possible 
but no later than 7 days after Sponsor receipt to DSMB, FDA and NIH/OBA. Concurrent copies of the FDA report will be reported to NIAID, 
to sites (PI, IRB, IBC). DSMB recommendations will be forwarded to sites (PI, IRB, IBC).  

56 
Version 12.0 
January 4, 2022 
13. Public Health Considerations 
Hematopoietic progenitors are transduced ex vivo in a closed culture system. The vector does not contain 
replication competent viruses, and will not be shed from transduced cells. The potential for transmission of 
vector sequences to other persons is therefore extremely small. 
 
14. Ethical considerations 
Good Clinical Practice 
The study will be conducted in accordance with the International Conference on Harmonization (ICH) 
for Good Clinical Practice (GCP) and the appropriate regulatory requirement(s).  Essential clinical 
documents will be maintained to demonstrate the validity of the study and the integrity of the data 
collected.  Master files will be established at the beginning of the study, maintained for the duration of 
the study and retained according to the appropriate regulations.   
 
Ethical Considerations 
The study will be conducted in accordance with ethical principles founded in the World Medical 
Association Declaration of Helsinki, Ethical Principles for Medical Research Involving Human Subjects 
(found at http://www.wma.net/e/).  The DSMB and local IRB will review all appropriate study 
documentation in order to safeguard the rights, safety and well-being of the patients. The protocol, 
Investigator’s Brochure(s), informed consent, written information given to the patients, safety updates, 
annual progress reports, and any revisions to these documents will be provided to the FDA by the 
Study Sponsor.  
 
Data Safety and Monitoring Plan (See also Manual of Procedures) 
Data and safety will be reviewed for each patient entered into this protocol by an independent Data 
Safety and Monitoring Board on at least a biannual basis. The Data Safety Monitoring Board is an 
independent group of experts who will advise the study investigators.  The members of the DSMB 
serve in an individual capacity and provide their expertise and recommendations.  The primary 
responsibilities of the DSMB are to 1) periodically review and evaluate the accumulated study data for 
participant safety, study conduct and progress, and when appropriate, efficacy and 2) make 
recommendations to the study investigators and regulatory agencies (IRB, IBC, FDA, etc.) concerning 
the continuation, modification, or termination of the trial. The review of data from each patient will occur 
by phone conference or in person  on at least a biannual basis for all patients enrolled in the study once 
a subject is enrolled, either in the companion European trial or in the United States. Any SAE will be 
reported to the DSMB as outlined above.     
 
Method to be Used in Procuring Consent of Subjects  
 
All prospective patients will have the study explained by the PI of the research team at each site.  The 
nature of the tests and procedures to be done will also be explained along with the potential hazards, 
possible adverse reactions and financial costs. The parent/guardian will be encouraged to ask further 
questions about the study to the Investigator or designee. Should a parent/guardian decide that the 
patient will participate they will be invited to sign the study consent form. Enrollment of each patient will 
be discussed with all site PIs, the study sponsor/grant PI, and grant co-PI after this consent is obtained; 
study sponsor will schedule the conference call to discuss eligibility within 48 hours of notification. 
 
Prior to the initiation of the study, defined as initiating any procedure for purposes of evaluating patient 
eligibility not otherwise a part of routine patient care, acknowledgement of the receipt of this information 
and the subject's freely tendered offer to participate will be obtained in writing from each subject in the 
study.  Those patients under the age of consent will voluntarily assent to the study under the same 
circumstances as their legal guardian and will sign the assent form.  
 

57 
Version 12.0 
January 4, 2022 
This protocol, informed consent, assent form, and any amendments to the protocol will be reviewed by 
the IRB prior to initiation.  The study will not be initiated without the approval of the IRB, whose 
operations must be in compliance with CFR 56; Title 21. 
 
Written notice that the protocol and informed consent/assent forms have been reviewed and approved 
by the IRB will be submitted to the Investigator and the Sponsor prior to study initiation. 
 
15. Administrative aspects  
 
15.1 Data Handling, Record Keeping, Sample Storage 
The Clinical and Translational Investigator Program (CTIP) will handle the data management for this 
trial.  All data will be collated, analyzed and stored by CTIP. CTIP will also analyze data shared from 
the European sites as specified including data for purpose of joint publication (Section 12, European 
data considerations). Data shared from European sites will be obtained according to local approved 
practices and transferred in a coded fashion. Sample storage will be accomplished at each site. 
Specimens will be held indefinitely at each institution. If for any reasons samples cannot be maintained 
at the local institution, they will be shipped to the Study Sponsor for continued storage.  
15.1.1 CRF completion 
Selected data will be recorded on study specific case report forms (CRF’s). Any paper CRFs will be 
completed in ink only, by personnel authorized to enter or change data on the CRF. Corrections can be 
made by striking out errors, with a single stroke, and not using correction fluid or obscuring the original 
entry. The correct entry must be entered by the side and initialled and dated by the person authorized 
to make the correction. Electronic CRFs will be completed in the InForm database. 
 
Clinical and Research laboratory results will be held electronically in the study files of each subject. The 
CRF will document when the patient samples have been taken and when tests have been carried out.  
15.1.2 Sample storage 
A record of retained body fluids / tissue samples will be completed every time a 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 gene transfer and at regular intervals post gene 
transfer, and kept indefinitely at each site.  Should it be required, samples will be disposed of in the 
appropriate manner according to local IRB guidelines, this will be detailed in the parent/guardian 
information sheet and consent obtained.  
15.1.3 Record retention 
Essential documents will be retained for a minimum of five years after completion of the trial. These 
documents will be retained for longer if required by the applicable regulatory requirements. 
 
15.2 Patient confidentiality 
In order to maintain patient privacy, all CRFs, IMP accountability records, study reports and 
communications will identify the patient by the assigned unique patient trial number.   
 
Direct access to the patient’s original medical records for verification of data gathered on the CRFs and 
to audit the data collection process will be permitted for trial-related monitoring & audits by the sponsor, 
IRB review, and regulatory inspection(s). 
 
15.3 Monitoring & Audit 
 

58 
Version 12.0 
January 4, 2022 
The trial will be monitored and audited according to the Standard Operating Procedures of the sponsor.  
 
15.4 Amendments to study documents  
Amendments are changes made to the research after approval by the IRB has been given.  
 
A ‘substantial amendment’ is defined as an amendment to the terms of the IRB or FDA application, or 
to the protocol or any other supporting documentation, that is likely to affect to a significant degree: 
 
- the safety or physical or mental integrity of the subjects of the trial; 
- the scientific value of the trial; 
- the conduct or management of the trial; or 
- the quality or safety of any intervention used in the trial. 
 
 All amendments, whether considered substantial or not, must be approved by the sites IRB prior to 
initiating the amended protocol and/or consent.  All amendments will be distributed to all sites for 
approval of local IRBs.  
 
15.5 Definition of end of study & end of study report 
The sponsor will notify the IRB and FDA 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 15 year follow-up of the last patient entered into the trial.  
 
In case the study is ended prematurely, the Study Sponsor will notify the IRB and FDA within 15 days, 
including the reasons for the premature termination. 
 
Within one year after the end of the study, the Study Sponsor will submit a final study report with the 
results of the study, including any publications/abstracts of the study to the FDA and IRBs.  
 
15.6 Insurance & indemnity 
Children’s Hospital Boston shall maintain the self-insurance program as approved by the Risk 
Management Foundation.  Children’s Hospital Boston is self-insured under CRICO, which has a rating 
equal to Best’s A-. 
 
15.7. Dissemination of results 
Results of this study will be disseminated by publication, oral presentation at scientific meetings, and by 
direct communication with regulatory agencies. 
 
16. Clinical facilities and arrangements 
Children’s Hospital Boston is a leading pediatric institution in the United States. CHB has an active 
Immunodeficiency Program within the Division of Immunology and an active Bone Marrow Transplant 
Program within the Division of Hematology/Oncology. Immunodeficiency patients are admitted to the 
Hematology/Oncology service at CHB and jointly managed by both services. Patients are cared for the 
Stem Cell Transplantation Unit that specializes in the care of immunocompromised patients.   
 
A GMP facility (managed by Dr. Jerome Ritz) is located in the Jimmy Fund Building of the Dana-Farber 
Cancer Institute and is connected to CHB and managed by the Joint Program in Transfusion Medicine, 
which supports CHB, Dana-Farber Cancer Institute and the Brigham and Women’s Hospital. The 
Connell and O'Reilly Families Cell Manipulation Core Facility (CMCF) at the DFCI is located in the 
Jimmy Fund Building-3 (JF-3).  Standard operating procedures (SOPs) for CMCF have been approved 
by the Laboratory Director and Assistant Medical Director and are reviewed at least annually. Specific 
procedures have been implemented to ensure the integrity of this product. The laboratory has adequate 
space for the orderly placement of equipment and materials within the facility and to ensure that only 

59 
Version 12.0 
January 4, 2022 
one product is processed in a given work space at a time.  The manufacturing facility is classified as an 
ISO 7 cleanroom. Appropriate process and environmental controls are in place to ensure the facility 
operated well within the guidelines for an ISO Class 7 clean room. The CMCF laboratory at Dana-
Farber is fully equipped for processing and cryopreservation of hematopoietic progenitor and other 
therapeutic cells as well as providing processing for other cellular products. The CMCF laboratory at 
the DFCI operates under the direction of Jerome Ritz, MD (Laboratory Director) and Grace Kao, MD 
(Assistant Medical Director).  The CMCF and their processes have been designed to meet the current 
good manufacturing practice (cGMP) and current Good Tissue Practice (cGTP) for hematopoietic cell 
and other cell processing as required by the Food and Drug Administration’s Center for Biologics 
Evaluation and Research (CBER), The Joint Commission and the Foundation for the Accreditation of 
Cell Therapy (FACT). 
 
17. Qualifications of responsible clinicians and investigators  
 
Dr. David Williams, Sponsor, is Chief of Hematology/Oncology, CHB and Professor of Pediatrics at 
Harvard Medical School and actively cares for children with non-malignant hematology diagnosis. He 
has served as PI, co-PI or sponsor of five human gene transfer trials in the past 15 years and has been 
active in gene transfer research since 1982. He currently serves as Editor-in-Chief of Molecular 
Therapy, the leading journal of gene transfer and serves on the NIH Recombinant DNA Advisory 
Committee.  
 
Dr. Sung-Yun Pai, Principal Investigator, is Associate Professor of Pediatrics, Harvard Medical School 
and a member of the Hematology/Oncology faculty at CHB and DFCI. Dr. Pai has extensive training in 
immunodeficiency, attends on the Bone Marrow Transplant service at CHB and coordinates the care of 
children with immunodeficiency with the Immunology and Hematology/Oncology programs.  
 
Dr. Wendy B. London, Co-Investigator, is the Director of Biostatistics and the Co-Director of the Clinical 
Translational Investigation Program.  Dr. London has extensive experience in the design, conduct, interim 
monitoring, and analysis of clinical trials.  In addition to being a faculty member in Hematology/Oncology at 
CHB and DFCI, she is a Senior Statistician in the Children’s Oncology Group (COG) which conducts biologic 
studies and Phase I, II, and III therapeutic trials for children with cancer. 
 
Dr. Jerome Ritz is Professor of Medicine, Harvard Medical School and Director of the Connell and 
O’Reilly Families Cell Manipulation Core, a GMP facility at the DFCI. Dr. Ritz has long-standing 
expertise in cell purification and cell manipulation and will oversee the purification and transduction of 
CD34+ cells.  
 
Ms. Colleen H. Dansereau, MSN, RN, CPN, has extensive experience in gene therapy clinical research 
trial coordination and clinical research related  activities. She is a board certified pediatric 
hematology/oncology registered nurse. She is responsible for the overall project management of this 
and other gene therapy trials at Boston Childrens Hospital. 
 
18. References cited 
 
1. 
Fischer A, Le Deist F, Hacein-Bey-Abina S, Andre-Schmutz I, Basile Gde S, de Villartay JP, 
Cavazzana-Calvo M. Severe combined immunodeficiency. A model disease for molecular immunology 
and therapy. Immunological reviews. 2005 Feb;203:98-109. 
2. 
Noguchi M, Nakamura Y, Russell SM, Ziegler SF, Tsang M, Cao X, Leonard WJ. Interleukin-2 
receptor ÀÀ chain: a functional component of the interleukin-7 receptor. Science (New York, NY. 
1993;262:1877-1880. 

60 
Version 12.0 
January 4, 2022 
3. 
Kovanen PE, Leonard WJ. Cytokines and immunodeficiency diseases: critical roles of the 
gamma(c)-dependent cytokines interleukins 2, 4, 7, 9, 15, and 21, and their signaling pathways. 
Immunological reviews. 2004 Dec;202:67-83. 
4. 
de Saint Basile G, Arveiler B, Oberle I, Malcolm S, Levinsky RJ, Lau YL, Hofker M, Debre M, 
Fischer A, Griscelli C, et al. Close linkage of the locus for X chromosome-linked severe combined 
immunodeficiency to polymorphic DNA markers in Xq11-q13. Proceedings of the National Academy of 
Sciences of the United States of America. 1987 Nov;84(21):7576-7579. 
5. 
Puck JM, Conley ME, Bailey LC. Refinement of linkage of human severe combined 
immunodeficiency (SCIDXI) to polymorphic markers in Xq13. Am J Hum Genet. 1993;53:176-184. 
6. 
Noguchi M, Yi H, Rosenblatt HM, Filipovich AH, Adelstein S, Modi WS, McBride OW, Leonard 
WJ. Interleukin-2 receptor ÀÀ chain mutation results in X-linked severe combined immunodeficiency in 
humans. Cell. 1993;73:147-157. 
7. 
Takeshita T, Asao H, Ohtani K, Ishii N, Kumaki S, Tanaka N, Munakata H, Nakamura M, 
Sugamura K. Cloning of the gamma chain of the human IL-2 receptor. Science (New York, NY. 1992 
Jul 17;257(5068):379-382. 
8. 
Orlic D, Girard LJ, Lee D, Anderson SM, Puck JM, Bodine DM. Interleukin-7R alpha mRNA 
expression increases as stem cells differentiate into T and B lymphocyte progenitors. Experimental 
hematology. 1997 Mar;25(3):217-222. 
9. 
Pepper AE, Buckley RH, Small TN, Puck JM. Two mutational hotspots in the interleukin-2 
receptor gamma chain gene causing human X-linked severe combined immunodeficiency. Am J Hum 
Genet. 1995 Sep;57(3):564-571. 
10. 
Puck JM, Pepper AE, Henthorn PS, Candotti F, Isakov J, Whitwam T, Conley ME, Fischer RE, 
Rosenblatt HM, Small TN, Buckley RH. Mutation analysis of IL2RG in human X-linked severe combined 
immunodeficiency. Blood. 1997 Mar 15;89(6):1968-1977. 
11. 
Peschon JJ, Morrissey PJ, Grabstein KH, Ramsdell FJ, Maraskovsky E, Gliniak BC, Park LS, 
Ziegler SF, Williams DE, Ware CB, Meyer JD, Davison BL. Early lymphocyte expansion is severely 
impaired in interleukin 7 receptor-deficient mice. The Journal of experimental medicine. 1994 Nov 
1;180(5):1955-1960. 
12. 
Suzuki H, Duncan GS, Takimoto H, Mak TW. Abnormal development of intestinal intraepithelial 
lymphocytes and peripheral natural killer cells in mice lacking the IL-2 receptor beta chain. The Journal 
of experimental medicine. 1997 Feb 3;185(3):499-505. 
13. 
Puel A, Ziegler SF, Buckley RH, Leonard WJ. Defective IL7R expression in T(-)B(+)NK(+) 
severe combined immunodeficiency. Nature genetics. 1998 Dec;20(4):394-397. 
14. 
Mrozek E, Anderson P, Caligiuri MA. Role of interleukin-15 in the development of human 
CD56+ natural killer cells from CD34+ hematopoietic progenitor cells. Blood. 1996 Apr 1;87(7):2632-
2640. 
15. 
Carson WE, Fehniger TA, Caligiuri MA. CD56bright natural killer cell subsets: characterization 
of distinct functional responses to interleukin-2 and the c-kit ligand. Eur J Immunol. 1997 
Feb;27(2):354-360. 
16. 
Lodolce JP, Boone DL, Chai S, Swain RE, Dassopoulos T, Trettin S, Ma A. IL-15 receptor 
maintains lymphoid homeostasis by supporting lymphocyte homing and proliferation. Immunity. 1998 
Nov;9(5):669-676. 
17. 
Kennedy MK, Glaccum M, Brown SN, Butz EA, Viney JL, Embers M, Matsuki N, Charrier K, 
Sedger L, Willis CR, Brasel K, Morrissey PJ, Stocking K, Schuh JC, Joyce S, Peschon JJ. Reversible 
defects in natural killer and memory CD8 T cell lineages in interleukin 15-deficient mice. The Journal of 
experimental medicine. 2000 Mar 6;191(5):771-780. 

61 
Version 12.0 
January 4, 2022 
18. 
Matthews DJ, Clark PA, Herbert J, Morgan G, Armitage RJ, Kinnon C, Minty A, Grabstein KH, 
Caput D, Ferrara P, et al. Function of the interleukin-2 (IL-2) receptor gamma-chain in biologic 
responses of X-linked severe combined immunodeficient B cells to IL-2, IL-4, IL-13, and IL-15. Blood. 
1995 Jan 1;85(1):38-42. 
19. 
Izuhara K, Heike T, Otsuka T, Yamaoka K, Mayumi M, Imamura T, Niho Y, Harada N. Signal 
transduction pathway of interleukin-4 and interleukin-13 in human B cells derived from X-linked severe 
combined immunodeficiency patients. J Biol Chem. 1996 Jan 12;271(2):619-622. 
20. 
Minegishi Y, Okawa H, Sugamura K, Yata J. Preferential utilization of the immature JH segment 
and absence of somatic mutation in the CDR3 junction of the Ig H chain gene in three X-linked severe 
combined immunodeficiency patients. Int Immunol. 1994 Nov;6(11):1709-1715. 
21. 
White H, Thrasher A, Veys P, Kinnon C, Gaspar HB. Intrinsic defects of B cell function in X-
linked severe combined immunodeficiency. Eur J Immunol. 2000 Mar;30(3):732-737. 
22. 
Ihle JN. Cytokine receptor signalling. Nature. 1995 Oct 19;377(6550):591-594. 
23. 
Taniguchi T, Miyazaki T, Minami Y, Kawahara A, Fujii H, Nakagawa Y, Hatakeyama M, Liu ZJ. 
IL-2 signaling involves recruitment and activation of multiple protein tyrosine kinases by the IL-2 
receptor. Ann N Y Acad Sci. 1995 Sep 7;766:235-244. 
24. 
Macchi P, Villa A, Giliani S, Sacco MG, Frattini A, Porta F, Ugazio AG, Johnston JA, Candotti F, 
O'Shea JJ, et al. Mutations of Jak-3 gene in patients with autosomal severe combined immune 
deficiency (SCID). Nature. 1995 Sep 7;377(6544):65-68. 
25. 
Russell SM, Tayebi N, Nakajima H, Riedy MC, Roberts JL, Aman MJ, Migone TS, Noguchi M, 
Markert ML, Buckley RH, O'Shea JJ, Leonard WJ. Mutation of Jak3 in a patient with SCID: essential 
role of Jak3 in lymphoid development. Science (New York, NY. 1995 Nov 3;270(5237):797-800. 
26. 
Leonard WJ, O'Shea JJ. Jaks and STATs: biological implications. Annual review of immunology. 
1998;16:293-322. 
27. 
Buckley RH, Schiff RI, Schiff SE, Markert ML, Williams LW, Harville TO, Roberts JL, Puck JM. 
Human severe combined immunodeficiency: genetic, phenotypic, and functional diversity in one 
hundred eight infants. The Journal of pediatrics. 1997 Mar;130(3):378-387. 
28. 
Jones AM, Clark PA, Katz F, Genet S, McMahon C, Alterman L, Cant A, Kinnon C. B-cell-
negative severe combined immunodeficiency associated with a common gamma chain mutation. 
Human genetics. 1997 May;99(5):677-680. 
29. 
DiSanto JP, Rieux-Laucat F, Dautry-Varsat A, Fischer A, de Saint Basile G. Defective human 
interleukin 2 receptor gamma chain in an atypical X chromosome-linked severe combined 
immunodeficiency with peripheral T cells. Proceedings of the National Academy of Sciences of the 
United States of America. 1994 Sep 27;91(20):9466-9470. 
30. 
Sharfe N, Shahar M, Roifman CM. An interleukin-2 receptor gamma chain mutation with normal 
thymus morphology. The Journal of clinical investigation. 1997 Dec 15;100(12):3036-3043. 
31. 
Ginn SL, Curtin JA, Kramer B, Smyth CM, Wong M, Kakakios A, McCowage GB, Watson D, 
Alexander SI, Latham M, Cunningham SC, Zheng M, Hobson L, Rowe PB, Fischer A, Cavazzana-
Calvo M, Hacein-Bey-Abina S, Alexander IE. Treatment of an infant with X-linked severe combined 
immunodeficiency (SCID-X1) by gene therapy in Australia. The Medical journal of Australia. 2005 May 
2;182(9):458-463. 
32. 
Stephan V, Wahn V, Le Deist F, Dirksen U, Broker B, Muller-Fleckenstein I, Horneff G, Schroten 
H, Fischer A, de Saint Basile G. Atypical X-linked severe combined immunodeficiency due to possible 
spontaneous reversion of the genetic defect in T cells. The New England journal of medicine. 1996 Nov 
21;335(21):1563-1567. 

62 
Version 12.0 
January 4, 2022 
33. 
Bousso P, Wahn V, Douagi I, Horneff G, Pannetier C, Le Deist F, Zepp F, Niehues T, Kourilsky 
P, Fischer A, de Saint Basile G. Diversity, functionality, and stability of the T cell repertoire derived in 
vivo from a single human T cell precursor. Proceedings of the National Academy of Sciences of the 
United States of America. 2000 Jan 4;97(1):274-278. 
34. 
Hendriks RW, Kraakman ME, Schuurman RK. Patterns of X chromosome inactivation in 
haematopoietic cells of female carriers of X linked severe combined immunodeficiency. 
Immunodeficiency. 1993;4(1-4):263-265. 
35. 
Candotti F, Oakes SA, Johnston JA, Giliani S, Schumacher RF, Mella P, Fiorini M, Ugazio AG, 
Badolato R, Notarangelo LD, Bozzi F, Macchi P, Strina D, Vezzoni P, Blaese RM, O'Shea JJ, Villa A. 
Structural and functional basis for JAK3-deficient severe combined immunodeficiency. Blood. 1997 Nov 
15;90(10):3996-4003. 
36. 
Gilmour KC, Cranston T, Loughlin S, Gwyther J, Lester T, Espanol T, Hernandez M, Savoldi G, 
Davies EG, Abinun M, Kinnon C, Jones A, Gaspar HB. Rapid protein-based assays for the diagnosis of 
T-B+ severe combined immunodeficiency. British journal of haematology. 2001 Mar;112(3):671-676. 
37. 
Antoine C, Muller S, Cant A, Cavazzana-Calvo M, Veys P, Vossen J, Fasth A, Heilmann C, 
Wulffraat N, Seger R, Blanche S, Friedrich W, Abinun M, Davies G, Bredius R, Schulz A, Landais P, 
Fischer A. Long-term survival and transplantation of haemopoietic stem cells for immunodeficiencies: 
report of the European experience 1968-99. Lancet. 2003 Feb 15;361(9357):553-560. 
38. 
Fischer A, Landais P, Friedrich W, Morgan G, Gerritsen B, Fasth A, Porta F, Griscelli C, 
Goldman SF, Levinsky R, et al. European experience of bone-marrow transplantation for severe 
combined immunodeficiency. Lancet. 1990 Oct 6;336(8719):850-854. 
39. 
Buckley RH. Molecular defects in human severe combined immunodeficiency and approaches 
to immune reconstitution. Annual review of immunology. 2004;22:625-655. 
40. 
Buckley RH, Schiff SE, Schiff RI, Markert L, Williams LW, Roberts JL, Myers LA, Ward FE. 
Hematopoietic stem-cell transplantation for the treatment of severe combined immunodeficiency. The 
New England journal of medicine. 1999 Feb 18;340(7):508-516. 
41. 
Buckley RH. The multiple causes of human SCID. The Journal of clinical investigation. 2004 
Nov;114(10):1409-1411. 
42. 
Cavazzana-Calvo M, Fischer A. Gene therapy for severe combined immunodeficiency: are we 
there yet? The Journal of clinical investigation. 2007 Jun;117(6):1456-1465. 
43. 
Abedi MR, Hammarstrom L, Ringden O, Smith CI. Development of IgA deficiency after bone 
marrow transplantation. The influence of acute and chronic graft-versus-host disease. Transplantation. 
1990 Sep;50(3):415-421. 
44. 
van Leeuwen JE, van Tol MJ, Joosten AM, Schellekens PT, van den Bergh RL, Waaijer JL, 
Oudeman-Gruber NJ, van der Weijden-Ragas CP, Roos MT, Gerritsen EJ, et al. Relationship between 
patterns of engraftment in peripheral blood and immune reconstitution after allogeneic bone marrow 
transplantation for (severe) combined immunodeficiency. Blood. 1994 Dec 1;84(11):3936-3947. 
45. 
Haddad E, Le Deist F, Aucouturier P, Cavazzana-Calvo M, Blanche S, De Saint Basile G, 
Fischer A. Long-term chimerism and B-cell function after bone marrow transplantation in patients with 
severe combined immunodeficiency with B cells: A single-center study of 22 patients. Blood. 1999 Oct 
15;94(8):2923-2930. 
46. 
Uchida N, Weissman IL. Searching for hematopoietic stem cells: evidence that Thy-1.1lo Lin- 
Sca-1+ cells are the only stem cells in C57BL/Ka-Thy1.1 bone marrow. The Journal of experimental 
medicine. 1992;175:175-184. 
47. 
Spangrude GJ, Heimfeld S, Weissman IL. Purification and characterization of mouse 
hematopoietic stem cells. Science (New York, NY. 1988;241:58-62. 

63 
Version 12.0 
January 4, 2022 
48. 
Morrison C, Smith GC, Stingl L, Jackson SP, Wagner EF, Wang ZQ. Genetic interaction 
between PARP and DNA-PK in V(D)J recombination and tumorigenesis. Nature genetics. 1997 
Dec;17(4):479-482. 
49. 
Abramson S, Miller RG, Phillips RA. The identification in adult bone marrow of pluripotent and 
restricted stem cells of the myeloid and lymphoid systems. The Journal of experimental medicine. 1977 
Jun 1;145(6):1567-1579. 
50. 
Dick JE, Magli MC, Huszar D, Phillips RA, Bernstein A. Introduction of a selectable gene into 
primitive stem cells capable of long-term reconstitution of the hemopoietic system of W/Wv mice. Cell. 
1985;42(1):71-79. 
51. 
Morrison SJ, Weissman IL. The long-term repopulating subset of hematopoietic stem cells is 
deterministic and isolatable by phenotype. J Immunol. 1994;1:661-673. 
52. 
Kondo M, Weissman IL, Akashi K. Identification of clonogenic common lymphoid progenitors in 
mouse bone marrow. Cell. 1997 Nov 28;91(5):661-672. 
53. 
Akashi K, Reya T, Dalma-Weiszhausz D, Weissman IL. Lymphoid precursors. Current opinion 
in immunology. 2000 Apr;12(2):144-150. 
54. 
Bhatia M, Bonnet D, Kapp U, Wang JC, Murdoch B, Dick JE. Quantitative analysis reveals 
expansion of human hematopoietic repopulating cells after short-term ex vivo culture. The Journal of 
experimental medicine. 1997;186(4):619-624. 
55. 
Cashman JD, Lapidot T, Wang JC, Doedens M, Shultz LD, Lansdorp P, Dick JE, Eaves CJ. 
Kinetic evidence of the regeneration of multilineage hematopoiesis from primitive cells in normal human 
bone marrow transplanted into immunodeficient mice. Blood. 1997;89(12):4307-4316. 
56. 
Larochelle A, Vormoor J, Hanenberg H, Wang JC, Bhatia M, Lapidot T, Moritz T, Murdoch B, 
Xiao XL, Kato I, Williams DA, Dick JE. Identification of primitive human hematopoietic cells capable of 
repopulating NOD/SCID mouse bone marrow: implications for gene therapy. Nat Med. 
1996;2(12):1329-1337. 
57. 
Goodell MA, Brose K, Paradis G, Conner AS, Mulligan RC. Isolation and functional properties of 
murine hematopoietic stem cells that are replicating in vivo. The Journal of experimental medicine. 
1996 Apr 1;183(4):1797-1806. 
58. 
Goodell MA, Rosenzweig M, H. K, Marks DF, DeMaria M, Paradis G, Grupp SA, Sieff CA, 
Mulligan RC, Johnson RP. Dye efflux studies suggest that hematopoietic stem cells expressing low or 
undetectable levels of CD34 antigen exist in multile species. Nature Medicine. 1997;3(12):1337-1345. 
59. 
Osawa M, Hanada Ki, Hamada H, Nakauchi H. Long-term lymphohematopoietic reconstitution 
by a single CD34-low/negative hematopoietic stem cell. Science (New York, NY. 1996;273:242-245. 
60. 
Zanjani ED, Almeida-Porada G, Livingston AG, Flake AW, Ogawa M. Human bone marrow 
CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ 
cells. Experimental hematology. 1998;26(4):353-360. 
61. 
Demaison C, Brouns G, Blundell MP, Goldman JP, Levinsky RJ, Grez M, Kinnon C, Thrasher 
AJ. A defined window for efficient gene marking of severe combined immunodeficient-repopulating cells 
using a gibbon ape leukemia virus-pseudotyped retroviral vector. Human gene therapy. 2000 Jan 
1;11(1):91-100. 
62. 
Galy A, Travis M, Cen D, Chen B. Human T, B, natural killer, and dendritic cells arise from a 
common bone marrow progenitor cell subset. Immunity. 1995 Oct;3(4):459-473. 
63. 
Holyoake TL, Nicolini FE, Eaves CJ. Functional differences between transplantable human 
hematopoietic stem cells from fetal liver, cord blood, and adult marrow. Experimental hematology. 1999 
Sep;27(9):1418-1427. 

64 
Version 12.0 
January 4, 2022 
64. 
Cao X, Shores EW, Hu-Li J, Anver MR, Kelsall BL, Russell SM, Drago J, Noguchi M, Grinberg 
A, Bloom ET, et al. Defective lymphoid development in mice lacking expression of the common 
cytokine receptor gamma chain. Immunity. 1995 Mar;2(3):223-238. 
65. 
DiSanto JP, Muller W, Guy-Grand D, Fischer A, Rajewsky K. Lymphoid development in mice 
with a targeted deletion of the interleukin 2 receptor gamma chain. Proc Natl Acad Sci USA. 
1995;92:377-381. 
66. 
Lo M, Bloom ML, Imada K, Berg M, Bollenbacher JM, Bloom ET, Kelsall BL, Leonard WJ. 
Restoration of lymphoid populations in a murine model of X-linked severe combined immunodeficiency 
by a gene-therapy approach. Blood. 1999 Nov 1;94(9):3027-3036. 
67. 
Otsu M, Anderson SM, Bodine DM, Puck JM, O'Shea JJ, Candotti F. Lymphoid development 
and function in X-linked severe combined immunodeficiency mice after stem cell gene therapy. Mol 
Ther. 2000 Feb;1(2):145-153. 
68. 
Soudais C, Shiho T, Sharara LI, Guy-Grand D, Taniguchi T, Fischer A, Di Santo JP. Stable and 
functional lymphoid reconstitution of common cytokine receptor gamma chain deficient mice by 
retroviral-mediated gene transfer. Blood. 2000 May 15;95(10):3071-3077. 
69. 
Cavazzana-Calvo M, Hacein-Bey S, de Saint Basile G, Gross F, Yvon E, Nusbaum P, Selz F, 
Hue C, Certain S, Casanova JL, Bousso P, Deist FL, Fischer A. Gene therapy of human severe 
combined immunodeficiency (SCID)-X1 disease [see comments]. Science (New York, NY. 
2000;288(5466):669-672. 
70. 
Hacein-Bey-Abina S, Le Deist F, Carlier F, Bouneaud C, Hue C, De Villartay JP, Thrasher AJ, 
Wulffraat N, Sorensen R, Dupuis-Girod S, Fischer A, Davies EG, Kuis W, Leiva L, Cavazzana-Calvo M. 
Sustained correction of X-linked severe combined immunodeficiency by ex vivo gene therapy. The New 
England journal of medicine. 2002 Apr 18;346(16):1185-1193. 
71. 
Gaspar HB, Parsley KL, Howe S, King D, Gilmour KC, Sinclair J, Brouns G, Schmidt M, Von 
Kalle C, Barington T, Jakobsen MA, Christensen HO, Al Ghonaium A, White HN, Smith JL, Levinsky 
RJ, Ali RR, Kinnon C, Thrasher AJ. Gene therapy of X-linked severe combined immunodeficiency by 
use of a pseudotyped gammaretroviral vector. Lancet. 2004 Dec 18;364(9452):2181-2187. 
72. 
Thrasher AJ, Hacein-Bey-Abina S, Gaspar HB, Blanche S, Davies EG, Parsley K, Gilmour K, 
King D, Howe S, Sinclair J, Hue C, Carlier F, von Kalle C, de Saint Basile G, le Deist F, Fischer A, 
Cavazzana-Calvo M. Failure of SCID-X1 gene therapy in older patients. Blood. 2005 Jun 
1;105(11):4255-4257. 
73. 
Baum C, von Kalle C, Staal FJ, Li Z, Fehse B, Schmidt M, Weerkamp F, Karlsson S, 
Wagemaker G, Williams DA. Chance or necessity? Insertional mutagenesis in gene therapy and its 
consequences. Mol Ther. 2004 Jan;9(1):5-13. 
74. 
Hacein-Bey-Abina S, Von Kalle C, Schmidt M, McCormack MP, Wulffraat N, Leboulch P, Lim A, 
Osborne CS, Pawliuk R, Morillon E, Sorensen R, Forster A, Fraser P, Cohen JI, de Saint Basile G, 
Alexander I, Wintergerst U, Frebourg T, Aurias A, Stoppa-Lyonnet D, Romana S, Radford-Weiss I, 
Gross F, Valensi F, Delabesse E, Macintyre E, Sigaux F, Soulier J, Leiva LE, Wissler M, Prinz C, 
Rabbitts TH, Le Deist F, Fischer A, Cavazzana-Calvo M. LMO2-associated clonal T cell proliferation in 
two patients after gene therapy for SCID-X1. Science (New York, NY. 2003 Oct 17;302(5644):415-419. 
75. 
Howe SJ, Mansour MR, Schwarzwaelder K, Bartholomae C, Hubank M, Kempski H, Brugman 
MH, Pike-Overzet K, Chatters SJ, de Ridder D, Gilmour KC, Adams S, Thornhill SI, Parsley KL, Staal 
FJ, Gale RE, Linch DC, Bayford J, Brown L, Quaye M, Kinnon C, Ancliff P, Webb DK, Schmidt M, von 
Kalle C, Gaspar HB, Thrasher AJ. Insertional mutagenesis combined with acquired somatic mutations 
causes leukemogenesis following gene therapy of SCID-X1 patients. The Journal of clinical 
investigation. 2008 Aug 7. 

65 
Version 12.0 
January 4, 2022 
76. 
Ott MG, Schmidt M, Schwarzwaelder K, Stein S, Siler U, Koehl U, Glimm H, Kuhlcke K, Schilz 
A, Kunkel H, Naundorf S, Brinkmann A, Deichmann A, Fischer M, Ball C, Pilz I, Dunbar C, Du Y, 
Jenkins NA, Copeland NG, Luthi U, Hassan M, Thrasher AJ, Hoelzer D, von Kalle C, Seger R, Grez M. 
Correction of X-linked chronic granulomatous disease by gene therapy, augmented by insertional 
activation of MDS1-EVI1, PRDM16 or SETBP1. Nat Med. 2006 Apr;12(4):401-409. 
77. 
Dave UP, Jenkins NA, Copeland NG. Gene therapy insertional mutagenesis insights. Science 
(New York, NY. 2004 Jan 16;303(5656):333. 
78. 
Woods NB, Bottero V, Schmidt M, von Kalle C, Verma IM. Gene therapy: therapeutic gene 
causing lymphoma. Nature. 2006 Apr 27;440(7088):1123. 
79. 
Pike-Overzet K, de Ridder D, Weerkamp F, Baert MR, Verstegen MM, Brugman MH, Howe SJ, 
Reinders MJ, Thrasher AJ, Wagemaker G, van Dongen JJ, Staal FJ. Gene therapy: is IL2RG 
oncogenic in T-cell development? Nature. 2006 Sep 21;443(7109):E5; discussion E6-7. 
80. 
Thrasher AJ, Gaspar HB, Baum C, Modlich U, Schambach A, Candotti F, Otsu M, Sorrentino B, 
Scobie L, Cameron E, Blyth K, Neil J, Abina SH, Cavazzana-Calvo M, Fischer A. Gene therapy: X-
SCID transgene leukaemogenicity. Nature. 2006 Sep 21;443(7109):E5-6; discussion E6-7. 
81. 
Shou Y, Ma Z, Lu T, Sorrentino BP. Unique risk factors for insertional mutagenesis in a mouse 
model of XSCID gene therapy. Proceedings of the National Academy of Sciences of the United States 
of America. 2006 Aug 1;103(31):11730-11735. 
82. 
Pike-Overzet K, de Ridder D, Weerkamp F, Baert MR, Verstegen MM, Brugman MH, Howe SJ, 
Reinders MJ, Thrasher AJ, Wagemaker G, van Dongen JJ, Staal FJ. Ectopic retroviral expression of 
LMO2, but not IL2Rgamma, blocks human T-cell development from CD34+ cells: implications for 
leukemogenesis in gene therapy. Leukemia. 2007 Apr;21(4):754-763. 
83. 
Kraunus J, Schaumann DH, Meyer J, Modlich U, Fehse B, Brandenburg G, von Laer D, Klump 
H, Schambach A, Bohne J, Baum C. Self-inactivating retroviral vectors with improved RNA processing. 
Gene Ther. 2004 Nov;11(21):1568-1578. 
84. 
Schambach A, Bohne J, Baum C, Hermann FG, Egerer L, von Laer D, Giroglou T. Woodchuck 
hepatitis virus post-transcriptional regulatory element deleted from X protein and promoter sequences 
enhances retroviral vector titer and expression. Gene Ther. 2006 Apr;13(7):641-645. 
85. 
Uetsuki T, Naito A, Nagata S, Kaziro Y. Isolation and characterization of the human 
chromosomal gene for polypeptide chain elongation factor-1 alpha. J Biol Chem. 1989 Apr 
5;264(10):5791-5798. 
86. 
Mizushima S, Nagata S. pEF-BOS, a powerful mammalian expression vector. Nucleic acids 
research. 1990 Sep 11;18(17):5322. 
87. 
Kim D, Uetsuki T, Kaziro Y, Yamaguchi N, Sugano S. Use of the human elongation factor 1 
alpha promoter as a versatile and efficient expression system. Gene. 1990;19(2):217-223. 
88. 
Zychlinski D, Schambach A, Modlich U, Maetzig T, Meyer J, Grassman E, Mishra A, Baum C. 
Physiological promoters reduce the genotoxic risk of integrating gene vectors. Mol Ther. 2008 
Apr;16(4):718-725. 
89. 
Modlich U, Bohne J, Schmidt M, von Kalle C, Knoss S, Schambach A, Baum C. Cell-culture 
assays reveal the importance of retroviral vector design for insertional genotoxicity. Blood. 2006 Oct 
15;108(8):2545-2553. 
90. 
Zychlinski D, Schambach A, Modlich U, Maetzig T, Meyer J, Grassman E, Mishra A, Baum C. 
Physiological Promoters Reduce the Genotoxic Risk of Integrating Gene Vectors. Mol Ther. 2008 Feb 
19. 

66 
Version 12.0 
January 4, 2022 
91. 
Modlich U, Bohne J, Schmidt M, von Kalle C, Knob S, Schambach A, Baum C. Cell culture 
assays reveal the importance of retroviral vector design for insertional genotoxicity. Blood. 
2006;108:2545-2553. 
92. 
Ryu BY, Evans-Galea MV, Gray JT, Bodine DM, Persons DA, Nienhuis AW. An experimental 
system for the evaluation of retroviral vector design to diminish the risk for proto-oncogene activation. 
Blood. 2008 Feb 15;111(4):1866-1875. 
93. 
Modlich U, Kustikova OS, Schmidt M, Rudolph C, Meyer J, Li Z, Kamino K, von Neuhoff N, 
Schlegelberger B, Kuehlcke K, Bunting KD, Schmidt S, Deichmann A, von Kalle C, Fehse B, Baum C. 
Leukemias following retroviral transfer of multidrug resistance 1 (MDR1) are driven by combinatorial 
insertional mutagenesis. Blood. 2005 Feb 15. 
94. 
Li Z, Dullmann J, Schiedlmeier B, Schmidt M, von Kalle C, Meyer J, Forster M, Stocking C, 
Wahlers A, Frank O, Ostertag W, Kuhlcke K, Eckert HG, Fehse B, Baum C. Murine leukemia induced 
by retroviral gene marking. Science (New York, NY. 2002 Apr 19;296(5567):497. 
95. 
Modlich U, Schambach A, Brugman MH, Wicke DC, Knoess S, Li Z, Maetzig T, Rudolph C, 
Schlegelberger B, Baum C. Leukemia induction after a single retroviral vector insertion in Evi1 or 
Prdm16. Leukemia. 2008 Aug;22(8):1519-1528. 
96. 
Will E, Bailey J, Schuesler T, Modlich U, Balcik B, Burzynski B, Witte D, Layh-Schmitt G, 
Rudolph C, Schlegelberger B, von Kalle C, Baum C, Sorrentino BP, Wagner LM, Kelly P, Reeves L, 
Williams DA. Importance of Murine Study Design for Testing Toxicity of Retroviral Vectors in Support of 
Phase I Trials. Mol Ther. 2007 Feb 13. 
97. 
Li Z, Schwieger M, Lange C, Kraunus J, Sun H, van den Akker E, Modlich U, Serinsoz E, Will E, 
von Laer D, Stocking C, Fehse B, Schiedlmeier B, Baum C. Predictable and efficient retroviral gene 
transfer into murine bone marrow repopulating cells using a defined vector dose. Experimental 
hematology. 2003 Dec;31(12):1206-1214. 
98. 
Ishii N, Asao H, Kimura Y, Takeshita T, Nakamura M, Tsuchiya S, Konno T, Maeda M, 
Uchiyama T, Sugamura K. Impairment of ligand binding and growth signaling of mutant IL-2 receptor 
gamma-chains in patients with X-linked severe combined immunodeficiency. J Immunol. 1994 Aug 
1;153(3):1310-1317. 
99. 
Mazzolari E, Forino C, Guerci S, Imberti L, Lanfranchi A, Porta F, Notarangelo LD. Long-term 
immune reconstitution and clinical outcome after stem cell transplantation for severe T-cell 
immunodeficiency. The Journal of allergy and clinical immunology. 2007 Oct;120(4):892-899. 
100.  Pai SY, Logan BR, Griffith LM, Buckley RH, Parrott RE, Dvorak CC, Kapoor N, Hanson IC, 
Filipovich AH, Jyonouchi S, Sullivan KE, Small TN, Burroughs L, Skoda-Smith S, Haight A, Grizzle A, 
Pulsipher MA, Chan KW, Fuleihan RL, Haddad E, Loechelt B, Aquino VM, Gillio A, Davis J, Knutsen A, 
Smith AR, Moore TB, Schroeder ML, Goldman FD, Connelly JA, Porteus MH, Xiang Q, Shearer WT, 
Fleisher TA, Kohn DB, Puck JM, Notarangelo LD, Cowan MJ, O’Reilly RJ. Transplant outcomes for 
severe combined immunodeficiency from 2000-2009 2014. Manuscript accepted.  
101.  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 
Ghonaium A, Ferster A, Duppenthaler A, Notarangelo L, Wintergerst U, Buckley RH, Bregni M, Marktel 
S, Valsecchi MG, Rossi P, Ciceri F, Miniero R, Bordignon C, Roncarolo MG. Gene therapy for 
immunodeficiency due to adenosine deaminase deficiency. N Engl J Med. 2009 ed. 2009 Jan 
29;360(5):447–58. 
102.  Candotti F, Shaw KL, Muul L, Carbonaro D, Sokolic R, Choi C, Schurman SH, Garabedian E, 
Kesserwan C, Jagadessh GJ, Fu PY, Gschweng E, Cooper A, Tisdale JF, Weinberg KI, Crooks GM, 
Kapoor N, Shah A, Abdel-Azim H, Yu XJ, Smogorzewska M, Wayne A, Rosenblatt HM, Davis CM, 
Hanson C, Rishi RG, Wang X, Gjertson D, Yang OO, Balamurugan A, Bauer G, Ireland JA, Engel BC, 

67 
Version 12.0 
January 4, 2022 
Podsakoff GM, Hershfield MS, Blaese RM, Parkman R, Kohn DB. Gene therapy for adenosine 
deaminase-deficient severe combined immune deficiency: clinical comparison of retroviral vectors and 
treatment plans. Blood. 2012 Nov 1;120(18):3635–46. 
103.  Bartelink IH, Boelens JJ, Bredius RGM, Egberts ACG, Wang C, Bierings MB, Shaw PJ, Nath 
CE, Hempel G, Zwaveling J, Danhof M, Knibee CAJ. Body weight-dependent pharmacokinetics of 
busulfan in paediatric haematopoietic stem cell transplantation patients: towards individualized dosing. 
Clin Pharmacokinet. 2012 May 1;51(5):331–45.  
104.  Savic RM, Cowan MJ, Dvorak CC, Pai S-Y, Pereira L, Bartelink IH, Boelens JJ, Bredius RGM, 
Wynn RE, Cuvelier GDE, Shaw PJ, Slatter MA, Long-Boyle J. Effect of weight and maturation on 
busulfan clearance in infants and small children undergoing hematopoietic cell transplantation. Biol 
Blood Marrow Transplant. 2013 Nov;19(11):1608–14.  
105. De Ravin SS, Wu X, Moir S, Anaya-O'Brien S, Kwatemaa N, Littel P, et al. Lentiviral hematopoietic 
stem cell gene therapy for X-linked severe combined immunodeficiency. Sci Transl Med. 2016 Apr 
20;8(335):335ra57.  
 

68 
Version 12.0 
January 4, 2022 
Appendix 1: Construction and sequence of SIN gammaretroviral vector pSRS11.EFS.IL2RG.pre* 
 
 
 
 
 
pSRS11 EFS IL2RGpre
5858 bps
1000
2000
3000
4000
5000
NdeI
BbeI
KasI
NarI
SfoI
AflII
SphI
MscI
NaeI
NgoMIV
BglII
BsaBI
SacII
SfiI
HpaI
Ecl136II
SacI
NotI
ClaI
MunI
Eco47III
PpuMI
Bpu1102I
BstXI
NcoI
SalI
Bst1107I
Sw aI
BsiWI
BstBI
HindIII
NheI
AscI
BssHII
XhoI
SapI
SspI
RSV Promotor
R U5
EFS
IL2RG
WPRE
3'SIN LTR

69 
Version 12.0 
January 4, 2022 
Appendix 2:  Outline of proposed criteria for enrollment 
 
 
 
 
 
 

70 
Version 12.0 
January 4, 2022 
Appendix 3: Transduction 
 

71 
Version 12.0 
January 4, 2022 
 
Appendix 4: Patient  Monitoring  Plan 
 
 
  
Time Points 
Lab category  
pre gene 
transfer
30 days 
60 days 
90 days 
120 days 
180 days 
9 months 
12 months 
18 months 
2 years 
2 ½ years 
3 years  
3 ½ years 
4 years 
4 ½ years 
5 years 
5 ½ years 
Yearly 6 to 
15 years 
CBC and differential 
X 
X7 
X 
X 
X 
X 
X 
X 
X 
X 
 
X 
 
X 
 
X 
 
X 
lymphocyte subsets 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
X 
 
X 
 
X 
 
X 
proliferation to PHA 
X 
  
  
X 
  
X 
  
X 
  
X 
 
X 
 
X 
 
X 
 
X 
Chemistries5 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
X 
 
X 
 
X 
 
X 
proliferation to tetanus 
  
  
  
  
  
  
  
  
X1 
X1 
 
X1 
 
X1 
 
X1 
 
X1 
IgG, IgA, IgM 
X 
  
  
  
  
X2 
  
X 
X 
X 
 
X 
 
X 
 
X 
 
X 
tetanus IgG 
  
  
  
  
  
  
  
  
X3 
X3 
 
X3 
 
X3 
 
X3 
 
X3 
Isohemagglutinins 
X 
  
  
  
  
  
  
X 
  
X 
 
X 
 
X 
 
X 
 
X 
γc expression and other 
research flow 
X 
  
  
X 
X 
X 
X 
X 
X 
X 
 
X 
 
X 
 
X 
 
X 
lineage specific gene marking 
  
  
  
  
X6 
X 
  
X 
X 
X 
 
X 
 
X 
 
X 
 
X 
lineage specific transgene 
integration and clonality 
analysis 
  
  
  
  
  
X 
  
X 
  
X4 
X4 
X4 
X4 
X4 
X4 
X4 
X4 
X 
TREC 
X 
  
  
  
  
X 
  
X 
  
X 
 
X 
 
X 
 
X 
 
X 
TCR Vβpanel 
  
  
  
  
  
X 
  
X 
  
X 
 
X 
 
X 
 
X 
 
X 
TCR spectratyping 
  
  
  
  
  
  
  
X 
  
X 
 
X 
 
X 
 
X 
 
X 
replication competent virus 
X 
  
  
X  
  
X 
  
X 
X 
X 
 
X 
 
X 
 
X 
 
X 
save serum 
X 
  
  
X 
  
X 
  
X 
X 
X 
 
X 
 
X 
 
X 
 
X 
save cells 
X 
  
  
X 
  
X 
  
X 
X 
X 
 
X 
 
X 
 
X 
 
X 

72 
Version 12.0 
January 4, 2022 
Study visits should occur at 30 days (+/- 3 days), 60 days (+/-7 days), 90 days (+/- 10 days), 120 days 
(+/- 14 days), 180 days (+/- 20 days), 9 months (+/- 30 days), 12 months (+/- 30 days), 18 months (+/- 
30 days), 2 Years (+/- 30 days) and thereafter +/- 90 days. 
 
1) T cell proliferation to tetanus will be checked at least 4 weeks after completing 3 vaccinations. 
 
2) IgG infusions will be given according to institutional standard, and in general are expected to be 
maintain trough IgG at least >500 mg/dL. IgA in all subjects and isohemagglutinins in subjects with 
blood type O, blood type A or blood type B will be checked at visits indicated above. If IgA is > 12 
mg/dL, and trough IgG is at least 500 mg/dL, IgG infusion may be stopped. IgG levels will be checked 
to document independence from IgG infusion according to standard clinical care. If criteria to stay off of 
IgG infusion are not met, the subject should go back on replacement. If IgG levels at least 2 months 
later are maintained or rising, the subject may stay off of IgG and vaccination including 3 doses of 
tetanus may be given according to published clinical standards (Griffith LM, Journal of Allergy and 
Clinical Immunology, 2009 vol. 124 (6) pp. 1152-60.e12). Prevaccination tetanus and other titers should 
be drawn as part of clinical care. Post-vaccination titer to tetanus will be drawn at the next study visit if it 
has not been drawn already as part of clinical care. Until protective responses to vaccines have been 
documented, the subject should remain on antibiotic prophylaxis. If the subject fails to mount a 
protective antibody response to tetanus, as determined by a titer of anti-tetanus antibodies >0.15 IU/ml 
4 weeks after the third immunization, replacement therapy with IVIG will be reinstituted. IVIG 
replacement therapy will also be reinstituted in the event that the subject develops infection requiring 
hospitalization while being off-IVIG and on antibiotic prophylaxis. 
 
3) IgG antibody to tetanus will be measured at least 4 weeks after completing 3 vaccinations. 
 
4) Assay run every six months in years 2-5 and then yearly until year 15. 
 
5) Chemistries include sodium, potassium, chloride, bicarbonate, blood urea nitrogen, creatinine, glucose, 
calcium, magnesium, phosphorus, asparate aminotransferase, alanine aminotransferase, total bilirubin, 
direct bilirubin. 
 
6) If CD3 count is deemed too low to perform lineage specific sorting, gene marking will be performed in 
peripheral blood mononuclear cell fraction. 
 
7) For patients receiving busulfan, CBC and differential will be monitored at least twice weekly until 
documentation of 3 consecutive days of absolute neutrophil count >500 cells/microliter after neutrophil 
nadir.  
 
 

73 
Version 12.0 
January 4, 2022 
Appendix 5: Busulfan dosing guidelines  
 
 
Busulfex doses will be adjusted based upon the area-under-the-curve calculations. AUC 
measurements at Boston Children’s Hospital are expressed as micromole-min/L, which can be 
converted into mg*h/L if divided by 244, a factor derived from the molecular weight of busulfan 246 
g/mole. For full myeloablation a total AUC of 23,200 micromole-min/L or 95 mg*h/L (equivalent to 
16 doses of every 6 hour busulfan with AUC of 1450 micromole-min/L) is the maximum tolerated 
with regard to incidence of veno-occlusive disease when busulfan is combined with fludarabine.  
 
The goal busulfan area-under-the-curve (AUC) for this protocol will be a total target exposure 
of 30 mg*h/L). If goal busulfan is exceeded, less than 3 doses may be given to achieve the 
goal total. 
 
Goal AUC for each of 2 doses is therefore 3050-4270 uM x minute. 
Dose adjustments must be discussed with the protocol site PI. 
 
 
References: 
 
Bartelink et al. Body weight-dependent pharmacokinetics of busulfan in paediatric haematopoietic 
stem cell transplantation patients: towards individualized dosing. Clin pharmacokinet (2012) vol. 51 
(5) pp. 
331-45. 
 
EBMT/ESID Guidelines for Haematopoietic Stem Cell Transplantation for Primary 
Immunodeficiencies, 2011. Available upon request. 
 
O'Donnell et al. Phase I study of dose-escalated busulfan with fludarabine and alemtuzumab as 
conditioning for allogeneic hematopoietic stem cell transplant: reduced clearance at high doses and 
occurrence of late sinusoidal obstruction syndrome/veno-occlusive disease. Leukemia & Lymphoma 
(2010) pp 879-885. 
 
 

74 
Version 12.0 
January 4, 2022 
Appendix 6: Changes to protocol 
 
*All dates are the IRB approval date from the Main site Children’s Hospital Boston 
 
Version 1 – Initial application (IRB approval date 07/13/2009) 
 
Version 2 - (IRB approval date 10/06/2010): 
 
Summary of Changes to Version 1  
 
Since the most recent renewal of the protocol “A multi-institutional phase I/II trial evaluating the 
treatment of SCID-X1 patients with retrovirus-mediated gene transfer,” (CHB 09-05-0254) the study has 
been extensively reviewed and critiqued by the NIAID/DAIT Clinical Research Committee. The majority 
of changes in the document were made in response to this review, and the study is now funded by 
NIAID (U01 AI087628). Additional changes were suggested by the DSMB and by the IRB at UCLA, a 
participating center. There are very few, if any, substantive changes. The changes are summarized 
below in order of occurrence. 
 
Front pages: Change in Co-PI at UCLA (De Oliveira) 
Clarification of roles on front page (per NIAID) 
 
Addition of version number (per NIAID) 
Addition of funding sponsor information and contacts (per NIAID) 
 
Listing of Steering Committee (per NIAID) 
 
Separate listing of European collaborators (per NIAID) 
 
Addition of Investigator signature page (per NIAID) 
Section 5.2.1 Clarification of vector type (per NIAID) 
Section 5.2.2 Addition that vector is certified (per NIAID) 
 
Clarification that local SOPs will be followed for cell infusion at different sites (per NIAID) 
Section 6 
Clarification of study design, this trial has 9 patients, parallel trials in Europe have 11 
patients (per NIAID) 
Section 7.1 
Clarification, 9 patients (per NIAID) 
Section 7.2 
Informed consent will be in accordance to each site’s IRB requirements (per UCLA IRB) 
Section 7.2 
Clarification that after consent, eligibility will be confirmed by all site PIs, study 
sponsor/grant PI and grant co-PI. 
Section 8.2.1 Clarification of CD34 cell number to proceed to transduction (per NIAID) 
 
Section 8.2.2 Clarification of release criteria, excluding fungus (per UCLA) 
 
Deletion of hanging phrase regarding RCR testing. 
 
Clarification of the Pre-Infusion PCR test requirement for GALV 
Section 9.1.1 Clarification of failure for efficacy definition at 6 months versus early failure defined at 
120 days (per DSMB) 
Section 9.2 
Modification of personnel and responsibilities for trial coordination. 
Section 9.2.3 Clarification of early failure definition at 120 days (per DSMB). 
Section 9.4 
Premature termination of study defined as suspension of enrollment pending review by 
DSMB and other agencies (per NIAID). 
Section 9.5 
Clarification of 9 patients on US trial, but 20 included for stopping rules (per NIAID). 
Clarification of statistical definition of failure (per DSMB). 
Section 9.5.1 Clarification of stopping rule (per NIAID). 
Section 11 
Adverse event reporting modified for change in personnel (CTIP and Project Manager to 
subsume responsibilities of DARCC). Clarification of events to be recorded and reported 
with regards to relatedness and expectedness. Clarification of the definition of clonal 

75 
Version 12.0 
January 4, 2022 
changes. Addition of Table for clarification. Clarification that NIAID funds will not be used 
for European data collection (per NIAID). 
 
 
Clarification of reporting protocol deviations and exceptions (per NIAID) 
Section 13 
DSMP has been deleted from Appendix 5 and is included instead in MOP. 
 
Detailed plan of verification of eligibility by PI committee after consent is obtained. 
 
Clarification that co-investigator may also consent patient. 
Section 14 
Modification to reflect changes in personnel from DARCC to CTIP. 
 
Section 16 
Addition of study staff qualifications. 
Appendix 4 
Modification of criteria for revaccination and modification of study test windows. 
 
Modification of lab tests, removing some tests from pre-gene transfer time point. 
 
Version 3 (IRB Deferred 11/8/2010): 
 
Summary of Changes to Version 2  
 
1) We propose a modification to add the possibility of a repeat bone marrow harvest and gene 
transfer procedure for the possible circumstance when the first bone marrow harvest is not 
successful due to contamination or other failure to meet release criteria. If cells fail to meet 
release criteria, they are not suitable for infusion. In this circumstance, the investigator will 
explain to the subject/guardian what happened, and the options open to the subject, 
including a) going through a second harvest 4-8 weeks after the first harvest and another 
attempt at gene transfer or b) coming off study and proceeding to allogeneic hematopoietic 
stem cell transplantation. (See page 21, 38, 45) We have reviewed past records and find 
that ~5-10% of bone marrow harvests are contaminated at our institution. The frequency at 
other institutions is not published, but it appears this level of contamination is compatible 
with what other programs experience. We will initiate at the same time a quality 
improvement process to reduce this incidence in our own program. The timing proposed is 
compatible with National Marrow Donor Program guidelines which call for a minimum of 4 
weeks between marrow harvests, and has been discussed with our DSMB. The maximum 
time of 8 weeks is proposed to limit the time during which the subject remains without a 
definitive therapeutic procedure, recognizing the time it would take to arrange alternative 
bone marrow transplant. The consent form has been modified to reflect this change.   
2) We propose a change in the minimum number of cells to infuse, based on available 
literature. Previously the protocol specified that a minimum of 2 x 10e6 CD34+ cells/kg 
would be required for infusion. Based on publications from the previous French trial, we 
propose to lower the minimum to 1 x 10e6 CD34+ cells/kg (Hacein-Bey-Abina et al. Efficacy 
of gene therapy for X-linked severe combined immunodeficiency. N Engl J Med (2010) vol. 
363 (4) pp. 355-64; Ginn et al. Treatment of an infant with X-linked severe combined 
immunodeficiency (SCID-X1) by gene therapy in Australia. Med J Aust (2005) vol. 182 (9) 
pp. 458-63). The protocol previously stated that a minimum of 4 x 10e6 CD34/kg must be 
available for transduction to proceed. Given the high variability in growth of CD34+ cells in 
culture, we propose to proceed with transduction regardless of the recovery.  (See page 37, 
38, 46).  
3) We clarified the definition of failure of gene transfer, which would lead to the subject coming 
off study. Subjects who have <0.1% of T cells and/or peripheral blood mononuclear cells 
containing vector sequences on 2 consecutive determinations will be defined as having 
failure of gene transfer and come off study (page 39,   42). We have also clarified that 
subjects who come off study because of early failure of immune reconstitution at day 120 (T 
cell count <200/microliter and/or absence of gene marking) would nevertheless be 

76 
Version 12.0 
January 4, 2022 
monitored for gene marking until failure of gene transfer with 2 consecutive measurements 
has been documented (page 42).   
4) Risk of possible blood transfusion because of anemia after bone marrow harvest was added 
to the protocol (page  45). This risk was already in the consent form. 
5) Given that subjects enrolled on this protocol may be ill with active infection and therefore 
require a high volume of blood to be drawn for clinical sampling, we propose to eliminate the 
blood drawing maximum of 7.5 ml/kg or 450 ml as it would be unrealistic to get the 
necessary safety studies within this limitation. We will however continue to track the amount 
of blood drawn for clinical and research purposes (page 45). The consent form has been 
modified to reflect this change. 
6) The consent form has been updated in the section regarding the occurrence of 
myelodysplastic syndrome in subjects undergoing gene transfer for chronic granulomatous 
disease, at the request of another site's IRB.   
7) A number of other minor spelling errors were corrected and clarifications made. 
 
Version 3 (IRB Approval date 11/22/2010): 
 
Summary of Changes to version 2 
 
The proposed modification to offer a second gene transfer procedure in case the first harvest product 
fails to meet release criteria potentially exposes the subject to additional risk including risks of second 
harvest (same risks as first harvest) and risk of remaining without immune reconstitution during the 4-8 
week period required for hematopoietic recovery between harvests. We believe it is appropriate to offer 
this option to already enrolled subjects because the subject has not received gene manipulated cells 
and therefore has not yet had the chance to benefit from the trial. The consent has been modified to 
inform subjects that second harvest might be a possibility. The subject would only proceed to second 
harvest after discussion of alternatives, and informed consent for the second procedure would be 
documented. 
The proposed modification to remove the limit on blood drawing increases the risk of subjects needing 
a blood transfusion for anemia. We believe this is justified because the study population may require 
clinical blood sampling that would make the drawing of blood for research purposes impractical within 
the limit and thus make it impossible to conduct the necessary safety monitoring. Thus we believe the 
risk of blood transfusion is less than the risk of failure to conduct safety monitoring.    
 
Version 4 (IRB Approval Date 01/26/2011): 
 
Summary Changes to Version 3 
 
1)  Section 9.2.2 – Change the wording of the RCR testing in order to reduce confusion. Used 
the words “Analyzed” and “Used” instead of “Withheld” as requested by the Indiana 
University Lab. 
2) Section 9.2.2 - Integration analysis frequency duration change to be in line with FDA 
guidance, this was a requested by FDA. 
3) Section 9.2.2 - Modify RCR testing to archive specimens, this is consistent with FDA 
guidance and simplifies process using a transient supernatant. 

77 
Version 12.0 
January 4, 2022 
4) Section 10.1.2 – Change clonal analysis monitoring 2X/year post-GT for the first 5 years. 
This change is now in line with the FDA guidance, and requested by FDA. 
5) Section 10.1.3 – Clarified that discarded blood samples will be archived for further studies 
relevant to this protocol. 
6) Section 10.2.1 – Clarification on what pre-treatment investigations need to be  recollected in 
case of a repeat bone marrow harvest. 
7) Section 10.2.3 – Removed the term “intent to treat”, this was requested by our DSMB.  
8) Section 10.5 – Change wording as DSMB requested that we clarify the protocol definitions 
of evaluability, success, failure and off study. 
9) Section 11.2 – Clarification on blood draws if the patient is deemed to be anemic. 
10) Section 12 – Clarification on AE reporting for events that do not meet the criteria for 
expedited reporting, this was a request of the NIH – NIAID. 
11) Appendix 4 – Updated to reflect the changes in the protocol (Points 2 and 3 of this list).  
12) ICF – Because data management is via our combined CHB/DFCI Clinical and Translational 
Investigation Program, we now include DFCI in the HIPAA section of our consent form 
13) Study Ad – The UCLA IRB requested some minor changes to make it clearer that the pre-
clinical safety data do not guarantee better clinical safety. 
Version 5 (IRB Approval Date 05/23/2011):  
 
Summary of changes to Version 4: 
 
Requested that the stopping rules for the study be changed because the current criteria is too 
restrictive and may lead to premature referral for allogenic transplant, which would be unsafe for the 
patients. The proposed revision was recommended to us by our DSMB, documentation is attached. 
With regard of the approved version in section 10.2.3 of the protocol (page 41), early failure of immune 
reconstitution is determine by the following. 
 
Current Approved Version: 
 
1) Poor recover of T lymphocyte number (CD3<200μl) and/or 
2) Absence of gene marking by 120 days 
 
Our Proposed Version: 
 
1) Lack of an increase in autologous T cell counts over baseline in at least two of three 
assessments at days 60, 90, and 120 
AND 
2) Lack of gene marking 

78 
Version 12.0 
January 4, 2022 
 
The Sponsor Dr. Williams, will no longer have a financial interest in the use of Retronectin. The protocol 
and consent have been updated for this change. 
 
 
Version 6 (Approved 11/14/2011):  
 
Summary of scientific review for this amendment  
 
This amendment broadens the inclusion criteria for the study.  
 
X-linked SCID is a fatal disease due to failure of T cell development and B cell function, which is usually 
treated by allogeneic hematopoietic cell transplantation (HCT), i.e. bone marrow transplantation (BMT). 
Outcome of BMT is best when an HLA matched sibling donor is available. For those who do not have a 
matched sibling donor, alternatives include T cell depleted BMT from a parent, often performed without 
conditioning (haplo-BMT) and matched unrelated donor BMT with conditioning (URD-BMT). Survival in 
single institution and large survey studies are similar for these two approaches, ranging from 66-77% 
[1, 2]. Younger infants, those less than 3.5 months in one single institution experience, have higher 
survival after unconditioned haplo-BMT of 94% [3].  
 
 
While survival overall is good, and survival in young infants is excellent, outcomes after alternative 
donor BMT for SCID are still not ideal. URD-BMT generally results in full T and B cell reconstitution but 
exposes small infants to chemotherapy conditioning [4]. Haplo-BMT without conditioning is nontoxic but 
sometimes requires repeat transplantation, frequently results in lack of B cell reconstitution (requiring 
life-long IVIG administration), and may be associated with waning immunity over time, though this last 
is controversial [5-9]. Either approach can be complicated by acute and/or chronic GVHD, and long-
term a percentage of children treated with BMT have ongoing complications [10]. Gene therapy would 
avoid GVHD entirely by using the patient as his own donor, and if performed without conditioning, 
would avoid exposure of infants to chemotherapy. Gene therapy trials in Paris and London enrolled 20 
subjects of whom 18 had successful and prompt T cell immune reconstitution ([11, 12] and slide 3). 
Unfortunately 5 of the 20 subjects developed T cell acute lymphoblastic leukemia due to insertion of the 
gene therapy retroviral vector into an oncogene, and 1 of these 5 children died [13-16]. The long-term 
outcome of the remaining 17 patients has been reported and all are infection free with no evidence of 
SCID ([17, 18] and slide 3). B cell reconstitution and function appear to be similar to patients 
undergoing unconditioned haplo-BMT [17, 18]. 
 
To maintain the excellent efficacy and reduce or eliminate the risk of leukemia, the retroviral vector was 
extensively modified (slide 4). In vitro data and murine in vivo data (not comparing directly to old clinical 
vector) were presented at the first review of this protocol at RAC on December 4, 2008. In the letter 
received on December 16, 2008 the following statement was made: 
 
Healthy infants with X-SCID who are less than 3.5 months old should be excluded because they should 
be treated with a haploidentical transplantation [i.e. parent] which offers proven efficacy at their age.  
 
Therefore the protocol inclusion criteria, exclusion criteria and informed consent document were revised 
to reflect this recommendation. The concerns cited included: 1. the risk of insertional mutagenesis, 2. 
the lack of efficacy data with this vector in humans, and 3. the established efficacy of alternative 
treatments. 
 

7 9 
Ver si o n 1 2. 0 
Ja n uar y 4, 2 0 2 2 
W e c o nti n u e t o m ai nt ai n t h at g e n e t h er a p y h a s t h e p ot e nti al t o b e e q u all y effi c a ci o u s a s st a n d ar d B M T 
w hil e a v oi di n g G V H D, a n d t h at wit h f ull i nf or m e d c o n s e nt aft er di s c u s si o n of alt er n ati v e t h er a pi e s a n d 
ri s k s, p ar e nt s of i nf a nt s of < 3. 5 m o nt h s of a g e m a y b e off er e d t h e c h oi c e t o e nr oll. H a vi n g e nr oll e d 1 
s u bj e ct i n B o st o n a n d l e ar n e d of e arl y effi c a c y i n 2 s u bj e ct s e nr oll e d i n P ari s, w e r e q u e st e d r e vi e w of a 
p ot e nti al a m e n d m e nt t o t h e pr ot o c ol a n d g a v e p u bli c r e vi e w at t h e R A C o n S e pt e m b er 1 3, 2 0 1 1. W e 
a d dr e s s e d t h e t hr e e c o n c er n s a b o v e.  
 
1.  
T h e ri s k of i n s erti o n al m ut a g e n e si s  
T h e o n s et of l e u k e mi a i n t h e pr e vi o u sl y e nr oll e d s u bj e ct s w a s l at e, 3 -5 y e ar s p o st g e n e t h er a p y; 
t h er ef or e it i s t o o s o o n t o c o m m e nt o n s af et y wit h t h e n e w v e ct or i n h u m a n s. H o w e v er, w e di d pr e s e nt 
k e y p orti o n s of e xt e n si v e n e w d at a t e sti n g t h e ol d cli ni c a l v e ct or ( M F C c) v er s u s n e w cli ni c al v e ct or 
h e a d t o h e a d i n b ot h i n vitr o h u m a n J ur k at c ell e x p eri m e nt s ( sli d e 5), a n d i n vi v o m uri n e b o n e m arr o w 
tr a n s pl a nt e x p eri m e nt s ( sli d e 6). I n all e x p eri m e nt s t h er e w a s l e s s e vi d e n c e of o n c o g e n e si s u si n g t h e 
n e w cli ni c al v e ct or.  
 
2.  T h e l a c k of effi c a c y d at a wit h t hi s v e ct or i n h u m a n s  
W e s h o w e d t h at t h e fir st p ati e nt e nr oll e d i n B o st o n o n t hi s tri al h a s e x pr e s si o n of t h e tr a n s g e n e ( c, 
sli d e 1 5), i m m u n e r e c o n stit uti o n a s g o o d ( T c ell) or b ett er ( N K c ell s) t h a n t h e pr e vi o u s tri al ( sli d e 1 6 a n d 
1 7), a n d cl e ar a n c e of t h er a p y -r e si st a nt i nf e cti o n ( sli d e 1 8, 1 9, 2 0). W e al s o s h o w e d t h at 2 p ati e nt s 
e nr oll e d i n P ari s h a v e e vi d e n c e of T c ell r e c o n stit uti o n, N K c ell r e c o n stit uti o n a n d i m pr o v e m e nt f or 1 
p ati e nt i n cli ni c al i nf e cti o n s ( sli d e 2 3, 2 4). T h u s 3 e nr oll e d s u bj e ct s h a v e e vi d e n c e of e arl y e ffi c a c y a n d 
2 of 3 h a v e cli ni c al i m pr o v e m e nt.  
 
3.  
T h e e st a bli s h e d effi c a c y of alt er n ati v e tr e at m e nt s  
W e c o m p ar e d c urr e nt s ur vi v al of 2 3 p ati e nt s w h o h a v e h a d g e n e t h er a p y c o m bi ni n g b ot h tri al s a n d 
s h o w t h at t h e cr u d e s ur vi v al i s c o m p ar a bl e t o p u bli s h e d a n d u n p u bli s h e d d at a of y o u n g c hil dr e n tr e at e d 
f or S CI D, wit h gr o u p s of 4 6-6 0 c hil dr e n ( sli d e 2 8). A d diti o n all y, w e s h o w e d u n p u bli s h e d d at a fr o m t h e 
E ur o p e a n l ar g e m ulti -i n stit uti o n al S C E TI D E r e gi str y s h o wi n g t h at 2 2 % of b a bi e s wit h T-B + S CI D ( m a n y 
of t h e s e ar e X -li n k e d S CI D) tr e at e d at a g e < 3. 5 m o nt h s wit h h a pl o-B M T h a d a c ut e G V H D a n d 9 % of 
t h e s e b a bi e s h a d c hr o ni c G V H D ( sli d e 3 1). T h u s g e n e t h er a p y a) c urr e ntl y h a s e q ui v al e nt s ur vi v al t o 
y o u n g i nf a nt s tr e at e d wit h B M T, a n d b) G V H D, a c o m pli c ati o n of B M T t h at i s a v oi d e d i n g e n e t h er a p y, 
i s pr e v al e nt i n t h e S CI D p ati e nt s tr e at e d wit h h a pl o-B M T.  
 
I n s u m m ar y w e pr e s e nt e d d at a t o s u p p ort o ur ar g u m e nt t h at i nf a nt s < 3. 5 m o nt h s w h o mi g ht ot h er wi s e 
b e tr e at e d wit h a h a pl o -B M T m a y b e off er e d t h e o p p ort u nit y t o e nr oll o n t hi s tri al of g e n e t h er a p y wit h 
a p pr o pri at e i nf or m e d c o n s e nt.  
 
S u m m ar y f or I R B of c h a n g e s t o pr ot o c ol v er si o n 5:  
C h a n g e s t o Pr ot o c ol:  
O n p a g e s 3 4, 3 5, a n d 3 6 t h e r el e v a nt c h a n g e s t o i n cl u si o n a n d e x cl u si o n crit eri a h a v e b e e n m a d e.  
 
Ot h er mi n or c h a n g e s i n cl u d e:  
--r e m o v al of “ti m e” fr o m t h e r e q uir e d i nf or m ati o n f or a d v er s e e v e nt r e p orti n g o n p a g e 4 8 
--cl arifi c ati o n of l a b or at or y st u di e s t o b e s e nt f or c h e mi stri e s i n A p p e n di x 4  
--c orr e cti o n of t y p o gr a p hi c al err or s o n p a g e 3 9 a n d i n A p p e n di x 4  
 
C h a n g e s t o c o n s e nt v er si o n 5:  
 
I n t h e l ett er r e c ei v e d o n S e pt e m b er 2 9, 2 0 1 1 t h e R A C st at e d: 
 

80 
Version 12.0 
January 4, 2022 
After reviewing your data, together with the data and opinions of two transplantation experts, the RAC 
concluded that it is reasonable to offer gene transfer to an infant less than 3 ½ months of age, even if 
that infant has the option of a related haploidentical transplant. 
 
The RAC further gave specific comments on modifications to the informed consent document to reflect 
the current status of alternative treatments. The following is a list of changes made to the informed 
consent document with specific reference to the letter from the RAC, to demonstrate how we have 
sought to incorporate the RAC recommendations in our amended protocol and ICF. Note that the RAC 
made comments based on the ICF activation date October 6, 2010. 
 
  
Version 7 (IRB approved 04/11/2012) 
 
Summary of changes to protocol version 6 
 
Section 1 – Removed the exclusion criteria patients less than 3.5 months old, the change was 
previously approved 
Section 6.2.4 – Clarified that autologous CD34+ cells undergo 2 or 3 rounds of retroviral transduction 
Section 10.1.2 – Clarified failure of gene transfer, i.e. <0.001 copies per cell 
Section 12 – The entire Adverse Event Reporting criteria has been updated to clarify what is reportable 
to the Sponsor. Because of the scrutiny this trial is under, the Sponsor is requesting additional reporting 
above and beyond what the FDA requires.  
Appendix 2 – A new outline of eligibility criteria for enrollment has been added to reflect previously 
approved changes 
Appendix 5 – The new Appendix for the summary of protocol changes has been added in order to keep 
the study team informed on the various changes the protocol has undergone.  
 
 Version 8 (IRB approved 08/01/2012) 
Summary of changes to protocol version 7 
 
Appendix 4 – At the May 2012 DSMB meeting, members recommended that the criteria for both 
discontinuation of IVIg and possible reinstatement of this supportive care be updated and clarified in the 
study protocol. 
 
    
 
Version 9 (IRB approved 03/11/2013)) 
 
Summary of changes to protocol version 8: 
 
Front Page: Addition of Wendy B. London, PhD as Co Investigator 
 
Changes throughout document to reflect Colleen Dansereau, RN as Project manager and Matthew 
Wladkowski as Regulatory Coordinator 
 
 
 Section 9.2.2 Testing of product prior to patient re-infusion 
Clarified testing done at at 0 hr  and end of incubation for proliferation and sterility (for bacteria, 
endotoxin and mycoplasma). 
 
10.1.2 Molecular characterization of gene transfer 

81 
Version 12.0 
January 4, 2022 
Based on discussions at the PI meeting in Versailles, France, we have decided to update the section of 
the protocol dealing with insertional mutagenesis. This is based on the following: 1) this section of the 
protocol was taken from previous protocols written shortly after the leukemia SAEs in the original SCID-
X1 trial and thus are quite old; 2) the experience in the gene therapy trial for thalassemia in Paris of 
non-leukemia related clonal skewing indicates that clonal skewing is not always predictive of 
leukemogenesis; 3) clinical and/or laboratory abnormalities are more likely to indicate possible 
leukemia. Thus we feel the new wording more accurately reflects the situation that might evolve in the 
setting of leukemia. The language change in this section was approved and recommended by the 
DSMB at the December 18, 2012 meeting.   
 
Section 10.1.3 
 Change in monitoring from EQUIP to Christine McNally – Dufort, RN, MBA 
 
Section 17 
Addition of qualifications of Colleen Dansereau, RN as Project Manager 
 
Version 10.0 (IRB approval date 8/21/2013) 
 
Section 8.1 
Deletion of previous gene transfer as exclusion criteria 
 
Section 10.2.3 
 
Definition of 2nd gene transfer procedure for patients that are deemed an early failure.  
 
Section 10.2.4 
 
Description of eligibility criteria for 2nd gene transfer procedure for patients who fail immune 
reconstitution. The eligibility criteria will be the same as the initial enrollment.  
 
Appendix 4 
 
Inclusion of timepoints for 2.5, 3.5, 4.5, and 5.5 years for insertional analysis samples.  
 
Version 11.0 (IRB 11/14/13) 
 
Section 15.1 Data Handling, Record Keeping, Sample Storage 
 
To clarify the plan for data analysis to include data transferred in a coded fashion from European sites, 
the following text was added. 
 
CTIP will also analyze data shared from the European sites as specified including data for purpose of 
joint publication (Section 12, European data considerations). Data shared from European sites will be 
obtained according to local approved practices and transferred in a coded fashion. 
 
The change does not require any modification of the consent form, per discussion with Susan 
Kornetsky—Director of Clinical Research Compliance, Boston Children’s Hospital Office of Clinical 
Investigation. 
 
Version 12.0 (IRB 8/22/16) 
 

82 
Version 12.0 
January 4, 2022 
Front page: replace Dr Notarangelo with Dr Sung-Yun Pai as PI 
 
Section 4.4.1 (p. 18) inclusion of data from PIDTC demonstrating effects of conditioning on HSCT 
outcomes in SCID patients. 
 
Section 4.4.2 (p. 20) inclusion of data from PIDTC demonstrating effects of conditioning on CD4+ 
counts in SCID patients. 
 
Section 5.3.1 (p. 23) inclusion of data from allogeneic transplants and previous gene transfer trials 
demonstrating effects of conditioning on engraftment.  
 
Section 7 (p. 36) clarification of enrollment numbers; 5 patients will receive busulfan conditioning. 
 
Section 8.1 (p. 37) clarification of eligibility for conditioning. 
 
Section 9.1.1 (p. 39) inclusion of mobilized peripheral blood leukapheresis as cell collection option. 
 
Section 9.1.2 (p. 39) inclusion of back-up collection for patients receiving busulfan. 
 
Section 9.2 (p. 39) addition of low dose busulfan conditioning for selected subjects. 
 
Section 9.3.3 and 9.3.4 (p. 41) infusion of transduced cells and criteria for infusion of back-up cells. 
 
Section 10.1.14 (p. 43) inclusion of additional assessments for patients receiving busulfan. 
 
Section 10.3 (p 45) procedure for failure of hematologic reconstitution in patients receiving busulfan. 
 
Section 11.4 (p.50) addition of side effects for busulfan conditioning. 
 
Tables 2a, 2b, 2c (p. 55-57): adverse event reporting requirements. 
 
Appendix 2: updated eligibility diagram. 
 
Appendix 5: busulfan dosing guidelines.