A Phase I/II Clinical Trial of Hematopoietic Stem Cell Gene Therapy for the Wiskott-Aldrich Syndrome
Phase: PHASE1
Conditions: Wiskott-Aldrich Syndrome (WAS)
Sponsor: Fondazione Telethon

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 1 of 119 
-- 
 
 
 
 
TITLE PAGE 
 
Information Type: 
Clinical Protocol 
 
Clinical Trial Protocol Title: 
A phase I/II clinical trial of hematopoietic stem cell gene 
therapy for the Wiskott-Aldrich Syndrome (TIGET-WAS) 
Study Number: 
TIGET-WAS 
Compound Number: OTL-103 (formerly known as GSK2696275 and Telethon003) 
Development Phase: I/II 
EudraCT number: 2009-017346-32 
Protocol Version: 12, 19 Sep 2023 
 
Rev. 
Description of the revisions 
Date 
1 
First issue 
15-10-2009 
2 
As requested by ISS: action to be taken in case of microbiological contamination 
22-01-2010 
3 
Editing and integration of amendment 1 related to Informed Consent Form: reporting a case of leukemia 
occurred during a study conducted in Germany with the retroviral vector encoding WASP 
14-10-2011 
4 
Editing, integration of new endpoints, better definition of time line for endpoints, and integration of 
amendment 4 related to Informed Consent Form: reporting 4 cases of leukemia 
10-05-2012 
Cytokine (SCF) production changed from Amgen to mobile Genix due to the interruption of the 
manufacturer production of cytokine (SCF) by Amgen. [ref: Amendment 5] 
28-02-2013 
5 
Follow up extension (from 3 years to 8 years); Promoter change from Fondazione Centro San Raffaele 
del Monte Tabor to Ospedale San Raffaele. [ref: CTA Amendment 6] 
10-04-2013 
6 
Change sample size (from 6 to 8), additional option for administration of MPB only, elimination of 
exclusion criterion 5 (revertant cell population) and change Financial Sponsor (from Fondazione 
Telethon to GSK R & D Ltd) [ref: CTA Amendment 7]; 
10-03-2014 
Communication of change in analytical method for testing WAS LVV QC [ref: Amendment 8]; 
25-09-2014 
7 
Discretional use of ATG in patients with clinical autoimmunity prior to gene therapy, addition of 
Plerixafor use for PBSC mobilization and change in Medicinal Product primary package (bag). Change 
of PI. [ref: CTA Amendment 9] 
09-01-2015 
Communication related to minor changes in Certificate of Analysis for SCF and Flt-3L [ref: 
Amendment 10] 
09-06-2015 
8 
Change regulatory sponsor (promoter) from Ospedale San Raffaele to GSK Research & Development 
Ltd) [ref: CTA Amendment 11] 
29-Jan-2016 
9 
Extended Long-Term Follow up (post-8 years); new assessment on platelet activation and morphology 
[ref: CTA Amendment 12] 
31-01-2018 
10 
Change of Sponsorship to Orchard Therapeutics 
27-06-2018 
11 
Entry to a separate observational long-term follow-up (OLTFU) study; simplified strategy for RCL 
testing; possibility to perform remote follow-up visits. 
22-05-2020 
Communication related to change in PV and medical monitor contact details; deletion of the mention of 
special interest for disease-related events 
14-07-2021 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 2 of 119 
-- 
 
 
 
 
 
Communication related to Medical Monitor and Sponsor 
16-02-2023 
Communication related to Medical Monitor and Sponsor Signatory 
04-08-2023 
12 
Change of Sponsorship (from Orchard Therapeutics to Fondazione Telethon ETS) 
19-09-2023 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 3 of 119 
-- 
 
 
 
 
 
Persons supplied with this information must understand that it is strictly confidential. 
Information contained herein cannot be disclosed, submitted for publication or used for any 
purpose other than that contemplated herein without the Sponsor’s prior written authorisation. 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 4 of 119 
-- 
 
 
 
 
PROTOCOL SYNOPSIS 
 
Title (Acronym) 
A phase I/II clinical trial of hematopoietic stem cell gene therapy for the 
Wiskott-Aldrich Syndrome (TIGET-WAS) 
Objectives 
Primary Objective 
Secondary Objectives 
Primary objectives: 
1) To evaluate the safety of the administration of autologous CD34+ cells 
transduced with a lentiviral vector containing the WASP gene in patients 
with WAS, after a reduced intensity conditioning regimen; 
2) To evaluate the long-term engraftment of WASP-expressing transduced 
cells; 
3) To evaluate the efficacy of gene therapy assessed as: 
3.1) Improvement of the patient’s immune function, specifically of 
T cell function and antigen-specific responses to vaccinations; 
3.2) Improvement of thrombocytopenia. 
Secondary objectives: 
To evaluate the efficacy of gene therapy in improving the patient’s clinical 
conditions assessed by a reduction in frequency of severe infections, and 
bleeding episodes and reduction of autoimmunity phenomena and eczema. 
Endpoints 
Primary Endpoints 
Secondary Endpoints 
Safety endpoints: 
Primary safety endpoints: 
1) Conditioning regimen-related safety, consisting in the absence of 
engraftment failure or delayed hematological reconstitution (prolonged 
aplasia) and surveillance of non hematological regimen related toxicity (for 
clinical features CTCAE ≥2, for metabolic/laboratory CTCAE ≥3); 
2) Safety of lentiviral gene transfer into HSC: 
- Short-term safety and tolerability of lentiviral-transduced cell infusion; 
- Long-term safety of lentiviral-transduced cell infusion (absence of 
Replication competent lentivirus (RCL) and abnormal clonal proliferation). 
Secondary safety endpoints 
1) Lack of immune response to transgene 
2) Overall safety of the treatment. 
Efficacy endpoints: 
Primary efficacy endpoints: 
1) Overall survival; 
2) Sustained engraftment of genetically corrected hematopoietic stem cells 
in peripheral blood and/or in bone marrow; 
3) Expression of vector-derived WASP; 
4) Improved T-cell functions; 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 5 of 119 
-- 
 
 
 
 
 
5) Antigen-specific responses to vaccination; 
6) Improved platelet count and MPV normalization. 
Secondary efficacy endpoints: 
1) Multiline age engraftment of genetically corrected cells; 
2) Reduced frequency of severe infections; 
3) Reduced bleeding episodes; 
4) Reduced autoimmunity phenomena; 
5) Improvement in eczema; 
6) Improved quality of life. 
Exploratory efficacy endpoint: 
1) Normalisation of platelet activation profile and morphology 
Criteria of assessment 
for: 
Safety 
Efficacy 
Safety assessment: 
1) For safety of LV gene transfer a monitoring plan and analyses of 
integrations will be performed. 
2) For immunogenicity, antibody response to WASP protein will be 
evaluated. 
3) For overall safety a descriptive analysis will be performed on AE, AR, 
SAE/SAR, UAR, SUSAR, and events will be classified for frequency, 
severity and body system involved. 
Efficacy assessment: 
1) Overall Survival; 
2) Engraftment of genetically corrected hematopoietic progenitors and/or 
differentiated cells in peripheral blood and/or in bone marrow; 
3) Expression of vector-derived WASP; 
4) Improved in vitro T cell functions; 
5) Improved platelet count and normalization of mean platelet volume 
(MPV); 
6) Reduced bleeding episodes; 
7) Antigen-specific responses to vaccinations; 
8) Reduced frequency of severe infections; 
9) Modification in eczema and autoimmunity phenomena; 
10) Improved quality of life; 
11) Platelet morphology and activation after stimulation with agonists. 
Protocol design 
Non randomized, open label, monocentric, phase I/II, prospective, 
involving a single infusion of autologous CD34 positive cells transduced 
with a 3rd generation VSV-G pseudo typed lentiviral vector in 8 patients 
affected by WAS. 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 6 of 119 
-- 
 
 
 
 
 
Description of the Protocol 
Five protocol phases are foreseen, after signature of the informed consent: 
1) Screening phase, during which the conditions required by the clinical 
protocol for patients’ enrollment will be assessed and fulfillment of the 
inclusion/exclusion criteria of the study will be evaluated; 
2) Baseline phase, carried from the end of the screening phase to the day 
before the harvest of peripheral blood stem cells, if performed, or the day 
before Rituximab administration (day -22); 
3) Treatment phase, from the end of the Baseline to day 0; 
4) Follow Up phase: the Follow Up phase is composed of an Initial 
Follow-Up period of at least 3 years, when patients are intended to reach 
the primary efficacy analysis timepoint, and then a further Long-Term 
Follow-Up period from 3 up to 8 years; 
5) Extended Long-Term Follow Up (ELTFU) phase: patients who complete 
the Follow up phase will then be contacted annually, in order to collect 
long-term safety and selected efficacy data, until a separate observational 
long-term follow-up (OLTFU) study is initiated and patients have enrolled 
in it. 
Once the OLTFU study is set up, patients will be invited to enter the 
OLTFU study at any time after they have completed a minimum of 5 years 
follow up in this study (WAS Pivotal, 201228). As part of the OLTFU 
study, patients will continue to be followed up for a total of 15 years from 
the date of treatment with gene therapy. 
Experimental drug 
description 
(dose/regimen/etc.) 
Medicinal Product 
The Medicinal Product consists of autologous CD34+ cells collected from 
the bone marrow and/or peripheral blood and transduced with a lentiviral 
vector encoding WASP controlled by WAS promoter sequences (w1.6W). 
Dosage indications 
A minimum dose of 2 x 106 CD34+ cells/Kg (maximum 20 x 106 CD34+ 
cells/Kg) and an optimal dose of 5-10 x 106 CD34+ cells/Kg will be infused 
i.v. 
Conditioning Regimen 
A reduced conditioning regimen will be administered before the autologous 
engineered stem cells infusion: 
- I.v. Busulfan: body weight-based and AUC targeted dose (range: 0,8-1,2 
mg/kg/dose), 8 (+ 1) doses administered every 6 hours from day -3 to day - 
1; 
- I.v. Fludarabine: 30 mg/sqm/day, on days -3 and -2; 
- I.v. Rituximab: 375 mg/sqm/day, on day -22. 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 7 of 119 
-- 
 
 
 
 
 
I.v. ATG Thymoglobuline may be used at PI discretion in patients with 
clinical autoimmunity before day-18. 
Inclusion criteria 
- Inclusion 
- Exclusion 
Inclusion Criteria 
1) Diagnosis of WAS defined by genetic mutation and at least one of the 
following criteria: 
- Severe WAS mutation; 
- Absent WASP expression; 
- Severe clinical score (Zhu clinical score ≥ 3). 
2) No HLA-identical sibling donor 
AND 
3.1) Negative search for a matched unrelated donor (10/10) or an adequate 
unrelated cord blood donor (6/6) within 4-6 months; 
OR 
3.2) Patients of > 5 years of age who are not candidate to unrelated 
allogeneic transplant based on clinical conditions. 
AND 
4) Parental/guardian/patient signed informed consent. 
Exclusion Criteria 
Patients who meet any of the following criteria will be excluded from study 
admission: 
1) Patients positive for HIV-infection; 
2) Patients affected by neoplasia; 
3) Patients with cytogenetic alterations typical of MDS/AML; 
4) Patients with end-organ functions or any other severe disease which, in 
the judgment of the investigator, would make the patient inappropriate for 
entry into this study; 
5) Patients who underwent an allogeneic hematopoietic stem cell 
transplantation in the previous 6 months; 
6) Patients who underwent an allogeneic hematopoietic stem cell 
transplantation with evidence of residual cells of donor origin. 
Sample size 
Eight patients will be treated in this study 
Accrual/duration 
timeline 
Patients will undergo regular follow up after gene therapy to assess the 
efficacy and safety of the treatment. 
1) For each subject the main study phase will be completed once the Initial 
Follow Up period of 3 years following treatment with OTL-103 is finished. 
2) A Long- Term Follow up period will then be conducted from 3 years up 
to 8 years after the reinfusion of transduced cells. 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 8 of 119 
-- 
 
 
 
 
 
3) For subjects who complete the Long-Term Follow Up phase, an 
ELTFU phase will then continue from 8 years after the reinfusion of 
transduced cells until a separate OLTFU is initiated 
and subjects have enrolled in it. As part of the OLTFU study, patients will 
continue to be followed up for a total of 15 years from the date of treatment 
with gene therapy. 
The end of the study is when all subjects have completed a minimum of 5 
years follow up and transitioned to a separate OLTFU study. Should a 
patient decide not to consent to participate in the ELTFU nor transition to 
the separate OLTFU study, then the subject will be considered to have 
completed this study and will be transitioned to standard of care according 
to local practice. 
Statistical design 
A descriptive analysis will be performed for all the endpoints (safety and 
efficacy). 
Percentage of subjects with sustained engraftment of genetically corrected 
hematopoietic stem cells at 1 year and percentage of those with detectable 
vector-derived WASP expression will be calculated, with 95% Confidence 
Intervals. The mean change between the pre-gene therapy and the 1-year 
values of vitro T cell proliferation will be calculated, with 95% CI. The 
percentage of subjects who improved the level of platelet count at one, two 
and three years after gene therapy compared to baseline, will be estimated, 
with 95% CI. 
When all the treated patients complete the 3-year regular follow up visit 
after gene therapy (study phase), a formal statistical analysis will be 
performed to assess the efficacy and safety of the investigational product 
(study phase analysis). A dedicated clinical study report will be generated 
based on all the data available at this time point. 
At the end of the study, a final statistical analysis will be conducted with all 
data available and the clinical study report will be updated accordingly. 
Rules for: 
Protocol modification: 
- 2 out of the first 3 patients suffering of prolonged aplasia; 
- 2 or more failures of engraftment of transduced cells. 
Early Stopping: 
- 2 transplant-related deaths; 
- 1 malignant proliferation related to gene therapy. 
In the interest of the patients’ safety, the third patient will be treated when: 
- at least 1 of the first 2 patients will achieve hematopoietic recovery; 
- at least 60 days after the first treatment. 
For the subsequent patients the above rules will be applied. 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 9 of 119 
-- 
 
 
 
 
TRIAL FLOW-CHART 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
PBSC mobilisation 
followed by 
leukapheresis for 
HSC collection for 
back-up and DP 
manufacture (if 
required) or BM 
harvest, 
chemotherapy, gene 
therapy schedule 
Evaluation of safety and 
efficacy, from +1 day up to 
8 years. The Follow Up 
phase is composed of an 
Initial Follow Up period of 
at least 3 years, when 
patients are intended to 
reach the primary efficacy 
analysis timepoint, and 
then a further Long-Term 
Follow Up period from 3 
up to 8 years. 
Evaluation of safety 
and efficacy, from 8 
years, up until a 
separate 
observational long- 
term follow up 
(OLTFU) study is 
available and 
patients have been 
enrolled in it. 
Treatment 
Extended Long- 
Term Follow Up 
(ELTFU) 
Follow-Up phase 
(Initial Follow Up period, 
Long-Term Follow Up 
period) 
Clinical laboratory 
and instrumental 
evaluation 
BM Back-up 
collection and 
storage (for patients not 
undergoing PBSC mobilization) 
Informed consent 
signature 
Preliminary 
evaluation and 
inclusion/exclusion 
criteria assessment 
Enrolment 
Baseline 
Screening phase 
Observational Long- 
Term Follow Up 
(OLTFU) study 
A separate observational 
long-term follow up 
(OLTFU) study will be set 
up, which patients will be 
invited to enter at any time 
after completing a 
minimum of 5 years follow 
up in this study 
(WAS Pivotal 201228) 
study. 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 10 of 119 
-- 
 
 
 
 
SPONSOR and PRINCIPAL INVESTIGATOR INFORMATION PAGE 
 
 
SPONSOR legal 
registered address 
Fondazione Telethon ETS 
Via Varese, 16b, 00185, Roma, Italia 
 
Principal Investigator 
Alessandro Aiuti, MD, PhD 
Deputy Director, SR-Tiget 
Director of Pediatric Immunohaematology Unit 
IRCCS San Raffaele Hospital 
Professor of Pediatrics "Vita – Salute” San Raffaele University 
Ospedale San Raffaele 
Via Olgettina 58/60 
20132 Milan, Italy 
Tel. +39 02.2643.4472 / Fax: +39 02.2643.6545, 
email: aiuti.alessandro@hsr.it 
 
 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 11 of 119 
-- 
 
 
 
 
 
 
 
SPONSOR SIGNATORIES 
 
 
Fondazione Telethon ETS 
 
 
 
 
 
 
 
Stefano Zancan 
 
 
 
 
 
 
 
Date 
Clinical Development Head  
 
 
 
 
 
 
Fondazione Telethon ETS

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 12 of 119 
-- 
 
 
 
 
 
LABORATORY INFORMATION PAGE 
 
Routine haematology 
Clinical chemistry 
Immunophenotyping 
Genetic analysis 
Microbiology 
SIMT, 
Servizio di Immunoematologia E Medicina Trasfusionale, 
Ospedale San Raffaele S.p.A, 
Via Olgettina 60, 
20132 Milano, 
Italy 
Servizio di Medicina di Laboratorio Ospedale 
San Raffaele S.p.A, 
Via Olgettina 60, 
20132 Milano, 
Italy 
PK analysis 
IRCCS Foundation Policlinico San Matteo, 
Dipartimento di Farmacologia, 
Pavia, 
Viale Golgi no.19, 27100, 
Italy 
Serum antibody levels to 
vaccinal antigens 
Immunologische Tagesklinik Biomedizinisches Institute, 
Schwarzspanierstraße 15, 1090 Wien, 
Austria 
RCL detection 
GenoSafe, 
1 rue de l'Internationale, 
91000 Evry, 
France 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 13 of 119 
-- 
 
 
 
 
INVESTIGATOR PROTOCOL AGREEMENT PAGE 
• I confirm agreement to conduct the study in compliance with the protocol. 
• I acknowledge that I am responsible for overall study conduct. I agree to personally conduct 
or supervise the described study. 
• I agree to ensure that all associates, colleagues and employees assisting in the conduct of the 
study are informed about their obligations. Mechanisms are in place to ensure that site staff 
receives the appropriate information throughout the study. 
 
 
 
Investigator Name: 
Investigator Signature 
Date 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 14 of 119 
-- 
 
 
 
 
TABLE OF CONTENTS 
TITLE PAGE ................................................................................................................................... 1 
PROTOCOL SYNOPSIS ................................................................................................................. 4 
TRIAL FLOW-CHART ................................................................................................................... 9 
 SPONSOR and PRINCIPAL INVESTIGATOR INFORMATION PAGE .................................. 10 
SPONSOR SIGNATORIES  ......................................................................................................... 11 
LABORATORY INFORMATION PAGE .................................................................................... 12 
INVESTIGATOR PROTOCOL AGREEMENT PAGE ............................................................... 13 
ABBREVIATIONS AND DEFINITIONS .................................................................................... 21 
1. 
BACKGROUND AND RATIONALE ....................................................................... 24 
1.1. 
Scientific Background and Rationale .......................................................................... 24 
1.1.1. 
WAS – Description of the condition ........................................................................... 24 
1.1.2. 
Molecular aspects of the disease ................................................................................. 25 
1.1.3. 
Clinical manifestations ................................................................................................ 25 
1.1.3.1. 
Thrombocytopenia and bleeding episodes .................................................................. 25 
1.1.3.2. 
Recurrent infections .................................................................................................... 26 
1.1.3.3. 
Eczema ........................................................................................................................ 26 
1.1.3.4. 
Autoimmunity ............................................................................................................. 26 
1.1.3.5. 
Cancer ......................................................................................................................... 27 
1.1.4. 
Classification of WAS patients ................................................................................... 27 
1.1.5. 
Current methods of treatment and limitations ............................................................. 28 
1.1.6. 
Rationale for GT in WAS ........................................................................................... 30 
1.1.6.1. 
Exploiting the selective advantage for WAS-expressing cells ................................... 31 
1.1.6.2. 
Choice of conditioning regimen and cell dose ............................................................ 32 
1.1.6.3. 
Choice of lentiviral vector ........................................................................................... 34 
1.1.7. 
Telethon-Généthon common efforts in the development of lentiviral vector 
gene therapy for WAS ................................................................................................. 34 
1.1.8. 
Ethical considerations ................................................................................................. 35 
2. 
PRODUCT MANUFACTURING AND CHARACTERIZATION ........................... 37 
2.1. 
Medicinal product definition ....................................................................................... 37 
2.2. 
Lentiviral vector encoding for WASP......................................................................... 37 
2.3. 
Establishment of MCBs .............................................................................................. 38 
2.4. 
Lentiviral Vector production ....................................................................................... 38 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 15 of 119 
-- 
 
 
 
 
2.5. 
Production of transduced CD34+ cells ....................................................................... 39 
2.5.1. 
Cell isolation ............................................................................................................... 39 
2.5.1.1. 
Isolation of BM CD34+ cells ...................................................................................... 39 
2.5.1.2. 
Isolation of mobilized peripheral blood CD34+ cells ................................................. 39 
2.5.2. 
Transduction of CD34+ cells ...................................................................................... 39 
2.6. 
Preclinical studies ....................................................................................................... 40 
2.6.1. 
Introduction ................................................................................................................. 40 
2.6.2. 
Preclinical studies performed with the proposed therapeutic vector .......................... 41 
2.6.2.1. 
In vitro studies ............................................................................................................. 41 
2.6.2.2. 
In vivo studies ............................................................................................................. 42 
2.7. 
Studies and considerations on safety related to the medicinal product ....................... 43 
2.7.1. 
Biodistribution and vector shedding ........................................................................... 43 
2.7.2. 
Germline transmission ................................................................................................ 45 
2.7.3. 
Genotoxicity and carcinogenicity ................................................................................ 46 
2.7.3.1. 
Integration analyses in other trials .............................................................................. 48 
2.7.4. 
Immunogenicity .......................................................................................................... 49 
2.7.5. 
Environmental risks .................................................................................................... 49 
2.8. 
Clinical trials performed in other countries with similar Drug Substance .................. 50 
2.8.1. 
Gene Therapy for Wiskott-Aldrich-Syndrome using a retroviral vector .................... 50 
2.8.2. 
Gene Therapy for Adrenoleukodystrophy using a lentiviral vector............................ 51 
3. 
OBJECTIVES ............................................................................................................. 52 
3.1. 
Primary Objectives ...................................................................................................... 52 
3.2. 
Secondary objectives ................................................................................................... 52 
4. 
INVESTIGATIONAL PLAN ..................................................................................... 53 
4.1. 
Study Design ............................................................................................................... 53 
4.1.1. 
Type of study .............................................................................................................. 53 
4.1.2. 
Study duration ............................................................................................................. 53 
4.1.3. 
Study population ......................................................................................................... 54 
4.2. 
Safety Endpoints ......................................................................................................... 54 
4.2.1. 
Primary safety endpoints ............................................................................................. 54 
4.2.1.1. 
Safety of reduced conditioning regimen ..................................................................... 54 
4.2.1.2. 
Safety of LV gene transfer into HSC .......................................................................... 55 
4.2.2. 
Secondary safety endpoints ......................................................................................... 55 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 16 of 119 
-- 
 
 
 
 
4.3. 
Efficacy Endpoints ...................................................................................................... 56 
4.3.1. 
Primary efficacy endpoints ......................................................................................... 56 
4.3.2. 
Secondary efficacy endpoints ..................................................................................... 57 
4.3.2.1. 
CTCAE grading scale ................................................................................................. 58 
4.3.3. 
Exploratory efficacy endpoint ..................................................................................... 59 
5. 
PATIENT SELECTION AND RECRUITMENT ...................................................... 59 
5.1. 
Study of population ..................................................................................................... 59 
5.1.1. 
Inclusion criteria ......................................................................................................... 59 
5.1.2. 
Exclusion criteria ........................................................................................................ 60 
5.1.3. 
Sample size ................................................................................................................. 60 
5.1.4. 
Patient withdrawal ....................................................................................................... 60 
5.1.4.1. 
Natural study termination for a single enrolled patient ............................................... 60 
5.2. 
Phases of study ............................................................................................................ 61 
6. 
TREATMENT PROTOCOL ...................................................................................... 64 
6.1. 
Treatment administration ............................................................................................ 64 
6.2. 
Stem cell back-up, collection and purification of mobilized CD34+ cells ................. 64 
6.3. 
Bone marrow harvest .................................................................................................. 66 
6.4. 
Cell manipulation and gene transfer with the lentiviral vector ................................... 66 
6.4.1. 
BM CD34+ cell purification ....................................................................................... 66 
6.4.2. 
Lentiviral vectors ........................................................................................................ 66 
6.4.3. 
Gene transfer of BM CD34+ cells .............................................................................. 66 
6.4.4. 
Gene transfer of MPB CD34+ cells ............................................................................ 67 
6.5. 
Conditioning regimen ................................................................................................. 67 
6.5.1. 
Busulfan ...................................................................................................................... 68 
6.5.2. 
Fludarabine .................................................................................................................. 69 
6.5.3. 
Rituximab .................................................................................................................... 70 
6.6. 
Safety considerations on the procedure and conditioning regimen............................. 70 
6.7. 
Packaging and labelling of the Medicinal Product ..................................................... 71 
6.8. 
Dose of the Medicinal Product.................................................................................... 72 
6.9. 
Infusion ....................................................................................................................... 72 
6.10. 
Hospitalization ............................................................................................................ 72 
6.11. 
Supportive therapy ...................................................................................................... 72 
6.12. 
Back-up reinfusion ...................................................................................................... 74 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 17 of 119 
-- 
 
 
 
 
6.13. 
Responsibilities ........................................................................................................... 74 
7. 
STUDY ASSESSMENTS ........................................................................................... 75 
7.1. 
Pre-treatment evaluation (screening and baseline) ..................................................... 75 
7.2. 
Post-treatment evaluation ............................................................................................ 77 
7.3. 
Extended Long-Term Follow Up ................................................................................ 78 
8. 
ASSESSMENT OF SAFETY ..................................................................................... 83 
8.1. 
Adverse Events ........................................................................................................... 83 
8.1.1. 
Adverse Event (AE) .................................................................................................... 83 
8.1.2. 
Adverse Reaction (AR) ............................................................................................... 83 
8.1.3. 
Serious adverse event (SAE) ....................................................................................... 83 
8.1.4. 
Unexpected adverse reaction (UAR) .......................................................................... 83 
8.1.5. 
Suspected unexpected serious adverse reaction (SUSAR) ......................................... 83 
8.2. 
Pharmacovigilance Procedure ..................................................................................... 83 
8.2.1. 
Reporting of Serious Adverse Events (SAEs) ............................................................ 83 
8.2.2. 
Regulatory Reporting Requirements for SAEs ........................................................... 84 
8.2.3. 
Adverse event recording ............................................................................................. 84 
8.2.4. 
Expected AE/AR ......................................................................................................... 85 
8.2.5. 
Follow up of AE / AR / SAE / SAR ........................................................................... 85 
8.2.6. 
Follow up procedure for withdrawn patients .............................................................. 86 
8.3. 
Assessment of safety endpoints .................................................................................. 86 
8.3.1. 
Safety of the conditioning regimen ............................................................................. 86 
8.3.2. 
Safety of the transduced cell infusion ......................................................................... 86 
8.4. 
Specific safety monitoring plan for gene therapy ....................................................... 86 
8.4.1. 
Replication competent lentivirus (RCL) ..................................................................... 87 
8.4.2. 
Abnormal clonal proliferation (ACP) ......................................................................... 87 
8.4.3. 
Immune response to transgene .................................................................................... 88 
9. 
ASSESSMENT OF EFFICACY ................................................................................. 90 
9.1. 
Sustained engraftment of genetically corrected cells in peripheral blood 
and/or in bone marrow ................................................................................................ 90 
9.2. 
Expression of vector-derived WASP .......................................................................... 90 
9.3. 
Improved T-cell functions ........................................................................................... 90 
9.4. 
Antigen-specific responses to vaccinations ................................................................ 90 
9.5. 
Improved platelet count and normalization of MPV .................................................. 91 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 18 of 119 
-- 
 
 
 
 
9.6. 
Reduced bleeding episodes ......................................................................................... 91 
9.7. 
Reduced frequency of severe infections ..................................................................... 91 
9.8. 
Modification of eczema and autoimmunity phenomena ............................................. 92 
9.9. 
Improved quality of life .............................................................................................. 92 
9.10. 
Platelet activation profile and morphology (exploratory endpoint) ............................ 92 
10. 
STUDY MONITORING ............................................................................................. 93 
10.1. 
Responsibilities of the Investigator(s) ......................................................................... 93 
10.2. 
Use and completion of Case Report Forms (CRFs) .................................................... 93 
10.3. 
Definition of Source Data ........................................................................................... 93 
10.4. 
Trial Monitoring .......................................................................................................... 94 
10.5. 
Qualifications of Monitors .......................................................................................... 94 
10.6. 
Monitoring Procedures ................................................................................................ 94 
10.7. 
Monitoring Report ...................................................................................................... 94 
11. 
ETHICAL CONSIDERATION .................................................................................. 95 
11.1. 
Informed Consent ........................................................................................................ 95 
11.2. 
Investigator's Brochure ............................................................................................... 95 
11.3. 
Minimizing distress ..................................................................................................... 96 
11.4. 
Minimizing Risk ......................................................................................................... 97 
12. 
STATISTICAL DESIGN ............................................................................................ 98 
12.1. 
Protocol modification and stopping rules ................................................................... 98 
12.2. 
Study Population and Timing of Analysis .................................................................. 98 
12.3. 
Safety Endpoints ......................................................................................................... 98 
12.4. 
Efficacy endpoints ....................................................................................................... 98 
12.5. 
Note on Confidence Interval calculation ..................................................................... 99 
12.6. 
Integration of results by other trials on GT ............................................................... 100 
12.7. 
Long-term follow up ................................................................................................. 100 
13. 
QUALITY ASSURANCE AND QUALITY CONTROL ........................................ 101 
13.1. 
Definitions ................................................................................................................. 101 
13.2. 
Monitoring ................................................................................................................ 101 
13.3. 
Deviation from study protocol. ................................................................................. 101 
13.4. 
Audits and inspections .............................................................................................. 101 
14. 
ADMINISTRATIVE RULES ................................................................................... 102 
14.1. 
Insurance ................................................................................................................... 102 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 19 of 119 
-- 
 
 
 
 
14.2. 
Financing ................................................................................................................... 102 
14.3. 
Final study report ...................................................................................................... 102 
15. 
BIBLIOGRAPHY ..................................................................................................... 103 
16. 
APPENDICES .......................................................................................................... 110 
16.1. 
Appendix 1 Summary of Significant Changes Introduced by Protocol 
Amendment Version 9 .............................................................................................. 110 
16.2. 
Appendix 2 Summary of Significant changes introduced by Protocol 
Amendment Version 8 .............................................................................................. 114 
16.3. 
Appendix 3: Summary of Significant Changes Introduced by Protocol 
Amendment Version 10 ............................................................................................. 118 
16.4. 
Appendix 4: Summary of Significant Changes Introduced by Protocol 
Amendments Version 11 and 11.1 ............................................................................ 119 
      16.5             Appendix 5: Summary of Significant Changes Introduced by Protocol 
Amendments Version 12 .................................................................................................................... 119 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 20 of 119 
-- 
 
 
 
 
 
LIST OF TABLES 
Table 1 
5-year survival after allogeneic HSCT in WAS patients according to donor 
type .............................................................................................................................. 29 
Table 2 
Efficacy of HSCT in a large cohort of WAS survivors .............................................. 29 
Table 3 
Overall survival (OS) and failure-free survival (FFS) 5 years after stem cell 
transplantation in WAS patients, according to donor type and age at the time 
of transplant ................................................................................................................. 29 
Table 4 
Overall survival (OS) 7 years after stem cell transplantation in WAS 
patients, according to donor type and age at the time of transplant ............................ 30 
Table 5 
Stratification of platelet count according to severity of thrombocytopenia 
(count x109/L) ............................................................................................................. 57 
Table 6 
Busulfan dosage according to patient’s weight ........................................................... 68 
Table 7 
Gene therapy schedule for WAS patients .................................................................. 69 
Table 8a 
Pre and post-treatment evaluation (up to 36 months after gene therapy) ................... 80 
Table 9 
WAS scoring system according to Zhu. ...................................................................... 82 
Table 10 
Safety follow up (from 0 to 3 years) ........................................................................... 89 
 
LIST OF FIGURES 
Figure 1 
Study Flow Chart ........................................................................................................ 63 
Figure 2 
PBSC Mobilization Protocol ....................................................................................... 65 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 21 of 119 
-- 
 
 
 
 
ABBREVIATIONS AND DEFINITIONS 
 
ADA-SCID 
Adenosine deaminase severe combined immunodeficiency 
AE 
Adverse event 
AIEOP 
Italian Association of Paediatric Hematology and Oncology 
AIHA 
Autoimmune haemolytic anaemia 
ALD 
Adrenoleukodystrophy 
ANC 
Absolute neutrophil count 
ASGT 
American Society of Gene & Cell Therapy 
ATG 
Anti-thymocyte globulin 
AUC 
Area under the curve 
AR 
Adverse reaction 
BMT 
Bone marrow transplantation 
CGD 
Chronic granulomatous disease 
CIS 
Common insertion sites 
CMV 
Cytomegalovirus 
CRF 
Case reports forms 
CTCAE 
Common Terminology Criteria for Adverse Events 
DC 
Dendritic cells 
DMSO 
Dimethyl sulfoxide 
DP 
Drug Product 
DS 
Drug Substance 
EBMT 
European Society for Blood and Marrow Transplantation 
eCRF 
Electronic case report form 
EBV 
Epstein Barr virus 
EMA 
European Medicines Agency 
FACS 
Fluorescence-activated cell sorting 
FFS 
Failure Free Survival 
GCP 
Good clinical practice 
G-CSF 
Granulocyte-colony stimulating factor 
GITMO 
Italian Bone Marrow Transplantation Group 
GMP 
Good manufacturing practice 
GTMPs 
Gene Therapy Medicinal Products 
GVHD 
Graft versus host disease 
ELTFU 
Extended long-term follow up 
HIV 
Human immunodeficiency virus 
HCP 
Healthcare Professional 
HSC 
Hematopoietic stem cells 
HSCT 
Hematopoietic stem cell transplantation 
HSPC 
Hematopoietic stem or progenitor cell 
HPC 
Hematopoietic progenitor cell 
SR-TIGET 
The San Raffaele Telethon Institute for Gene Therapy 
IB 
Investigators brochure 
IC 
Informed consent 
IMPD 
Investigational Medicinal Product Dossier 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 22 of 119 
-- 
 
 
 
 
ISCT 
International Society for Cellular Therapy 
IV 
Intravenous 
IVIG 
Intravenous immunoglobulin 
ITP 
Idiopathic thrombocytopenic purpura 
JACIE 
Joint Accreditation Committee ISCT-Europe & EBMT 
LAM-PCR 
Linear-amplification mediated polymerase chain reaction 
LM-PCR 
Ligation- mediated polymerase chain reaction 
LTR 
Long terminal repeats 
LV 
Lentiviral vectors 
mAb 
monoclonal Antibody 
MCB 
Master Cell Bank 
MLD 
Metachromatic leukodystrophy 
MLV 
Moloney leukemia virus 
MMFD 
Mismatched family donor 
MMUD 
Mismatched unrelated donor 
MNC 
Mononuclear cell 
MND 
Myeloproliferative sarcoma virus enhancer, Negative control region, 
Deleted, dl587rev primer-binding site substituted 
MOI 
Multiplicity of Infection 
MPB 
Mobilized peripheral blood 
MPV 
Mean platelet volume 
MSD 
Matched sibling donor 
MUD 
Matched unrelated donor 
NCI 
National Cancer Institute 
NK 
Natural killer 
OLTFU 
Observational long-term follow up 
OOS 
Out Of Specification 
OS 
Overall Survival 
PB 
Peripheral blood 
PBSC 
Peripheral blood stem cells 
PID 
Primary immune deficiency 
PLT 
Platelet 
PPD 
Pharmaceutical Pharma Development 
qPCR 
Quantitative polymerase chain reaction 
RCL 
Replication-competent lentivirus 
SAE 
Severe adverse event 
SAR 
Serious adverse reaction 
SCID 
Severe combined immune deficiency 
SCID-X1 
X-linked severe combined immune deficiency 
SUSAR 
Suspected unexpected serious adverse reaction 
TRECs 
T cell-receptor excision circles 
TCR 
T cell-receptor 
TPO 
Thrombopoietin 
TU 
Transduction Unit 
UAR 
Unexpected adverse reaction 
UCB 
Umbilical Cord Blood 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 23 of 119 
-- 
 
 
 
 
VCN 
Vector copy number 
VSV-G 
Vesicular stomatitis virus glycoprotein 
WAS 
Wiskott-Aldrich syndrome 
WASP 
Wiskott-Aldrich syndrome protein 
WIP 
WASP-interacting protein 
XLN 
X-linked neutropenia 
XLT 
X-linked thrombocytopenia 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 24 of 119 
-- 
 
 
 
 
1. 
BACKGROUND AND RATIONALE 
1.1. 
Scientific Background and Rationale 
1.1.1. 
WAS – Description of the condition 
Wiskott-Aldrich Syndrome (WAS) is a rare X-linked primary immunodeficiency disease with an 
estimated incidence of 1:250.000 males and characterized by micro-thrombocytopenia, recurrent 
infections and eczema; this syndrome is associated with a high incidence of autoimmunity 
phenomena and lymphoid malignancies (Burns et al. 2004). 
The disease is due to mutations in the WAS gene which encodes the cytoskeletal regulator 
Wiskott-Aldrich Syndrome protein (WASP) (Derry et al. 1994). WASP is expressed exclusively 
in hematopoietic cells where it functions as a regulator of actin reorganization by linking various 
signals received at the cell surface to Arp2/3-mediated actin nucleation. 
WASP is an effector of cdc42 and controls actin nucleation for the formation of filopodia and 
membrane protrusions at the leading edge of hematopoietic cells. Consequently, WASP plays a 
pivotal role in cell migration and establishment of cell-to-cell contacts such as immunological 
synapses (Notarangelo and Ochs 2003). Defective WASP expression has been reported to cause 
functional impairments in hematopoietic precursors, platelets, T cells, B cells, Natural Killer 
(NK) cells, dendritic cells (DC), monocytes/macrophages, neutrophils and mast cells. 
In T cells, WASP is an essential component of the signal transduction cascade initiated by T cell- 
receptor (TCR) engagement contributing to the establishment of an immunological synapse. 
WASP is recruited to lipid rafts and involved in lipid raft clustering to the site of the 
immunological synapse following TCR and CD28 triggering (Dupre et al. 2002). WASP 
deficiency in T cells is associated to reduced activation and to downstream defects of cytokine 
production and proliferation (Trifari et al. 2006). 
In highly motile cells such as immature DC, WASP is essential for motility and for the formation 
of podosomes, which are actin-rich, highly dynamic cytoskeletal structures. In phagocytic cells 
such as macrophages and DC, clearance of apoptotic cells is accompanied by the recruitment of 
WASP to the phagocytic cup. A failure in apoptotic cell clearance due to WASP deficiency may 
perpetuate tissue inflammation and facilitate autoimmunity (Leverrier et al. 2001). 
Thrombocytopenia is the most frequent clinical manifestation of WAS: platelet number can be 
drastically reduced in the blood of WAS patients, and the residual circulating platelets are 
morphologically abnormal (reduced in size). Patients with a milder form of the disease (X-linked 
thrombocytopenia, XLT) are characterized mainly by thrombocytopenia. Peripheral destruction 
of the platelets in the spleen contributes to the thrombocytopenia, since a correction of platelet 
count and size can be reached after splenectomy. Accelerated destruction of platelets could be 
the consequence of either intrinsic platelet morphological abnormalities or of an autoimmune 
reaction. 
B cells defects include abnormalities of migration, homing, and also deficient production of 
antibodies, particularly against polysaccharide antigens. This may reflect a marked depletion of 
marginal zone splenic B cells (Meyer-Bahlburg et al. 2008;Westerberg et al. 2008). Microvilli 
expressed on WAS deficient B cells appear to be shorter and less dense in cell contacts than in 
wild type cells. 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 25 of 119 
-- 
 
 
 
 
The absence of WASP has been associated with numerous hematopoietic cell defects including 
motility, antigen uptake by endocytosis, adhesion, and activation (Burns, Cory et al. 2004), 
(Ochs and Notarangelo, 2005). The numerous and distinct cellular defects in hematopoietic cells 
from WAS patients are related to a high degree of diversity of clinical manifestations of the 
disease. It is reasonable to think that specific clinical manifestations arise from specific intrinsic 
cellular defects. However, the complexity of the physiopathology of WAS suggests that 
mechanisms requiring the interplay of different hematopoietic cell subsets are defective. 
 
1.1.2. 
Molecular aspects of the disease 
WAS gene is composed of 12 exons, encoding a 502 amino acids (a.a.) protein (WASP) with an 
expression restricted to hematopoietic cells. WASP is located in the cytoplasmic compartment 
with the highest density along the cell membrane. WASP is a member of a family of cytoskeletal 
regulator proteins (WASP/SCAR) involved in cytoskeleton reorganization by activating Arp 2/3 
mediated actin nucleation. 
The C-terminal domain of WASP activates the actin nucleation activity of Arp 2/3 complex by 
bringing Arp 2 in the proximity of Arp 3. The N-terminal region constitutively interacts with 
WIP (WASP-interacting protein), which plays the role of a chaperon controlling the localization 
and level of WASP within the cell. WASP interacts with the small Rho GTPase Cdc42, which 
has the capacity to induce a conformational activation resulting in interaction with Arp 2/3. 
Indeed, in the absence of stimuli, WASP exists in a folded auto-inhibited conformation. WASP 
also interacts with signalling proteins and tyrosine kinases (Nck, Fyn, Btk, Grb2) that regulate its 
activity. Additional modifications of WASP play a role in modulating whether the protein is in 
its auto-inhibitory or activatory state. Tyr 291 phosphorylation by different tyrosine kinases 
(Lyn, Btk, Hck) directly activates WASP, independently of Cdc42, and stabilizes WASP active 
conformation. On the contrary, constitutive binding of WIP stabilizes the inactive conformation. 
Overall, WASP is involved in receptor-mediated signalling cascades that activate Arp 2/3 and 
actin remodelling. Receptors with a link to WASP include TCR, BCR, co-stimulatory receptors 
and chemokine receptors. Actin remodelling plays an important role in cellular response 
allowing change in shape or motility, endocytosis, vesicular trafficking, and membrane 
reorganization. 
1.1.3. 
Clinical manifestations 
 
1.1.3.1. 
Thrombocytopenia and bleeding episodes 
Platelets are the cells that display the most drastic numeric reduction in the blood of WAS 
patients and the most profound morphological abnormalities (reduced mean platelet volume). 
However, it is still unclear how mutations in the WAS gene (independently of the severity of the 
mutation) alter platelet numbers and size. There are contrasting reports on the capacity of 
megakaryocytes from WAS/XLT patients to produce pro-platelets to normal levels. WASP- 
defective platelets show defects of filopodia formation and F-actin reorganization upon adhesion. 
In addition, platelets from XLT/WAS patients express high level of phosphatidylserine, which is 
a signal for macrophage mediated platelet destruction. Thrombocytopenia has also been 
proposed to result from accelerated destruction via an autoimmune mechanism, as observed in 
the murine model of the disease (Prislovsky et al. 2008). Indeed, WAS/XLT patients consistently 
present high levels of antibodies adherent to the surface of platelets. As a consequence of the 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 26 of 119 
-- 
 
 
 
 
platelet defect, bleeding episodes are a common manifestation in WAS (>80% incidence). The 
number of life-threatening episodes of bleeding (intracranial haemorrhage, intestinal bleeding) 
appears to be higher in WAS patients as compared to the XLT patients. This covers up to 20% of 
the death cases and severity of these episodes appears to be related to the number of platelets 
(severe episodes mainly in patients with platelets <10.000/µl, less severe episodes in patients 
with platelets = 50.000 - 100.000/µl) (Imai et al. 2004). 
 
1.1.3.2. 
Recurrent infections 
WAS patients have a high susceptibility to develop infections from numerous micro-organisms 
including bacteria, viruses and fungi (Imai, Morio et al. 2004). Bacterial infections due to 
encapsulated bacteria are the most frequent cause of morbidity, since WAS patients produce 
IgG2 against polysaccharide antigens at reduced levels. Respiratory infections, together with 
systemic infections (meningitis and sepsis), are commonly observed. Among viral infections, the 
most commonly reported are due to Herpes simplex viruses (type 1 and 2), which can be severe 
and disseminated. Infections due to cytomegalovirus (CMV) can cause encephalitis and hepatitis 
in WAS patients. These patients also develop Molluscum contagiosum infections and warts due 
to Papilloma viruses, at a relatively high frequency. Fungal infections are also common and 
mainly due to Candida and Aspergillus, while opportunistic pulmonary infections due to 
Pneumocystis jirovecii are also frequent. 
1.1.3.3. 
Eczema 
Eczema affects 80% of WAS patients in the course of the disease and the typical skin lesions 
resemble acute or chronic eczema in appearance and distribution (Imai, Morio et al. 2004). XLT 
patients also frequently develop eczema, although at a minor grade and for reduced periods, as 
compared to WAS patients. It is not known what the link is between WASP deficiency and 
eczema. In WAS patients, eczema is often associated to high IgE levels and could be of an 
allergic nature or due to an imbalance in Th2 cytokine production. It is also possible that other 
genetic or environmental factors contribute to this clinical manifestation. 
 
1.1.3.4. 
Autoimmunity 
The incidence of autoimmune diseases associated with classical WAS is high (40% and 72% in 2 
independent patient cohorts) and is not necessarily correlated with the overall disease severity or 
complete absence of WASP expression (Dupuis-Girod et al. 2003; Imai, Morio et al. 2004). 
Indeed, this complication can also develop in patients with otherwise milder disease. In addition, 
WAS patients often suffer from more than one autoimmune manifestation at the same time (25 to 
36%). The most common manifestations (80%) are autoimmune haemolytic anaemia (first 
autoimmune manifestation to appear in the course of the disease), cutaneous vasculitis, IgA 
nephropathy (also common in XLT patients) and arthritis. 
Less common autoimmune manifestations in WAS patients are: chronic inflammatory intestinal 
disease and idiopathic thrombocytopenic purpura (as a consequence of splenectomy). WAS 
patients with autoimmune manifestations have a 25% risk to develop tumors (as compared to 5% 
for WAS patients without autoimmunity). 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 27 of 119 
-- 
 
 
 
 
In addition, 75% of patients developing tumors had signs of autoimmunity in earlier follow up. 
Thus, autoimmunity is associated with an increased risk of mortality and this is particularly true 
for autoimmune haemolytic anaemia and idiopathic thrombocytopenic purpura. 
Despite being an important cause of morbidity and mortality, the mechanisms of WAS- 
associated autoimmunity are still poorly understood. WASP may be required for normal thymic 
maturation and theoretically, self-reactive T cells may escape negative selection because of 
defective TCR-induced apoptosis, as described for WASP-deficient murine lymphocytes. 
Impaired function of WASP-deficient T cells, B cells, macrophages, or DC could all destabilize 
important mechanisms participating in the maintenance of normal tolerance. In addition, recent 
studies have demonstrated defects in homeostasis and suppressor functions of natural regulatory 
T cells (Marangoni et al. 2007), which may at least partly account for the autoimmune disorders 
associated with WAS. 
1.1.3.5. 
Cancer 
Together with severe hemorrhages and autoimmune manifestations, tumors are the major 
complication of WAS. They occur in 13-22% of the patients with a median age of onset of 9.5 
years (Imai, Morio et al. 2004). This reported frequency most probably underestimates the 
current situation since the median survival of WAS patients has been improving over the last 
decades, thereby increasing the risk to develop tumors. WAS associated-tumors are mainly 
lymphoreticular malignancies, since leukemia, myelodysplasia and lymphoma cover 90% of the 
cases. This is most probably linked to the restricted expression and function of WASP in 
hematopoietic cells. Immunodeficiency is likely to contribute as a proportion of malignancies are 
associated with Epstein-Barr virus (EBV) and there may be specific immune-surveillance defects 
(such as impaired NK or cytotoxic T cell cytolysis). However, this is unlikely to be the full 
explanation, as the highest lymphoma risk (44%) may be conferred by a single splice site 
mutation that is otherwise associated with a mild clinical phenotype. Interestingly, there also 
appears to be a significant incidence of myelodysplasia in severe WAS patients. Although the 
mechanisms of malignancy remain undetermined, there may be a role for WASP in human cell 
division and maintenance of genomic stability (Moulding et al. 2007) as has been reported for 
the WASP homolog in fission yeast. 
 
1.1.4. 
Classification of WAS patients 
Historically, the clinical classification of WAS patients arose from a large cohort of patients 
classified into 5 severity scores (Zhu et al. 1997). This was based on the following clinical 
findings: microthrombocytopenia, eczema, immunodeficiency, autoimmune disorders/cancer 
(Table 9). 
Approximately 300 unique mutations have been reported in the WAS gene (WASPbase: 
http://pidj.rcai.riken.jp/WASPbase/). Studies in large cohorts of patients have recently shown 
that the disease severity correlates with the severity of WASP expression defect, which in turn 
correlates with the type of mutation in the WAS gene. Therefore, direct measurement of WASP 
residual expression by Western blot or by flow cytometric analysis is useful for predicting the 
phenotypic outcome of WAS (Imai, Morio et al. 2004; Jin et al. 2004; Lutskiy et al. 2005). 
Missense mutations usually result in residual expression of a mutated protein and are associated 
with a mild clinical form of the disease (XLT), in which only platelet defects are present (Villa et 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 28 of 119 
-- 
 
 
 
 
al. 1995). In contrast, nonsense mutations usually totally impair protein expression and are 
associated with the WAS severe clinical phenotype consisting in thrombocytopenia and recurrent 
infections and eventually autoimmunity and cancer. 
Therefore, the expression of WASP has been proposed as a main prognostic parameter by the 
Italian Association of Paediatric Haematology and Oncology (AIEOP), which has adopted 
guidelines, which characterize WAS patients based on WASP expression in combination with 
clinical signs. 
 
1.1.5. 
Current methods of treatment and limitations 
Clinical management of patients with WAS represents significant challenges, particularly in 
attenuated phenotypes where the natural history of disease progression is unclear. Splenectomy, 
by decreasing excessive sequestration and elimination of platelets, is an effective treatment for 
thrombocytopenia, but carries a significant long-term risk of sepsis and is therefore probably 
indicated only in severe disease where there is no prospect for curative intervention. Moreover, it 
may not be effective in the setting of autoimmune thrombocytopenia in addition to intrinsic 
platelet defects. At present, hematopoietic stem cell transplantation (HSCT) from an HLA- 
identical sibling is the treatment of choice for WAS since it has been reported to lead to 82 to 
88% long-term survival in different European and American centers (Filipovich et al. 2001; 
Kobayashi et al. 2006; Ozsahin et al. 2008), with an average of 87% in the American study 
(Table 1). However, the majority of the patients do not have such a donor available. As an 
alternative, WAS patients have been receiving HSCT from HLA-matched unrelated donors 
(MUD). Following this procedure, the survival rate had been previously estimated to be 71% 
(Filipovich, Stone et al. 2001), with a higher success rate when performed before the age of 5 
(Table 2). Recent studies have reported improved survival rates (80-81%) after MUD HSCT, 
possibly due to the availability of a detailed HLA-matching system for unrelated donors, with 
further indication that prompt diagnosis and intervention is crucial for successful outcome 
(Kobayashi et al. 2006; Pai et al. 2006) (Table 3). The success of MUD transplantation is 
however hampered by a risk to develop autoimmune complications when complete chimerism is 
not obtained (Ozsahin et al. 2008). HSCT from alternative donors (partially HLA-matched 
relatives) has led to disappointing results, with 5-year overall survival of only 55%, which 
dropped to 25% when patients over 2 years of age were analyzed (Table 4). When successful, 
HSCT can lead to restoration of platelet counts and immune functions. In case of stable mixed 
chimerism, clinical conditions can also be improved, although a number of these patients show 
recurrence of clinical symptoms later on. In contrast to SCID, in which severe impairment of 
hematopoietic precursor differentiation conveys space and a selective advantage for engrafted 
progenitors with the potential for normal differentiation, the differentiation of hematopoietic 
cells in WAS is not blocked since all mature subsets are present in the blood of patients, although 
at reduced levels (platelets, B cells and T cells). Therefore, successful HSCT in WAS patients 
requires myeloablative conditioning before transplantation, usually performed with a 
combination of alkylating agents (Busulfan + Cyclophosphamide + ATG, Fludarabine + 
Melphalan + ATG/Alemtuzumab). 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 29 of 119 
-- 
 
 
 
 
Table 1 
5-year survival after allogeneic HSCT in WAS patients according to donor 
type 
 
Parameter 
HLA-Identical Siblings 
Mismatched related donor 
MUD 
Overall Survival 
87% 
52% 
71% 
Overall survival according to age at the time of HSCT 
<5 Years 
90% 
53% 
84% 
>5 Years 
79% 
38% 
<30% 
Overall survival according to performance score at the time of HSCT 
< 90% 
94% 
41% 
70% 
≥ 90% 
96% 
61% 
71% 
Overall survival according to pre-HCST infection at the time of transplant 
No 
84% 
59% 
71% 
Yes 
100% 
35% 
55% 
from (Filipovich, et al. 2001) 
Table 2 
Efficacy of HSCT in a large cohort of WAS survivors 
 
 
Disease Status 
HLA-Identical Siblings 
Mismatched related donor 
MUD 
Cured 
73% 
57% 
73% 
Improved 
20% 
24% 
27% 
Unchanged 
7% 
5% 
0% 
from (Filipovich, Stone et al. 2001) 
Table 3 
Overall survival (OS) and failure-free survival (FFS) 5 years after stem cell 
transplantation in WAS patients, according to donor type and age at the time 
of transplant 
 
OS 
FFS 
HLA-Identical Siblings (n= 67) 
81.8% 
64.3% 
Mismatched related donor 
37.5% 
37.5% 
MUD BMT 
80% 
75.2% 
MUD CORD 
80% 
71.4% 
<5 Years 
79.8% 
72% 
>5 Years 
53.8% 
46.2% 
From (Kobayashi, Ariga et al. 2006) 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 30 of 119 
-- 
 
 
 
 
Table 4 
Overall survival (OS) 7 years after stem cell transplantation in WAS 
patients, according to donor type and age at the time of transplant 
 
OS 
HLA-Identical Siblings (n=45) 
88% 
MUD BMT (n=32) 
71% 
≤2 Years 
82% 
>2 Years 
55% 
Other relative (n= 19) 
55% 
≤2 Years 
75% 
>2 Years 
25% 
From (Ozsahin, Cavazzana-Calvo et al. 2008) 
In recent years, survival rates after HSCT have improved, in particular in the MUD setting. 
In a recent retrospective collaborative study (Moratto et al., 2011), long-term outcome and donor 
cell engraftment were analysed in 194 patients with WAS, who have been treated by HSCT in 
the past 30 years. Donor type included matched sibling donor (MSD), mismatched family donors 
(MMFD), MUD, and umbilical cord blood (UCB). Overall survival was 84.0%, resulting higher 
for those treated after year 2000 (89.1% 5-year survival). This is probably a result of recent 
improvement of outcome after transplantation from mismatched family donors and for patients 
who received HSCT from an unrelated donor at an older age (> 5 years). Younger age was 
associated to best 5-year survival in patients treated with MUD HSCT (91.9% for pts < 2 years 
old at time of HSCT; 73.3% for pts > 5 years of age), but it did not significantly affect survival 
in patients treated by MSD or MMFD. An increase in survival after HSCT from MMFD was also 
observed in most recent transplant, but the number of patients is relatively small to draw 
conclusions. Mixed chimerism was associated with an increased risk of incomplete lymphocyte 
count reconstitution and development of autoimmunity after HSCT. Moreover, persistent 
thrombocytopenia after HSCT was strongly associated with low or null myeloid chimerism, 
suggesting that robust and stable engraftment of donor-derived myeloid cells is required to 
correct this defect, because of the lack of selective advantage for WASp-positive cells in the 
myeloid compartment. 
In another recent paper by Shin et al. (Shin at al., 2012), the outcome of HSCT in 47 WAS 
patients treated at a single center since year 1990 has been reported. Donor type included MSD, 
MUD, mismatched unrelated donors (MMUD) and UCB. Outcome improvement after year 2000 
was observed also in this study, from a 5-year OS of 62.5% for patients transplanted during years 
1990-2000 to 90.8% and 2001-2009, respectively. In multivariate analysis, age was only 
marginally significant, and the extent of HLA-mismatch did not significantly affect the incidence 
of acute GVHD, chronic GVHD or survival. Post-transplant autoimmune cytopenias were 
frequently diagnosed (55% patients), but their occurrence was associated with donor type, acute 
or chronic GVHD or mixed chimerism. 
1.1.6. 
Rationale for GT in WAS 
Allogeneic HSCT from an HLA-identical donor can cure all the pathological aspects related to 
WAS. However, in the absence of an HLA-matched bone marrow donor, currently available 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 31 of 119 
-- 
 
 
 
 
alternative strategies are to be considered unsatisfying. In fact it has been clearly demonstrated 
that results with HLA-mismatched, especially T-cell depleted, parental grafts are discouraging, 
with an unacceptable incidence of transplant related mortality (Filipovich, Filipovich, Stone et al. 
2001; Kobayashi, Ariga et al. 2006). Consequently, for patients affected by WAS who lack a 
suitable stem cell donor, ex vivo gene therapy represents an alternative approach, since it is in 
principle applicable to all patients. In principle, gene therapy has several advantages over 
allogeneic transplant, including the lack of graft-versus-host disease and reduced toxicity. 
In recent years gene therapy via autologous hematopoietic stem cells (HSC) has been used as an 
alternative therapeutic option in several immunodeficiencies. The successful use of 
oncoretroviral vectors derived from murine leukemia virus (MLV) to treat patients affected with 
X-linked severe combined immunodeficiency (Hacein-Bey-Abina et al. 2002) and Adenosine 
deaminase deficiency (Aiuti et al. 2002; Aiuti et al., 2009) has opened the perspective for 
application of gene therapy to other immunodeficiencies, including WAS. Indeed, primary 
immunodeficiencies are the best candidates for gene correction because of the possibility to use 
HSC as targets, the potential in vivo selective advantage of the corrected cells and the possibility 
to correct the immune defect even when partial engraftment of engineered cells occurs. There is 
a consensual agreement that also in the case of WAS, the optimal cell target to deliver the 
healthy copy of the WAS gene is the hematopoietic stem cell. Indeed, the multilineage progeny 
of HSC engineered to express the normal WAS gene should be functional and able to expand in 
the treated patients, and to correct the phenotype of WAS. 
Preclinical studies suggest that lentiviral vectors (LV) are to be considered superior to the 
retroviral vectors both for efficacy and safety reasons (Kay et al. 2001). In general, LV derived 
from HIV provide stable long-term gene expression in vitro and in vivo and they present a 
number of advantages over gamma-retroviral vectors for the transduction of haematopoietic stem 
cells. From the safety point of view, LV are based on a self-inactivating configuration in order to 
minimize the risk of producing replication-competent lentivirus (RCL), they can be adapted to 
incorporate the use of a physiological gene promoter, and are likely to possess a safer integration 
profile as compared to retroviral vectors. Lentiviral vectors have recently entered the clinic with 
wide-ranging applications, as several trials are ongoing or are beginning in Europe and US to 
treat HIV infection, neurodegenerative syndromes or genetic diseases such as thalassemia. 
 
1.1.6.1. 
Exploiting the selective advantage for WAS-expressing cells 
It is commonly accepted that a selective advantage for the transduced cells is a key factor for the 
success of gene therapy and various lines of evidence suggest that restoration of WASP gene 
expression by lentiviral vectors in WASP-deficient cells may also confer a selective advantage. 
In heterozygous females with a nonsense WASP mutation, the pattern of X chromosome 
inactivation occurs in a non-random manner during the migration of early haematopoietic 
progenitor cells from fetal liver to bone marrow, suggesting a chemotactic (or survival) 
advantage for haematopoietic progenitors and stem cells with a normal WASP expression 
(Lacout et al. 2003). In addition, recently, it has been described that WASP expression confers a 
selective advantage to the development of hematopoiesis dependent on the receptor c-Kit (Mani 
et al., 2009). This finding suggests that WASP transduced progenitor cells, which are exposed 
during gene transfer to stem cell factor (c-Kit ligand), may preferentially survive and/or expand 
in vitro and in vivo. 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 32 of 119 
-- 
 
 
 
 
Furthermore, somatic mosaicism has been reported to spontaneously occur in T lymphocytes 
(including regulatory T cells) and more unusually in B and NK cells of WAS patients, leading to 
a milder clinical phenotype (Wada et al. 2001; Konno et al. 2004; Lutskiy, Rosen et al. 2005; 
Humblet-Baron et al. 2007). A selective advantage of gene-corrected cells over the null cells has 
also been observed in T lymphocytes both in vitro and in bone marrow reconstitution 
experiments in mice (Klein et al. 2003; Dupre et al. 2004). Thus, a proliferative advantage is 
expected to be conferred by WASP expression in the lymphoid compartment following gene 
therapy in WAS patients. Further indirect evidence for a strong survival or proliferative 
advantage of WASP-expressing lymphocytes comes from studies of mixed chimera status in 
WAS patients after HSCT (Yamaguchi et al. 2002). 
A recent analysis of the possible advantage for WASP-expressing cells over WASP-negative 
cells in a competitive setting of WASP +/- mice has been documented by Westerberg et al 
(Westerberg, de la Fuente et al. 2008) providing insight into which lineages are more likely to be 
corrected by gene therapy. 
The authors demonstrate that WASP is important for the development and maturation of T-cell 
and B-cell lineage cells, but the selection for WASP-expressing myeloid cells appears less robust 
than in other lineages, although in humans expressions of WASP appears to confer a significant 
advantage already in hematopoietic progenitor cells (HPCs), as previously shown in carrier 
females. 
Although the lack of donor myeloid engraftment is associated with increased risk of persistent 
thrombocytopenia after allogeneic HSCT (Yamaguchi, Ariga et al. 2002; Pai, DeMartiis et al. 
2006), data in heterozygous WASP +/- mice have shown that a moderate excess of WASP+ 
platelets was present in the circulation. While it remains to be seen whether such increase reflects 
advantage in the number and/or differentiation of WASP-expressing megakaryocytes or an 
increased peripheral consumption of WASP-negative platelets, these data represent an 
encouraging result in view of the potential use of gene therapy in WAS. 
1.1.6.2. 
Choice of conditioning regimen and cell dose 
We envisage to administer a reduced intensity conditioning regimen assuming that a stable 
mixed chimerism (with gene corrected and uncorrected cells) in bone marrow and peripheral 
blood will be sufficient to provide clinical benefit with a low regimen-related toxicity. We 
estimate that partial engraftment of corrected cells in the bone marrow, resulting from a reduced 
intensity conditioning, will lead to an engraftment of corrected cells in the peripheral 
compartment sufficient to provide improvement of immune and hematological functions. 
Based on our experience in combining ex vivo gene therapy with low doses of intravenous 
Busulfan in children affected by ADA-SCID, we will adopt iv Busulfan as myelotoxic drug 
(Aiuti et al., 2002). 
The total dose of Busulfan will be increased as compared to the ADA-SCID trial in which lower 
levels of corrected myeloid cells are sufficient to lead to clinical benefit. In order to achieve 
correction of the lymphoid compartment, and especially T cells, we will complete the 
conditioning regimen with Fludarabine to target lymphoid progenitors and mature lymphocytes. 
Indeed, the mechanisms of selective advantage in WAS are different than the ones in ADA- 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 33 of 119 
-- 
 
 
 
 
SCID. In ADA-SCID patients treated by gene therapy, the selective advantage takes place during 
differentiation of HSC into mature B cells and T cells. 
In contrast, the studies on WAS patients with mixed chimerism following HSCT or with 
revertant mutation in the T cell compartment indicate that the selective advantage for WASP+ 
cells might take place mainly through peripheral expansion upon antigen triggering rather than 
during thymic differentiation (Wada, Schurman et al. 2001; Yamaguchi, Ariga et al. 2002). 
An additional contribution to the selective advantage of corrected T cells might be the previously 
reported susceptibility to apoptosis of WASP-deficient cells (Rawlings et al. 1999). 
Based on these considerations, a low dose of Fludarabine will be optimal to break the 
homeostasis in the compartment of early lymphoid progenitors in order to boost, at an early stage 
of differentiation, the expansion of genetically corrected T cell precursors. 
In addition, through its activity on the naïve T cell compartment, Fludarabine will favor the 
establishment of a pool of corrected naïve T cells in the periphery. The selective advantage 
described above should favor the expansion of genetically corrected memory T cells, during 
encounter with antigens. 
We will also introduce in the conditioning anti-CD20 monoclonal antibody (Rituximab), a 
biological agent that depletes peripheral B cells and has been shown to provide a clinical benefit 
in several autoimmune disorders. Anti-CD20 will serve as depleting agent for B cells and 
particularly of autoreactive cells, thereby facilitating the engraftment and expansion of gene 
corrected B cells expressing WASP. In addition, anti-CD20 will act as pre-emptive treatment for 
lymphoproliferative disorder due to EBV, which represents a high risk factor for the 
development of lymphoma in WAS patients. 
Another important consideration is the presence of self-reactive T cells responsible for 
autoimmune complications in WAS patients. For patients with autoimmune manifestations, the 
use of ATG Thymoglobuline as an additional lympho-depleting agent may be considered to 
eliminate auto-reactive memory T cells. 
Based on these considerations, we plan to use iv Busulfan (a total dose of 6.4-9.6 mg/kg adjusted 
for patient’s weight and AUC targeted) and Fludarabine (at total dose of 60 mg/sqm) as 
preparative regimen prior to gene therapy, associated to a single dose of Rituximab (at the dose 
of 375 mg/sqm). 
A detailed description of the conditioning regimen is presented in Section 10.5 ,Table 7 . 
In case the conditioning regimen described above will not produce mixed chimerism associated 
with clinical benefit, the protocol will be amended to include fully myeloablative conditioning. 
This may include re-treatment of the patients originally conditioned with the reduced intensity 
protocol one year after the first gene therapy treatment. The choice of the regimen will depend 
also on the results of the parallel trials which will be adopted different full conditioning 
regimens: the UK trial for WAS gene therapy and the TIGET trial for gene therapy in 
metachromatic leukodystrophy (MLD). 
A direct correlation between cell dose and engraftment is a well-known phenomenon in 
hematopoietic transplant medicine and has been established also for ADA-SCID gene therapy 
trial (Aiuti et al., 2009). A minimum of 2 x 106 CD34+ cells/kg is usually required to safely 
perform an autologous hematopoietic stem cell transplant. Therefore, a dose of 2 x 106 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 34 of 119 
-- 
 
 
 
 
transduced CD34+ cells/kg is the minimum target dose at infusion in our study, and we will aim 
at infusing 5-10 x 106 CD34+ cells/Kg. The goal is to collect a slightly higher number of cells 
than usually recommended for a conventional un-manipulated transplantation because of the 
subsequent procedures (mononuclear cell (MNC) purification, positive selection, transduction 
and QC). Therefore, we will harvest approximately 20-30 bone marrow (BM) ml/kg (max 35 
ml/kg) patient body weight with a minimum target CD34+ cell count (before any manipulation) 
of 5 x 106/kg. In the case of use of mobilized peripheral blood (MPB), leukapheresis will be 
performed to achieve a minimum target CD34+ cell count (before any manipulation) of 5 x 
106/kg. 
1.1.6.3. 
Choice of lentiviral vector 
Initial studies using oncoretroviral MLV-based vectors for exvivo gene therapy of WAS have 
demonstrated the feasibility of gene transfer in WAS patient cells (Candotti et al. 1999; Dupre, 
Aiuti et al. 2002; Strom et al. 2003) or in hematopoietic progenitor cells of WAS null mice 
(Klein, Nguyen et al. 2003; Strom, Turner et al. 2003). However, the requirement for high- 
frequency HSC transduction positions lentiviral vectors as prime candidates for HSC gene 
transfer in gene therapy for WAS. Indeed, LV offer a unique combination of advantages for the 
proposed application: i) they integrate efficiently into a variety of cell types ex vivo and in vivo; 
ii) they allow stable and robust transgene expression; iii) they significantly alleviate the safety 
concerns associated with RV integrations. Because of these features, LV enable efficient gene 
marking of mouse and human HSC (the latter ones being assayed in mouse xenografts as non- 
obese diabetic/severe combined immunodeficient (NOD/SCID) mice with minimal in vitro 
manipulation and cell perturbation, allowing for full maintenance of stem cell properties and 
multiclonal repopulation of chimeric hosts (Miyoshi et al. 1999; Guenechea et al. 2000; 
Guenechea et al. 2001; Ailles et al. 2002). As detailed below, LV have recently been approved 
for clinical testing. 
The vector selected for the WAS gene therapy trial contains as internal promoter the 1.6 kb-long 
sequence of the WAS gene endogenous promoter. In the context of a LV, the WAS promoter 
appears weaker in driving WAS mRNA expression than other internal promoters, such as EF1- 
alfa. Nevertheless, it leads to optimal expression of the protein (Charrier et al. 2007), thereby 
suggesting an optimal regulation of expression by the WAS promoter. This regulatory element 
restricts transgene expression mainly to hematopoietic cells (Dupre, Trifari et al. 2004), thus 
limiting the possibility of transgene toxicity to this system. 
In summary, the lack of satisfactory therapeutic options for many patients affected by WAS with 
no HLA identical donor, together with experimental proofs of concept and a relatively good 
understanding of the molecular basis of this disease, provide a strong rationale for designing a 
gene therapy approach to WAS based on lentiviral vector technology. 
1.1.7. 
Telethon-Généthon common efforts in the development of lentiviral vector gene 
therapy for WAS 
The Medicinal Product was developed to the clinical application under a collaborative agreement 
between the non-profit funding agencies Telethon and Généthon. Généthon is an Institute located 
in Evry (Paris) sponsored by the non-profit organization AFM. Généthon has obtained Orphan 
Drug Designation from EMA (EU/3/05/345, 24/01/2006 “lentiviral vector containing the human 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 35 of 119 
-- 
 
 
 
 
WASP gene”). The manufacturing, purification process and the specifications of the vector 
developed at MolMed (for Telethon) are very similar to the ones developed at Généthon. We 
acknowledge the contribution of preclinical data from investigators at Généthon (Anne Galy) and 
Great Ormond Street Hospital (Adrian Thrasher) for this application. A different clinical trial 
sponsored by Généthon is being conducted at Necker Hospital, Paris (PI: Marina Cavazzana 
Calvo), UCL Institute of Child Health, London (PI: Bobby Gaspar) and Children’s Hospital 
Boston (PI: David Williams) with the same lentiviral vector. A myeloablative conditioning will 
be used to prepare the patient to the infusion of transduced CD34+ cells. The results of these 
clinical trials will be useful to compare the effects of the different conditioning protocols and 
obtain crucial information that could be used to modify the clinical trial in case of toxicity or 
lack of engraftment. 
Following the submission of this protocol, Fondazione Telethon has received its own orphan 
drug designation (ODD) for Telethon003 (Autologous CD34+ cells transfected with lentiviral 
vector containing WAS cDNA ODD#10-3043) in the US on April 30th 2010 for the treatment of 
WAS. An ODD for the same medical product was also obtained in Europe on 06/06/2012 by 
EMA (European Medicines Agency). 
1.1.8. 
Ethical considerations 
The rare nature of the condition, the pediatric target population and the specificity the gene 
therapy treatment pose critical ethical questions that were addressed following the recent 
guidelines issued by the ad hoc group of pediatric clinical trials for the implementation of the 
EMA Directive 2001/20/EC (2008). 
Based on the above considerations, the clinical course and therapeutic options in WAS indicate 
that the objective of developing alternative therapies for WAS appears not only to be justified, 
but desirable in ethical terms. Although the transplantation of allogeneic HSC is a curative 
therapy, this option is limited to those patients who have access to a compatible donor. In 
addition, allogeneic HSC transplant is associated to severe risks for the patient and, therefore, is 
usually limited to those patients with a severe clinical course of the disease. 
Moreover, there is general consensus that gene therapy is an ethically justified approach for the 
treatment of severe diseases, considering the previous successful attempts in primary 
immunodeficiencies. 
The results of the preclinical studies with the medicinal product, as well as the preliminary 
results of a clinical trial with similar products (same vector for a different condition or different 
vector for the same condition) provide evidence of the biological activity and efficacy of 
genetically corrected autologous HSC transduced with a lentiviral vector, therefore supporting a 
reasonable expectation of a benefit for the individual participating in the study. 
The risks associated with the procedures have also been carefully analyzed. The risks associated 
with the procedure (bone marrow harvest, conditioning, cell reinfusion) are not superior to those 
expected from a standard conditioning regimen for transplant of allogeneic HSC, and actually are 
expected to be lower, considering the reduced conditioning regimen. The specific risks related to 
the use of lentiviral vector transduced cells are discussed extensively below in a dedicated 
session. The vector technology and vector design employed in this study is believed to be the 
most updated, reliable and safe currently available method for stable vector integration in the 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 36 of 119 
-- 
 
 
 
 
human genome. The extensive preclinical studies conducted in vitro and in mice for long-term 
period of observations by our group and our collaborators have not shown any adverse event, 
including clonal proliferation. Nevertheless, the patient and/or patient’s proxy will be extensively 
informed about the potential risks of gene transfer, which are inherent to the use of integrating 
vector. 
In summary, the experimental treatment proposed can be classified under the category of 
“Greater than minor increase over minimal risk with benefit for the individual that is especially 
favorable in relation to available alternative approaches for the individual”, according to the ad 
hoc group guidelines for the implementation of pediatric clinical trials of the EMA. In the case of 
the proposed WAS gene transfer protocol, the risk-benefit evaluation is in favor of the 
enrollment and treatment of patients with a severe condition, in the absence of a suitable donor 
for allogeneic HSC. 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 37 of 119 
-- 
 
 
 
 
2. 
PRODUCT MANUFACTURING AND CHARACTERIZATION 
2.1. 
Medicinal product definition 
Drug product (DP) is defined as autologous CD34+ cells genetically modified with a lentiviral 
vector (LV) encoding for the human WAS cDNA resuspended in their final formulation medium. 
Medicinal product manufacturing and release and LV supernatant production will be performed 
at MolMed S.p.A. (Molecular Medicine S.p.A., Via Olgettina 58, 20132 Milano), a GMP (Good 
Manufacturing Practice) certified facility authorized by Italian Ministry of Health according to 
D.L.vo 166/2003 for the production of sterile medicinal products, the production of specific 
medicinal products obtained with particular technologies (i.e. gene therapy), and for primary 
packaging operations. 
2.2. 
Lentiviral vector encoding for WASP 
According to the draft revision of Annex I Part IV of 2001/83 Directive, the drug substance (DS) 
is defined as autologous CD34+ cell enriched population that contains haematopoietic stem and 
progenitor cells (HSPC) transduced ex vivo using a lentiviral vector encoding the human (WAS) 
gene. 
The LV proposed for this application is a pseudo-type vector made by a core of HIV-1 structural 
proteins and enzymes, the envelope of the VSV and a genome containing HIV-1 cis-acting 
sequences, no viral genes and one expression cassette for the WAS transgene under the control 
of the WAS 1.6Kb promoter. The 3 vector components (core, envelope and genome) are 
transiently expressed in vector producer cells by 4 different constructs: 2 core packaging 
constructs, the envelope construct and the transfer vector construct. Only the transfer vector 
transcript is transferred and integrated into the target cells. The latter has been optimized for 
maximal transduction efficiency and stable constitutive transgene expression in target cells. 
The proposed LV is replication-defective by design. No viral genes are transferred to target cells. 
Because the vector is pseudotyped by the envelope of an unrelated virus, wild-type HIV cannot 
be generated by recombination among the constructs used to make vectors. Moreover, the lack of 
homology between the envelope and the core packaging sequences makes recombination highly 
unlikely between these constructs (Vigna and Naldini 2000). The main safety concerns 
associated with ex vivo administration of LV are: i) contamination by replication competent 
lentiviral recombinant, ii) mobilization and recombination of the vector with wild-type virus in 
infected hosts, iii) insertional mutagenesis. 
The combination of the third-generation packaging system with a SIN vector expressed in 
producer cells by a chimeric 5’LTR (long terminal repeat) provides an advanced multi-split 
design (four constructs are used to make vector) expressing only a fractional set of HIV-1 genes 
(three out of nine in the wild-type virus: gag, pol and rev) with two built-in safety mechanisms 
that would interfere with the generation of an unlikely RCL: expression of the gag pol gene 
conditional on trans-complementation by a separately expressed Rev protein, and self- 
inactivation of the vector LTR. 
Concerning the risk of insertional mutagenesis, several factors suggest a lower risk for LV than 
for conventional onco- or gamma-retroviral vectors. Firstly, despite the extensive replication of 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 38 of 119 
-- 
 
 
 
 
HIV-1 in chronically infected immuno-compromised hosts, there is no evidence of increased 
oncogenesis directly linked to viral integration. 
Secondly, HIV-1 integration site selection does not show a bias for the promoter regions of 
transcriptionally active genes or for gene subsets involved in growth control and oncogenesis as 
observed for MLV-based vector (Schroder et al. 2002; Wu et al. 2003; Mitchell et al. 2004). 
These features suggest a lower chance for LV to activate oncogenes at the integration site (De 
Palma et al. 2005). 
Thirdly, the lack of transcriptionally active LTR, combined with a moderately active internal 
promoter to drive transgene expression, is likely to be a major safety advantage of the proposed 
vector. As described below in the preclinical studies of this application, we have challenged the 
predicted safety of the proposed vector design by transducing HSPC from a tumor-prone mouse 
model in conditions representative of those proposed for the clinical trial with either the SIN- 
PGK LV or prototypical MLV-based RVs and provided direct experimental evidence that 
integration of a SIN LV (with an internal PGK promoter) has low to undetectable genotoxic 
potential. 
2.3. 
Establishment of MCBs 
Vector plasmids are produced by incorporation into E. coli and a Master Cell Bank (MCB) is 
generated for each plasmid after selection. Each E. coli MCB is tested for a number of 
parameters among which viability, microbial purity, identity, and plasmid retention. Each 
plasmid preparation is suitably characterized to confirm identity, sterility, purity and to assess the 
level of contaminants and the stability. 
A subclone of the human kidney 293T cell line (from Stanford University) originally selected at 
the Salk Institute (La Jolla, CA, USA) for its high yield performance in production of LV by 
transient transfection is used for MCB establishment. 293T cells were chosen as the cell line for 
LV production because of the vastly superior performance (10-fold) as compared to 293 cells in 
pilot experiments performed at SR-TIGET, MolMed and Genethon laboratories. 
The MCB was generated at Genethon; the entire cell banking procedure, including formulation 
of the culture media, equipment cleaning, and all cell manipulations, was performed in GMP 
conditions, according to Standard Operating Procedures (SOP). The MCB was tested for the 
absence of microbial and viral contaminants, and for purity according to international guidelines 
(PTC in the characterization of cell lines used to produce biological – 1993, ICH Topic Q5D, 
Note for guidance on the quality, preclinical and clinical aspects of gene transfer medicinal 
products – EMA/CPMP/BWP/3088/99). 
2.4. 
Lentiviral Vector production 
The LV supernatant is produced by transient quadri-transfection of the producer cells 293T with 
GMP plasmid preparations encoding for two core packaging constructs (gagpol and rev), the 
envelope construct (VSV-G) and the transfer vector construct, followed by a vector purification 
process involving DNase (Benzonase) treatment, anion exchange chromatography, concentration 
by ultra-filtration, gel filtration and final sterilizing filtration. Each batch of LV will be 
characterized for viral particles and titer, infectivity, viral proteins, vector integrity and identity, 
for the presence of selected impurities related to the producer cells or the manufacturing process 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 39 of 119 
-- 
 
 
 
 
and for the absence of microbial contaminants (bacteria, fungi, mycoplasma and viruses) and 
RCL. 
LV production is performed at Molmed S.p.A., as described in the investigational medicinal 
product dossier (IMPD). 
 
2.5. 
Production of transduced CD34+ cells 
The final medicinal product is made of LV-transduced CD34+ cells expressing human WASP 
resuspended in the final formulation medium. CD34+ cells will be purified from bone marrow 
aspirates and/or from mobilized peripheral blood. 
Transduced cell production is performed at Molmed S.p.A., as described in the IMPD. The 
production of WASP transduced CD34+ cells will be divided into the following steps. 
2.5.1. 
Cell isolation 
 
2.5.1.1. 
Isolation of BM CD34+ cells 
Bone marrow harvest (minimum 5, maximum 20 x106 CD34+/kg) will be diluted, stratified with 
lymphoprep and centrifuged in order to separate blood fractions. A cell suspension primarily 
made of mononuclear cells (MNC) will be collected. The cells will be tested for viability and cell 
concentration. MNC thus obtained will be labeled with magnetic beads by 30 min. incubation at 
5°C mixing 200 µl CD34+ reagent (CD34 Microbeads, Milteny Biotec) with 100x106 MNC/ml. 
At the end of the incubation, the cells will be washed and MNC loaded on the magnetic column 
device. CD34+ cell positive and negative fractions will be then eluted from the column and 
assayed for sterility, viability and cell number, purity and clonogenic potential (see Investigator’s 
Brochure). 
 
2.5.1.2. 
Isolation of mobilized peripheral blood CD34+ cells 
Autologous leukoapheresis product will be transferred in two or more 250 ml tubes and then 
centrifuged at 1000±50 rpm for 30±5 minutes at room temperature to remove plateles from 
mononuclear cells (MNC). The cell pellet, primarily made of mononuclear cells (MNC), will be 
re-suspended and tested for viability and cell concentration. MNC thus obtained will be labeled 
with magnetic beads by 30 min. incubation at room temperature mixing 200 µl CD34+ reagent 
(CD34 Microbeads, Milteny Biotec) with 100x106 MNC/ml. At the end of the incubation the 
cells will be washed and MNC loaded on the magnetic column device. CD34+ cell positive and 
negative fractions will be then eluted from the column and assayed for sterility, viability and cell 
number, purity and clonogenic potential. 
 
2.5.2. 
Transduction of CD34+ cells 
On day -3 of the production process, purified CD34+ cells will be seeded at final concentration 
of 1x106/ml in serum free medium (Cell Gro) supplemented with the cytokines SCF, TPO, 
FLT3-L and IL-3, in gas-permeable bags coated with retronectin, and incubated for 24 hours at 
37°C in 5% CO2 atmosphere. On day -2 and -1, the LV (1x108TU/ml, multiplicity of infection 
(MOI) 100) will be added to the cell suspension and incubated for 16 hours (over-night) (first 
and second round of transduction). On day 0, at the end of the transduction, cells will be washed 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 40 of 119 
-- 
 
 
 
 
and re-suspended in the final formulation medium to obtain the final Drug Product. Aliquots will 
be retained for the Quality Control characterization (see Investigator’s Brochure for detailed list 
of QC tests and specifications). Since the medicinal product is freshly infused, the interval time 
prior to infusion does not allow the execution of all Quality Control (QC) tests, therefore the 
results for some of them will be obtained only after patient treatment. In order to maximize the 
number of QC tests results available at the time of infusion, the manufacturing process is put on 
hold for few hours at the Drug Substance stage and aliquots for most of the QC tests are retained 
at this stage. Results for viability, immunophenotype, endotoxin and mycoplasma tests will be 
available before infusion (see the Investigator’s Brochure). 
The outlined QC strategy has been designed taking into consideration the peculiar nature of the 
manufacturing process, and the definitions of the Drug Substance and the Drug Product reported 
above. In this light, the manufacturing process can be considered as a single intervention starting 
from the collection of the cells from the patient up to the re-injection and, therefore, the DP can 
be considered equivalent to the DS as only a change in the formulation medium is performed. 
If an Out Of Specification (OOS) occurs, responsible physicians will be immediately informed. 
Before starting the production of clinical batches, the validation of the manufacturing process 
will be completed repeating three times the whole production process. 
2.6. 
Preclinical studies 
2.6.1. 
Introduction 
A large body of safety and efficacy preclinical data has been generated in the past decade for the 
use of retroviral and lentiviral vectors encoding the Wiskott-Aldrich syndrome protein (WASP). 
A summary of the available preclinical safety and efficacy studies is provided below. 
Studies conducted using onco-retroviral vectors encoding WASP under either an internal 
promoter or the LTR demonstrated in vitro correction of TCR-driven activation, immunological 
synapse assembly, restoration of WASP expression and actin cytoskeleton architecture in both T 
and B cells. In vivo studies conducted in was-/- mouse model resulted in the differentiation of 
WASP positive lymphocytes with restored TCR-driven proliferation. Furthermore, an in vivo 
selective advantage for gene-corrected lymphocytes was also observed (Klein, Nguyen et al. 
2003). 
More recently, a number of third generation lentiviral vectors encoding WASP under the control 
of constitutive or autologous promoters have been tested in vitro and in vivo in the was-/- mouse 
model. 
Re-expression of WASP was demonstrated in vitro in different patient hematopoietic cell 
lineages (T, B and DC). T cell proliferation, IL-2 secretion, F-actin polarization and correction of 
DC cytoskeleton defects in DC, as well as improvement of colitis were observed in vivo along 
with a confined expression of WASP to the hematopoietic compartment, when expressed under 
the autologous promoter, and a selective advantage for corrected lymphocytes. Overall, the 
results obtained from these preliminary studies along with the absence of any short and long term 
adverse reactions in treated animals indicate that gene therapy treatment for WASP is a feasible, 
well tolerated approach and prompted the selection of the final WASP vector for preclinical 
studies. 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 41 of 119 
-- 
 
 
 
 
2.6.2. 
Preclinical studies performed with the proposed therapeutic vector 
 
2.6.2.1. 
In vitro studies 
 
2.6.2.1.1. T cells 
Over the past few years, we have tested the efficacy of WAS gene transfer with the w1.6W LV in 
T cells from numerous WAS patients. Transduction levels ranged from 20 to 80% depending on 
vector concentration used for transduction (MOI 10 = 1x107 TU/ml to MOI 100 = 1x108 TU/ml). 
Transduction levels were comparable for T cells of each patient. Importantly, the percentage of 
transduced T cells expressing WASP progressively increased during long-term (9-30 weeks) 
culture indicating that WASP+ T cells have a selective growth advantage over WASP- T cells in 
vitro. This phenomenon was very reproducible since it was observed in 11 out of 11 transduced 
cell lines kept in long-term cultures. WASP expression in transduced cells reached normal levels 
and no over-expression was observed. Correction of functional defects including TCR-driven 
proliferation and IL-2 production was reproducibly achieved after transduction at the dose of 
1x108 TU/ml, which resulted in approximately 1-3 vector copy per cell. Clones of transduced 
CD4+ T cells were generated to determine the relationship between number of integrated vectors, 
WASP expression levels and functional correction. One to two vector copies of the WASP- 
encoding vectors were sufficient to restore WASP expression and IL-2 production. Importantly, 
no evidence for toxicity (abnormal proliferation, IL-2 independent growth, abnormal 
morphology) could be observed in long-term cultures of transduced T cell clones or bulk cell 
lines (up to 30 weeks). 
 
2.6.2.1.2. B cells 
Our collaborator A. Galy (Généthon) tested WAS transgene expression in WASP-negative EBV- 
transformed B cell lines from 3 WAS patients following transduction with the w1.6W LV. Cells 
were analyzed 8 days post-transduction to evaluate WASP mRNA and protein expression using 
respectively a quantitative real-time RT-PCR assay, intracellular fluorescence-activated cell 
sorting (FACS) detection or Western blotting. Levels of WAS mRNA and protein expression 
correlated with the number of integrated copies per cell, leading to restoration of WASP 
expression in B cell lines. 
 
2.6.2.1.3. Dendritic cells 
Our collaborators at Généthon also tested WAS transgene expression in monocyte-derived DC. 
These cells were prepared from the blood of 2 WAS patients by culture of adherent blood cells in 
the presence of GM-CSF and IL-4 for 2 weeks. Cells were transduced with the w1.6W vector at 
the dose of 1x108 TU/ml. Quantitative RT-PCR showed that w1.6W vector induced WAS mRNA 
expression in these preparations of cells compared to untransduced cells. Functional correction 
was documented by the restoration of podosome formation in transduced DC. 
Collectively, these data demonstrate that transduction of T cells, B cells and DC from WAS 
patients with the w1.6W vector leads to WASP expression and to functional correction. 
Importantly no toxic effect or event of transformation was documented in any of the models 
tested. 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 42 of 119 
-- 
 
 
 
 
2.6.2.1.4. CD34+ cells 
As a prerequisite for the development of a gene therapy protocol for WAS, the safety and 
efficacy of WAS gene transfer into HSC isolated from WAS patients had to be validated. This 
has been reported recently using both a retroviral vector and a lentiviral vector (Dewey et al. 
2006; Charrier et al. 2007) and confirmed with GMP-grade LV. Results with the w1.6W 
lentiviral vector indicate that WAS HSC can be normally purified from the bone marrow and can 
be transduced at high levels, while retaining ex vivo survival rates and ability to form colonies 
similar to those of control HSC. The restoration of WASP expression was demonstrated in the 
differentiated progeny of WAS HSC by Western Blot. To assess if WASP over-expression could 
be toxic in HSC, we tested the effect of vector-driven WASP expression in normal HSC in vitro. 
No perturbation of in vitro differentiation of hematopoietic stem cells from healthy donors was 
observed following transduction. In addition to hematopoietic cells, we found that WASP was 
expressed also in normal human endothelial cells (which can express the CD34 antigen), but 
transduction with w1.6W LV did not result in increased expression of the endogenous WASP. 
Collectively, these results demonstrate the feasibility of lentiviral vector-mediated ex vivo WAS 
gene transfer into hematopoietic cells. 
 
2.6.2.2. 
In vivo studies 
In vivo proof of principle data were obtained using 2 different WAS KO mouse models. 
Experiments were carried out in collaboration between TIGET and Généthon. Murine Lin– cells 
transduced at different viral MOI with 1 or 2 hit transduction procedure were re-infused in sex- 
mismatched animals. Mice were followed for up to 12 months post-transplantation and 
monitored for a number of different functional parameters. 
2.6.2.2.1. Long-term engraftment and WASP expression 
We have generated solid evidence of long-term engraftment and efficacy of the gene therapy 
approach based on the w1.6W lentiviral vector in Was-/- transplanted mice (Marangoni et al., 
2009). Fifty transplanted mice were followed for up to 12 months. Engraftment and transduction 
efficiency, vector copy number, functional correction and blood counts were evaluated at 
different time points throughout the observation period. A full size transgenic WAS protein was 
produced in mice cells as assessed by Western Blot and FACS analyses. High, stable and 
multilineage donor cell engraftment and WASP expression were achieved up to 12 months after 
gene therapy. Noteworthy, a selective advantage for T and B lymphocytes expressing transgenic 
WASP was observed. 
2.6.2.2.2. Safety in the mouse model 
Autopsy and histopathology examination were performed at sacrifice. None of the analyzed 
parameters revealed side effects related to the treatment. Survival rate of the treated mice was 
within the normal range and the observed hematopoietic tumors (4) were of host origin and did 
not contain vector sequences. Such events were also seen in KO mice treated with untransduced 
Lin- cells and were therefore not attributable to a side effect of the gene therapy procedure. 
In order to further evaluate if the WASP-encoding vector is associated with in vivo 
tumorigenesis, secondary transplantation experiments were designed and performed at our 
collaborating site (Généthon). A total of 32 recipient mice were irradiated and infused with bone 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 43 of 119 
-- 
 
 
 
 
marrow cells obtained from recipients of primary transplants with transduced cells. Mice were 
observed up to 6 months. Multi-lineage and long-term persistence of the w1.6W vector could be 
measured, attesting that long-term hematopoietic stem cells were initially transduced with the 
vector and that the vector did not prevent them to function for extended periods of time. No 
tumors of donor/transduced cell origin were observed. In conclusion, we documented successful 
engraftment of transduced cells in primary and secondary transplantation protocols. The study 
design and analytical methods allowed the detection of tumors in the mice, and there was no 
evidence of clonal expansion or malignancy induced by the treatment with the WASP-encoding 
vector, no evidence of hematopoietic toxicity from the WASP-encoding vector, and no evidence 
of toxicity from the WAS transgene as expressed in this system. 
 
2.6.2.2.3. Efficacy in the mouse model 
Ex vivo gene therapy in the was-/- murine model allowed long-term correction the T cell defects, 
restoring anti-CD3 proliferation and production of various Th1 and Th2 cytokines (Marangoni et 
al., 2009). B cell-counts were improved in mice receiving Lin- cells transduced at high MOI. 
Moreover, ex vivo gene therapy resulted in correction of the defect in B cell-migration in 
response to CXCL13 and improvement of antibody response to polysaccharides (Pneumococcal 
vaccine) at 4-5 months after gene therapy as compared to untreated was-deficient mice. 
Importantly, mice treated with LV gene therapy showed restoration of cytoskeletal functions in 
myeloid cells, as assessed by the presence of podosome-forming cells in dendritic cells 
differentiated in vitro, at levels of 40-100% of normal. Although Was-/- mice show only a 
moderate defect in platelet count, we evaluated the effect of gene therapy on platelets. Transplant 
of Lin- cells transduced at high MOI, but not low MOI, resulted in significant improvement in 
platelet count to levels achieved with transplant of wild type Lin- cells. 
Mice deficient in WASP in the 129/Sv background develop a TH2-mediated autoimmune colitis, 
with a severe colitis developing after irradiation and transplant of WAS-KO cells. Data generated 
at Gènethon showed that the colon of mice transplanted with cells transduced with the WASP- 
encoding vector was significantly ameliorated and only focal or mild defects persisted in some 
mice; in one set of experiments diarrhea was measured and a reduction was observed in the 
animals treated with gene therapy. 
In summary, cumulative results from gene therapy in the murine model show that the proposed 
w1.6W vector is capable of significant biological correction of multiple disorders. 
 
2.7. 
Studies and considerations on safety related to the medicinal product 
2.7.1. 
Biodistribution and vector shedding 
It is expected that the biodistribution of the autologous transduced CD34+ cells reflects that of 
untransduced HSPC, which migrate to the lympho-hematopoietic organs of conditioned 
transplant recipients. In addition, the hematopoietic progeny of the engrafted HSC will also 
infiltrate non-hematopoietic tissues to become resident or migrating extra-vascular hematopoietic 
lineage cells. Biodistribution studies were performed in RAG2-/-IL-2rγc-/- neonate mice, which 
represent a suitable model to study engraftment and differentiation of human HSPC (Traggiai 
Chicha et al. 2004). Human derived umbilical cord blood (UCB) CD34+ cells were transduced 
with clinical grade vector according to the manufacturing procedure established for the clinical 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 44 of 119 
-- 
 
 
 
 
trial. The transduction efficiency was on average 72% as assessed in a CFU-C assay and the 
mean integrated VCN on cultured cells was 1.5. After transduction, 0.3x106 human CD34+ UCB 
cells were transplanted into each RAG2-/-IL2r-γc-/- neonate mouse. This dose corresponds to 
1x108 CD34+ cells/kg, thus largely exceeds the 5x106/kg average dose that will be administered 
to patients. Control animals were transplanted with untransduced (UT) or unmanipulated (UM) 
UCB cells, transplanted in equal doses. Mice were euthanized 2 months post-infusion and their 
lympho-hematopoietic organs (BM, liver, spleen, thymus) were collected for further studies. In 
addition, brains and gonads (as representative non-hematopoietic organs) were collected from 
male mice after perfusion with saline solution. 
We found no differences in the biodistribution of the UT, UM and transduced human UCB cells, 
including CD34+ cells in the BM. A normal differentiation pattern into T cells was observed in 
the thymus and differentiated B cells were normally represented in the BM and spleen of mice 
treated with transduced human HSPC. qPCR (quantitative polymerase chain reaction) 
documented the presence of LV in hematopoietic organs of all mice transplanted with transduced 
CD34+ cells. The results obtained from both brain and gonads for LV and human genome 
sequences were below the sensitivity threshold of the assay (< 16 copies in 105 cells). 
Upon transduction, the vector becomes stably integrated as a provirus within the chromatin of 
target cells. Because of the replication-defective nature of the vector, there is no possibility of 
spontaneous mobilization of the provirus from the transduced cells unless helper functions are 
also provided in the transduced cells either by recombination during vector production or by 
super-infection with wild-type HIV in an infected host. Both of these possibilities are extremely 
unlikely, because: 1) the multi-split design of the packaging system and the lack of homology 
between the envelope and transfer vector genome make extremely unlikely that a replication- 
competent recombinant be generated during production; 2) the self-inactivating feature of the 
vector LTR prevents transcription from the proviral LTR and thus prevents vector mobilization 
even in the case of recombination with the packaging constructs or superinfection of the 
transduced cell by wild-type HIV-1, as we have previously reported (Bukovsky et al. 1999). 
There is however the concern that some infectious vector particles remain associated with the 
transduced cells and that can be subsequently released to transduce bystander cells as shown with 
our laboratory-grade vector stocks in CD34+ cells cultured with stromal cells and other Authors 
using mouse HSPC (Pan et al. 2007). Whereas the amount of vector being released is 
conceivably limited and thus the chance of off-target transduction is low, the potential release of 
some infectious vector from the transduced cells after in vivo infusion may raise concerns on its 
biodistribution and the potential transduction of unwanted cell types which are typically 
associated with in vivo vector administration. We have set up experiments specifically aimed at 
assessing the extent of vector release from previously transduced human HPSC. Our results show 
that: 1) vector release and bystander transduction can occur but at substantially lower level when 
using vector stocks from the large-scale production and purification process used for GMP 
production, as compared to laboratory grade stocks. This difference may be due to the fact that 
the latter are concentrated by ultracentrifugation without further purification and may contain 
larger amount of impurity and aggregated particles; 2) exposure of the transduced HSPC to 
autologous human plasma significantly inhibits bystander transduction, so that it becomes 
virtually undetectable when using large scale LV stocks. These findings suggest that in the 
clinical setting, in which the transduced cells are inoculated into the bloodstream of human 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 45 of 119 
-- 
 
 
 
 
patients, the potential occurrence of bystander cell transduction may be reduced to negligible 
levels. 
Moreover, to directly assess the eventual occurrence of bystander cell transduction upon in vivo 
infusion of transduced cells, we have exploited the vector integration studies performed in the 
organs of Rag2-/-, Il2r-gamma chain-/- mice transplanted with transduced HSPC. DNA 
extracted from the organs of the transplanted mice was analyzed for the content of vector DNA 
and human genome by qPCR as described above. In the experiment performed we have observed 
corresponding estimates for: 1) the extent of human cell repopulation estimated by FACS 
analysis for cells expressing the human hematopoietic marker CD45 and the proportion of 
human genome over mouse genome in the total DNA extracted from the organ (as estimated by 
qPCR for human telomerase and mouse b-actin); 2) the vector content per human genome in the 
different organs (as estimated by qPCR for vector sequence versus human telomerase). The 
results obtained with this strategy, indicate that in the mouse organs tested the vector distributes 
together with the human cells and that at least the vast majority of the detectable vector genome 
remains associated with the human genome in stable proportions, thus without showing 
indication of mobilization or bystander transduction of mouse cells. 
In parallel, another PCR able to discriminate whether LV DNA integrated in mouse genome was 
set up, in order to exclude that a mobilization from human to mouse cells had occurred. This 
PCR amplifies vector DNA integrated in the proximity of mouse repetitive and highly 
interspersed B2 SINE family elements (Follenzi et al. 2002). As these sequences constitute 
approximately 0.7% of total mouse genomic DNA (estimates ranging from 2500 to 100000) and 
are only found at low abundance in humans (< 50), it is likely that most integrants in the mouse 
genome are found at a PCR-amplifiable short distance from one such element. The results of 
these studies have allowed to establish that bystander transduction does not occur above a 
threshold detection level in several tissues of the mice transplanted with transduced HSPC. 
2.7.2. 
Germline transmission 
Because the risk of inadvertent germ line transmission associated with the administration of ex 
vivo transduced human cells is considered “low” or “minimal” according to EMA and ICH, 
respectively, and animal testing of human cells may be difficult or not meaningful, non-clinical 
germ line transmission studies of human genetically modified cells are not recommended (EMA 
Guideline on non-clinical testing for inadvertent germline transmission of gene transfer vectors, 
EMA/273974/2005 and CHMP/ICH/469991/2006). 
Moreover, no evidence of germ line transmission nor of transgene expression in germ cells after 
in vivo delivery of RV and LV into experimental animals have been reported to date. In a study 
of testicular tubule injection of LV in mice, the sperm collected from recipient testes were able to 
generate normal pups and none of the offspring carried the transgene (Ikawa et al. 2002). Rat 
spermatogonial stem cells, however, can be effectively transduced ex vivo by LV and generate 
viable transgenic offspring upon re-implantation into rat testes. Moreover, LV can transduce in 
vitro fertilized eggs from a variety of species, including mouse, rat and pig. Indeed, LV are 
becoming common tools for the generation of transgenic animals by in vitro oocyte transduction 
followed by in utero implant (Park 2007). 
Thus, given the concerns for residual infectious vector associated with the transduced HSPC, we 
have expanded the biodistribution study of vector and HSPC progeny in Rag2-/-, Il2r-gamma 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 46 of 119 
-- 
 
 
 
 
chain-/- mice described above to test for the presence of LV genome in the gonads at the time of 
sacrifice, 2 months post-transplant. For this experiment, we extracted DNA from the gonads and 
performed: 1) qPCR for vector and human genome content, as described above; 2) nested B2- 
specific PCR for vector integrated within the mouse genome, as described above. The assays are 
designed in a way that if any vector DNA is detected in the gonads it should be associated with 
the human genome, and in corresponding ratio as observed for the other organs of the mouse. 
Our results show no detectable vector in the gonads of those mice by qPCR, which was also 
confirmed by nested B2-specific PCR. 
Last but not least, we should remind that the concerns for germ line transmission of the genetic 
modification are unfortunately further mitigated by the severe disability and short life expectancy 
of the patients to be enrolled in the trial, and by the administration of conditioning regimen 
which is often associated to the induction of sterility. 
2.7.3. 
Genotoxicity and carcinogenicity 
Insertional oncogenesis is a possible consequence of vector integration into the human genome. 
Several decades of studies of gamma- or onco-retrovirus induced lymphomas/leukemias (Lund et 
al. 2002; Suzuki et al. 2002; Uren et al. 2005) in mice have shown that proviral insertions near or 
within proto-oncogenes can trigger their over-expression and/or aberrant transcription and lead 
to cell transformation. Both mechanisms of oncogene activation are dependent on the strong 
transcriptional enhancer/promoter elements contained in the viral Long Terminal Repeat (LTR) 
(Uren, Kool et al. 2005). Initially, the gamma retrovirus-derived vectors (gRV) proposed for 
gene therapy applications were considered to pose a much lower risk of insertional mutagenesis 
compared to gamma-retrovirus infection, given the lack of replication and the limited number of 
integrations occurring during the ex vivo transduction procedure. Unfortunately, however, gRV- 
induced activation of the LMO2 oncogene and/or other oncogenes resulting in leukemogenesis 
has now been reported in 5 out of 20 X-linked SCID patients treated with HSPC gene therapy 
(Hacein-Bey-Abina et al. 2003; Pike-Overzet et al. 2007). Similarly, clonal expansions resulting 
in myelodysplastic syndrome were ascribed to gRV insertion into the EVI1, PRDM16 or SETBP1 
(Ott et al. 2006; Ott et al. 2007) proto-oncogenes in 3 CGD patients treated with HSPC gene 
therapy. 
In light of these serious adverse events, the oncogenic risk of vector integration in HSPC had to 
be re-evaluated (Baum and Fehse 2003; von Kalle et al. 2004) and a great effort went into 
defining the integration pattern of the different vectors and its impact on endogenous gene 
transcription (Schroder, Shinn et al. 2002; Wu, Li et al. 2003; Hematti et al. 2004; Lewinski et al. 
2006). A better understanding of the risk associated with gRV and LV in HSPC gene therapy is 
emerging from a growing number of pre-clinical studies which have modeled genotoxicity in in 
vitro cell immortalization assays and in in vivo murine models of leukemia/lymphoma 
development (Williams 2006; Will et al. 2007) and from transcriptional studies of gene de- 
regulation at the vector integration sites in cells transduced in vitro or retrieved from transplanted 
mice and patients (Recchia et al. 2006; Ryu et al. 2008). 
Direct comparison of gRV and LV in these assays demonstrates that prototypical LV have a 
substantially lower oncogenic potential than gRV (Montini et al. 2006; Modlich et al. 2008). 
Important features contributing to the lower genotoxicity of LV as compared to conventional 
gRV are likely to be: 1) the self-inactivating design, which prevents LTR-dependent effects on 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 47 of 119 
-- 
 
 
 
 
the transcription of genes neighboring the integration site; 2) the use of a vector internal 
promoter to drive transcription of the therapeutic transgene, which is chosen among those with 
moderate transcriptional activity as compared to gRV LTR (these aspects are discussed in more 
details in paragraph 6.4.). 
Moreover, it has been demonstrated that both gRV and LV display a non random genomic 
distribution pattern, which is characteristic for each vector and that may thus influence the safety 
profile of each vector differently. Indeed, both gRV and LV integrate more frequently within 
genes than expected of random insertion throughout the genome, gRV preferentially integrate 
near the promoters and CpG islands of actively transcribed genes (Wu et al. 2003; Bushman et 
al. 2005). This preference makes it more likely for the LTR to interfere with regulation of the 
genes near the integration site (Schroder et al. 2002; Wu, Li et al. 2003; Bushman, Lewinski et 
al. 2005; De Palma et al. 2005). Furthermore, when hot spots of vector integration were 
identified in the mouse and human HSPC genome, striking differences were observed between 
gRV and LV (Montini, Cesana et al. 2006; Aiuti et al. 2007; Bushman 2007; Cattoglio et al. 
2007; Deichmann et al. 2007; Schwarzwaelder et al. 2007). gRV, but not LV, integrations 
occurred at proto-oncogenes and genes involved in the control of cell proliferation with a 
frequency significantly higher with respect to the expected random frequency. LV integrations 
appeared to occur more broadly across gene classes and hot spots were found at low frequency 
and in locations characterized by high density of expressed genes. 
Indeed, the unique propensity of gRV to integrate near potentially dangerous genes may be an 
important factor determining genotoxicity, and the lack of such preference in the case of LV may 
well contribute to the improved safety profile demonstrated in preclinical gene therapy models. 
Beside the intrinsic features of the vector, however, other factors may contribute significantly to 
the risk of oncogenesis in gene therapy treated patients (Woods et al. 2006), including disease- 
related conditions that may increase the susceptibility to develop tumors (such as the 
accumulation of immature progenitors or immune deficiency) and the biological activity of the 
therapeutic transgene itself. 
In order to detect possible genotoxic effect of the WASP LV, a recently developed assay was 
performed by Gènèthon. This test detects the presence of clonal dominance of insertional 
mutants which expand in initially polyclonal cultures of primary murine hematopoietic cells 
within two weeks after gene transfer and acquire serial replating ability, the extent of which 
provides a measure of clonal fitness (Modlich et al., 2006). The test has some limitations, since it 
detects essentially only genotoxicity related to EVI1 insertions and is based on a mouse in vitro 
transformation after in vitro cytokine expansion. In none of the three assays conducted with 
WASP LV, induction of replating activity was observed. As expected, a positive control 
represented by gRV containing a strong myeloid viral promoter (SFFV) showed a high replating 
activity. Thus, the WAS vector did not exhibit any genotoxic potential, confirming its safety also 
in this context. 
To efficiently analyze high numbers of lentiviral insertion sites in the DNA of transduced cells, 
Généthon has developed an improved high-throughput method called vector integration tag 
analysis (VITA), described in (Mantovani et al. 2006). VITA is based on the identification of 
genomic tags associated to the insertion sites, which are used as signatures of the integration 
events. We analyzed the integration sites following WASP-encoding w1.6W LV transduction of 
murine WKO cells and compared the pattern of integration sites obtained in a population of 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 48 of 119 
-- 
 
 
 
 
hematopoietic progenitor cells cultured in vitro with that of cells capable of engraftment in vivo 
(Mantovani et al., 2009). Lineage-negative hematopoietic progenitor cells were prepared from 
WKO mice and were transduced ex vivo with the w1.6W LV and transplanted into lethally 
irradiated WKO mice. Extracts from the Lin- cells (pre-infusion), as well as blood and BM of 
mice after gene therapy were used to prepare genomic DNA and were compared by VITA 
technique to analyze the vector insertion sites. 
A high number of unique junctions was found in the cultured cells (more than 90 unique 
junctions in a first approach without concatenation, more than 300 in a second approach with 
concatenation). In blood and BM samples, the numbers of unique junctions were significantly 
lower with respectively 17 to 28 unique sites in blood samples and 8 to 13 unique sites in BM 
samples, depending on the animal analyzed. In contrast to what observed with the cultured cell 
sample, the number of unique sites found in the blood/BM samples reached a plateau as number 
of sequences increased (80-120 junctions sequenced for each point). As previously reported by 
others, we found a bias toward insertion of the vector into genes, but no obvious bias for the 
distribution of insertions into specific categories of genes. No common integration sites between 
the in vitro and in vivo samples and no evidence of integrations shared by different mice were 
detected. 
Altogether, the analysis of the w1.6W LV integration sites in murine cells revealed insertions 
into a polyclonal population, no hotspot and no insertion of the vector into previously identified 
CIS. 
2.7.3.1. 
Integration analyses in other trials 
LV-based HSPC gene therapy was recently tested in ALD patients. The vector tested was a third- 
generation LV similar to the one proposed for this trial, except for the internal promoter driving 
the therapeutic cDNA, which was derived from the strong MND LTR. A clear clinical benefit 
has been reported in the two treated patients together with stable polyclonal hematopoietic 
repopulation by the transduced HSPC and no evidence for skewing in genomic distribution or 
specific gene classes by comparing vector integration sites in pre- and post-transplant samples 
(Cartier N. et al., 2009). These data provide compelling evidence that LV provide for efficient, 
safe and long-term marking of human hematopoiesis as predicted by a large amount of pre- 
clinical studies. 
In another recent trial of LV-based HSC gene therapy for beta-thalassemia, a self-limiting 
expansion of a clone bearing a LV insertion putatively leading to a gain-of-function in growth 
control was recently reported in a beta-thalassemia patient (Cavazzana-Calvo M. et al. Nature 
2010). Further investigations are needed to clarify the potential contribution of this insertion to 
the observed clonal expansion and its benign or pre-malignant nature. Moreover, several 
additional factors may have contributed to the findings in the thalassemia trial, including a 
complex vector design and the use of non-purified low titer vector that resulted in the 
transplantation of very few transduced HSPC and a long-delayed in vivo marking rate. 
In summary, our studies and recent preliminary findings from clinical trials indicate that 
integration of prototypical SIN LV has low genotoxic potential and, together with the high gene 
transfer efficiency and the therapeutic efficacy demonstrated in pre-clinical models, provide a 
major scientific rationale for advancing WAS LV to clinical experimentation. 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 49 of 119 
-- 
 
 
 
 
2.7.4. 
Immunogenicity 
Even though immunogenicity is considered in international guidelines for GTMPs, it is unlikely 
that the proposed therapeutic approach may elicit an immune response either against the 
therapeutic gene product or against the viral vector. 
The infusion of genetically modified cells occurs, in the proposed clinical protocol, following the 
administration of a non-myeloablative conditioning regimen known to establish a certain degree 
of tolerance to donor encoded antigens in the context of allogeneic transplant. 
Further, the drug product consists of CD34+ genetically modified cells exposed to the viral 
vector ex-vivo. It is recognized that this procedure has a lower anti-vector immunogenic risk 
when compared to in vivo administration of viral vectors. In addition, WASP is an intracellular 
protein, its expression in the vector is regulated by the endogenous, physiological promoter, and 
WAS protein levels are regulated by the WIP molecule. 
For these reasons, no in vivo pre-clinical studies for immunogenicity have been performed. 
Moreover, there was no evidence of immune mediated elimination of WASP transduced cells in 
the murine model of gene therapy, since transduced cells persisted long term with evidence of 
selective advantage. 
Nonetheless, a careful assessment of any immune response will be performed by monitoring 
anti-WASP and anti-HIV proteins (anti-p24) antibodies every 6 months for the first year, then 
once a year during the clinical trial follow up period. 
 
2.7.5. 
Environmental risks 
The gene treatment planned in this clinical trial does not involve particular risks in terms of 
public health. The cell infusion will occur in a dedicated room of the authorized area (Impianto 
Area for MOGM Class -MI/IC/IMP.2/01-002 Rev.2) in the Hematology and Bone Marrow 
Transplantation Unit, notified for the use of the specific GMOs. 
The procedures and programs for the cleaning, disinfection and decontamination of the ward, the 
operating theater (if it is used) and the equipment do not differ from the standard ones in force 
for the management of patients who undergo bone marrow transplantation, according to 
Institutional SOPs. 
All the solid and liquid waste contaminated by biological and/or potentially infected material are 
disposed of in compliance with the hospital regulations in force. All members of the staff, 
wearing gloves and a protection mask, are adequately educated and trained. In particular, the 
staff is shown how to apply the regulations in force to patients who receive multiple transfusions, 
are immunosuppressed and at high risk of infections. 
In a recent review of the literature, no RCR was detected in 103 patients treated with ex vivo 
retrovirally transduced cells, nor in 342 patients treated in vivo with retroviral vectors (Schenk- 
Braat et al. 2007). Overall, in more than 18 years of clinical trials, there has been no report of 
cultural or molecular RCR test positivity, in more than 300 clinical trials conducted worldwide 
with retroviral vectors and a very high number of people considering healthcare staff, family 
members and other people. In addition, no RCL has been detected in the clinical trials currently 
conducted with SIN lentiviral vector (Cartier N et al., Science 2009). Even in the unlikely event 
of RCL, the risk of transmission is expected to be typical of a lipid membrane enveloped virus. 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 50 of 119 
-- 
 
 
 
 
Therefore, risks for healthcare staff and others should be mostly limited to accidental 
venipuncture and accidental mucosal or parenteral exposure to body fluids. An emergency plan 
is active in the Hospital for this circumstance. 
The patients will be followed to exclude the possible development of RCL infection (see above). 
Samples will be collected before treatment (at baseline) and after 1, 3, 6, 12 and 24 months, and 
then yearly after treatment, as suggested by FDA guidelines (Guidance for Industry, November 
2006). In case the samples collected during the first two years result always negative for the 
screening tests HIV p24, VSV-G DNA and HIV-pol RNA, the subsequent yearly samples will be 
stored, but not analyzed. If one of the above- mentioned tests will result positive, the screening 
evaluations will be repeated at the next planned follow up visit; in the case two of the three 
screening test will result positive, a confirmatory RCL culture tests will be performed. Of note, 
HIV-derived RCL would conceivably be sensitive to – and treatable by – conventional anti-HIV 
therapy targeting all pol-derived enzymes (RT, protease and integrase). We can estimate that the 
risk of acquiring an infection by wild type HIV for the patients enrolled in the clinical study is 
superimposable to that of the population of the same age not under study. 
 
2.8. 
Clinical trials performed in other countries with similar Drug 
Substance 
2.8.1. 
Gene Therapy for Wiskott-Aldrich-Syndrome using a retroviral vector 
Since 2006, a gene therapy clinical trial has been conducted in Hannover Medical School by Dr. 
C. Klein. The preliminary results have been presented at the 2008 American Society of Gene & 
Cell Therapy (ASGT) meeting and an update was given at the 2009 European Society for Blood 
and Marrow Transplantation (EBMT) Inborn Error meeting in Cambridge (UK). Ten patients 
were treated with infusion of autologous mobilized peripheral blood CD34+ cells transduced 
with a gammaretroviral vector encoding WASP, under the control of a viral promoter. All 
patients are alive, one patient underwent a bone marrow transplant due to failure of engraftment, 
associated with low dose of cells infused. The first two patients have reached >24 months of 
follow up and showed evidence of long-term gene marking in myeloid and lymphoid cells. Gene 
marking in myeloid cells is about 10-30%, in NK cells 50-80%, in T cells 80-95%. In addition, 
the majority of peripheral thrombocytes showed evidence of WASP expression and platelet 
counts have substantially improved. Functional reconstitution was documented in dendritic cells 
(podosome formation), T cells (proliferation in response to CD3-signaling), and NK cells 
(formation of immunological synapse). Of note, they did not develop any severe infections or 
bleeding after gene therapy. In the first patient eczema and autoimmune haemolytic anaemia 
have resolved, while anti-neutrophil and anti-PLT antibodies have disappeared. In the second 
patient eczema has resolved. Clinically, patients are in good conditions more than two years after 
gene therapy (Boztug K et al, 2010). These results are very encouraging and confirm our 
hypothesis that the mixed chimerism associated with reduced conditioning, also thanks to the 
occurrence of selective advantage, is sufficient to allow biological efficacy and clinical benefit. 
However, the majority of WAS patients treated with the retroviral vector encoding WASP have 
developed leukaemia during the study conducted in Germany (M. Schmidt, K Boztug personal 
communication). As of January 2014, 7 out of the 10 treated patients developed acute leukaemia 
(one acute myeloid leukemia (AML), four T cell acute lymphoblastic leukemia (T-ALL), and 
two primary T-ALL with secondary AML, with all six cases of T-ALL associated with a 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 51 of 119 
-- 
 
 
 
 
dominant clone with vector integration at the LMO2) (Braun et al, 2014). It should be noted that 
the lentiviral vector used in the present study has a different origin and it does not contain the 
part of the retroviral vector that is potentially responsible of the leukaemia. While more 
extensive studies and longer follow up will be needed to determine the overall safety profile of 
gene therapy with gamma-retroviral vector, this finding further reinforces the choice of a 
potentially safer platform, based on HIV vector for gene transfer in WAS. 
 
2.8.2. 
Gene Therapy for Adrenoleukodystrophy using a lentiviral vector 
As mentioned above, a clinical trial of gene therapy based on HSC and LV for the treatment of 
cerebral Adrenoleukodystrophy (ALD) was started at the Hôpital Saint-Vincent de Paul and 
Hôpital Necker in Paris (Cartier N et al, Science 2009). X-linked adrenoleukodystrophy (X- 
ALD), a genetic disorder secondary to alterations in the ABCD1 (ATP-binding cassette, sub- 
family D [ALD], member 1) gene, results in defective peroxisomal oxidation and the 
accumulation of very long chain fatty acids (VLCFA) in all tissues. The disorder primarily 
affects the adrenal cortex and the nervous system. 
Up to now, three patients suffering from cerebral ALD have been included. Autologous CD34+ 
progenitor cells were collected from MPB and transduced ex vivo for 18 hours with a 3rd 
generation LV encoding the ALD protein cDNA under the control of the MND promoter. The 
LV used in this trial differs from the Drug Substance described in the present protocol for some 
aspects, including the presence in the transfer construct of a different promoter driving 
therapeutic gene expression, and the protocol of large scale GMP production. The gene transfer 
procedure of CD34+ cells was based on a similar cytokine combination to the one used for the 
preparation of the Medicinal Product described in the present protocol. Transduced cells were 
frozen after transduction to perform all tests planned before infusion. After thawing and prior to 
re-injection, 20-40% of the transduced CD34+ cells expressed the ALD protein with a mean of 
0.7 copies of integrated provirus per cell. Transduced cells were re-infused into 
pharmacologically conditioned patients (receiving a full myeloablative regimen consisting of 
Busulfan and Cyclophosphamide). The first results of this study have shown that genetically 
modified CD34+ progenitor cells transduced with the ALD protein-encoding LV are able to 
differentiate in all hematopoietic lineages (granulocytes, monocytes, B and T lymphocytes) in 
vivo. The percentage of corrected lymphocytes and monocytes in the peripheral blood of treated 
patients remained stable from day 30 to the longest follow up (30 and 24 months for Pt 1 and 
Pt2, respectively). From 10% to 15% of CD14+, CD3+, CD19+ and CD3-CD56+ cells expressed 
the ALD protein. Test for vector-derived RCL and vector mobilization were negative up to the 
last follow up. Clinical and instrumental evaluations revealed stability of the disease and a course 
comparable to that of patients undergoing allogeneic HSC transplantation. 
These results indicate that HSC gene therapy using HIV-1-derived LV is not associated with the 
emergence of RCL and vector mobilization, and that LV allow safe and efficient transduction of 
human HSC and maintenance of gene expression in vivo. 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 52 of 119 
-- 
 
 
 
 
3. 
OBJECTIVES 
The objective of the present gene therapy clinical trial is to evaluate the safety of the transduced 
stem cells infusion in patients affected by WAS after a reduced-intensity conditioning regimen 
and its efficacy in improving the patient’s immune-function and thrombocytopenia. 
 
3.1. 
Primary Objectives 
1) To evaluate the safety of the administration of autologous CD34+ cells transduced with a 
lentiviral vector containing the WASP gene in patients with WAS, after a reduced intensity 
conditioning regimen. 
The short-term and long-term safety of LV gene transfer into HSC will be evaluated on the basis 
of adverse event reporting and monitoring for the theoretical risk of systemic reactions to cell 
infusion, abnormal clonal proliferation and immune response to the transgene. Hematological 
reconstitution after non-fully myeloablative conditioning regimen will be closely monitored. 
2) To evaluate the long-term engraftment of WASP-expressing transduced cells. 
The proportion of multilineage WASP-expressing cells, indicating engraftment of repopulating 
transduced stem cells, will be evaluated at different time points after transplant. 
3) To evaluate the efficacy of gene therapy assessed as: 
3.1) Improvement of the patient’s immune function, specifically of T cell function 
and antigen-specific responses to vaccinations 
Improvement of in vitro T-cell functions and in vivo antibody production will be 
analyzed as part of the post-gene therapy follow up. 
3.2) Improvement of thrombocytopenia 
Increase in platelet count compared to baseline and normalization of mean platelet 
volume (MPV) at 1-year post treatment will be assessed according to the severity of 
thrombocytopenia before gene therapy. 
 
3.2. 
Secondary objectives 
A) To evaluate the efficacy of gene therapy in improving the patient’s clinical conditions, 
assessed by reduction in frequency of severe infections, and bleeding episodes and 
reduction of autoimmunity phenomena and eczema. 
A complete clinical and laboratory assessment of the patient after the proposed treatment is 
scheduled at different time points, to provide an overall evaluation of the clinical benefit. 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 53 of 119 
-- 
 
 
 
 
4. 
INVESTIGATIONAL PLAN 
This clinical study is based on ex vivo gene therapy for Wiskott-Aldrich syndrome using 
autologous hematopoietic stem/progenitor cells (purified CD34+ cells). Genetically modified 
cells will be re-infused to patients following a reduced intensity conditioning regimen (see 
chapter 10 for details), dispensed to eliminate the defective cells and favor the engraftment of 
gene corrected cells expressing a normal WASP. The eligibility of the patients will be evaluated 
according to enrolment criteria, which include patients suffering from a severe clinical condition 
or severe WAS phenotype, without a suitable matched donor for allogeneic transplant or 
ineligible for HSCT because of age > 5 years or for clinical features. 
In this study, we plan to treat a total of 8 patients in a period of 5 years, which will be followed 
for the safety and efficacy endpoint measures. There is a Follow Up phase composed of an 
Initial Follow Up period of at least 3 years, when subjects are intended to reach the primary 
efficacy timepoint, and then a further Long-Term Follow Up period from 3 years up to 8 years. 
For patients who complete the Follow Up phase, from year 8 onwards there will be an additional 
extended long-term follow up (ELTFU) phase. During the ELTFU phase, patients will be 
contacted annually, in order to collect long-term safety and selected efficacy data, until a 
separate observational long-term follow-up (OLTFU) study is initiated, and patients have 
enrolled in it. Once the OLTFU study is set up, patients will be invited to enter the OLTFU study 
at any time after they have completed a minimum of 5 years follow up in this study (WAS 
Pivotal 201228). 
The treatment will be administered at the Pediatric Immunohematology and Bone Marrow 
Transplantation Unit at the San Raffaele Scientific Institute in Milan, Italy. The investigators and 
the manufacturer of the product have a specialized expertise in hematopoietic stem cells 
manipulation, LV production and gene transfer procedure according to GMP standards. Clinical 
trials using the same lentiviral vectors have been implemented at the Institute for Child Health in 
London, Necker Hospital in Paris and Children’s Hospital in Boston (see above). These clinical 
trials have adopted similar criteria for the enrolment of patients and similar safety and efficacy 
endpoints, but the conditioning regimen is different. The Genethon sponsored trial have adopted 
a myeloablative conditioning regimen based on the use of Busulfan 12 mg/kg, associated to 
Fludarabine (120 mg/sqm), possibly associated to anti-CD20 mAb and/or anti-Thymocyte 
globulin in the setting of autoimmunity. Information deriving from the preliminary results of the 
two trials will be exchanged and discussed in order to allow to define an optimal safe and 
efficacious conditioning for gene therapy in WAS patients. 
4.1. 
Study Design 
4.1.1. 
Type of study 
This is a phase I/II, open label, non-randomized, prospective, single centre study. 
 
4.1.2. 
Study duration 
Enrolment phase: 5 years. 
Starting date: April 2010 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 54 of 119 
-- 
 
 
 
 
Follow Up phase: the Follow Up phase is composed of an Initial Follow Up period of at least 3 
years after the reinfusion of transduced cells, when subjects are intended to reach the primary 
efficacy analysis endpoint, and then a further Long-Term Follow Up period from 3 up to 8 years 
after the reinfusion of transduced cells. In the Initial Follow Up period, patients will undergo 
regular follow up assessments to assess the efficacy and safety of the treatment. 
Extended Long-Term Follow Up phase (ELTFU): patients who complete the Follow Up phase 
will then be contacted annually from year 8 onwards in order to collect long-term safety and 
selected efficacy data until a separate OLTFU study is initiated and patients have enrolled in it. 
Once the OLTFU study is set up, patients will be invited to enter the OLTFU study at any time 
after they have completed a minimum of 5 years follow up in this study (WAS Pivotal 201228). 
 
4.1.3. 
Study population 
Patients of pediatric or adult age affected by WAS, with severe clinical phenotype and/or severe 
genetic mutation, who meet the inclusion/exclusion criteria described below. 
 
4.2. 
Safety Endpoints 
4.2.1. 
Primary safety endpoints 
 
4.2.1.1. 
Safety of reduced conditioning regimen 
a) Hematological reconstitution. 
The absence of engraftment failure and prolonged aplasia (defined as ANC <500/µl at day +60, 
with no evidence of bone marrow recovery and requiring back-up administration) is assumed as 
proving the safety of reduced intensity conditioning regimen associated with the infusion of ex 
vivo transduced CD34+ cells. 
In case the first two patients present prolonged aplasia, modification of the conditioning regimen 
is introduced in the third patient and the rule is again applied for this patient and the following 
two. 
b) Regimen related non-hematopoietic toxicity. 
A surveillance of clinical and laboratory parameters (for clinical features CTCAE ≥2, for 
metabolic/laboratory CTCAE ≥3) of the treated patients (CTCAE will be applied during the first 
100 days after transplant in order to assess the degree of morbidity associated to the reduced 
conditioning regimen). 
Stopping rule: in case of 2 deaths occurring within 180 days from transplant (not superior to 
mortality reported from mismatched related donors, see Table 1, Table 2, Table 3 and Table 4). 
For detailed follow up and timing of clinical and laboratory surveillance see Table 8a, Table 8b 
and Table 10. 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 55 of 119 
-- 
 
 
 
 
4.2.1.2. 
Safety of LV gene transfer into HSC 
a) Short-term safety and tolerability of lentiviral-transduced cell infusion 
This will be evaluated on the basis of adverse events reporting and monitoring of the systemic 
reactions to cell infusion (fever, tachycardia, nausea and vomiting, joint pain, skin rash). 
The short-term safety of lentiviral-transduced cells infusion consists in the absence of serious 
adverse reactions within 48 hours from infusion. 
b) Long-term safety of lentiviral-transduced cell infusion 
This will consist in the absence of: 
• Replication competent lentivirus (RCL). 
• Molecular monitoring of RCL will be assessed via ELISA for HIV p24 antigen in serum. 
A positive HIV p24 test result will be subject to confirmation. If confirmed, second level 
testing will include a) DNA PCR for VSV-G envelope (cells) and b) RT-PCR for HIV- 
pol RNA (serum). Should one of these second level tests also provide a positive result, an 
appropriate confirmatory test may be conducted. 
The tests will be performed at baseline and after 1, 3, 6, 12 months and 24 months. 
Samples will be stored every year thereafter. If one of the above mentioned tests has a 
positive result, the screening evaluations will be repeated at the next planned follow up 
visit; if two of the three screening test have a positive result, a confirmatory RCL culture 
tests will be performed. 
• Abnormal clonal proliferation. Abnormal hematopoietic clonal expansion will be 
monitored by clinical and laboratory surveillance, repertoire study, and bone marrow 
examination. 
Stopping rule: 1 event of malignant proliferation related to gene transfer. 
 
4.2.2. 
Secondary safety endpoints 
a) Lack of immune response to transgene 
The lack of immune response towards the transgene will be measured by antibodies to WASP 
(immunoblot analyses). 
b) Overall safety surveillance 
This will be evaluated on the basis of clinical and laboratory parameters recorded and monitored 
during the follow up of the treated patients, according to the assessment plan. A descriptive 
analysis will be performed on AE, AR, SAE/SAR, UAR, SUSAR, and events will be classified 
for frequency, severity and body system involved. 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 56 of 119 
-- 
 
 
 
 
 
4.3. 
Efficacy Endpoints 
4.3.1. 
Primary efficacy endpoints 
a) Overall survival 
From day +1 to 3 years after gene therapy, and then monitored as part of the long-term follow 
up. 
b) Sustained engraftment of genetically corrected hematopoietic stem cells in peripheral blood 
and/or in bone marrow 
Presence of gene corrected cells assessed by quantitative PCR on peripheral blood and bone 
marrow cells at 1 year after gene therapy. Adequate engraftment is defined as ≥0.04 VCN/cell in 
BM CD34+ (equivalent to 4% assuming a VCN of 1) or ≥0.1 VCN/cell in peripheral blood T 
lymphocytes (equivalent to 10% assuming a VCN of 1). 
c) Expression of vector-derived WASP 
Presence of detectable vector-derived WASP expression at 1 year after gene therapy by FACS 
analyses and/or Western Blot. 
In case of failure of engraftment of transduced cells in two or more patients, intensification of 
conditioning dose and increase in the minimal cell dose will be considered after an amendment 
to the study. 
d) Improved T-cell functions 
Improvement in in vitro T-cell proliferation upon stimulation with anti-CD3i mAbs at 1, 2, and 3 
years after gene therapy as compared to pre-gene therapy values. 
e) Antigen-specific responses to vaccinations 
- Ability to mount a protective humoral response to nominal antigens, including antibodies to at 
least 4 out of 5 T cell dependent antigens tested (Tetanus Toxoid, Diphtheria, Hepatitis B, 
Pertussis, Haemophilus B) measured after the end of the vaccination schedule (foreseen >1 year 
after gene therapy). In case results are available on n<5 antigens, the rule of at least n-1 will be 
applied for defining success. 
- Ability to mount a protective humoral response to unconjugated polysaccharide antigens 
(Pneumococcus), measured after the end of the vaccination schedule (foreseen >1 year after gene 
therapy). 
- Positive cellular response to Tetanus Toxoid after vaccination, measured by in vitro 
proliferative response >1 year after gene therapy. 
f) Improved platelet count and normalization of MPV 
Sustained increase in platelet count compared to baseline during the 3 year follow up, analyzing 
the individual longitudinal profile, according to the planned follow up visit and including platelet 
counts collected for clinical reasons during follow up (in transfusion independent patients). 
Patients will be also stratified according to the severity of thrombocytopenia before gene therapy 
in 5 groups (A, B, C, D and E, Table 5) and the patient will be defined as improved if there is a 
shift from one group to the subsequent. 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 57 of 119 
-- 
 
 
 
 
Table 5 
Stratification of platelet count according to severity of thrombocytopenia 
(count x109/L) 
 
A 
B 
C 
D 
E 
< 20 
20- 49 
50 - 99 
100 – 149 
≥150 
In addition, normalization of MPV at 1-year post treatment compared to baseline will be 
evaluated. 
The need for a platelet infusion will be evaluated by the Investigators based on several factors 
(see Section 6.11). 
4.3.2. 
Secondary efficacy endpoints 
a) Longitudinal analyses of sustained engraftment of genetically corrected hematopoietic stem 
cells in peripheral blood and/or in bone marrow and expression of vector derived WASP 
Presence of gene corrected cells and WASP expression will be assessed at the time points shown 
in Table 8a and Table 8b, as described above. 
b) Analyses of sustained multilineage engraftment of genetically corrected cells in peripheral 
blood and/or in bone marrow 
Presence of gene corrected cells assessed by quantitative PCR on all the available peripheral 
blood and/or bone marrow cell subpopulations (BM subpopulations: GlyA+, CD15+, CD3+, 
CD19+, CD56+; PB subpopulations: CD15+, CD19+, CD56+) at 1, 2 and 3 years after gene 
therapy. The availability of all the subpopulations depends on the amount of samples and on the 
technical procedures. 
Adequate engraftment is defined as ≥0.04 VCN/cell (equivalent to 4% assuming a VCN of 1). 
c) Reduced frequency of severe infections 
Decrease in number of severe infections as evaluated starting from the first year follow up visit 
and thereafter until the 3-year visit after the treatment, by , complete physical examinations, 
hematological and microbiological tests compared to clinical history from the 12 months prior to 
GT. Infections will be assigned a CTCAE grading (as described in Section 4.3.2.1) by the 
Investigators, identical to how severity grading was used in clinical history. 
d) Reduced bleeding episodes 
Reduction in bleeding manifestations when present, as assessed starting from the first year 
follow up visit and thereafter until the 3-year visit, by clinical monitoring, compared to clinical 
history from 12 months prior to GT. Information about bleeding will be recorded through follow 
up by collecting data from parents, clinical reports periodically sent by local doctors following 
the patients at home and by clinical evaluations performed at each follow up visit at our center. 
Data will be recorded in the patients’ clinical charts. Bleeding events will be given CTCAE 
grading (as described in Section 4.3.2.1) by the Investigators, identical to how severity grading 
was used in clinical history. 
e) Reduced autoimmunity phenomena and eczema 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 58 of 119 
-- 
 
 
 
 
Reduction in laboratory markers (number and titer of antibody when available) and/or clinical 
manifestations of autoimmunity, as evaluated starting from the first year follow up visit and 
thereafter until the 3-year visit, by organ-specific and systemic autoantibodies, imaging and 
clinical follow up, compared to clinical history 
Reduction in eczema, as evaluated by clinical score according to Imai et al. 2004. Specifically, 
eczema will be scored by the Investigators based on the following scale: 
1. None 
2. Transient (mild): Asymptomatic or mild symptoms; additional medical intervention over 
baseline not indicated (consistent with CTCAE grade 1). 
3. Moderate: topical or oral intervention indicated; additional medical intervention over baseline 
indicated (consistent with CTCAE grade 2). 
4. Severe: medically significant but not immediately life threatening; intravenous intervention 
indicated (consistent with CTCAE grade 3). 
Eczema scores over time will be presented in a shift table relative to the baseline score. 
f) Improved quality of life 
Improved quality of life, measured after the first year of treatment by reduced hospitalization, 
reduced requirement of drugs, school attendance and social activities. The evaluation will be 
performed according to patients’ age (in particular, social ability with peers will not be evaluated 
before 2 years of age and sport practice will not be considered before 4 years of age). 
 
4.3.2.1. 
CTCAE grading scale 
The severity of the infection and bleeding events will be assigned by the Investigators using the 
National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE) 
definitions. The CTCAE displays Grades 1 through 5 with unique clinical descriptions of 
severity for each event. Grade 1 and Grade 2 events are respectively considered as mild and 
moderate. An event graded 3 or above will be considered for analysis. 
If an infection or bleeding event is not listed as a unique clinical description in the CTCAE, 
clinical judgement will be applied to grade the event. 
For infections, a Grade 3 event is one that requires intravenous antibiotics; and/or antifungal 
intervention; and/or antiviral intervention and/or invasive intervention is indicated. A Grade 4 
event is an event that has life-threatening consequences, and/or urgent intervention indicated. A 
Grade 5 event is an event that results in death. 
For bleeding events, a Grade 1 event is one that presents with mild symptoms; intervention not 
indicated. A Grade 2 event is one that presents with moderate symptoms (limiting age- 
appropriate instrumental activities of daily living); intervention indicated. A Grade 3 event is one 
that requires platelets or red blood cells transfusions; and/or invasive intervention; and/or 
hospitalization. A Grade 4 event is an event that has life-threatening consequences, and/or urgent 
intervention indicated A Grade 5 event is an event that results in death. 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 59 of 119 
-- 
 
 
 
 
4.3.3. 
Exploratory efficacy endpoint 
a)  Platelet activation profile and morphology 
Platelet activation after stimulation with agonists will be assessed after ≥3y post gene therapy by 
measuring the expression of activation markers on the cell surface by FACS analysis. Platelet 
morphology will be assessed at the same time using transmission electron microscopy. 
 
5. 
PATIENT SELECTION AND RECRUITMENT 
Our Centre is a reference at national and international level for diagnostic referral of WAS 
patients. The chance of recruitment will be supported by our activity of molecular screening, 
clinical follow up and co-operation within national (AIEOP) and international (EBMT) 
networks. In particular, our centre participates to the CSS-ID network of the Italian Paediatrics 
Oncology-Haematology Association (AIEOP), a national committee on primary 
immunodeficiencies. 
Based on the incidence of the disease and the potential recruitment of candidate patients SR- 
TIGET expected enrolment rate is of about 2 patients per year for a total of eight patients. 
 
5.1. 
Study of population 
5.1.1. 
Inclusion criteria 
1 
Diagnosis of WAS defined by genetic mutation and at least one of the following criteria: 
Severe WASP mutation 
Absent WASP expression 
Severe clinical score (Zhu clinical score ≥ 3) 
2 
No HLA-identical sibling donor 
AND 
3.1 
Negative search for a matched unrelated donor (10/10) or an adequate unrelated cord blood 
donor (6/6) within 4-6 months 
OR 
3.2 
Patients with age > 5 years who are not candidate to unrelated allogeneic transplant based on 
clinical conditions 
AND 
4 
Parental/guardian/patient signed informed consent 
The severity of the genetic mutation will be defined by literature data (genotype-phenotype 
studies) and the level of WASP expression will be assessed by FACS. The identification of a 
MUD donor will be established based on high-resolution allele-level DNA matching for 10/10 
alleles (HLA-A, -B, -C, -DRB, and -DQB1 loci). For cord blood transplantation, both cell dose 
(>2.5 x 107 nucleated cells/kg body weight) and HLA matching (low-resolution HLA-A, -B, and 
high-resolution DRB1) will be considered. 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 60 of 119 
-- 
 
 
 
 
5.1.2. 
Exclusion criteria 
Patients who meet any of the following criteria will be excluded from study admission: 
 
1 
Patients positive for HIV-infection 
2 
Patients affected by neoplasia 
3 
Patients with cytogenetic alterations typical of MDS/AML 
4 
Patients with end-organ functions or any other severe disease which, in the judgement 
of the investigator, would make the patient inappropriate for entry into this study 
5 
Patients who underwent an allogeneic haematopoietic stem cell transplantation in the 
previous 6 months 
6 
Patients who underwent an allogeneic haematopoietic stem cell transplantation with 
evidence of residual cells of donor origin 
 
5.1.3. 
Sample size 
Given the low frequency of disease and the degree of novelty of the proposed experimental 
approach, we plan to treat overall 8 patients, in an estimated 5-year period. 
5.1.4. 
Patient withdrawal 
The patients will leave the study in case of impossibility to perform the planned procedures and 
treatment before gene therapy. 
In addition, the following circumstance is to be considered sufficient to withdraw from the study 
an enrolled patient: 
• Withdrawal of consent by the parent/legal tutor or the patient. 
• Any AE/SAE or clinical condition that prevents the long-term continuation of follow 
up. 
 
5.1.4.1. 
Natural study termination for a single enrolled patient 
The natural study termination, for a single enrolled patient, is when the patient has completed a 
minimum of 5 years follow up in this study (WAS Pivotal, 201228) and has transitioned and has 
transitioned to a separate OLTFU study. As part of the OLTFU study, patients will continue to 
be followed up for a total of 15 years from the date of treatment with gene therapy. Should a 
patient decide not to consent to participate in the ELTFU nor transition to the OLTFU study, 
then the subject will be considered to have completed this study and will be transitioned to 
standard of care according to local practice. This study will be considered to have ended once all 
subjects have transitioned into the separate OLTFU study or transitioned to standard of care 
according to local practice. 
In addition, the following circumstances are to be considered as natural study termination: 
a) Treatment of the patient with allogeneic hematopoietic stem cell transplantation 
b) Loss to follow up 
c) Death of patient 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 61 of 119 
-- 
 
 
 
 
All patients will be included for evaluation until withdrawal. 
 
5.2. 
Phases of study 
Starting from the signature of the informed consent by patients’ legal tutors, the subjects will be 
enrolled in the clinical trial. 
Five protocol phases are foreseen after enrolment (see Figure 1, Study flow chart): 
1) Screening phase, during which the conditions required by the clinical protocol for 
patient’s enrolment will be assessed and fulfillment of the inclusion/exclusion criteria of 
the study will be evaluated. 
2) Baseline phase, carried from the end of the screening phase to the day before PBSC 
mobilization, if performed, or the day before Rituximab administration (day -22). 
3) Treatment phase, from the end of the Baseline to day 0. 
4) Follow Up phase: the Follow Up phase is composed of an Initial Follow Up period of at 
least 3 years, when patients are intended to reach the primary efficacy timepoint, and then 
a further Long Term Follow Up period from 3 up to 8 years. 
5) ELTFU phase: patients who complete the Follow up phase will then be contacted 
annually from year 8 onwards, in order to collect long-term safety and selected efficacy 
data, until a separate OLTFU study is initiated and patients have enrolled in it. 
Once the OLTFU study is set up, patients will be invited to enter the OLTFU study at any time 
after they have completed a minimum of 5 years follow up in this study (WAS Pivotal, 201228). 
If the subject and family are unable to attend clinic visits (due to, for instance, the health of the 
subject, or the inability to travel to the clinical site), but are willing to continue participation in 
the study, remote follow-up visits, i.e. visits conducted at the local healthcare professional (HCP) 
or healthcare facility close to where the subject resides may be considered as an alternative 
method of ensuring assessment of efficacy and safety while reducing the burden on the subject 
and subject’s family. This alternative will be discussed with the PI, the medical monitor and the 
sponsor. 
• Remote follow up visits should be avoided prior to the 3-year primary endpoints. 
Thereafter, remote follow-up visits may be conducted provided they are reviewed and 
agreed by the sponsor. 
• The communication between the local HCP and the clinical site will be documented 
in a dedicated plan. This plan will also describe the arrangements of the remote 
follow-up visits and the responsibilities of the local HCP. The local HCP will have to 
record and report all observations pertinent to the subject’s follow-up, including all 
safety events, and provide the source data to the subject's original treatment center. 
The Sponsor or the Principal Investigator may provide HCPs with tools to aid data 
collection. The Principal Investigator remains ultimately responsible for the conduct 
of the study and for the causality assessment of all potential safety events. 
• Collection of biological samples might be performed at the local HCPs, in accordance 
with local regulations. If blood samples are collected during a remote follow-up visit, 
the volume of blood drawn should not exceed the volume required for scheduled 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 62 of 119 
-- 
 
 
 
 
laboratory assessment. Local HCPs will not have access to the electronic case report 
form (eCRF) or any study database. Data entry will be performed by appropriately 
delegated personnel at the original treatment center. 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 63 of 119 
-- 
 
 
 
 
Figure 1 
Study Flow Chart 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Preliminary 
evaluation and 
inclusion/exclusion 
criteria assessment 
Screening phase 
Observational Long-Term 
Follow Up (OLTFU) study 
Treatment 
Extended Long- 
Term Follow Up 
(ELTFU) 
Follow Up phase 
(Initial Follow Up 
period, Long-Term 
Follow Up period) 
Clinical laboratory 
and instrumental 
evaluation 
BM Back-up 
collection and 
storage (for patients not 
undergoing PBSC mobilization) 
Informed consent 
signature 
Baseline 
Enrolment 
Evaluation of 
safety and 
efficacy, from 8 
years, up until a 
separate 
observational 
long-term follow 
up (OLTFU) study 
is available and 
patients have been 
enrolled in it. 
Evaluation of safety and 
efficacy, from +1 day up 
to 8 years. The Follow 
Up phase is composed of 
an Initial Follow Up 
period of at least 3 
years, when patients are 
intended to reach the 
primary efficacy analysis 
timepoint, and then a 
further Long-Term 
Follow Up period from 3 
up to 8 years. 
PBSC mobilisation 
followed by 
leukapheresis for HSC 
collection for back-up 
and DP manufacture 
(if required) or BM 
harvest, 
chemotherapy, gene 
therapy schedule 
A separate observational 
long-term follow up 
(OLTFU) study will be set 
up, which patients will be 
invited to enter in at any time 
after completing a minimum 
of 5 years follow up in this 
study (WAS Pivotal 201228). 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 64 of 119 
-- 
 
 
 
 
6. 
TREATMENT PROTOCOL 
6.1. 
Treatment administration 
The Medicinal product consists of autologous stem/progenitor cells from the bone marrow 
and/or mobilized peripheral blood transduced ex vivo with LV encoding the WAS cDNA to be 
administered intravenously. 
The procurement of BM/PBSC and treatment will be performed at Paediatric Immuno- 
haematology and Bone Marrow Transplantation Unit, which are part of the San Raffaele Stem 
Cell Programme (Director: Prof. F. Ciceri), Milan, Italy, accredited by the Italian Bone Marrow 
Transplantation Group (GITMO) and currently under JACIE (Joint Accreditation Committee 
ISCT (International Society for Cellular Therapy-Europe) & EBMT) accreditation. 
Clinical Investigators and Clinical Staff will receive a training to provide education on the 
contained use of microorganism genetically modified (GMOs), according to Legislative Decree 
206/01 (National Implementation of EU Directive 98/81/CE). 
 
6.2. 
Stem cell back-up, collection and purification of mobilized CD34+ 
cells 
A central venous catheter will be inserted to allow infusion of blood products, chemotherapy, 
parenteral nutrition and leukapheresis under general anesthesia. Before the procedure the patient 
may receive either intravenous immunoglobulin (IVIG) or platelets transfusion(s) in order to 
achieve a safe platelet count. About 5-10 weeks before the planned BM harvesting, a back-up of 
PBSC will be collected following G-CSF+Plerixafor stimulation (in case of foreseen use of MPB 
as only stem cell source, CD34+ cell will also be collected for future transduction). High doses 
of G-CSF administered twice daily will be used (10-12.5 mcg/kg in 2 divided doses) since they 
appear to mobilize more CD34+ cells. Moreover, starting from day 3 of G-CSF administration, 
an additional mobilizing agent, Plerixafor (AMD3100), will be administered in order to enhance 
PBSC mobilization (Vose et al, 2009; Yannaki et al. 2013; Gardellini et al. 2013; Aabiden et al. 
2011; Vettenranta et al. 2012). The starting day of the Plerixafor administration could be shifted 
to day 4 or day 5 depending on WBC and CD34+ cell count/mcl in patient’s PB. Patients will 
receive Plerixafor once daily at the dose of 0.24 mg/kg sub-cutaneously approximately 8-10 
hours before standard leukapheresis is performed (see Figure 2– PBSC Mobilization Protocol) in 
agreement with internal procedure of the SR Stem Cells Program. G-CSF and Plerixafor 
administration will be repeated until sufficient mobilized peripheral blood (MPB) CD34+ cells 
are harvested (up to a maximum of 3 leukapheresis on 3 consecutive days or 7 days of G-CSF 
administration). According to clinical needs, an additional central or peripheral venous catheter 
can be inserted. During treatment with G-CSF + Plerixafor, complete blood cell count will be 
obtained daily and PB CD34+ cell count from the third day. Leukapheresis will be performed as 
soon as MPB CD34+ cell count reaches an adequate level and collections will be performed 
using a COBE Spectra blood under standard procedures. The optimum target number of CD34+ 
cells to collect for back-up is 5x106/kg, although a minimum of ≥3x106 autologous CD34+ 
cells/kg will be considered sufficient for safe transplantation. In case of concomitant collection 
of CD34+ cells for future transduction, an additional cell amount 5x106 CD34+ cells/kg will be 
collected. 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 65 of 119 
-- 
 
 
 
 
Figure 2    PBSC Mobilization Protocol 
 
 
 
 
 
Failure of the procedure (poor mobilization) will be defined if PB CD34+ cell count remains <10 
cells/μl in spite of mobilization, or an insufficient yield is reached after the third apheresis. 
The procedure may be repeated to achieve an adequate cell dose for back-up and/or treatment. 
An unmanipulated apheresis fraction containing at least 3x106 CD34+ cells/kg with dimethyl 
sulfoxide (DMSO) at 10% final concentration will be stored in liquid nitrogen. Samples from 
each bag will be sent for detecting bacterial contamination. 
The remaining fraction of the apheresis will be used to purify CD34+ cells by the Milteny 
ClinicaMACS immunomagnetic device. The purified stem cells, resuspended in 10% DMSO, 
will be stored in liquid nitrogen in at least two aliquots. 
The MPB CD34+ cells may be used as cell source of genetically modified cells. 
The stored back-up, together with the negative fraction following selection procedure, will be 
infused in case of engraftment failure or lack of reinfusion of the medicinal product, if 
conditioning regimen has been already started (see 10.12). In case the patients cannot undergo a 
collection of PBSC for clinical reasons (ex. patient’s age, weight, unsuitability) or in case of 
poor mobilization, a back-up BM harvest targeting a minimum level of ≥1x108 total nucleated 
cells per kg of body weight will be performed. 
In the case of a dedicated collection of autologous umbilical cord blood performed at birth, this 
may be considered as a source of HSC for back-up, if adequate for cell number and quality. 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 66 of 119 
-- 
 
 
 
 
6.3. 
Bone marrow harvest 
BM will be collected in the operating room from the iliac crests under sterile conditions and 
using general anesthesia, on day –3. The bone marrow collection procedure is described in an 
internal SOP. The total volume of the required amount of bone marrow will be estimated 
according to the content of bone marrow CD34+ cells evaluated by previous bone marrow 
aspiration. On average approximately 20-30 BM ml/kg patient body weight (max 35 ml/kg) will 
be collected considering the subsequent need of manipulations (MNC selection, centrifugation, 
washing, positive selection, transduction). 
The BM target CD34+ cell count before any manipulation is 5x106 CD34+ cells/kg to achieve a 
minimum dose of 2x106 CD34+ transduced cells/kg. Hemoglobin values <7.0-7.5 g/dl will be 
corrected by irradiated and filtered red blood cells transfusion. 
The harvested bone marrow, collected in a dedicated bag, sealed and identified, will be then 
transferred in a biohazard container to MolMed S.p.A, located near San Raffaele Hospital, by the 
research nurse or physician participating to the study. 
BM harvest is not performed if MPB is used as the only stem cell source for transduction. 
 
6.4. 
Cell manipulation and gene transfer with the lentiviral vector 
6.4.1. 
BM CD34+ cell purification 
Patients’ HSC manipulation will be performed by MolMed S.p.A. in GMP conditions. The 
collected marrow will be diluted, stratified with lymphoprep and centrifuged in order to separate 
blood fractions. Final cell suspension primarily made of MNC will be collected. Positive 
selection on BM will be performed using immunomagnetic beads (CliniMACS, Miltenyi) and an 
immunomagnetic enrichment device. WBC counts will be performed in basal BM samples from 
each marrow bag, after buffy coat collection and both in the positive and negative cell fraction. 
Phenotypic expression of surface molecules (CD34) will be determined by direct labeling using 
standard methodology. After centrifugation to reduce volume, the negative fraction will be 
collected in divided bags with DMSO, frozen and stored in liquid nitrogen for possible infusion 
if engraftment failure will occur. Samples from each bag will be sent for detecting bacterial 
contamination. 
6.4.2. 
Lentiviral vectors 
Autologous bone marrow CD34+ cells will be transduced with a clinical grade LV containing 
WAS gene. In this construct (w1.6W), the WAS gene is designed under the control of WAS 
autologous promoter. The w1.6W vector is further modified by introducing a mutation in the 
WPRE element. Viral supernatants will be produced in a GMP facility of the biotech company, 
MolMed S.p.A. (Milan, Italy). 
6.4.3. 
Gene transfer of BM CD34+ cells 
Purified CD34+ cells will be seeded in retronectin coated bags and further incubated for 24 hours 
at 37°C in CO2 in serum free medium (Cell Grow) supplemented with the cytokines SCF, TPO, 
FLT3-L and IL-3. At the end of the incubation period, the cells will be transduced in the 
presence of cytokines with the LV-WASP vector. Two rounds of transduction are foreseen, each 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 67 of 119 
-- 
 
 
 
 
lasting about 16 hours in the presence of 1x108TU/ml of LV (corresponding to a Multiplicity of 
Infection – MOI - of 100), with a washout time of 12 hours, for a total of about 68 hours of in 
vitro culture. Since the cells will be freshly infused, only tests for endotoxin, mycoplasma, large 
T antigen DNA, and immunophenotype characterization will be completed and results will be 
available at the infusion time. The validated Hospital Sterility test (BacT/ALERT) is performed 
to allow a shorter response time and intermediate results will be available at the time of infusion. 
The detailed tests and specifications of the drug substance and drug product are reported in the 
investigator’s brochure. At the end of the transduction procedure, the transduced CD34+ cells 
will be harvested, washed with saline and resuspended in a minimum of 20 ml saline solution at 
the concentration of 10x106/ml. The final drug product will consist of a cell suspension 
containing the transduced CD34+ cells in a sterile syringe(s) or bag(s) of the suitable volume. 
If the cells are detected to be contaminated, the medicinal product will not be returned to the 
patient and the patient will receive the unmanipulated back-up. If a positive result is obtained for 
the sterility test after the infusion, the patient will receive a specific antibiotic treatment and will 
be strictly monitored. 
6.4.4. 
Gene transfer of MPB CD34+ cells 
Mobilized peripheral blood (MPB) CD34+ cells may be used as cell source of genetically 
modified cells. Transduction will be performed as described above for BM CD34+ cells, starting 
from the previously stored CD34+ cells obtained from leukapheresis. 
MPB may be used as single cell source: 
1) in case of older patients (> 5 years old) to increase the dose of infused cells, if the 
expected collection from bone marrow is predicted not be sufficient to achieve the 
optimal target dose or for clinical reasons. 
or 
2) in case the patient has initiated conditioning and the transduced bone marrow CD34+ 
cells cannot be released for infusion. In this case the day of infusion will be postponed 
accordingly. 
A mixture of MPB and BM CD34+ cells can be administered if required to obtain a sufficient 
cells dose. 
 
6.5. 
Conditioning regimen 
A reduced intensity-conditioning regimen will be administered before the autologous-engineered 
cells infusion based on the rationale described in chapter 5. 
We will combine iv Busulfan (bodyweight-based and AUC targeted dose, see below, 8 +/- 1 
doses administered consecutively from days –3 to –1), Fludarabine (total dose 60 mg/sqm, on 
days –3 and –2) and Rituximab (375 mg/sqm in single dose, on day -22). 
Iv ATG Thymoglobuline may be used at PI discretion in patients with clinical autoimmunity 
before day-18 as an additional lympho-depleting agent to eliminate auto-reactive memory T 
cells. 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 68 of 119 
-- 
 
 
 
 
Clinical data from the ADA-SCID trial indicate that the dose of administered CD34+ cell is one 
of the important factors in determining the engraftment of transduced progenitors, and thus the 
outcome of gene therapy. HSC freezing is associated to a significant reduction of the overall cell 
number, further increased by the mortality consequent to transduction. Thus, with the aim of 
preserving the maximum absolute number of CD34+ cells, we will avoid freezing the transduced 
cells. 
Since our protocol is based on the transduction and re-infusion of freshly isolated CD34+ cells, 
the window of time between harvesting and transplantation will be limited to the minimum. The 
interval between the last infusion of drug and cell infusion will be sufficient to reduce drug 
concentration to safe levels for Busulfan (24-26 hours wash out) and Fludarabine (50-54 hours 
wash out). 
6.5.1. 
Busulfan 
Based on our experience in combining ex vivo gene therapy with reduced intensity in the 15 
patients enrolled in the ADA-SCID gene therapy trial and previous pilot studies, we will adopt 
intravenous Busulfan (Busilvex) as myelotoxic drug. The alkylant will be administered at a 
higher dose than in ADA-SCID trial, because a significant level of myeloid engraftment can be a 
requirement for platelet correction in WAS patients. 
Patients will receive one of five bodyweight-based doses of intravenous busulfan, according to 
the following scheme: 
Table 6 
Busulfan dosage according to patient’s weight 
 
 
< 9 kg 
9 to < 16 kg 
16-23 kg 
> 23 – 34 kg 
> 34 kg 
1 mg/kg/dose 
1.2 mg/kg/dose 
1.1 mg/kg/dose 
0.95 mg/kg/dose 
0.8 mg/kg/dose 
Patients will receive a total of 8 (+1) doses, given every 6 hours from day -3 to day -1. The dose 
of Busulfan and the number of doses may be adjusted to reach the target AUC (see below). 
The mean half-life of i.v. Busulfan ranges between 2.83 to 3.90 hours. 
We will monitor Busulfan plasmatic levels by temporized sampling following infusion after the 
first and the sixth dose. 
The exposure to Busulfan, based on pharmacokinetic profiling, will be defined by the 
Pharmacological Institute of the University of Pavia. The dose of Busulfan will be adjusted to 
avoid excessive toxicity or insufficient exposure, aiming at a target AUC of 6000 ng/ml*h, 
equivalent to a cumulative target AUC of 48000 ng/ml*h (Malar R, et al. 2011). This value is 
equivalent to 53% of the myeloablative dose exposure (90000 ng/ml*h). Thus, in order to 
achieve this target cumulative dose of Busulfan, the fifth and the following doses will be 
adjusted according to the AUC obtained after the first dose. In particular, in case the estimated 
total target AUC ± 4000 ng/ml*h is achieved already after the 4th dose, the administration of the 
following doses will be stopped. In case a lower AUC is detected at the first dose, the fifth and 
next doses should be increased both to reach the target AUC as well to replenish the reduced 
Busulfan exposure in the first four doses. 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 69 of 119 
-- 
 
 
 
 
A dose adjustment will be performed if total predicted AUC shows more than 10% deviation 
from the target (total predicted AUC is < 43200 ng/ml*h or > 52800 ng/ml*h). 
Dose adjustment calculation: D5 = D1*(target AUC – AUC1*4)/(AUC1*4) 
D5 = 5th dose (mg) 
D1 = 1st dose (mg) 
AUC1 = AUC 0-∞ after first dose 
In case the total cumulative target is not reached after 8 doses, an additional dose may be 
administered according to the following calculation: 
D9 = D5*[target AUC – (AUC1*4+AUC6*4)/AUC6 
D9 = 9th dose (mg) 
AUC6 = AUC 0-6h after sixth dose 
In case of administration of a ninth dose of Busulfan, the cell infusion will be scheduled to allow 
a wash-out time of 24 hours from the last dose. 
6.5.2. 
Fludarabine 
Fludarabine is a nucleoside analogue, currently included in non-myeloablative preparative 
regimens for HSCT as a drug inducing abrogation of lymphoid precursors in the bone marrow. 
Fludarabine is commonly used in conditioning regimen with a dose scheme ending at day -3 or - 
2 before reinfusion of donor HSC at a total dose of 60 mg/sqm. In the peripheral compartment, 
Fludarabine targets both resting and proliferating lymphocytes, producing a significant 
lymphopenia of B and T cells. It has been described that CD20+ B cells, CD4+ T cells (mainly 
naive (CD4+CD45RA+) T cells) and CD8+ T cells decrease profoundly upon Fludarabine 
treatment. Its half-life is 9.6 hours (3.1-26) measured in adult patients undergoing transplant 
(Jacobson 2005). 
Table 7 
Gene therapy schedule for WAS patients 
 
DAY 
* 
-22 
-20 
-19 
-18 
-03 
-02 
-01 
0 
PBSC back-up ± CD34+ cell collection 
BM harvest *** 
iv RITUXIMAB 
375 mg/sqm/day 
iv BUSULFAN 
Dose: see below 
iv FLUDARABINE 
30 mg/sqm/day 
ATG THYMOGLOBULINE ** 
2,5 mg/kg/day 
Infusion of transduced CD34+ cells 
Busulfan bodyweight-based and AUC targeted dose, to be given every 6 hours for 8 doses (+/- 1) as described above 
(see Table 6 ). 
*Usually performed about 5-10 weeks before treatment. BM collection is performed, if PBSC mobilization is not 
suitable. 
**Iv ATG Thymoglobuline may be used at PI discretion in patients with clinical autoimmunity. 
*** BM harvest is not performed if MPB is used as the only cell source for transduction 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 70 of 119 
-- 
 
 
 
 
6.5.3. 
Rituximab 
Rituximab, a monoclonal antibody directed against the CD20 molecule found on pre-B cells and 
mature B cells, was introduced in the late 1990s for the treatment of non-Hodgkin's lymphoma. 
Recently, this antibody has been used to treat autoimmune diseases, especially those associated 
with a prominent humoral component and with potentially pathogenic autoantibodies. Small 
cohort studies have indicated that rituximab could have an important role in the management of 
these disorders. Rituximab has also been utilized in the transplant setting, to diminish levels of 
alloreactive antibodies in highly sensitized patients, to manage ABO-incompatible transplants, 
and to treat rejection associated with B cells and antibodies. The exact mechanism by which 
rituximab exerts its effects in autoimmunity and transplantation remains unclear, as specific 
autoantibody or alloantibody levels often seem not to diminish in parallel with clinical 
improvement. A role for rituximab in depleting B cells and compromising their antigen- 
presenting function seems likely; rituximab might also inhibit T-cell activation. Rituximab has 
been successfully used to treat lymphoproliferative disorder following solid organ 
transplantation and is commonly employed as pre-emptive therapy for EBV-induced 
lymphoproliferation in the context of allogeneic HSCT. Rituximab will be used at 375 mg/sqm 
in a single dose on day -22. 
 
6.6. 
Safety considerations on the procedure and conditioning regimen 
Patients will undergo blood tests before and during treatment and in the follow up phase. In case 
blood removal will occur through venipuncture, the risks of these procedures are pain, formation 
of hematoma and minimal risk of infection at the puncture site. Blood tests will be planned on 
different days to minimize the risks of anemia due to blood loss. 
Standard risks associated with implantation of a central venous catheter, collection of 
stem/progenitor cells by means of apheresis, and BM harvest under general anesthesia are to be 
taken into consideration (Moog et al. 2001; Nishimori et al. 2001; Mattioli et al. 2007). In 
particular, the risks of central catheter implantation include bleeding, formation of hematoma, 
pain, infections and rarely pneumothorax. The possible side effects of G-CSF administration for 
mobilization of CD34+ cells in the peripheral blood include fever, pain, and rarely allergic type 
reactions. Overall the safety record of G-CSF is very good, apart from extremely rare reports of 
spontaneous splenic ruptures. Plerixafor is registered for use in adults. The most commons 
adverse reactions reported in patients in conjunction with G-CSF were diarrhea, nausea, fatigue, 
injection site reactions, headache, arthralgia, dizziness and vomiting. The safety and efficacy of 
its use in children has not been studied in controlled clinical trials; several single centers studies 
reported no major adverse events with plerixafor administration in children (Pham HP et al. 
2012; Son MH et al. 2013; Hong KT et al. 2012; Sevilla J et al. 2012). 
Adverse reactions to leukapheresis can derive from transient volume loss, and lowering of 
calcium levels by the citrate anticoagulant. BM harvest can be associated with bleeding, 
formation of haematoma, pain, infections and those correlated with general anesthesia. 
The risk of bleeding and bruising is expected to be increased in thrombocytopenic patients and 
will be minimized by the infusion of IVIG and/or platelets’ transfusions before the procedure. 
The risks associated with reduced intensity busulfan treatment are expected to be decreased 
compared to standard dose treatment. Busulfan toxicity includes myelosuppression, nausea, 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 71 of 119 
-- 
 
 
 
 
vomiting, hair loss, mucositis, hepatotoxicity, seizures, hypo/hypertension, and lung fibrosis. 
Prolonged exposure to Busulfan may result in cataract formation, secondary tumors and sterility. 
Fludarabine is a drug currently used in paediatric non-myeloablative preparative regimen in 
HSCT. Fludarabine toxicity includes bone marrow aplasia, nausea, vomiting, mucositis, and 
transient increase of liver enzymes. If given at high doses, Fludarabine can induce a progressive 
neurotoxicity due to demyelinating damage as main side effect. The patients enrolled in our 
clinical trial will receive lower doses of i.v. Fludarabine (standard dose in transplant: 100-150 
mg/sqm). During a prolonged exposure to Fludarabine, transient visual disturbances and 
alterations of consciousness might occur: these scheduled events are described in literature as 
revertible with the drug’s suspension. 
ATG is an immunosuppressive drug approved in conventional conditioning as an agent inducing 
a selective decrease in T-cell count. Prior to and during ATG administration, adequate pre- 
medication will be dispensed to prevent anaphylaxis episodes or fever and chills, mainly 
described as occurring during initial infusions. Other common adverse effects include 
thrombocytopenia, leukopenia, headache, abdominal pain, nausea, diarrhoea, dyspnoea, and 
dizziness, reversible upon withdrawal. In addition, patients receiving ATG might develop severe 
infections resulting from over-immunosuppression. Particular caution will be adopted 
considering post-transplant lymphoproliferative disease. Thus, EBV-positive patients showing 
signs of viral reactivation after gene therapy, will receive appropriate pre-emptive therapy 
(Rituximab). Potentially increased risk of malignancy (non-Hodgkin’s lymphoma, myeloma) has 
been reported as complication in long-term exposure of the drug. 
The risks associated with Rituximab infusion include infusion reactions (that can be rarely fatal), 
nausea, vomiting, headache, wheezing, allergic reactions, severe muco-cutaneous reactions, 
LDH increase, hypo/hypertension. Cases of HBV reactivation and progressive multifocal 
leukoencephalopathy have been described after the use of this drug. Prior to Rituximab 
administration, adequate pre-medication will be dispensed to prevent anaphylaxis episodes or 
fever and chills, mainly described as occurring during initial infusions. 
In case of microbiological contamination of bone marrow CD34+ cells, detected after the 
initiation of the conditioning regimen, culture will be stopped and the lot not released. A new lot 
will be prepared from mobilized CD34+ cells previously cryopreserved. Cells will be thawed, 
transduced and re-infused, as described in the IMPD, 3-7 days after the foreseen date. 
Considering that the drug product consists of autologous cells, which are washed before cell 
infusion, no short-term toxicity is expected to occur. In this regard, it should be borne in mind 
that the infusion of autologous CD34+ cells from bone marrow or mobilized peripheral blood in 
children affected by haematological or oncological disorders usually does not cause side effects. 
6.7. 
Packaging and labelling of the Medicinal Product 
The final Medicinal Product will consist of a cell suspension containing the CD34+ cells 
transduced with the LV-WASP vector, resuspended in saline, in one or more sterile syringe(s) or 
bag(s) of the suitable volume. 
The Medicinal Product will be labeled with a unique identification code of the patient and of the 
product, released by MolMed S.p.A, according to cGMP. The Medicinal Product will be released 
to an Investigator or another member of the investigating team designated by the Principal 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 72 of 119 
-- 
 
 
 
 
Investigator and then transferred in a biohazard container at room temperature to the clinical 
ward for infusion, according to San Raffaele Stem Cell Program internal operating procedures. 
6.8. 
Dose of the Medicinal Product 
Each patient will receive a minimum dose of BM and/or MPB transduced CD34+ cells of 2 x 106 
/ Kg, maximum dose of transduced CD34+ cells of 20 x 106 / Kg, with a target of 5 - 10 x 106 / 
Kg, depending on the yield of cells available after transduction. 
Transduced mobilized peripheral blood cells will be used in addition to BM in case of 
insufficient recovery of BM cells (< 2 x 106 CD34+ cells/Kg) or in place of BM in case 
transduced CD34+ cells are not released for infusion. 
6.9. 
Infusion 
At day 0, patients will be prepared for the infusion of the transduced cells. Before the infusion, 
all other i.v. drugs will be interrupted. After pre-medication (15-30 min before infusion) with 
chlorpheniramine (0.25 mg/kg, max dose 10 mg), transduced cells will be infused i.v. through 
the central venous catheter in about 20 minutes. At the end of infusion, saline solution will be 
used to wash the syringe(s)/bag(s) and the line. After the whole procedure, if needed, furosemide 
(0.2-1 mg/kg, max dose 20 mg) could be administered. Vital signs (BP, HR, SO2) and symptoms 
will be monitored at baseline, then every ten minutes during infusion and every hour, for 3 hours 
after the infusion. 
 
6.10. 
Hospitalization 
The patients enrolled in the gene therapy trial will be followed at the Pediatric Clinical Research 
Unit/Pediatric Immuno-haematology and Bone Marrow Transplantation Unit, (within the San 
Raffaele Stem Cell Program), Milan, Italy, a certified AIEOP and GITMO center, currently 
under JACIE accreditation. At present, the Unit consists of: 3 single rooms for pediatric 
transplant in a dedicated and confined area; inpatients beds in the department of Pediatrics; day- 
hospital rooms; outpatient clinics. 
During baseline, the patients will be hospitalized for implantation of a central venous catheter, 
for 3-5 days or according to clinical conditions. During treatment, the patient will be hospitalized 
for an average of 60 days during chemotherapy, gene therapy infusion and short-term follow up. 
Thereafter, patients will be followed as outpatients unless invasive procedures are required or 
complications will occur. 
6.11. 
Supportive therapy 
• During hospitalization, patients will be cared for in an isolation unit. 
• Beginning before conditioning until recovery from severe neutropenia (ANC<500/µl) 
or discharge: oral decontamination and mouth care according to local standards. 
• Pentamidine (nebulized) as first line Pneumocystis jirovecii prophylaxis from day -30 
until complete immunological reconstitution. 
• Antibacterial and antifungal prophylaxis according to local standards. 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 73 of 119 
-- 
 
 
 
 
• Acyclovir as Herpes Simplex-prophylaxis until complete immunological 
reconstitution. 
• Intravenous substitution to maintain immunoglobulin IgG >5g/l. 
• Hydration of 3L/m2 will be given during the conditioning phase (from day -4 to 24 
hours after Busulfan). 
• CMV (PCR), EBV (PCR) and adenovirus (PCR) viral load will be determined weekly 
till day +90. If repeatedly negative, after day +90, the interval of testing could be 
prolonged. Pre-emptive therapy with Ganciclovir or Foscarnet will be administered, 
according to local standards, if CMV positive. 
• Pre-emptive therapy with Rituximab will be administered to EBV-positive patients 
after gene therapy in case of repeated increase of EBV viral load, according to local 
standards. 
• Erythrocyte and thrombocyte concentrates will be substituted according to local 
standard. The decision to administer erythrocytes or platelets transfusions should 
incorporate individual clinical characteristics of the patients and not simply be a 
reflexive reaction to the platelet count. All blood products will be leukocyte depleted 
and irradiated. 
• The need for a platelet infusion will be evaluated by the Investigators based on 
several factors, including: 
o platelets <20 x 109/L; 
o 
the presence of active bleeding; 
o stability of thrombocytopenia; 
o concurrent infection and febrile condition; 
o immune thrombocytopenic purpura; 
o use and response to thrombopoietin (TPO) receptor agonists; 
o presence of risk factors for bleeding (such as an upcoming invasive surgical 
procedure); 
o toxicity which may lead to an increased risk of bleeding; 
o subjects at risk of fall and trauma; and 
o overall clinical condition of the subject. 
• Several factors are considered in deciding whether the interruption of IVIG can be 
stopped: 
o stable trough levels of IgG (immunoglobulin G) remaining at or above 5 g/L; 
o absence of symptoms of infections or positivity at laboratory tests for 
pathogens; 
o 
the presence of adequate levels of B cells in the peripheral blood; 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 74 of 119 
-- 
 
 
 
 
o presence of other risk factors, for example, IVIG is rarely stopped during 
winter, as winter is considered a period of increased risk for developing upper 
respiratory tract infections such as influenza. 
Based on these factors, the Investigators will consider first prolonging the interval of 
infusion of IVIG. If trough levels remain stable, IVIG will be stopped with the aim to 
start vaccinations after 3 months. During this period, IgG will be monitored to check 
the stability of their levels in serum. 
• Antiemetic prophylaxis and analgesic according to local standards. 
• Antiepileptic drugs will be dispensed for a short period to prevent seizures due to 
Busulfan administration. 
• G-CSF in case of absolute neutrophil counts <500/microl, at day +45, following bone 
marrow evaluation. 
• Parenteral nutrition will be introduced in case of severe loss of body weight or 
clinical decision. 
The administration of other drugs will be allowed by the Investigators of the study depending on 
the clinical findings of the patient enrolled in this study. 
 
6.12. 
Back-up reinfusion 
In case of engraftment failure, the stored back-up, with or without the CD34 negative fraction 
following marrow manipulation, will be infused according to local standards. For the purposes of 
this study, engraftment failure is defined as failure to reach an absolute neutrophil count (ANC) 
> 500 neutrophils/1 with no evidence of BM recovery (i.e. hypocellular marrow) on day +60. 
The infusion of the stored back-up could be anticipated in case of lack of evidence of progression 
to engraftment and presence of a severe life-threatening complication. 
6.13. 
Responsibilities 
The Investigator, or other personnel allowed to store and dispense the treatment, will be 
responsible for ensuring that the medicinal product used in the study is securely maintained as 
specified in Investigator’s Brochure and in accordance with the applicable regulatory 
requirements. 
All the treatment shall be dispensed in accordance with the Investigator's prescription and it is 
the Investigator's responsibility to ensure that an accurate record of medicinal product (issued 
and returned) is maintained. 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 75 of 119 
-- 
 
 
 
 
7. 
STUDY ASSESSMENTS 
7.1. 
Pre-treatment evaluation (screening and baseline) 
After having obtained Informed Consent from parents or guardians, eligible patients will 
undergo a clinical, laboratory and instrumental pre-treatment work-up including also disease- 
specific evaluations (extensive immunological, microbiological and autoimmunity assessment). 
A BM aspirate will be obtained in order to evaluate morphology, cellularity, CD34+ cell content, 
and clonogenic activity. 
Clinical evaluation, laboratory tests and imaging will be performed before treatment as 
scheduled in Table 8a and Table 8b: 
a) Clinical evaluation. Medical history, physical examination with assessment of 
performance status (Lansky), including eczema assessment (according to Imai et al. 
2004). 
b) Zhu score assignment (Table 9 ). 
c) WAS mutation and WASP expression. A search for WASP gene mutation will be 
performed if not available in the clinical records. The mutation will be classified as 
severe/non severe based on literature data (Imai et al., 2004) database information and 
prediction studies. The residual levels of WAS protein will be assessed by FACS 
analyses on peripheral blood and classified as either: a) present, b) reduced, c) absent. 
The presence of a population expressing normal levels of WASP and representing >5% 
of lymphocytes will be classified as: d) revertant. 
d) HLA-typing (to exclude family donor or adequate unrelated donor at screening), if not 
previously performed; negative search for unrelated donor performed in the last two 
years, performed for at least for 4-6 months, will be considered a valid inclusion criteria. 
e) Specialist examination: immunological, anesthesiological, ophthalmological, dental, 
cardiological evaluation. 
f) Diagnostic imaging and instrumental tests: Imaging n°1: Chest X-ray, ECG, abdomen 
US scan, echocardiogram, Rx of left hand-wrist for evaluation of bone age, respiratory 
functional test (if children older than 5 years old). Imaging n°2: chest CT scan. 
g) Routine laboratory n°1: blood count with differential (absolute count), including 
platelet count, MPV, CRP, LDH, AST, ALT, bilirubin (total, direct and indirect), blood 
glucose, BUN (Blood Urea Nitrogen), creatinine, creatine phosphokinase (CPK), ALP, 
GGT, electrolytes (Na, K, Ca, Mg), protein content, urine analysis. Routine laboratory 
n°2: ESR, protein electrophoresis, blood iron, transferrin, ferritin, reticulocytes, blood 
gases analyses, thyroid balance, coagulation (XDP, PT, APTT, FG). 
h) Bone marrow evaluation: needle aspirate with immunophenotype: CD3+, CD3+/CD4+, 
CD3+/CD8+, CD19+, CD20+, CD22+, CD20+/CD22+, CD2+, CD16+/CD56+, 
CD19+/IgM+, CD19+/kappa, CD19+/lambda, CD71+/Glycophorin A+, CD15+, CD13+, 
CD38+/CD138+, CD61+, CD34+ (ISHAGE), clonogenic progenitors (CFU-C), 
morphology, and karyotype. In case of previous allogeneic HSCT, a donor chimerism 
evaluation will be performed. Pre-therapy bone marrow evaluation will include CD34+ 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 76 of 119 
-- 
 
 
 
 
cell purification and transduction of a small volume of BM aspirate (qPCR). Pre- 
treatment levels of genetically modified cells will be assessed in BM mononuclear cells. 
The BM aspirate will be planned on the occasion of other procedures requiring sedation, 
if possible. 
i) Microbiological evaluation: n°1: Search for bacteria and fungi in nasal and pharyngeal 
swabs; CMV and EBV DNA search by molecular tests on PB plasma, stool culture, 
search for bacteria in urine. n°2: Search for Legionella antigen in urine. Search for 
Bacteria, Mycobacteria, fungi, Pn. Jirovecii, Legionella, Mycoplasma pn, Chlamydia pn 
in deep pharyngeal aspirates. Search for Respiratory Syncytial Virus (RSV) in nasal 
wash. Search for VZV, adenovirus and HSV1, HSV2, HHV6, HHV7 genomes in the PB 
plasma (molecular test); search for parasites and enteroviruses in the stools. n°3: Search 
for HCV, HBV, HIV genomes in PB plasma (molecular test), TPHA. 
j) Immunological evaluation n°1: Immunophenotype with lymphocyte subpopulations 
(CD45+, CD14+, CD3+, CD3+/CD4+, CD3+/CD8+, CD19+, CD2+, CD16+/CD56+, 
percentage and absolute count. Serum immunoglobulins (IgG, IgA, IgM, total IgE). n°2: 
Immunophenotype with lymphocyte subpopulations (CD3+, CD4+, CD8+, CD16+, 
CD56+, CD14+, CD45+, TCR1+ (alfa/beta), TCR2+ (gamma/delta), CD4+/CD45RA+ 
&CD45RO+, CD8+/CD45RA+ & CD45RO+, CD4+/DR+, CD8+/DR+, CD4+/CD25+, 
CD8+/CD25+, CD3+/CD4+, CD3+/CD8+, CD19+, CD2+, CD16+/CD56+, kappa+, 
lambda+) percentage and absolute count. Serum immunoglobulins (IgG, IgA, IgM, total 
IgE). n°3: Proliferative response to mitogens (PHA, anti-CD3i, anti-CD3i+anti-CD28, 
IL-2, PWM). n°4: Proliferative response to Candida and alloantigens. Dose response of 
proliferation after anti-CD3i stimulation on PBMC and/or T-cell lines. Proliferative 
response to tetanus toxoid (if patient was vaccinated). TCR repertoire by FACS. n°5 In 
patients who are not receiving routinely IVIG: serum antibody levels to vaccinal antigens 
(tetanus toxoid, diphtheria, hepatitis B, Pneumococcus, Pertussis, haemophilus B) and 
antibodies to pathogens (EBV VCA, measles, rubeola, CMV, HSV, VZV). 
k) Autoimmunity evaluation, searching organ-specific and systemic antibodies in the 
serum: anti-platelet antibodies, anti-neutrophil cytoplasmic antibodies (p-ANCA. c- 
ANCA), anti-DNA antibodies (ADNA), anti-mitochondrial antibodies (AMA), anti- 
smooth muscle antibodies (ASMA), anti-Liver-Kidney-Muscle (LKM), Coombs tests 
(direct/indirect) and anti-nuclear antibodies (ANA). If ANA positive, anti-extractable 
nuclear antigens antibodies (ENA) should be evaluated. If ENA positive, the type should 
be specified (Anti-RNP, Anti SCL-70, Anti-Sm, Anti SSA, Anti SSB, Anti Jo1). 
l) Archiving of samples. Samples from peripheral blood and bone marrow (serum, plasma, 
cell pellets, viable cells in DMSO, cell lines, DNA) will be archived stored at -80° at SR- 
TIGET, according to Institutional guidelines. 
Repeat or unscheduled samples may be taken, or tests performed, for safety reasons or due to 
technical issues with the samples or tests. 
For ethical reasons, under exceptional circumstances, tests or exams performed before patient’s 
enrolment may be considered valuable for patient’s pre-gene therapy evaluation. 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 77 of 119 
-- 
 
 
 
 
7.2. 
Post-treatment evaluation 
Patient’s assessment will include complete medical examination, diagnostic imaging, 
microbiological tests, routine laboratory tests, immune profiling evaluation and specific 
immunological tests. 
Clinical evaluation, laboratory tests and imaging will be performed after treatment as scheduled 
in Table 8a and Table 8b. For clinical evaluation and tests to be performed at time points after 
day +120, a timeframe of +/- 10% will be accepted, while for time points after 1 year, a 
timeframe of ± 30 days will be accepted. 
a) Clinical examination. Anamnesis, physical examination with assessment of performance 
status (Lansky), including eczema assessment (Imai). 
b) Zhu score will be re-assigned every year (Table 9). 
c) WASP expression. Assessed by FACS analyses on peripheral blood. 
d) Specialist examinations: cardiological, immunological, ophthalmological. 
e) Diagnostic imaging and instrumental tests: Imaging n°1: Chest X-ray, ECG, abdomen 
US scan, echocardiogram, Rx of left hand-wrist for evaluation of bone age, respiratory 
functional test (if children older than 5 years old). Imaging n°2: chest CT scan. Imaging 
n°3: ECG, abdomen US scan, echocardiogram, Rx of left hand-wrist for evaluation of 
bone age, respiratory functional test (if children older than 5 years old). In addition, at 5th 
year: Chest X-ray or CT scan if clinical indication, and at 8th year: chest CT scan. 
Imaging n°4: ECG, abdomen US scan, echocardiogram, respiratory functional test (if 
children older than 5 years old). 
f) Routine laboratory n°1: CBC with differential (absolute count), including platelet 
count, MPV (every year), CRP, LDH, AST, ALT, bilirubin, blood glucose, BUN, 
creatinine, creatine phosphokinase, ALP, GGT, electrolytes, protein content, urinalysis. 
Routine laboratory n°2: ESR, protein electrophoresis, blood iron, transferrin, ferritin, 
reticulocytes, blood gases analyses, thyroid balance, coagulation (XDP, PT, APTT, FG). 
g) Bone marrow evaluation: needle aspirate with immunophenotype: CD3+, CD3+/CD4+, 
CD3+/CD8+, CD19+, CD20+, CD22+, CD20+/CD22+, CD2+, CD16+/CD56+, 
CD19+/IgM+, CD19+/kappa, CD19+/lambda, CD71+/Glycophorin A+, CD15+, CD13+, 
CD38+/CD138+, CD61+, CD34+(ISHAGE), clonogenic progenitors (CFU-C), 
morphology, and karyotype. PCR analysis on transduced cells will be performed 
separately, including the assessment of %LV+ colony-forming cells (CFC) from patient’s 
BM-derived clonogenic progenitors. 
h) Microbiological evaluation: n°1: Search for bacteria and fungi in nasal and pharyngeal 
swabs; CMV and EBV DNA search by molecular tests on PB plasma, stool culture, 
search for bacteria in the urine. N°2: Search for Legionella antigen in urine. Search for 
Bacteria, Mycobacteria, fungi, Pn. jirovecii, Legionella, Chlamydia pn. and Mycoplasma 
pn. in deep pharyngeal aspirates. Search for Respiratory Syncytial Virus (RSV) in nasal 
wash. Search for adenovirus in the blood (molecular test); search for parasites and 
enteroviruses in the stools. N°3 Search for HSV1, HSV2, HHV6, HHV7, HCV, HBV, 
HIV genomes in the PB plasma (molecular test). 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 78 of 119 
-- 
 
 
 
 
i) Immunological evaluation n°1: Immunophenotype with lymphocyte subpopulations 
(CD45+, CD14+, CD3+, CD3+/CD4+, CD3+/CD8+, CD19+, CD2+, CD16+/CD56+, 
percentage and absolute count. Serum immunoglobulins (IgG, IgA, IgM, total IgE). n°2: 
Immunophenotype with lymphocyte subpopulations (CD3+, CD4+, CD8+, CD16+, 
CD56+, CD14+, CD45+, TCR1+ (alfa/beta), TCR2+ (gamma/delta), CD4+/CD45RA+ & 
CD45RO+, CD8+/CD45RA+ & CD45RO+, CD4+/DR+, CD8+/DR+, CD4+/CD25+, 
CD8+/CD25+, CD3+/CD4+, CD3+/CD8+, CD19+, CD2+, CD16+/CD56+, kappa+, 
lambda+) percentage and absolute count. Serum immunoglobulins (IgG, IgA, IgM, total 
IgE). n°3: Proliferative response to mitogens (PHA, anti-CD3i, anti-CD3i+anti-CD28, 
IL-2, PWM). n°4: Proliferative response to Candida and alloantigens. Proliferative 
response to tetanus toxoid (if patient was vaccinated). Dose response of proliferation after 
anti-CD3i stimulation on PBMC and/or T-cell lines. TCR repertoire studies in the 
peripheral blood by FACS (Vbeta repertoire). n°5 In patients who discontinued IVIG (as 
described in Section 6.11), a vaccination program will be started. Serum antibody levels 
to vaccinal antigens (tetanus toxoid, diphtheria, hepatitis B, Pneumococcus, Pertussis, 
Haemophilus B) or pathogens (EBV VCA, CMV, measles, rubeola, VZV, HSV) will be 
measured. 
j) Autoimmunity evaluation, searching organ-specific and systemic antibodies in the 
serum: anti-platelet antibodies, anti-nuclear antibodies (ANA); anti-extractable nuclear 
antigens antibodies (ENA), if ANA positive; anti-neutrophil cytoplasmic antibodies (p- 
ANCA. c-ANCA); anti-DNA antibodies (ADNA); anti-mitochondrial antibodies (AMA); 
anti smooth muscle antibodies (ASMA), anti-Liver-Kidney-Muscle (LKM); Coombs’ 
tests (direct/indirect). 
k) Evaluation of the presence of genetically modified cells. Vector copy number 
(measured by quantitative PCR as VCN/cells) will be done after gene therapy in total 
nucleated cells (day +14), and afterwards in PB lymphocyte subpopulations (CD3+ 
and/or CD4+ and CD8+ T lymphocytes, B lymphocytes, NK cells) and granulocytes and 
in bone marrow cell subpopulations (CD15+, GlyA+, CD3+, CD19+, CD56+, CD34+). 
l) Archiving of samples. Samples from peripheral blood and bone marrow (serum, plasma, 
cell pellets, viable cells in DMSO, cell lines, DNA) will be archived stored at -80° at SR- 
TIGET, according to Institutional guidelines. 
m) Platelet activation and morphology: For platelet activation, the expression of activation 
markers on the surface of peripheral blood-derived platelets after stimulation with 
agonists will be assessed. For platelet morphology, the platelet perimeter, area and 
ultrastructure will be assessed by transmission electron microscopy. 
Repeat or unscheduled samples may be taken, or tests performed, for safety reasons or due to 
technical issues with the samples or tests. 
 
7.3. 
Extended Long-Term Follow Up 
Both European and US regulatory agencies have expectations for the long-term follow up of 
patients who have been treated using gene therapy (EMA/CAT/GTWP/671639/2008 and 
revisions; EMEA/CHMP/GTWP/60436/2007; EMA/CAT/852602/2018; FDA LTFU guidance 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 79 of 119 
-- 
 
 
 
 
January 2020; FDA, Considerations for the Design of Early-Phase Clinical Trials of Cellular and 
Gene Therapy Products, June 2015). 
Patients who complete 8 years of follow up from the initial infusion of transduced stem cells (as 
part of the Follow Up phase) will then continue to be followed up as part of the ELTFU phase. 
During the ELTFU phase, patients will be contacted annually (+/- 3 months) by telephone, email 
or letter in an effort to collect long-term safety and selected efficacy data until a separate OLTFU 
study is available and patients have enrolled in it (see Table 8c). As part of the OLTFU study, 
patients will continue to be followed up for a total of 15 years from the date of treatment with 
gene therapy. Should a patient decide not to consent to participate in the ELTFU nor transition to 
the OLTFU study, then the subject will be considered to have completed this study (WAS 
Pivotal 201228) and will be transitioned to standard of care according to local practice. 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 80 of 119 
-- 
 
 
 
 
Table 8a 
Pre and post-treatment evaluation (up to 36 months after gene therapy) 
 
Parameter 
Clinical Exam.*** 
Routine Lab. N° 1 
Routine Lab. N° 2 
Bone Marrow+ qPCR 
Microbiology N° 1 
Microbiology N° 2 
Microbiology N° 3 
Immunology N° 1 
Immunology N° 2 
Immunology N° 3 
Immunology N° 4 
Immunology N° 5 
Zhu score 
HLA typing and donor search 
WASP Expression** 
Autoimmunity 
PCR Transduced cells (PB) 
Archiving Blood/BM sample 
Specialist Examination 
Imaging N° 1 
Imaging N° 2 
AEs/SAEs recording 
Platelet activation and morphology 
Time point 
Screening 
X X 
X* 
X 
X 
X X X 
X 
Baseline 
X X 
X 
X 
X 
X X X X X 
X X 
X X X X 
Day -14 
X X 
X 
Day 0 
X X 
X 
X 
X 
+ 7 days 
X X 
X 
X 
X 
+ 14 days 
X X 
X 
X 
X X 
X 
+ 21 days 
X X 
X 
X 
X 
+ 30 days 
X X 
X X 
X 
X 
X 
X X 
X 
+ 60 days 
X X 
X 
X 
X 
X X 
X 
+ 90 days 
X X 
X X X 
X 
X X 
X 
X X 
X 
+ 180 days 
X X 
X 
X 
X X X 
X X X X 
X 
+ 1 y 
X X 
X X X 
X X 
X X X X X 
X X X X X X 
X 
+ 1,5 y 
X X 
X 
X 
X 
X X 
X 
+ 2 y 
X X 
X X X 
X X 
X X X X X 
X X X X X X 
X 
+ 2,5 y 
X X 
X 
X 
X 
X X 
X 
+ 3 y 
X X 
X X X 
X X 
X X X X X 
X X X X X X X X Xp 
* if the qPCR is not performed at this time point it may be performed on bone marrow collected for back-up or 
harvested for preparation of medical product. 
** WAS mutation search carried out at screening if not available 
*** Eczema will be evaluated as part of the clinical examination (according to Imai et al. 2004) 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 81 of 119 
-- 
 
 
 
 
P Platelet activation and morphology will be assessed at at least one time point ≥ 3y post-GT 
Repeat or unscheduled samples may be taken, or tests performed, for safety reasons or due to 
technical issues with the samples or tests. 
Table 8b 
Long-term follow up evaluation 
 
Parameter 
Clinical Exam.*** 
Routine Lab. N° 1 
Routine Lab. N° 2 
Bone Marrow+ qPCR 
Microbiology N° 1 
Microbiology N° 2 
Microbiology N° 3 
Immunology N° 2 
Immunology N° 3 
Immunology N° 4 
Immunology N° 5 
Zhu score 
WASP Expression 
Autoimmunity 
PCR Transduced cells (PB) 
Archiving Blood/BM sample 
Specialist Examination 
Imaging N° 3 
Imaging N° 4 
AEs/SAEs recording 
Platelet activation and morphology 
Time 
point 
+ 4 y 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Xp 
+ 5 y 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Xp 
+ 6 y 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Xp 
+ 7 y 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Xp 
+ 8 y 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Xp 
*** Eczema will be evaluated as part of the clinical examination (according to Imai et al. 2004) 
P Platelet activation and morphology will be assessed at at least one time point ≥ 3y post GT 
Repeat or unscheduled samples may be taken, or tests performed, for safety reasons or due to 
technical issues with the samples or tests. 
Table 8c 
Extended Long-Term Follow Up evaluation 
 
Parameter 
Year 9+ p.a. (+/- 3 
months) 
Social Life 
Is the patient living in a protected environment? 
Is the patient attending kindergarten/school or in employment 
(according to age)? - Please comment. 
How is the patient’s social ability with peers (if applicable for age)? - If 
abnormal please specify. 
Does the patient practice any sport (if applicable for age)? - Please 
comment. 
X 
General 
Number of platelet infusions per year 
Complete Blood Count (CBC) including Platelet count/MPV 
X 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 82 of 119 
-- 
 
 
 
 
Table 8c 
Extended Long-Term Follow Up evaluation (Continued) 
 
Parameter 
Year 9+ p.a. (+/- 3 
months) 
Specialist laboratory assessments 
Integration Site Analysis (ISA) 
X 
Safety* 
SAEs 
AEs (related to bleeds, infections, autoimmunity, eczema, suspected 
malignancies) 
X 
Ig usage** 
Intravenous Ig 
Subcutaneous Ig 
X 
* SAE and AEs related to bleeds, infections, autoimmunity, eczema, suspected malignancies will be forwarded to 
TIGET as per current practice by the physician overseeing the patient in their home country. This includes the 
event, labs, and any other relevant data. In addition patients/family may also provide such information directly to 
TIGET. All such AEs should be entered into the eCRF within 30 days of awareness. SAEs, and any suspected 
malignancies, should be reported within 24 hours of awareness. 
** Frequency to be specified if possible. 
Table 9 
WAS scoring system according to Zhu. 
 
Clinical scores 
XLT 
WAS 
0.5 
1 
2 
3 
4 
5A 
5M 
Thrombocytopenia 
+/- 
+ 
+ 
+ 
+ 
+ 
+ 
Eczema 
- 
- 
+/- 
+ 
++ 
++/- 
++/- 
Immunodeficiency 
- 
- 
+/- 
+ 
++ 
++/- 
++/- 
Autoimmunity 
- 
- 
- 
- 
- 
+ 
- 
Malignancy 
- 
- 
- 
- 
- 
- 
+ 
The score is based on Zhu and colleagues (Zhu et al. 1997), with subsequent refinements 
(Bosticardo et al., 2009). 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 83 of 119 
-- 
 
 
 
 
8. 
ASSESSMENT OF SAFETY 
8.1. 
Adverse Events 
8.1.1. 
Adverse Event (AE) 
Any untoward medical occurrence in a patient or clinical trial subject administered a medicinal 
product and which does not necessarily have a causal relationship with this treatment. 
8.1.2. 
Adverse Reaction (AR) 
All untoward and unintended responses to an investigational medicinal product related to any 
dose administered. 
 
8.1.3. 
Serious adverse event (SAE) 
Any untoward medical occurrence or effect that at any dose results in death, is life-threatening, 
requires hospitalization or prolongation of existing hospitalization, results in persistent or 
significant disability or incapacity, or is a congenital anomaly or birth defect. 
 
8.1.4. 
Unexpected adverse reaction (UAR) 
An adverse reaction, the nature or severity of which is not consistent with the applicable product 
information (e.g. investigator's brochure for an unauthorized investigational product or summary 
of product characteristics for an authorized product). 
 
8.1.5. 
Suspected unexpected serious adverse reaction (SUSAR) 
An adverse event assessed as serious and unexpected and for which there is a reasonable 
suspected causal relationship with an investigational medical product. 
8.2. 
Pharmacovigilance Procedure 
8.2.1. 
Reporting of Serious Adverse Events (SAEs) 
The Investigator shall report all SAEs to the Sponsor or its designee within 24 hours after 
becoming aware of the SAE. 
SR-TIGET should inform the Sponsor of the following events within three days of gaining 
awareness: 
• Positive result from replication competent lentivirus (RCL) testing 
• Test results suggesting abnormal clonal proliferation 
Events meeting the Hy’s law criteria (ALT ≥ 3xULN and bilirubin ≥ 2xULN (>35% direct 
bilirubin) or ALT ≥ 3xULN and INR>1.5, if INR measured) will be reported as SAEs. 
Patients with liver function abnormalities should be monitored until liver chemistries resolve, 
stabilise or return within baseline. Tests to define the aetiology of the abnormal liver function 
tests should be conducted as relevant. 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 84 of 119 
-- 
 
 
 
 
SAEs should be reported using the paper SAE reporting form. After completion, the form should 
be scanned and emailed to the SAE contacts (Medical Monitor and Sponsor’s 
pharmacovigilance designee) found at the beginning of the protocol on the Sponsor/Medical 
Monitor Contact Information page. 
In rare circumstances if email communication is not available, notification by facsimile is 
acceptable. 
 
8.2.2. 
Regulatory Reporting Requirements for SAEs 
Prompt notification by the Investigator of SAEs related to study treatment is essential so that 
legal obligations and ethical responsibilities towards the safety of subjects and the safety of a 
product under clinical investigation are met. 
The Sponsor has a legal responsibility to notify both the local regulatory authority and other 
regulatory agencies about the safety of a product under clinical investigation. The Sponsor will 
comply with country specific regulatory requirements relating to safety reporting to the 
regulatory authority, Institutional Review Board (IRB)/Independent Ethics Committee (IEC) and 
investigators. 
Investigator safety reports are prepared for suspected unexpected serious adverse reactions 
according to local regulatory requirements and Sponsor policy and are forwarded to Investigators 
as necessary. 
An Investigator who receives an investigator safety report describing a SAE(s) or other specific 
safety information (e.g., summary or listing of SAEs) from the Sponsor will file it with the IB 
and will notify the IRB/IEC, if appropriate according to local requirements. 
 
Suspected Unexpected Serious Adverse Reaction (SUSAR) 
In case of SAR that is unexpected, the Investigator/Sponsor must report the reaction as soon as 
possible (and in any case no later than 7 days for reactions that are fatal or life-threatening, and 
no later than 15 days for all other SUSARs) to the competent Regulatory Authorities and to the 
competent Ethics Committee (and that relevant follow up information is subsequently 
communicated per the applicable regulation/guideline). 
8.2.3. 
Adverse event recording 
All AE/AR, regardless of seriousness or relationship to Medicinal Product or expectedness, are 
to be recorded on the corresponding page(s) included in the Case Report Forms. Whenever 
possible, symptoms should be grouped as a single syndrome or diagnosis. The Investigator 
should specify the date of onset, maximal intensity, action taken with respect to Medicinal 
Product, corrective therapy given, outcome and his/her opinion as to whether there is a 
reasonable possibility that the event was caused by the study Medicinal Product. 
The investigator shall keep detailed records of all adverse events, which are reported. Events 
occurring from enrolment to the administration of the autologous gene modified cells (Medicinal 
Product), including the preparatory phase of conditioning, will be recorded as Concomitant 
Diseases in the CRF. These may include events related to the procedures (e.g. CVC positioning, 
bone marrow harvest, mobilization and collection), chemotherapy, and other drugs. These 
reports must be provided to the competent Authorities if they so request. 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 85 of 119 
-- 
 
 
 
 
8.2.4. 
Expected AE/AR 
The following reactions are expected following G-CSF + Plerixafor administration, conditioning 
regimen, other concomitant drug administration, and blood products and will be recorded if ≥2 
CTCAE grade for clinical manifestations, ≥ 3 CTCAE grade for laboratory parameters: 
• Vomiting 
• Nausea 
• Stomatitis 
• Mucositis 
• Diarrhea 
• Hair loss 
• Fever of undetermined origin during neutropenia 
• Fatigue 
• Loss of appetite 
• Cytopenia 
• Bleeding 
• Headache 
• Hypotension 
• Hypertension 
• Vasomotor effects, including flushing 
• Dizziness 
• Arthralgias 
• Injection site reactions 
• Allergic reactions (to transfusions or drugs different from the medicinal product) 
• Electrolytes disturbances* 
• Liver and renal function alterations* 
 
*Abnormal laboratory values or test results constitute adverse events only if they induce clinical 
signs or symptoms or require therapy. 
 
8.2.5. 
Follow up of AE / AR / SAE / SAR 
The Investigator should take all appropriate measures to ensure the safety of the patients, notably 
he/she should follow up the outcome of any event (clinical signs, laboratory values or other, 
etc…) until the return to normal or until consolidation of patient conditions. 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 86 of 119 
-- 
 
 
 
 
In the case of any Serious Adverse Event or Serious Adverse Reaction, the patient must be 
followed up until clinical recovery is complete and laboratory results have returned to normal, or 
until progression has been stabilized. When the clinical recovery lasts longer than thirty (30) 
days the Investigator will provide to the Ethics Committee an Additional Safety Report. This 
may imply that follow up will continue after the patient left the study and that additional 
investigations may be requested by the Ethical Committee. 
 
8.2.6. 
Follow up procedure for withdrawn patients 
All withdrawn patients should be recorded by the Investigator in the appropriate pages of the 
CRF when considered as confirmed. If possible, the patients should be assessed using the 
procedure planned for the end of study visit. 
For the withdrawn patients, according to Italian regulations on gene therapy, the follow up will 
continue just for the safety aspects. 
8.3. 
Assessment of safety endpoints 
8.3.1. 
Safety of the conditioning regimen 
Conditioning regimen-related safety consists in the absence of failure of hematological recovery 
and/or delayed hematopoietic reconstitution, which is defined as ANC<500 at +60 days after 
transplantation, with aplastic bone marrow and requirement for back-up infusion. 
8.3.2. 
Safety of the transduced cell infusion 
Short-term safety of LV-transduced cell infusion consists in the absence of severe adverse 
reactions after infusion. 
Microbiological contamination of the drug product: although all manipulations will be 
performed under validated GMP procedures, collection of bone marrow cells and their 
manipulation ex vivo is potentially associated with contamination. Standard operating procedures 
aimed at reducing this risk will be implemented during collection. The final results of the 
sterility test will be available after patient infusion. If a contamination is detected, the patient will 
receive a specific antibiotic treatment and a strict monitoring. 
Long-term safety of LV-transduced cell infusion consists in the absence of RCL, abnormal 
clonal proliferation and immune responses against the transgene. 
The overall safety of the experimental treatment will be evaluated on the basis of clinical and 
laboratory parameters recorded and monitored during the follow up of the treated patients, based 
on the assessment plan. A descriptive analysis will be performed on AE, AR, SAE/SAR, UAR, 
SUSAR, and events will be classified for frequency, severity and body system involved. 
 
8.4. 
Specific safety monitoring plan for gene therapy 
As per gene therapy regulations, patients who received gene therapy with lentiviral vectors will 
be followed up for 15 years after treatment. The first three years of safety follow up (during the 
Initial Follow Up period) are described in Table 10 . After completion of this Initial Follow Up 
period, patients enrolled and treated in this study will continue to be followed from 3 years up to 
8 years as part of the Long-Term Follow Up period. 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 87 of 119 
-- 
 
 
 
 
Patients who complete the Long-Term Follow Up period in this study(i.e. 8 years of follow-up) 
will then be contacted annually from year 8 onwards (as part of the ELTFU phase) in order to 
collect long-term follow up safety and selected efficacy data, until a separate OLTFU study is 
initiated and patients have enrolled in it. Once the OLTFU study is set up, patients will be invited 
to enter the OLTFU study at any time after they have completed a minimum of 5 years of follow 
up in this study (WAS Pivotal 201228) . As part of the OLTFU study, patients will be followed 
up for a total of 15 years following treatment with gene therapy. In case no detectable 
engraftment of LV-transduced cells will be observed for >1 year, patients will be followed only 
for safety aspects. 
 
8.4.1. 
Replication competent lentivirus (RCL) 
Molecular monitoring of RCL will be assessed via ELISA for HIV p24 antigen in serum. 
A positive HIV p24 test result will be subject to confirmation. If confirmed, second level testing 
will include a) DNA PCR for VSV-G envelope (cells) and b) RT-PCR for HIV-pol RNA 
(serum). Should one of these second level tests also provide a positive result, an appropriate 
confirmatory test may be conducted. 
The tests will be performed at baseline, after 1, 3, 6, 12 months and 24 months. The samples will 
be stored every year thereafter. If one of the above mentioned tests are positive, the screening 
evaluations will be repeated at the next planned follow up visit; in the case two of the three 
screening tests are positive, a confirmatory RCL culture test will be performed. 
 
8.4.2. 
Abnormal clonal proliferation (ACP) 
For safety monitoring of abnormal clonal proliferation, the following plan will be implemented 
during the first 3 years of follow up: 
A. First level screening (every 6 months) 
1. Clinical evaluation 
2. Blood count with differential to detect abnormal proliferation or cytopenia 
3. Routine biochemical tests to detect abnormal organ functions 
4. Serum immunoglobulins and protein serum electrophoresis, for the presence of 
altered or monoclonal immunoglobulin components 
5. Flow cytometry on peripheral blood for lymphocyte subpopulations (CD2, CD3, 
CD4, CD8, CD56, CD16, TCR alfa/beta, TCR gamma/delta, CD19, Ig-kappa, Ig- 
lambda) 
6. Flow cytometry with monoclonal antibodies for TCR Vbeta families 
7. PCR analysis for measuring vector copy number (VCN). 
B. Second level screening (every year): 
1. Bone marrow aspirate, including morphological examination, cytogenetic analysis 
and detailed immunophenotyping 
2. Cytogenetic analyses on lymphocytes (karyotype) 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 88 of 119 
-- 
 
 
 
 
3. Archive pellets and viable cells of BM and/or PB. 
C. Additional tests (if above tests suggest monoclonal expansion, to be performed on recent 
and archived samples): 
1. BCR clonality (PCR) 
2. Clonality analysis for TCR (PCR) 
3. Establishment of cell lines 
4. Molecular tests for identifying proviral integrants ((Linear-amplification mediated) 
LAM-PCR or (ligation-mediated) LM-PCR) 
5. RT-PCR for expression of vector and genes next to the integration 
6. All other appropriate imaging and laboratory tests. 
Tests (1) to (5) are available as research grade at TIGET 
Test of first and second level screening (I, II) are already included in the efficacy evaluation in 
Table 8a and Table 8b. 
For the long-term follow up (from 3 years up to 8 years) the ACP safety-monitoring tests will be 
performed as follows: 
1) First level screening will be performed every year from the 4th to 8th year of follow up. 
2) Second level screening will be performed as follows: 
a. Bone marrow aspirate on year 5 and 8 
b. Cytogenetic analyses on lymphocytes (karyotype): every year from the 4th to 8th year 
follow up 
c. Archive pellets and viable cells of BM and/or PB: every year from the 4th to 8th year 
follow up 
As part of the overall safety evaluation, a detailed analysis of LV integrations will be performed 
on PBMC and BM cells, to monitor the nature and distribution vector integration sites. These 
studies will help defining the clonal composition of the transduced cell graft and the eventual 
occurrence of clonal expansion. 
8.4.3. 
Immune response to transgene 
Patients will be monitored for antibodies anti-WASP for the lack of immune response to 
transgene every 6 months for the first year, then at year 2 and year 3. Moreover, anti-HIV 
antibodies (anti-p24) will be monitored at the same time points. 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 89 of 119 
-- 
 
 
 
 
Table 10 
Safety follow up (from 0 to 3 years) 
 
Parameter 
RCL 
ACP 
Immune response to transgene 
Time point 
Baseline 
X 
X 
X 
+  30 days 
X 
+  90 days 
X 
+ 180 days 
X 
X 
X 
+  1 
y 
X 
X 
X 
+  1,5 y 
X 
+  2 
y 
X 
X 
X 
+  2,5 y 
X 
+  3 
y 
Archive 
X 
X 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 90 of 119 
-- 
 
 
 
 
9. 
ASSESSMENT OF EFFICACY 
9.1. 
Sustained engraftment of genetically corrected cells in peripheral 
blood and/or in bone marrow 
Positive engraftment will be assumed as detectable gene corrected cells on peripheral blood and 
bone marrow cells at 1 year after gene therapy. Adequate engraftment is defined as ≥ 4% CD34+ 
cells (equivalent to 0.04 VCN/cell) in the bone marrow or ≥ 10% peripheral blood T 
lymphocytes or peripheral blood mononuclear cells (when cells are not sufficient) (equivalent to 
0.1 VCN/cell). This value is based on clinical experience in other gene therapy trials, which has 
demonstrated 4% average long-term engraftment of autologous hematopoietic stem/progenitor 
cells transduced with retroviral vectors in the bone marrow of paediatric patients with ADA- 
SCID and receiving reduced intensity, non-myeloablative conditioning (Aiuti et al., 2009; 
unpublished results). 
To further confirm results, the PB T cell or PB MNC cells and the pre-GT sample will be 
performed at MolMed S.p.A. 
In addition, the multilineage engraftment will be evaluated in BM and/or PB subpopulations 
(BM subpopulations: GlyA+, CD15+, CD3+, CD19+, CD56+; PB subpopulations: CD15+, 
CD19+, CD56+). If data on PB CD3+ are not available, the average of PB CD4+ and PB CD8 T 
cells will be calculated and reported as T-cell engraftment. Adequate multilineage engraftment is 
defined as ≥ 4% in all the available cells (equivalent to 0.04 VCN/cell). 
Assessment parameter: PCR for transduced cells (specific for lentiviral vector sequences). 
 
9.2. 
Expression of vector-derived WASP 
Positive WASP expression will be assumed by presence of WASP protein on peripheral blood or 
bone marrow cells, as investigated by FACS analyses or Western Blot. 
Assessment parameters: analyses of WASP expression (FACS or Western Blot with antibodies 
specific for WASP). 
 
9.3. 
Improved T-cell functions 
Improvement in T-cell proliferation upon stimulation with anti-CD3i mAbs ≥1 year after gene 
therapy (as compared to pre-gene therapy values) on PBMC and/or T-cell lines. The degree of 
correction will be evaluated with respect to normal controls. 
Assessment parameters: proliferative responses in response to anti-CD3i mAb stimulation in 
vitro. 
 
9.4. 
Antigen-specific responses to vaccinations 
Ability to mount a humoral response to nominal antigens including antibodies to T-cell 
dependent antigens (Tetanus Toxoid, Diphtheria, Hepatitis B, Pertussis, Haemophilus B) and 
unconjugated polysaccharide antigens (Pneumococcus), measured after vaccination (foreseen >1 
year after gene therapy). In case of failure of immunization with unconjugated polysaccharide 
antigens after vaccination, the patients will receive a conjugated polysaccharide vaccine. Positive 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 91 of 119 
-- 
 
 
 
 
cellular response to Tetanus Toxoid after vaccination will be measured by in vitro proliferative 
response >1 year after gene therapy. 
Assessment parameters: 4 out of 5 protective antibody titers after vaccination and antigen 
proliferative responses to tetanus toxoid. In case results are available on n<5 antigens, the rule of 
at least n-1 will be applied for defining success. 
Protective antibody titer after unconjugated vaccination to Pneumococcus (the determination of 
this antibody titer will be performed by the laboratory of Dr. M. Eibl, Immunologische 
Tagesklinik, Wien, Austria). 
9.5. 
Improved platelet count and normalization of MPV 
Increase in platelet count compared to baseline, considered as the mean of the (at least three) 
determinations performed in the 3 months before beginning of treatment. Patients will also be 
evaluated according to a stratification of the severity of thrombocytopenia in 5 groups (see 
Section 8.3.1, Table 5). 
The individual longitudinal profile will be analyzed according to the planned follow up visit and 
including platelet counts collected for clinical reasons during follow up (in transfusion 
independent patients). 
The determination of platelet count following procedures that may alter platelet number 
(thrombocyte infusion, TPO agonists etc.) will not be considered for evaluation. 
Normalization of MPV compared to baseline will be evaluated at 1 year after gene therapy. 
Assessment parameters: Platelet count and MPV. 
9.6. 
Reduced bleeding episodes 
Reduction in severity and/or frequency of bleeding manifestations when present, starting from 
the first year follow up visit and thereafter until the 3-year visit compared to clinical history from 
the 12 months prior to GT. Information about bleeding will be recorded through follow up by 
collecting data from parents, clinical reports periodically sent by local doctors following the 
patients at home and by clinical evaluations performed at each follow up visit at our center. Data 
will be recorded in the patients’ clinical charts. Bleeding events will be given CTCAE grading 
(as described in Section 4.3.2.1). 
Assessment parameters: clinical evaluation. 
 
9.7. 
Reduced frequency of severe infections 
Decrease in number of severe infections (defined by CTCAE criteria , as described in 
Section 4.3.2.1) will be evaluated starting from the first year follow up visit and thereafter until 
the 3-year visit by clinical history, complete physical examinations, and microbiological tests, 
compared to clinical history from the 12 months prior to GT. 
Assessment parameters: Clinical evaluation, laboratory tests, microbiology. 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 92 of 119 
-- 
 
 
 
 
9.8. 
Modification of eczema and autoimmunity phenomena 
Modification of eczema and of autoimmunity laboratory markers and/or clinical manifestations 
(if present before treatment), as evaluated starting from the first year follow up visit and 
thereafter until the 3-year visit by organ-specific and systemic autoantibodies, imaging and 
clinical follow up, as compared to clinical history. 
Assessment parameters: clinical evaluation including eczema score (Imai, 2004) and 
autoimmunity laboratory tests. 
 
9.9. 
Improved quality of life 
Improved quality of life, measured after the first year of treatment by reduced hospitalization, 
reduced requirement of drugs, school attendance, social activities. 
Assessment parameters: clinical evaluation, performed according to patients’ age (ex. social 
ability with peers will not be evaluated before 2 years of age and sport practice will not be 
considered for evaluation before 4 years of age). 
 
9.10. 
Platelet activation profile and morphology (exploratory endpoint) 
Activation of platelets will be assessed after stimulation with different concentrations of the 
platelet activation agonists ADP and thrombin. 
Assessment parameters: platelet activation will be assessed by cytofluorimetric measurement of 
surface activation markers P-selectin and GPIIbIIIa after stimulation with various concentrations 
of ADP and thrombin. The dose response curves will be compared with healthy controls. 
Platelet morphology will be assessed by transmission electron microscopy. 
Assessment parameters: platelet perimeter, area and ultrastructure. Morphology will be 
compared with healthy controls. 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 93 of 119 
-- 
 
 
 
 
10. 
STUDY MONITORING 
10.1. 
Responsibilities of the Investigator(s) 
The Investigator(s) undertake(s) to perform the study in accordance with this Protocol, Good 
Clinical Practice and the applicable regulatory requirements. 
The Investigator(s) should permit monitoring and auditing by the sponsor, and inspection by the 
appropriate regulatory Authority(ies). 
The Investigator is required to ensure compliance with the investigational product schedule, 
visits schedule and procedures required by the protocol. 
The Investigator agrees to provide all information requested in the Case Report Form in an 
accurate and legible manner. 
 
10.2. 
Use and completion of Case Report Forms (CRFs) 
It is the responsibility of the Investigator to maintain adequate and accurate CRFs to record all 
observations and other data pertinent to the clinical investigation. 
Under SR-TIGET sponsorship paper CRFs were utilized; these should be completed in their 
entirety in a neat, legible manner to ensure accurate interpretation of data; a black ball point pen 
should be used to ensure the clarity of reproduced copy of all CRFs. Should a correction be 
made, the information to be modified must not be overwritten. The corrected information will be 
transcribed by the authorized person next to the previous value and dated. 
An electronic CRF will be implemented; all data previously collected on paper CRF and all data 
collected following sponsorship transfer will be entered into defined CRFs. All these data will be 
transmitted electronically to the Sponsor or designee and combined with data provided from 
other sources in a validated data system. 
Management of clinical data will be performed in accordance with applicable Sponsor standards 
and data cleaning procedures to ensure the integrity of the data, e.g., removing errors and 
inconsistencies in the data. 
Adverse events and concomitant medications terms will be coded using MedDRA (Medical 
Dictionary for Regulatory Activities) CRFs (including queries and audit trails) will be retained 
by the Sponsor, and copies will be sent to the investigator to maintain as the investigator copy. 
Subject initials will not be collected or transmitted to the Sponsor according to the Sponsor 
policy. 
10.3. 
Definition of Source Data 
Source data include all the documents related to the patient during the trial. These include (but is 
not limited to) the medical records which support the SAE and SUSAR report forms, and may 
include patient's medical files, appointment book, original laboratory records, discharge 
summary, death certificate, autopsy report if available, as they are related to the trial and support 
the data in the CRFs. 
All parameters asked for in the case report form should be documented in the source documents. 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 94 of 119 
-- 
 
 
 
 
10.4. 
Trial Monitoring 
The purposes of trial monitoring are to verify that: 
• the rights and well-being of human subjects are protected; 
• the reported trial data are accurate, complete, and verifiable from source documents; 
• the trial will be conducted in compliance with the currently approved 
protocol/amendment(s), with GCP, and with the applicable regulatory requirement(s). 
 
10.5. 
Qualifications of Monitors 
Monitors should be appointed by the sponsor. 
Monitors should be appropriately trained, and should have the scientific and/or clinical 
knowledge needed to monitor the trial adequately. 
Monitors should be thoroughly familiar with the investigational product(s), the protocol, written 
informed consent form and any other written information to be provided to subjects, the sponsors 
SOPs, GCP, and the applicable regulatory requirement(s). 
 
10.6. 
Monitoring Procedures 
The monitor should follow the sponsor’s established written SOPs as well as those procedures 
that are specified by the sponsor for monitoring a specific trial. 
 
10.7. 
Monitoring Report 
The monitor should submit a written report to the sponsor after each trial-site visit or trial-related 
communication. 
Reports should include the date, site, name of the monitor, and name of the investigator or other 
individual(s) contacted. 
Reports should include a summary of what the monitor reviewed and the monitor's statements 
concerning the significant findings/facts, deviations and deficiencies, conclusions, actions taken 
or to be taken and/or actions recommended to secure compliance. 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 95 of 119 
-- 
 
 
 
 
11. 
ETHICAL CONSIDERATION 
This trial complies with the principles laid down by the Declaration of Helsinki (last version) and 
the ICH GCP guidelines. 
This Protocol and its conduction comply with in use laws and guidelines of the Italian Ministry 
of Health. 
 
11.1. 
Informed Consent 
The Principal Investigator, according to applicable regulatory requirements, or a person 
designated by the Principal Investigator, should fully inform the patient and/or parents or legal 
representatives of all pertinent aspects of the clinical trial. 
The Informed Consent Form and its updates used by the Investigator for obtaining the patient's 
informed consent must be reviewed and approved by Ethics Committee. Informed consent will 
be performed by the Principal Investigator, or a person designated by the Principal Investigator, 
at the original treatment center, and may take place remotely. 
In case of the patient is minor (<18 years of age), the Informed Consent of the parents or legal 
representatives must be obtained; consent must represent the minor’s presumed will and may be 
revoked at any time, without detriment to the minor. 
The parents or legal representatives should have an interview with the investigator, or another 
member of the investigating team designated by the Principal Investigator, in which they are 
given the opportunity to understand the objectives, risks and inconveniences of the trial and the 
conditions under which it is to be conducted. 
The minor should receive information according to its capacity of understanding, from staff with 
experience with minors, regarding the trial, the risks, and the benefits. 
The person not able to give informed legal consent should receive information according to 
his/her capacity of understanding the trial, the risks, and the benefits. 
Prior to a patient’s participation in the clinical trial, the Informed Consent Form should be signed 
and dated by the patient and/or the parents or legal representatives, and by the person who 
conducted the informed consent discussion. 
The patient and/or parents or legal representatives should receive an original copy of the signed 
and dated written informed consent form. 
During the participation in the trial, the subject or legal representatives should receive a copy of 
the signed and dated consent form updates and an original copy of any amendments to the 
written information provided to subjects. 
The patient and/or the parents or legal representatives may at any time, and without giving any 
explanation, withdraw from the trial. In any case the doctors will continue to follow the patient 
with proper attention and care. 
11.2. 
Investigator's Brochure 
The Investigator’s Brochure (IB) should provide the investigators and others involved in the trial 
with the information to facilitate their understanding of the rationale for, and their compliance 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 96 of 119 
-- 
 
 
 
 
with, many key features of the protocol, such as the dose, dose frequency/interval, methods of 
administration, and safety monitoring procedures. 
The IB should contain the following sections, with appropriate references for each chapter (ICH 
Topic E6 GCP): 
- Table of Contents 
- Summary 
- Introduction 
- Physical, Chemical, and Pharmaceutical Properties and Formulation 
- Nonclinical Studies 
- Nonclinical Pharmacology 
- Pharmacokinetics and Product Metabolism in Animals 
- Toxicology 
- Effects in Humans 
- Pharmacokinetics and Product Metabolism in Humans 
- Safety and Efficacy 
- Marketing Experience 
- Summary of Data and Guidance for the Investigator 
- Reference on 1. Publications 
2. Reports 
 
11.3. 
Minimizing distress 
Repeated invasive procedures may be painful or frightening. This trial is designed to minimize 
the discomfort. The study is designed and conducted by investigators experienced in the 
treatment of paediatric/vulnerable subjects/patients. 
The protocol and investigations have been designed specifically for the paediatric population and 
approved by an Ethical Committee expert in paediatric/vulnerable subjects/patients. 
Practical considerations to ensure that participants’ experiences in clinical studies are positive 
and to minimize discomfort and distress include the following: 
- 
personnel skilled in dealing with the paediatric population and its age-appropriate needs, 
including skill in performing paediatric procedures; 
- 
an environment with furniture, play equipment, activities, and food appropriate for age; 
- 
the conduction of studies in a familiar environment such as the hospital or clinic where 
participants normally receive their care; 
- 
approaches to minimize procedures related discomfort, such as: 
o local anaesthesia to place peripheral venous catheters; 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 97 of 119 
-- 
 
 
 
 
o indwelling catheters (central or peripheral) rather than repeated venipunctures for 
blood sampling; 
o collection of protocol-specific blood samples in occasion of routine blood 
sampling, performed for clinical reasons. 
 
11.4. 
Minimizing Risk 
Every effort has been made to anticipate and reduce known hazards. Investigators are fully aware 
and trained on all relevant preclinical and clinical toxicity of the medicinal product. To minimize 
risk in paediatric/vulnerable subjects clinical studies, investigators are experienced in studying 
the paediatric/vulnerable subjects population, including the evaluation and management of 
potential adverse events. 
In designing studies, every attempt was made to minimize the number of procedures, consistent 
with good study design. Mechanisms are in place to ensure that the study can be put on hold or 
modified should an unexpected hazard be noted. 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 98 of 119 
-- 
 
 
 
 
12. 
STATISTICAL DESIGN 
12.1. 
Protocol modification and stopping rules 
As already described in section 8.2 for relevant endpoints, rules for protocol modification and 
stopping serve as guidelines for discussing the continuation/modification of the study. The rules 
are defined below. 
Protocol modification: 
- 2 out of the first 3 patients suffering of prolonged aplasia; 
- 2 or more failures of engraftment of transduced cells. 
Early Stopping: 
- 2 transplant-related deaths; 
- 1 malignant proliferation related to gene therapy. 
In the interest of the patients’ safety, the third patient will be treated when: 
- at least 1 of the first 2 patients will achieve hematopoietic recovery; 
- at least 60 days after the first treatment. 
For the subsequent patients the above rules will be applied. 
 
12.2. 
Study Population and Timing of Analysis 
All study patients will be included for evaluation until the earliest time point of the following: 
completion of a minimum of 5 years follow up in this study (WAS Pivotal, 201228) and 
transition to a separate OLTFU study, death, withdrawal or loss to follow up. Patients who, at 1 
year after gene therapy, show absence of transduced cell engraftment in peripheral blood and 
bone marrow will be subsequently evaluated for safety aspects only. 
At the end of the study, when all subjects have completed a minimum of 5 years follow up and 
transitioned to a separate OLTFU study, a final statistical analysis will be performed with all 
data available up to the transition to the OLTFU study, and a final clinical study report will be 
compiled. 
 
12.3. 
Safety Endpoints 
A descriptive analysis will be performed for all primary and secondary safety endpoints. 
 
12.4. 
Efficacy endpoints 
Primary efficacy endpoints 
Overall survival 
The Kaplan-Meier estimate of survival at 3 years from gene therapy will be obtained, with 95% 
Confidence Intervals, based on deaths by any causes related to disease. Survival will be also 
monitored as part of the long-term follow up. 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 99 of 119 
-- 
 
 
 
 
Sustained engraftment of genetically corrected haematopoietic stem cells in peripheral blood 
and/or in bone marrow 
The percentage of subjects who present at one year after gene therapy with adequate engraftment 
defined as ≥ 4% CD34+ in the bone marrow or ≥ 10% in peripheral blood T lymphocytes will be 
estimated and the 95% Confidence Intervals (CI) of this proportion will also be calculated. The 
description of the longitudinal profile over time of the percentage of gene corrected cells on both 
bone marrow and peripheral blood might be included as additional information. 
Expression of vector-derived WASP 
The percentage of subjects who present at one year after gene therapy with detectable vector- 
derived WASP expression will be estimated, with 95% CI. 
Improved T-cell functions 
The mean change between the pre-gene therapy and the 1-year values of in vitro T-cell 
proliferation upon stimulation with anti-CD3i will be calculated, with 95% CI, if appropriate. 
The description of the longitudinal profile over time of the levels of the in vitro T-cell 
proliferation might be included as additional information. The degree of correction (reached at 1, 
2 and 3 years from gene therapy) with respect to normal control levels will also be described. 
Antigen-specific responses to vaccinations 
The percentage of subjects who respond to vaccination (see Section 9.4) performed > 1 year after 
gene therapy will be estimated, with 95% CI. 
Improved platelet count 
The percentage of subjects who improved the level of platelet count at 1, 2 and 3 years after gene 
therapy compared to baseline, shifting from one category to the subsequent as defined in 
Section 9.5, will be estimated, with 95% CI. The relative mean change in platelet count with 
respect to pre-gene therapy value will also be given. The description of the longitudinal profile 
over time of the platelet count might be included as additional information. 
Secondary efficacy endpoints 
The percentage of subjects who present at one year after gene therapy with adequate multilineage 
engraftment defined as ≥ 4% in bone marrow and in peripheral blood cell subpopulations (BM 
subpopulations: GlyA+, CD15+, CD3+, CD19+, CD56+; PB subpopulations: CD15+, CD19+, 
CD56+) will be estimated and the 95% Confidence Intervals (CI) of this proportion will also be 
calculated. 
A descriptive analysis of the other secondary efficacy endpoints will be provided. 
 
12.5. 
Note on Confidence Interval calculation 
The Confidence Intervals for proportions will be calculated according to the Wilson approach 
(Brown et al., 2001), while an exact Poisson procedure will be adopted for deriving CIs for 
incidence rates. Confidence Intervals for the means or mean changes in continuous variables will 
be derived with standard parametric methods, if appropriate. 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 100 of 119 
-- 
 
 
 
 
12.6. 
Integration of results by other trials on GT 
A meta-analysis with data coming from similar trials (the studies sponsored by Généthon to our 
current knowledge and others that may be implemented in the near future) is envisaged. This 
would be useful, based on a pooled estimate of safety and efficacy, to: 
a) assess whether there is a meaningful heterogeneity in results between different trials; 
b) compare outcome after gene-therapy with outcome after different types of conditioning 
regimen and cell preparation; 
c) consider the opportunity for the clinical development of a phase II-III trial. 
Any changes in the planned statistical methods will be documented in the study report. 
12.7. 
Long-term follow up 
Individual patient annual data, collected from the 3rd up to the 8th year after infusion of 
transduced stem cells (Table 8b), and at the end of the study, will be summarized by means of 
descriptive summary statistics or frequency tables as appropriate, as described above. 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 101 of 119 
-- 
 
 
 
 
13. 
QUALITY ASSURANCE AND QUALITY CONTROL 
13.1. 
Definitions 
Audit: A systematic and independent examination of trial related activities and documents to 
determine whether the evaluated trial related activities were conducted, and the data were 
recorded, analysed and accurately reported according to the protocol, sponsor's standard 
operating procedures (SOPs), Good Clinical Practice (GCP), and the applicable regulatory 
requirement(s). 
Monitoring: The act of overseeing the progress of a clinical trial, and of ensuring that it is 
conducted, recorded, and reported in accordance with the protocol, Standard Operating 
Procedures (SOPs), Good Clinical Practice (GCP), and the applicable regulatory requirement(s). 
Quality Assurance (QA): All those planned and systematic actions that are established to ensure 
that the trial is performed and the data are generated, documented (recorded), and reported in 
compliance with Good Clinical Practice (GCP) and the applicable regulatory requirement(s). 
Quality Control (QC): The operational techniques and activities undertaken within the quality 
assurance system to verify that the requirements for quality of the trial-related activities have 
been fulfilled. 
 
13.2. 
Monitoring. 
A comprehensive quality control can be ordered in the form of monitoring. It might include 
checking the whole course of the study, the management of documentation, the management of 
data, the management of subject enrolled, the management of experimental drug and the 
management of biological sample. The sponsor of the study will be in charge for monitoring. 
The investigators will assure the monitoring of the clinical study to assure conformance to 
protocol as well as the completeness, correctness, and plausibility of the completed case report 
forms (CRF). 
13.3. 
Deviation from study protocol. 
Every deviation from the trial protocol must be specified and documented separately for each 
patient. The investigator must consult with the monitor and discuss the type and extent of 
deviation, as well as the possible consequences for further participation of the patient in the 
study. If the evaluability of a patient is questionable the coordinating investigator will be 
consulted. 
13.4. 
Audits and inspections 
If necessary, a comprehensive quality control could be ordered in the form of an audit. It might 
include checking the whole course of the study, the documentation, statistical analysis, and the 
investigators. 
The sponsor guarantees the availability for the inspections from the regulatory agencies. 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 102 of 119 
-- 
 
 
 
 
14. 
ADMINISTRATIVE RULES 
14.1. 
Insurance 
The conduct of the study is covered by an insurance policy. 
The insurance policy is in accordance with local laws and requirements. A copy of the insurance 
certificate will be archived in the Trial Master File. 
14.2. 
Financing 
Up to January 2014 Telethon Foundation was the source of monetary support of this clinical 
trial, i.e.Telethon Foundation funded SR-TIGET to conduct the clinical trial at the Pediatric 
Clinical Research Unit and Stem Cell Program at Ospedale San Raffaele. An agreement 
(Convenzione) between Ospedale San Raffaele (via Olgettina, 60 – Milano) and Telethon 
Foundation (via G. Saliceto, 5/a – Roma) assigned a grant to Pediatric Clinical Research Unit 
TIGET. This grant covered the costs of the clinical trial. 
As of January 2014, GlaxoSmithKline Research and Development Ltd, 980 Great West Road, 
Brentford London TW8 9GS, United Kingdom was the source of monetary support of the 
clinical trial. 
 
As of April 2018, Orchard became the source of monetary support of the clinical trial. 
 
As of December 2023, Fondazione Telethon became the source of monetary support of the clinical 
trial. 
 
14.3. 
Final study report 
The sponsor will be responsible for preparing formal interim clinical study reports and the final 
clinical study report. 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 103 of 119 
-- 
 
 
 
 
15. 
BIBLIOGRAPHY 
Aabiden K, Anoop P, et al. (2011). "The feasibility of plerixafor as a second-line stem cell 
mobilizing agent in children." J Pediatr Hematol Oncol 2011 Jan;33(1):65-7. 
Ailles, L., M. Schmidt, et al. (2002). "Molecular evidence of lentiviral vector-mediated gene 
transfer into human self-renewing, multi-potent, long-term NOD/SCID repopulating 
hematopoietic cells." Mol Ther 6(5): 615-26. 
Aiuti A, Cattaneo F, et al. (2009). "Gene therapy for immunodeficiency due to adenosine 
deaminase deficiency." N Engl J Med. 29;360(5):447-58. 
Aiuti, A., B. Cassani, et al. (2007). "Multilineage hematopoietic reconstitution without clonal 
selection in ADA-SCID patients treated with stem cell gene therapy." J Clin Invest 117(8): 2233- 
40. 
Aiuti, A., S. Slavin, et al. (2002). "Correction of ADA-SCID by stem cell gene therapy combined 
with nonmyeloablative conditioning." Science 296(5577): 2410-3. 
Baum, C. and B. Fehse (2003). "Mutagenesis by retroviral transgene insertion: risk assessment 
and potential alternatives." Curr Opin Mol Ther 5(5): 458-62. 
Bosticardo M, Marangoni F, et al. (2009) .Recent advances in understanding the 
pathophysiology of Wiskott-Aldrich syndrome. Blood. 18;113(25):6288-95. 
Boztug K, Dewey RA, et al (2006) Development of hematopoietic stem cell gene therapy for 
Wiskott-Aldrich syndrome.Curr Opin Mol Ther. 8:390-5. 
Boztug K, Schmidt M, et al. (2010). "Stem-cell gene therapy for the Wiskott-Aldrich syndrome." 
N Engl J Med 363(20): 1918-27. 
Braun CJ, Boztug K, et al (2014). "Gene Therapy for Wiskott-Aldrich Syndrome - Long-term 
Efficacy and Genotoxicity." Sci Trans Med 6(227): 1-14. 
Brown LD, Cai TT, DasGupta A. Interval estimation for a binomial proportion. Statistical 
Science 2001;16(2):101-133 
Bukovsky, A. A., J. P. Song, et al. (1999). "Interaction of human immunodeficiency virus- 
derived vectors with wild-type virus in transduced cells." J Virol 73(8): 7087-92. 
Burns, S., G. O. Cory, et al. (2004). "Mechanisms of WASp-mediated hematologic and 
immunologic disease." Blood 104(12): 3454-62. 
Bushman, F., M. Lewinski, et al. (2005). "Genome-wide analysis of retroviral DNA integration." 
Nat Rev Microbiol 3(11): 848-58. 
Bushman, F. D. (2007). "Retroviral integration and human gene therapy." J Clin Invest 117(8): 
2083-6. 
Candotti, F., F. Facchetti, et al. (1999). "Retrovirus-mediated WASP gene transfer corrects 
defective actin polymerization in B cell lines from Wiskott-Aldrich syndrome patients carrying 
'null' mutations." Gene Ther 6(6): 1170-4. 
Cartier, N., S. Hacein-Bey-Abina,et al. (2009). "Hematopoietic stem cell gene therapy with a 
lentiviral vector in X-linked adrenoleukodystrophy." Science. Nov 6;326(5954):818-23. 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 104 of 119 
-- 
 
 
 
 
Cattoglio, C., G. Facchini, et al. (2007). "Hot spots of retroviral integration in human CD34+ 
hematopoietic cells." Blood 110(6): 1770-8. 
Cavazzana-Calvo, M., E. Payen, et al. (2010). "Transfusion independence and HMGA2 
activation after gene theraphy of human β-thalassaemia." Nature. Sep 16;467(7313):318-22. 
Charrier, S., L. Dupre, et al. (2007). "Lentiviral vectors targeting WASp expression to 
hematopoietic cells, efficiently transduce and correct cells from WAS patients." Gene Ther 
14(5): 415-28. 
De Palma, M., E. Montini, et al. (2005). "Promoter trapping reveals significant differences in 
integration site selection between MLV and HIV vectors in primary hematopoietic cells." Blood 
105(6): 2307-15. 
Deichmann, A., S. Hacein-Bey-Abina, et al. (2007). "Vector integration is nonrandom and 
clustered and influences the fate of lymphopoiesis in SCID-X1 gene therapy." J Clin Invest 
117(8): 2225-32. 
Derry, J. M., H. D. Ochs, et al. (1994). "Isolation of a novel gene mutated in Wiskott-Aldrich 
syndrome." Cell 78(4): 635-44. 
Dewey, R. A., I. Avedillo Diez, et al. (2006). "Retroviral WASP gene transfer into human 
hematopoietic stem cells reconstitutes the actin cytoskeleton in myeloid progeny cells 
differentiated in vitro." Exp Hematol 34(9): 1161-9 
Dupre, L., A. Aiuti, et al. (2002). "Wiskott-Aldrich syndrome protein regulates lipid raft 
dynamics during immunological synapse formation." Immunity 17(2): 157-66 
Dupre, L., S. Trifari, et al. (2004). "Lentiviral vector-mediated gene transfer in T cells from 
Wiskott-Aldrich syndrome patients leads to functional correction." Mol Ther 10(5): 903-15. 
Dupuis-Girod, S., J. Medioni, et al. (2003). "Autoimmunity in Wiskott-Aldrich syndrome: risk 
factors, clinical features, and outcome in a single-center cohort of 55 patients." Pediatrics 111(5 
Pt 1): e622-7. 
European Union.(2008) "Ethical considerations for clinical trials on medicinal products 
conducted with the paediatric population." Eur J Health Law 15(2): 223-50. 
Filipovich, A., J. Stone, et al. (2001). "Impact of donor type on outcome of bone marrow 
transplantation for Wiskott-Aldrich syndrome: collaborative study of the International Bone 
Marrow Transplant Registry and the National Marrow Donor Program." Blood 97: 1598-1603. 
Follenzi, A., G. Sabatino, et al. (2002). "Efficient gene delivery and targeted expression to 
hepatocytes in vivo by improved lentiviral vectors." Hum Gene Ther. 13(2): 243-60. 
Galy A, Roncarolo MG et al. (2008) Development of lentiviral gene therapy for Wiskott Aldrich 
syndrome. Expert Opin Biol Ther.8:181-90. 
Gardellini A., Babic A., et al. (2013). "Successful mobilisation of peripheral blood stem cells in 
children using plerixafor: a case report and review of the literature." Blood Transfus 2013; 11: 
308-10. 
Guenechea, G., O. I. Gan, et al. (2001). "Distinct classes of human stem cells that differ in 
proliferative and self-renewal potential." Nat Immunol 2(1): 75-82. 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 105 of 119 
-- 
 
 
 
 
Guenechea, G., O. I. Gan, et al. (2000). "Transduction of human CD34+ CD38- bone marrow 
and cord blood-derived SCID-repopulating cells with third-generation lentiviral vectors." Mol 
Ther 1(6): 566-73. 
Hacein-Bey-Abina, S., F. Le Deist, et al. (2002). "Sustained correction of X-linked severe 
combined immunodeficiency by ex vivo gene therapy." N Engl J Med 346(16): 1185-93. 
Hacein-Bey-Abina, S., C. von Kalle, et al. (2003). "A serious adverse event after successful gene 
therapy for X-linked severe combined immunodeficiency." N Engl J Med 348(3): 255-6. 
Hematti, P., B. K. Hong, et al. (2004). "Distinct genomic integration of MLV and SIV vectors in 
primate hematopoietic stem and progenitor cells." PLoS Biol 2(12): e423. 
Hong KT, Kang HJ, et al. (2012). "Successful mobilization using a combination of plerixafor 
and G-CSF in pediatric patients who failed previous chemomobilization with G-CSF alone and 
possible complications of the treatment. J Hematol Oncol. 2012 Mar 30; 5:14. 
Humblet-Baron, S., B. Sather, et al. (2007). "Wiskott-Aldrich syndrome protein is required for 
regulatory T cell homeostasis." J Clin Invest 117(2): 407-18. 
Ikawa, M., V. Tergaonkar, et al. (2002). "Restoration of spermatogenesis by lentiviral gene 
transfer: offspring from infertile mice. ." Proc Natl Acad Sci U S A 99(7524-9). 
Imai, K., T. Morio, et al. (2004). "Clinical course of patients with WASP gene mutations." Blood 
103(2): 456-64. 
Jacobson PA, Rogosheske R, et al (2005) "Fludarabine Pharmacokinetics in Nonmyeloablative 
Hematopoietic Cell Transplantation (HCT): Association with Engraftment and Neurotoxicty." 
Blood, 106, 11, Abstract #3673 
Jin, Y., C. Mazza, et al. (2004). "Mutations of the Wiskott-Aldrich Syndrome Protein (WASP): 
hotspots, effect on transcription, and translation and phenotype/genotype correlation." Blood 
104(13): 4010-9. 
Kay, M. A., J. C. Glorioso, et al. (2001). "Viral vectors for gene therapy: the art of turning 
infectious agents into vehicles of therapeutics." Nat Med 7(1): 33-40. 
Klein, C., D. Nguyen, et al. (2003). "Gene therapy for Wiskott-Aldrich syndrome: rescue of T- 
cell signaling and amelioration of colitis upon transplantation of retrovirally transduced 
hematopoietic stem cells in mice." Blood 101(6): 2159-66. 
Kobayashi, R., T. Ariga, et al. (2006). "Outcome in patients with Wiskott-Aldrich syndrome 
following stem cell transplantation: an analysis of 57 patients in Japan." Br J Haematol 135(3): 
362-6. 
Konno, A., T. Wada, et al. (2004). "Differential contribution of Wiskott-Aldrich syndrome 
protein to selective advantage in T- and B-cell lineages." Blood 103(2): 676-8. 
Lacout, C., E. Haddad, et al. (2003). "A defect in hematopoietic stem cell migration explains the 
nonrandom X-chromosome inactivation in carriers of Wiskott-Aldrich syndrome." Blood 102(4): 
1282-9. 
Leverrier, Y., R. Lorenzi, et al. (2001). "Cutting edge: the Wiskott-Aldrich syndrome protein is 
required for efficient phagocytosis of apoptotic cells." J Immunol 166(8): 4831-4. 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 106 of 119 
-- 
 
 
 
 
Lewinski, M. K., M. Yamashita, et al. (2006). "Retroviral DNA integration: viral and cellular 
determinants of target-site selection." PLoS Pathog 2(6): e60. 
Lund, A. H., G. Turner, et al. (2002). "Genome-wide retroviral insertional tagging of genes 
involved in cancer in Cdkn2a-deficient mice." Nat Genet 32(1): 160-5. 
Lutskiy, M. I., F. S. Rosen, et al. (2005). "Genotype-proteotype linkage in the Wiskott-Aldrich 
syndrome." J Immunol 175(2): 1329-36. 
Malär R, Sjöö F, et al. (2011). "Therapeutic drug monitoring is essential for intravenous busulfan 
therapy in pediatric hematopoietic stem cell recipients", Pediatr Transplant 15(6): 580-8. 
Mani, M. Venkatasubrahmanyam S. et al., (2009) " Wiskott-Aldrich syndrome protein is an 
effector of Kit signaling", Blood July 30 (advanced publication) 
Mantovani, J., N. Holic, et al. (2006). "A high throughput method for genome-wide analysis of 
retroviral integration." Nucleic Acids Res 34(19): e134. 
Mantovani J, Charrier S, et al (2009) "Diverse genomic integration of a lentiviral vector 
developed for the treatment of Wiskott-Aldrich syndrome. J Gene Med. 11(8):645-54. 
Marangoni, F., S. Trifari, et al. (2007). "WASP regulates suppressor activity of human and 
murine CD4+CD25+FOXP3+ natural regulatory T cells." J Exp Med 204(2): 369-80. 
Marangoni, F.,Marita Bosticardo, et al 2009 Evidence for long-term efficacy and safety of gene 
therapy for Wiskott-Aldrich syndrome in preclinical models, Mol Ther. 2009 Jun;17(6):1073-82 
Mattioli, C., M. Gemma, et al. (2007). "Sedation for children with metachromatic 
leukodystrophy undergoing MRI." Paediatr Anaesth 17(1): 64-9. 
Meyer-Bahlburg, A., S. Becker-Herman, et al. (2008). "Wiskott-Aldrich syndrome protein 
deficiency in B cells results in impaired peripheral homeostasis." Blood 112(10): 4158-69. 
Mitchell, R. S., B. F. Beitzel, et al. (2004). "Retroviral DNA integration: ASLV, HIV, and MLV 
show distinct target site preferences." PLoS Biol 2(8): E234. 
Miyoshi, H., K. A. Smith, et al. (1999). "Transduction of human CD34+ cells that mediate long- 
term engraftment of NOD/SCID mice by HIV vectors." Science 283(5402): 682-6. 
Modlich, U., Bohne, J. et al. (2006). "Cell-culture assay reveal the importance of retrorviral 
vector design for insertional genotoxicity." Blood 108(8): 2545-53. 
Modlich, U., A. Schambach, et al. (2008). "Leukemia induction after a single retroviral vector 
insertion in Evi1 or Prdm16." Leukemia 22(8): 1519-28. 
Montini, E., D. Cesana, et al. (2006). "Hematopoietic stem cell gene transfer in a tumor-prone 
mouse model uncovers low genotoxicity of lentiviral vector integration." Nat Biotechnol 24(6): 
687-96. 
Moog, R. (2001). "Adverse events in peripheral progenitor cell collection: a 7-year experience." 
J Hematother Stem Cell Res 10(5): 675-80. 
Moratto, D. (2011). "Long-term outcome and lineage-specific chimerism in 194 patients with 
Wiskott-Aldrich syndrome treated by hematopoietic cell transplantation in the period 1980-2009: 
an international collaborative study." Blood 118: 1675-1684. 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 107 of 119 
-- 
 
 
 
 
Moulding, D. A., M. P. Blundell, et al. (2007). "Unregulated actin polymerization by WASp 
causes defects of mitosis and cytokinesis in X-linked neutropenia." J Exp Med 204(9): 2213-24. 
Nishimori, M., A. Tateoka, et al. (2001). "Self-reported recovery time of daily activity after bone 
marrow harvesting from healthy donors." J Anesth 15(1): 1-5. 
Notarangelo, L. D. and H. D. Ochs (2003). "Wiskott-Aldrich Syndrome: a model for defective 
actin reorganization, cell trafficking and synapse formation." Curr Opin Immunol 15(5): 585-91. 
Ochs, H. D. and L. D. Notarangelo (2005). "Structure and function of the Wiskott-Aldrich 
syndrome protein." Curr Opin Hematol 12(4): 284-91. 
Ott, M. G., M. Schmidt, et al. (2006). "Correction of X-linked chronic granulomatous disease by 
gene therapy, augmented by insertional activation of MDS1-EVI1, PRDM16 or SETBP1." Nat 
Med 12(4): 401-9. 
Ott, M. G., R. Seger, et al. (2007). "Advances in the treatment of Chronic Granulomatous 
Disease by gene therapy." Curr Gene Ther. 7(3): 155-61. 
Ozsahin, H., M. Cavazzana-Calvo, et al. (2008). "Long-term outcome following hematopoietic 
stem-cell transplantation in Wiskott-Aldrich syndrome: collaborative study of the European 
Society for Immunodeficiencies and European Group for Blood and Marrow Transplantation." 
Blood 111(1): 439-45. 
Pai, S. Y., D. DeMartiis, et al. (2006). "Stem cell transplantation for the Wiskott-Aldrich 
syndrome: a single-center experience confirms efficacy of matched unrelated donor 
transplantation." Bone Marrow Transplant 38(10): 671-9. 
Pan, Y. W., J. M. Scarlett, et al. (2007). "Prolonged adherence of human immunodeficiency 
virus-derived vector particles to hematopoietic target cells leads to secondary transduction in 
vitro and in vivo." J Virol 81: 639-49. 
Park, F. (2007). "Lentiviral vectors: are they the future of animal transgenesis? ." Physiol 
Genomics 31: 159-73. 
Pham HP, Patel N, et al. (2012) "The use of plerixafor in hematopoietic progenitor cell collection 
in pediatric patients: a single center experience." Cytotherapy. Apr; 14(4): 467-72. 
Pike-Overzet, K., D. de Ridder, et al. (2007). "Ectopic retroviral expression of LMO2, but not 
IL2Rgamma, blocks human T-cell development from CD34+ cells: implications for 
leukemogenesis in gene therapy." Leukemia 21(4): 754-63. 
Prislovsky, A., B. Marathe, et al. (2008). "Rapid platelet turnover in WASP(-) mice correlates 
with increased ex vivo phagocytosis of opsonized WASP(-) platelets." Exp Hematol 36(5): 609- 
23 
Rawlings, S. L., G. M. Crooks, et al. (1999). "Spontaneous apoptosis in lymphocytes from 
patients with Wiskott-Aldrich syndrome: correlation of accelerated cell death and attenuated bcl- 
2 expression." Blood 94(11): 3872-82. 
Recchia, A., C. Bonini, et al. (2006). "Retroviral vector integration deregulates gene expression 
but has no consequence on the biology and function of transplanted T cells." Proc Natl Acad Sci 
U S A 103(5): 1457-62. 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 108 of 119 
-- 
 
 
 
 
Ryu, B. Y., M. V. Evans-Galea, et al. (2008). "An experimental system for the evaluation of 
retroviral vector design to diminish the risk for proto-oncogene activation." Blood 111(4): 1866- 
75. 
Schenk-Braat, E. A., M. M. van Mierlo, et al. (2007). "An inventory of shedding data from 
clinical gene therapy trials." J Gene Med 9(10): 910-21. 
Shin, C.R. (2012). "Outcomes following hematopoietic cell transplantation for Wiskott-Aldrich 
syndrome." Bone Marrow Transplantation 47, 1428 - 1435. 
Schroder, A. R., P. Shinn, et al. (2002). "HIV-1 integration in the human genome favors active 
genes and local hotspots." Cell 110(4): 521-9. 
Schwarzwaelder, K., S. J. Howe, et al. (2007). "Gammaretrovirus-mediated correction of SCID- 
X1 is associated with skewed vector integration site distribution in vivo." J Clin Invest 117(8): 
2241-9. 
Sevilla J, Schiavello E, et al. (2012). "Priming of hematopoietic progenitor cells by plerixafor 
and filgrastim in children with previous failure of mobilization with chemotherapy and/or 
cytokine treatment." J Pediatr Hematol Oncol 2012 Mar;34(2):146-50. 
Son MH, Kang ES, et al. (2013). "Efficacy and toxicity of plerixafor for peripheral blood stem 
cell mobilization in children with high-risk neuroblastoma." Pediatr Blood Cancer. Aug; 
60(8):E57-9. 
Strom, T. S., S. J. Turner, et al. (2003). "Defects in T-cell-mediated immunity to influenza virus 
in murine Wiskott-Aldrich syndrome are corrected by oncoretroviral vector-mediated gene 
transfer into repopulating hematopoietic cells." Blood 102(9): 3108-16. 
Suzuki, T., H. Shen, et al. (2002). "New genes involved in cancer identified by retroviral 
tagging." Nat Genet 32(1): 166-74. 
Traggiai, E., L. Chicha, et al. (2004). "Development of a human adaptive immune system in cord 
blood cell-transplanted mice." Science 304(5667): 104-7. 
Trifari, S., G. Sitia, et al. (2006). "Defective Th1 cytokine gene transcription in CD4+ and CD8+ 
T cells from Wiskott-Aldrich syndrome patients." J Immunol 177(10): 7451-61. 
Uren, A. G., J. Kool, et al. (2005). "Retroviral insertional mutagenesis: past, present and future." 
Oncogene 24(52): 7656-72. 
Vettenranta K, Möttönen M. et al. (2012). "The use of plerixafor in harvesting autologous stem 
cells in the pediatric setting." Pediatr Blood Cancer Jul 15;59(1):197-8. 
Vigna, E. and L. Naldini (2000). "Lentiviral vectors: excellent tools for experimental gene 
transfer and promising candidates for gene therapy." J Gene Med 2(5): 308-16. 
Villa, A., L. Notarangelo, et al. (1995). "X-linked thrombocytopenia and Wiskott-Aldrich 
syndrome are allelic diseases with mutations in the WASP gene." Nature Genet. 9: 414-417. 
von Kalle, C., B. Fehse, et al. (2004). "Stem cell clonality and genotoxicity in hematopoietic 
cells: gene activation side effects should be avoidable." Semin Hematol. 41(4): 303-18. 
Vose JM, Ho AD, Coiffier B, et al (2009) Advances in mobilization for the optimization of 
autologous stem cell transplantation. Leuk Lymphoma. 14:1-10. 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 109 of 119 
-- 
 
 
 
 
Wada, T., S. H. Schurman, et al. (2001). "Somatic mosaicism in Wiskott--Aldrich syndrome 
suggests in vivo reversion by a DNA slippage mechanism." Proc Natl Acad Sci U S A 98(15): 
8697-702. 
Westerberg, L. S., M. A. de la Fuente, et al. (2008). "WASP confers selective advantage for 
specific hematopoietic cell populations and serves a unique role in marginal zone B-cell 
homeostasis and function." Blood 112(10): 4139-47. 
Will, E., J. Bailey, et al. (2007). "Importance of murine study design for testing toxicity of 
retroviral vectors in support of phase I trials." Mol Ther 15(4): 782-91. 
Williams, D. A. (2006). "Gene therapy advances but struggles to interpret safety data in small 
animal models." Mol Ther 13(6): 1027-8. 
Woods, N. B., V. Bottero, et al. (2006). "Gene therapy: therapeutic gene causing lymphoma." 
Nature 440(7088): 1123. 
Wu, X., Y. Li, et al. (2003). "Transcription start regions in the human genome are favored targets 
for MLV integration." Science 300(5626): 1749-51 
Yamaguchi, K., T. Ariga, et al. (2002). "Mixed chimera status of 12 patients with Wiskott- 
Aldrich syndrome (WAS) after hematopoietic stem cell transplantation: evaluation by flow 
cytometric analysis of intracellular WAS protein expression." Blood 100(4): 1208-14. 
Yannaki E., Karponi G., et al. (2013). "Hematopoietic stem cell mobilization for gene therapy: 
superior mobilization by the combination of granulocyte-colony stimulating factor plus 
plerixafor in patients with β-thalassemia major." Hum Gene Ther. Oct; 24(10): 852-60. 
Zhu, Q., C. Watanabe, et al. (1997). "Wiskott-Aldrich Syndrome/X-linked thrombocytopenia: 
WASP gene mutations, protein expression and phenotype." Blood 90: 2680-2689. 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 110 of 119 
-- 
 
 
 
 
16. 
APPENDICES 
16.1. 
Appendix 1 Summary of Significant Changes Introduced by Protocol 
Amendment Version 9 
The following changes were made to the protocol: 
1. Page 1. Version number, description of the revision and date added. 
2. Page 3. Rationale for amendment updated as follows: 
The purpose of the current amendment is to extend the follow up period post-gene therapy. This 
period is referred to as Extended Long-Term Follow Up. The overall objective of this change is 
to allow for the collection of long-term safety and selected efficacy data in support of a 
marketing authorisation application for the gene therapy treatment. 
In addition, a new assessment on platelet activation and morphology has been added to the 
schedule of assessments. These assays have recently been developed and validated at SR-Tiget, 
hence they can now be used within this clinical trial. Abnormal platelet function and morphology 
are common features in WAS patients, hence it is important to understand whether platelets are 
able to return to a more normal phenotype after gene therapy. 
In addition, bruising has been removed from the secondary objectives, CD61+ has been removed 
from the bone marrow analysis after gene therapy for the evaluation of the presence of 
genetically modified cells, and a small number of typographical errors have been corrected. 
A list of all changes contained in this amendment is included in Appendix 1. 
3. Page 3. Bruising removed from secondary objectives in protocol synopsis. Bruising is not 
recorded as a separate item in the CRF. Any severe bruising that is present will be recorded 
as an AE. 
In addition, based upon observations within the programme, data collection about bruising 
occurrences prior to screening has been identified as being subject to recall bias, due to the high 
frequency of bruising occurring in WAS patients. 
Wording has also been updated throughout the rest of the protocol synopsis and in Section 3.2 , 
Section 4.3.2 and Section 9.6 in order to be consistent with this update. 
4. Page 4. Exploratory efficacy endpoint added to protocol synopsis as follows: 
1) Normalisation of platelet activation profile and morphology 
In order to be consistent with this update, wording has also been updated throughout the rest of 
the protocol synopsis and additional wording added in Section 4.3.3 as follows: 
4.3.3. Exploratory efficacy endpoint 
a) 
Platelet activation profile and morphology 
Platelet activation after stimulation with agonists will be assessed after ≥3y post gene therapy by 
measuring the expression of activation markers on the cell surface by FACS analysis. Platelet 
morphology will be assessed at the same time using transmission electron microscopy. 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 111 of 119 
-- 
 
 
 
 
In order to be consistent with this update, wording has also been added to Section Section 7.2 as 
follows: 
m) 
Platelet activation and morphology: For platelet activation, the expression of activation 
markers on the surface of peripheral blood-derived platelets after stimulation with agonists will 
be assessed. For platelet morphology, the platelet perimeter, area and ultrastructure will be 
assessed by transmission electron microscopy. 
Again, in order to be consistent with this update, wording has also been added in Section 9.10 as 
follows: 
9.10. Platelet activation profile and morphology (exploratory endpoint) 
Activation of platelets will be assessed after stimulation with different concentrations of the 
platelet activation agonists ADP and thrombin. 
Assessment parameters: platelet activation will be assessed by cytofluorimetric measurement of 
surface activation markers P-selectin and GPIIbIIIa after stimulation with various 
concentrations of ADP and thrombin. The dose response curves will be compared with healthy 
controls. 
Platelet morphology will be assessed by transmission electron microscopy 
Assessment parameters: platelet perimeter, area and ultrastructure. Morphology will be 
compared with healthy controls. 
In addition, Table 8a and Table 8b have been updated accordingly. 
5. Page 6. The number of protocol phases has been updated from four to five and a new fifth 
phase has been added as follows: 
5) Extended Long-Term Follow Up phase: after completion of 8 years of follow up after gene 
therapy, patients will be contacted annually in order to collect long-term safety and selected 
efficacy data, until a registry is available and patients have been enrolled in it. 
In order to be consistent with this update, wording has also been updated throughout the rest of 
the protocol synopsis and in Section 4, Section 4.1.2, Section 5.2 and Section 8.4 . 
In addition, wording has been added to Section Section 7.3 as follows: 
7.3. 
Extended Long-Term Follow Up 
Both European (EMEA/CHMP/GTWP/60436/2007) and US regulatory agencies (Food and 
Drug Administration (FDA)) have expectations for the long-term follow up of patients who have 
been treated using gene therapy. 
After completion of a total of 8 years of follow up from the initial infusion of transduced stem 
cells, patients will be contacted annually (+/- 3 months) by telephone, email or letter in an effort 
to collect long-term safety and selected efficacy data, until a registry is available and patients 
have enrolled in it (see Table 8c). This part of the study is known as Extended Long-Term Follow 
Up (ELTFU). Should a patient decide not to consent to participate in the ELTFU nor transition 
to the registry, then the subject will be considered to have completed the protocol and will be 
transitioned to standard of care according to local practice. 
In addition, Table 8c has been added to Section 7.3 as follows: 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 112 of 119 
-- 
 
 
 
 
Table 8c – Extended Long-Term Follow Up evaluation 
 
Parameter 
Year 9+ p.a. (+/- 3 
months) 
Social Life 
Is the patient living in a protected environment? 
Is the patient attending kindergarten/school or in employment 
(according to age)? - Please comment. 
How is the patient’s social ability with peers (if applicable for age)? - If 
abnormal please specify. 
Does the patient practice any sport (if applicable for age)? - Please 
comment. 
X 
General 
Number of platelet infusions per year 
Complete Blood Count (CBC) including Platelet count/MPV 
X 
Specialist laboratory assessments 
Integration Site Analysis (ISA) 
X 
Safety* 
SAEs 
AEs of Special Interest (such as bleeds, infections, autoimmunity, 
eczema, suspected malignancies) 
X 
Ig usage** 
Intravenous Ig 
Subcutaneous Ig 
X 
* SAE and AE’s of special interest will be forwarded to TIGET as per current practice by the physician overseeing 
the patient in their home country. This includes the event, labs, and any other relevant data. In addition 
patients/family may also provide such information directly to TIGET. All SAEs, and any suspected malignancies, 
should be reported to Sponsor within 24 hours of awareness. Other AEs of Special Interest should be entered into 
the eCRF within 30 days of awareness. 
** Frequency to be specified if possible. 
In addition, the trial flow chart has been updated (see 7 below). 
6. Page 8. The end of the study has been updated as follows: 
The end of the study is when all subjects have completed a minimum of 8 years follow up and 
transitioned to a registry. Should a patient decide not to consent to participate in the ELTFU nor 
transition to a registry, then the subject will be considered to have completed the protocol and 
will be transitioned to standard of care according to local practice. 
In order to be consistent with this update, wording has also been updated in Section 5.1.4.1 , 
Section Section 12.2 and Section Section 12.7 . 
7. Page 10. The trial flow chart has been updated as follows: 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 113 of 119 
-- 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
In order to be consistent with this update, this has also been updated in Figure 1 in Section 5.2 . 
8. Page 52. Data and reference from a previous clinical trial has been updated in Section 2.8.1 , 
with the removal of previous out of date references, as follows: 
As of January 2014, 7 out of the 10 treated patients developed acute leukemia (one acute 
myeloid leukemia (AML), four T cell acute lymphoblastic leukemia (T-ALL), and two primary T- 
ALL with secondary AML, with all six cases of T-ALL associated with a dominant clone with 
vector integration at the LMO2) (Braun et al, Sci Trans Med 2014). 
The bibliography has been updated accordingly. 
9. Page 77. Wording has been updated for the bone marrow evaluation after gene therapy in 
Section 7.2 as follows: 
PCR analysis on transduced cells will be performed separately, including the assessment of 
%LV+ colony-forming cells (CFC) from patient’s BM-derived clonogenic progenitors. 
10. Page 78. CD61+ has been removed from the bone marrow analysis after gene therapy for the 
evaluation of the presence of genetically modified cells in Section 7.2 . 
This has been removed due to the identification of technical issues related to the purification of 
the CD61+ cell fraction from patients’ bone marrow. 
Enrolment 
Screening phase 
Baseline 
PBSC mobilisation 
followed by 
leukapheresis for 
HSC collection for 
back up and DP 
manufacture (if 
required) or BM 
harvest, 
chemotherapy, gene 
therapy schedule 
Evaluation of safety 
and efficacy, from 
+1 day to 8 years 
Treatment 
Follow-up 
Informed consent 
signature 
Preliminary 
evaluation and 
inclusion/exclusion 
criteria assessment 
Clinical laboratory 
and instrumental 
evaluation 
BM Back-up 
collection and 
storage (for patients not 
undergoing PBSC mobilization) 
Evaluation of safety 
and efficacy, from 
8 years, up until a 
registry is available 
and patients have 
been enrolled in it 
Extended Long- 
Term Follow Up 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 114 of 119 
-- 
 
 
 
 
In order to be consistent with this update, this has also been updated in Section 4.3.2 , 
Section 9.1 and Section 12.4 . 
11. Page 82. Wording has been updated for reporting of SAEs to GSK in Section 8.2.1 as 
follows: 
SR-TIGET should inform GSK of the following events within three days of gaining awareness: 
• 
Positive result from replication competent lentivirus (RCL) testing 
• 
Test results suggesting abnormal clonal proliferation 
Events meeting the Hy’s law criteria (ALT ≥ 3xULN and bilirubin ≥ 2xULN (>35% direct 
bilirubin) or ALT ≥ 3xULN and INR>1.5, if INR measured) will be reported as SAEs. 
Patients with liver function abnormalities should be monitored until liver chemistries resolve, 
stabilise or return within baseline. Tests to define the aetiology of the abnormal liver function 
tests should be conducted as relevant. 
12. Page 97. Wording has been updated for study population and timing of analysis in 
Section 12.2 as follows: 
When all the treated patients complete the 8-year follow up visit after gene therapy, a statistical 
analysis will be performed with all data available and a clinical study report will be compiled 
accordingly. 
At the end of the study, a final statistical analysis will be performed with all data available and a 
final clinical study report will be compiled. 
13. Page 101. Wording has been updated for final study report in Section 14.3 as follows: 
The sponsor will be responsible for preparing formal interim clinical study reports and the final 
clinical study report. 
14. Minor typographical and table numbering errors have been corrected throughout. 
 
16.2. 
Appendix 2 Summary of Significant changes introduced by Protocol 
Amendment Version 8 
The following changes were made to the protocol: 
1. Page 1. TIGET logo removed. 
2. Page 1. GSK study number added 
3. Page 1. GSK compound number added 
4. Page 1. Version date amended and Protocol Amendment number added. 
5. Page 2. Rationale for amendment added as follows: 
Protocol Amendment Version 8: Rationale 
In November 2013, GSK in-licenced GSK2696275 (previously described as Telethon 003) from 
Fondazione Telethon and SR-TIGET. While SR-TIGET currently remained the regulatory 
sponsor of the clinical study protocol at this time, GSK replaced Fondazione Telethon as the 
Financial Sponsor. 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 115 of 119 
-- 
 
 
 
 
The purpose of the current protocol amendment is to formally register GSK as the new 
regulatory sponsor for the clinical trial and align data management and serious adverse event 
reporting methods with company processes. Where appropriate background information has 
been updated to reflect this change. 
The overall objective of the changes described are to allow the existing all clinical data and to 
be collated by GlaxoSmithKline R&D in support of a marketing application for the gene therapy 
treatment. 
A list of all changes contained in this amendment is included in Appendix 1 . 
1. Protocol Synopsis moved into cover page section (pages 3-7) 
2. Page 9: Medical monitor/Sponsor Information Page added. 
Of note, Medical Monitor details remain unchanged from the prior amendment and version. A 
new GSK SAE contact has been added given GSK’s role as regulatory sponsor. 
3. Page 10: Sponsor Legal Registered Address has been added. 
4. Page 11: GSK Sponsor signatory page has been added. 
5. Page 12: Investigator Protocol Agreement Page added 
6. Section 1. Background, the following text was removed: 
The promoter of the study is Ospedale San Raffaele. The Ospedale San Raffaele is recognized by 
the Italian Ministry of Health as a Scientific Institute, carrying out biomedical research and 
clinical activities of relevant national interest (IRCCS). 
Fondazione Telethon is a major italian non-profit foundation responsible for raising and 
distributing funds for biomedical research in Italy, primarily in universities, public and non- 
profit research institutes. Telethon’s mission is to advance research toward the cure of muscular 
dystrophies and other rare genetic diseases. 
The San Raffaele-Telethon Institute for Gene Therapy of Genetic Diseases (SR-TIGET) was 
established a joint venture between Fondazione San Raffaele and Fondazione Telethon for the 
implementation of basic and clinical research on gene therapy for genetic diseases. The mission 
of the Institute is to perform cutting edge science in the field of gene and cellular therapy and to 
promote the translation of basic discoveries into therapeutic advances. 
7. The following text from Section 4.1.1 has been removed from the protocol. 
Section 4.1.1. Type of Study 
Principal investigators: Alessandro Aiuti, MD, PhD. 
Treatment Center: Paediatric Clinical Research Unit, SR-TIGET/Paediatric Immunohematology 
and Bone Marrow Transplantation Unit-OSR. 
Personnel: physicians, nurses and paramedical staff working in the Paediatric 
Immunohematology and Bone Marrow Transplantation Unit-OSR are fully qualified to assist 
patients affected by critical haematological diseases, such as WAS, and to conduct this clinical 
trial in accordance with applicable laws and regulations, as well as recognized good clinical 
practice. 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 116 of 119 
-- 
 
 
 
 
8. Global Changes made to protocol include: Study promoter changed from ‘Ospedale San 
Raffaele’ to ‘Sponsor’ (which is GSK). Where ‘HSR-TIGET’appeared in text, the 
abbreviation was changed to ‘SR-TIGET.’ 
9. Page 75: 8.1.3 . or Serious Adverse Reaction (SAR) removed from heading: the definition 
for SAR is not included in the text section. 
10. Page 76: Section 8.2 . Pharmacovigilance Procedure 
The following text has been removed from the protocol: 
8.2.1. Serious Adverse Events (SAEs): SAEs will be reported according to the law and 
regulations in force. 
The following text replaces section 8.2.1: 
8.2.1 Reporting of Serious Adverse Events (SAEs) to GSK 
The Investigator shall report all SAEs to GSK within 24 hours after becoming aware of the SAE. 
SAEs should be reported to GSK using the paper SAE reporting form. After completion, the form 
should be scanned and emailed to the SAE contacts (Medical Monitor and GSK Italy 
pharmacovigilance) found at the beginning of the protocol on the Sponsor/Medical Monitor 
Contact Information page. 
In rare circumstances if email communication is not available, notification by telephone is 
acceptable, with a copy of the SAE form sent by overnight mail. 
The following is added to the protocol: 
8.2.2. Regulatory Reporting Requirements for SAEs 
Prompt notification by the Investigator to GSK of SAEs related to study treatment (even for non- 
interventional post-marketing studies) is essential so that legal obligations and ethical 
responsibilities towards the safety of subjects and the safety of a product under clinical 
investigation are met. 
GSK has a legal responsibility to notify both the local regulatory authority and other regulatory 
agencies about the safety of a product under clinical investigation. GSK will comply with 
country specific regulatory requirements relating to safety reporting to the regulatory authority, 
Institutional Review Board (IRB)/Independent Ethics Committee (IEC) and investigators. 
Investigator safety reports are prepared for suspected unexpected serious adverse reactions 
according to local regulatory requirements and GSK policy and are forwarded to Investigators 
as necessary. 
An Investigator who receives an investigator safety report describing a SAE(s) or other specific 
safety information (e.g., summary or listing of SAEs) from GSK will file it with the IB and will 
notify the IRB/IEC, if appropriate according to local requirements. 
The following text has been removed from the protocol: 
8.2.3. Suspected Unexpected Serious Adverse Reaction (SUSAR) I n case of SAR that is 
unexpected, the Investigator/Sponsor must report the reaction as soon as possible (and in any 
case no later than 7 days for reactions that are fatal or life-threatening, and no later than 15 
days for all other SUSARs) to the competent Regulatory Authorities and to the competent Ethics 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 117 of 119 
-- 
 
 
 
 
Committee (and that relevant follow up information is subsequently communicated within an 
additional eight days). 
8.2.4. Periodic reporting Once a year throughout the trial, the Investigator should provide to 
Competent Regulatory Authorities and the Ethics Committee an Annual Safety Report, that is a 
listing of all suspected SAR, which have occurred over this period and a report of the subjects’ 
safety. 
11. Page 86: Section 10.2 . Documentation of data in the case report form (CRF) 
The following wording has been removed from the protocol: 
It is the responsibility of the Investigator to maintain adequate and accurate CRFs to record all 
observations and other data pertinent to the clinical investigation. All CRFs should be completed 
in their entirety in a neat, legible manner to ensure accurate interpretation of data; a black ball 
point pen should be used to ensure the clarity of reproduced copy of all CRFs. Should a 
correction be made, the information to be modified must not be overwritten. The corrected 
information will be transcribed by the authorized person next to the previous value and dated. 
The following wording has been added to the protocol: 
It is the responsibility of the Investigator to maintain adequate and accurate CRFs to record all 
observations and other data pertinent to the clinical investigation. 
Under TIGET sponsorship paper CRFs will be utilized; these should be completed in their 
entirety in a neat, legible manner to ensure accurate interpretation of data; a black ball point 
pen should be used to ensure the clarity of reproduced copy of all CRFs. Should a correction be 
made, the information to be modified must not be overwritten. The corrected information will be 
transcribed by the authorized person next to the previous value and dated. 
Under GSK sponsorship an electronic CRF will be implemented; all data previously collected on 
paper CRF and all data collected following sponsorship transfer will be entered into GSK 
defined CRFs. All these data will transmitted electronically to GSK or designee and combined 
with data provided from other sources in a validated data system. 
Management of clinical data will be performed in accordance with applicable GSK standards 
and data cleaning procedures to ensure the integrity of the data, e.g., removing errors and 
inconsistencies in the data. 
Adverse events and concomitant medications terms will be coded using MedDRA (Medical 
Dictionary for Regulatory Activities) and an internal validated medication dictionary, GSKDrug. 
CRFs (including queries and audit trails) will be retained by GSK, and copies will be sent to the 
investigator to maintain as the investigator copy. Subject initials will not be collected or 
transmitted to GSK according to GSK policy. 

Protocol version 12, 19 Sep 2023  
 
Fondazione Telethon ETS – 
CONFIDENTIAL 
Page 118 of 119 
-- 
 
 
 
 
16.3. 
Appendix 3: Summary of Significant Changes Introduced by Protocol 
Amendment Version 10 
A list of all changes contained in this amendment are included below: 
1. Date and protocol amendment number has been changed 
2. Medical Monitor/Sponsor Information has been changed. An Orchard Therapeutics Medical 
Monitor and PPD SAE contact have been added given Orchard Therapeutics role as 
regulatory sponsor. 
3. Sponsor Legal Registered Address has been added 
4. Orchard Therapeutics Sponsor signatory page has been added 
5. Orchard Therapeutics investigator protocol agreement page has been added 
6. PPD has been added to the abbreviation/acronym list 
7. Global changes made to protocol include: 
a. The acronym GSK has been changed to Orchard Therapeutics as regulatory sponsor. 
b. For the reporting of SAEs section, the acronym GSK has been changed to PPD. 
c. For the section on collection of pregnancy information, the acronym GSK has been 
changed to PPD. 
d. The protocol header number has been removed and name has been changed to Orchard 
Therapeutics. 

-- 
 
 
16.4. 
Appendix 4: Summary of Significant Changes Introduced by 
Protocol Amendments Version 11 and 11.1 
A list of significant changes contained in version 11 are listed below. 
Throughout: text has been amended to allow all subjects to enter a separate observational long-term 
follow-up (OLTFU) study once it is set up and subjects been followed up for a minimum of 5 years, not 
just after subjects enter the (extended long-term follow-up) ELTFU phase. As part of the OLTFU study, 
patients will continue to be followed up for a total of 15 years from the date of treatment with gene 
therapy. 
Throughout: text has been added to allow patients to attend remote visits (upon agreement by PI, 
medical monitor and sponsor,) and text has been added to describe these remote visits. Text has also been 
added to allow informed consent (performed by the PI or a person designated by the PI) to take place 
remotely. 
Throughout: the severity assessments used for clinical events including bleeding, infections and eczema 
have been clarified upon FDA feedback. 
8.4.1. Replication competent lentivirus (RCL): the strategy for RCL testing was simplified. In the 
previous version of the protocol, 3 assays (ELISA for HIV p24 antigen on serum, DNA PCR for VSV-G 
envelope on cells, and RT-PCR for HIV-pol RNA on serum) were used for first line detection of RCL. In 
the amended text, one test is performed in the first instance (HIV p24 antigen detection in the serum by 
ELISA), in line with EMA guideline CHMP/BWP/2458/03, which defines the best assays for the 
determination of RCL. Detection of HIV p24 antigen in serum by ELISA is identified as a validated and 
sensitive test, which is also routinely used in the clinic to determine HIV positivity in case of suspected 
viral infection by the wild type lentivirus. Should this test be positive, further analyses will be performed, 
including the two remaining tests mentioned above (DNA PCR for VSV-G envelope on cells, and RT- 
PCR for HIV-pol RNA on serum). If a positive result is obtained from at least one of the additional tests, 
an appropriate confirmatory test may be performed. This change is required since some of the 
commercially available tests to detect RNA-Pol HIV (e.g. Roche COBAS Ampliprep/COBAS Taqman 
HIV-1 v2.0 Test), are unable to differentiate between the DNA of gene corrected cells and HIV infection, 
potentially leading to false positive results. 
Minor readability and typographical errors were corrected in the text 
The following changes are contained in version 11.1: 
Medical Monitor/ Sponsor information page: Contact details for the Medical Monitor and for 
pharmacovigilance SAE reporting have been updated to reflect changes in personnel and vendor. 
Throughout: PPD (former pharmacovigilance vendor) was replaced by Sponsor/ Sponsor’s 
pharmacovigilance designee. 
Table 8c: The mention of ‘special interest’ was removed from the definition of events such as bleeds, 
infections, autoimmunity, eczema and suspected malignancies, which are disease related and do not meet the 
criteria for events of special interest in terms for PV safety analysis and reporting. 
 
16.5. 
 Appendix 5: Summary of Significant Changes Introduced by 
Protocol Amendments Version 12 
 
A list of significant changes contained in version 12 are listed below. 
a. 
the name of the previous Sponsor (Orchard Therapeutics) has been replaced with the name of the new 
Sponsor (Fondazione Telethon ETS) in the text. 
 
Fondazione Telethon ETS – CONFIDENTIAL 
Page 119 of 119