A Phase 1/2 Open Label Study Evaluating the Safety and Efficacy of Gene Therapy in Subjects With β-Thalassemia Major by Transplantation of Autologous CD34+ Cells Transduced Ex Vivo With a Lentiviral βA-T87Q-Globin Vector (LentiGlobin® BB305 Drug Product)
Phase: PHASE1
Conditions: β-thalassemia Major
Sponsor: Genetix Biotherapeutics Inc.

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Protocol HGB-204 Version 5.0 
Page 3 of 67 
29 June 2015  
 
bluebird bio, Inc. 
 
Section of 
current 
document 
Rationale 
Change  
3M provides only data on short-term 
progenitors, and has no value for 
predicting integration patterns in long-
term progenitors  
Section 6.2.7 
Provided more guidance on medical 
history to be collected, especially on 
past transfusion parameters 
The medical history is to include all prior and 
current medical history, including age of 
first transfusion, age of starting regular 
transfusions (i.e., age at which transfusion 
needs stabilized to between once every 4 to 
8 weeks), age of starting iron chelation 
treatment, and spleen size (cm below 
coastal margin), and additional 
transfusion details  at enrollment.  
Section 4.5, 
Section 6.2.22.1 
Clarified AEs follow-up requirements 
during study and after discontinuation 
Section 4.5: Clarified follow-up rules for 
subjects who withdraw, either before or after 
drug product treatment 
Section 6.2.21.1: Removed rules for follow-
up of AEs after discontinuation from study, 
and cross referenced Section 4.5. 
Section 6.2.21 
Simplified the presentation of AE 
information for improved readability  
All sections on AEs included as subheadings 
in a single section 6.2.21 
Appendix 10.2, 
Section 5.2.1 
Increased experience with apheresis of 
subjects with β-thalassemia major has 
led to more detailed advice on 
mobilization and apheresis procedures. 
Updated with current recommendations for 
hemoglobin levels during mobilization and 
apheresis, and updated general 
recommendations for cell collection by 
apheresis 
 
In addition, several minor changes were made to correct typographical errors, and to improve 
clarity. 

Protocol HGB-204 Version 5.0 
Page 4 of 67 
29 June 2015  
 
bluebird bio, Inc. 
 
CLINICAL STUDY PROTOCOL SYNOPSIS 
Protocol Title: 
A Phase 1/2, Open Label Study Evaluating the Safety and Efficacy of Gene Therapy 
in Subjects with -Thalassemia Major by Transplantation of Autologous CD34+ 
Stem Cells Transduced Ex Vivo with a Lentiviral A-T87Q-Globin Vector 
(LentiGlobin BB305 Drug Product) 
Protocol Number: 
HGB-204 
Objectives: 
 Evaluate the safety of treatment with LentiGlobin BB305 Drug Product in subjects 
with β-thalassemia major 
 Evaluate the efficacy of treatment with LentiGlobin BB305 Drug Product in 
subjects with β-thalassemia major 
Study Design: 
This is a non-randomized, open label, multi-site, single dose, Phase 1/2 study in up 
to 18 subjects (including at least 3 adolescents ≥ 12 < 18 years of age) with 
β-thalassemia major who receive at least 100 mL/kg/year of packed red blood cells 
(pRBCs) or ≥8 transfusions of pRBCs per year in each of the 2 years preceding 
enrollment. The study will evaluate the safety and efficacy of autologous 
hematopoietic stem cell (HSC) transplantation (HSCT) using LentiGlobin BB305 
Drug Product (autologous CD34+ HSCs transduced with LentiGlobin BB305 
lentiviral vector encoding the human βA-T87Q-globin gene and resuspended in 
cryopreservative solution in the final immediate container for the intended medical 
use).  
Initially, treatment will be staggered. The second subject will begin myeloablative 
conditioning only after the first subject 1) engrafts (defined as an absolute neutrophil 
count [ANC] ≥ 0.5 × 109/L for 3 consecutive days); and 2) has no LentiGlobin 
BB305 Drug Product treatment-related serious adverse event (SAE) unexpected to 
occur with autologous HSCT. After Subject 2 meets these same criteria, parallel 
drug product treatment can occur with additional subjects.  
The study has 4 distinct stages, as follows. 
Stage 1:Screening to determine eligibility 
Stage 2: Autologous CD34+ cell collection, LentiGlobin BB305 Drug Product 
manufacture and disposition  
Within the period of 30 days prior to HSC collection, the subject’s transfusion 
regimen may be adjusted to maintain a minimum of 10 g/dL of hemoglobin (Hb) in 
order to suppress dyserythropoiesis, which can impede the isolation of CD34+ cells 
enriched for undifferentiated HSC. 
Each subject will undergo HSC mobilization with filgrastim and plerixafor. 
Peripheral blood mononuclear cells (PBMCs) will be collected by apheresis using 
standard methods. A total of 2 mobilization cycles may be performed if needed. 
Each mobilization cycle may include up to 5 apheresis procedure days, but only 2 
consecutive apheresis procedure products may be sent for transduction from any 
mobilization cycle. The other apheresis procedure products should be used for 
rescue cells, if they meet minimum requirements as a rescue product. If 
2 mobilization cycles are needed to collect sufficient HSCs to meet the requirement 
of a total dose of ≥ 3.0 × 106 CD34+ cells/kg, then 2 transductions will be 
performed: one on the cells collected during Mobilization Cycle 1, and one on the 
cells collected during Mobilization Cycle 2. Each transduced product from each 
Mobilization Cycles 1 and 2 is an independent drug product. Thus, subjects who 
require 2 mobilization cycles to produce the minimum cell dose will be treated with 
2 drug products in total. Mobilization cycles must be separated by at least 2 weeks. 
A bone marrow harvest is also allowed, but only to procure cells for rescue.  
The harvested cells to be used for transduction will be selected for the CD34+ 
marker to enrich for HSCs, transduced with LentiGlobin BB305 Lentiviral Vector, 
and stored under the vapor phase of liquid nitrogen while testing is ongoing. 

Protocol HGB-204 Version 5.0 
Page 5 of 67 
29 June 2015  
 
bluebird bio, Inc. 
 
Stage 3: Myeloablative conditioning and infusion of LentiGlobin BB305 Drug 
Product 
After the transduced cells are dispositioned for clinical use, the subject will undergo 
myeloablative conditioning with busulfan. Busulfan will be administered 
intravenously (IV) at a starting dose of 3.2 mg/kg/day or 0.8 mg/kg every 6 hours 
for 4 consecutive days. The dose of busulfan will be adjusted based upon first dose 
busulfan pharmacokinetics in order to maintain appropriate levels for myeloablation 
(area under the curve [AUC] goal of 1000 [range 900 to 1200] µM*min for an every 
6 hours [q6h] dosing regimen, or 4000 [range 3600 to 5000] µM*min for a once 
daily [qd] dosing regimen). Clinical sites that use a test dose of busulfan several 
days before beginning myeloablation to pre-determine busulfan dose may also do so 
in this protocol. After completion of the 4-day course of busulfan, there must be a 
minimum of 72 hours of busulfan washout before drug product infusion.   
On Day 0, after thawing, the LentiGlobin BB305 Drug Product(s) will be 
administered via IV infusion at a dose of ≥ 3.0 × 106 CD34+ cells/kg.  Subjects who 
undergo 2 mobilizations (and subsequent transduction of those cells) to achieve a 
total dose of ≥ 3.0 × 106 CD34+ cells/kg will have 2 LentiGlobin BB305 Drug 
Products, which should be administered in sequence, with the second administered 
immediately after the first. 
Stage 4: Follow-up, through engraftment and 24 months after drug product infusion 
Subjects will be followed daily in the transplant unit for adverse events (AEs), and 
laboratory parameters will be followed to monitor bone marrow engraftment. The 
subject may be discharged from the transplant unit once: 
1) engraftment occurs (defined as an ANC ≥ 0.5 × 109/L for 3 consecutive days); 
and 
2) the subject is considered medically stable.  
After discharge, subjects will be followed in this protocol for a minimum of 
24 months after LentiGlobin BB305 Drug Product infusion.  
Data Monitoring 
Committee:  
A Data Monitoring Committee (DMC) comprised of members with appropriate 
scientific and medical expertise to monitor the study will be convened before the 
study is opened.  
The DMC will be charged with review of all unexpected (i.e., unexpected in nature 
and / or in severity) LentiGlobin BB305 Drug Product treatment-related adverse 
events, following notification by the Sponsor. 
The DMC may recommend that the Sponsor stop the study at any time due to 
concerns for the safety of the subjects.  
Number of Subjects 
Planned: 
Up to 18 subjects, including at least 3 adolescents (≥ 12 < 18 years of age) will be 
treated with drug product. Replacement subjects may be added if subjects withdraw 
prior to conditioning.  
Inclusion Criteria: 
1. Subjects between 12 and 35 years of age, inclusive, at the time of consent or 
assent (as applicable), and able to provide written consent (adults, or legal 
guardians, as applicable) or assent (adolescents). 
2. Diagnosis of β-thalassemia major and a history of at least 100 mL/kg/year of 
pRBCs or ≥8 transfusions of pRBCs per year for the prior 2 years. 
3. Documented baseline, or pretransfusion, Hb level ≤7 g/dL. 
4. Clinically stable, have a Karnofsky performance status of ≥ 60, and eligible to 
undergo HSCT. 
5. Treated and followed for at least the past 2 years in a specialized center that 
maintained detailed medical records, including transfusion history.  
Exclusion Criteria: 
1. Positive for presence of human immunodeficiency virus type 1 or 2 (HIV-1 or 
HIV-2), hepatitis B virus (HBV), or hepatitis C virus (HCV). (Note that subjects 

Protocol HGB-204 Version 5.0 
Page 6 of 67 
29 June 2015  
 
bluebird bio, Inc. 
 
who are positive for anti-HBV antibody [to either core or envelope proteins] or 
for anti-HCV antibody are eligible as long as they have a negative HBV or HCV 
viral load by quantitative polymerase chain reaction [qPCR]). Where clinically 
and/or regionally indicated, one or more of the following tests may be performed, 
in which case positive results would exclude the subject from participating: 
human T-lymphotrophic virus-1 (HTLV-1) or HTLV-2, syphilis (RPR), 
toxoplasmosis, Trypanosoma cruzi, or West Nile Virus. 
2. Active bacterial, viral, fungal, or parasitic infection. 
3. A white blood cell (WBC) count < 3 × 109/L, and / or platelet 
count < 100 × 109/L if not due to hypersplenism. 
4. Uncorrected bleeding disorder. 
5. Any prior or current malignancy or myeloproliferative or immunodeficiency 
disorder 
6. Immediate family member with a known or suspected Familial Cancer Syndrome 
(including but not limited to hereditary breast and ovarian cancer syndrome, 
hereditary non-polyposis colorectal cancer syndrome and familial adenomatous 
polyposis). 
7. Prior HSCT. 
8. Advanced liver disease, defined as: 
a. 
Baseline alanine transaminase or direct bilirubin value >3 × the upper limit of 
normal (ULN), or 
b. Liver biopsy demonstrating cirrhosis, any evidence of bridging fibrosis, or 
active hepatitis. 
9. Baseline estimated glomerular filtration rate (eGFR) < 70 mL/min/1.73 m2, as 
determined using the Chronic Kidney Disease Epidemiology Collaboration 
(CKD-EPI) creatinine equation) for ≥ 18 years of age, and Bedside Schwartz 
equation calculator for < 18 years of age. (see 
http://www.kidney.org/professionals/kdoqi/gfr_calculator.cfm 
10. Uncontrolled seizure disorder. 
11. Diffusion capacity of carbon monoxide (DLco) < 50% of predicted (corrected for 
hemoglobin and/or alveolar volume, as clinically indicated). 
12. A cardiac T2* < 10 ms by magnetic resonance imaging (MRI).  
13. Any other evidence of severe iron overload that, in the investigator’s opinion, 
warrants exclusion. 
14. Clinically significant pulmonary hypertension, as defined by the requirement for 
ongoing pharmacologic treatment or the consistent or intermittent use of 
supplemental home oxygen. 
15. Participation in another clinical study with an investigational drug within 30 days 
of Screening. 
16. Failure to obtain appropriate informed consent / assent. 
17. Any other condition that would render the subject ineligible for HSCT, as 
determined by the attending transplant physician or investigator 
18. Contraindications to the conditioning regimen. 
19. Prior receipt of gene therapy. 
20. Diagnosis of significant psychiatric disorder of the subject that could seriously 
impede the ability to participate in the study. 
21. Pregnancy or breastfeeding in a postpartum female or absence of adequate 
contraception for fertile subjects. Females of child-bearing potential are required 
to use effective contraception from Screening through at least 6 months after 
drug product infusion. Male subjects are required to use effective contraception 

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Protocol HGB-204 Version 5.0 
Page 8 of 67 
29 June 2015  
 
bluebird bio, Inc. 
 
Statistical Methods: 
The primary populations for analysis of both efficacy and safety will consist of those 
subjects who initiate any study procedures (intent-to-treat; ITT), beginning with 
mobilization, and those who undergo LentiGlobin BB305 Drug Product treatment 
(transplant population; TP), should this be a smaller number of subjects. Subjects in 
the ITT or TP population will be considered treatment failures in the primary 
endpoint analysis, should they have less than 6 months follow-up after drug product 
infusion. To evaluate the efficacy of LentiGlobin BB305 Drug Product in subjects 
who are successfully transplanted and are followed for sufficient time to evaluate 
clinical benefit, an evaluable population will be defined as those subjects who 
successfully engraft and have sufficient study visit compliance to acquire primary 
efficacy data through 24 months after drug product infusion.  
Tabulations will be produced for appropriate demographic, baseline, efficacy, and 
safety parameters. Descriptive summary statistics will be presented. For categorical 
variables, summary tabulations of the number and percentage within each category 
(with a category for missing data) of the parameter will be presented. For continuous 
variables, the mean, median, standard deviation, minimum and maximum values 
will be presented. Two-sided 90% confidence intervals will be calculated. 
By-subject listings of data for all completed and discontinued subjects will be 
provided.  
For change from baseline analyses, baseline will be defined as the value closest to, 
but prior to drug product infusion. Longitudinal data (collected serially over time on 
study and follow-up) will be presented by appropriate time intervals, such as 
monthly, quarterly and so forth, depending on the nature of the data. 
AEs will be tabulated separately for those that occur 1) after signing the informed 
consent and prior to conditioning; 2) from the start of conditioning until Day 0 
(immediately before the start of LentiGlobin BB305 Drug Product infusion); 3) from 
the start of LentiGlobin BB305 Drug Product infusion on Day 0 through 42 days 
post-infusion; 4) from the start of LentiGlobin BB305 Drug Product infusion on Day 
0 through 12 months post-infusion (≥Grade 2 AEs); and 5) from the start of 
LentiGlobin BB305 Drug Product infusion on Day 0 through the entire 24-month 
study period (SAEs and LentiGlobin BB305 Drug Product-related AEs). Safety 
parameters will be summarized for each 3-month period, and survival status, AE, 
SAEs, laboratory abnormalities (values outside of normal ranges) and insertional 
oncogenesis (insertional mutagenesis resulting in oncogenesis) events will be 
tabulated. 
For clinical events and transfusion requirements, each subject will serve as their own 
control in that 2 years of pre-drug product infusion data will be compared with 
post-infusion values. 

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Protocol HGB-204 Version 5.0 
Page 11 of 67 
29 June 2015  
 
bluebird bio, Inc. 
 
TABLE OF CONTENTS 
SUMMARY OF CHANGES .................................................................................................... 2 
CLINICAL STUDY PROTOCOL SYNOPSIS ........................................................................ 4 
LIST OF ABBREVIATIONS ................................................................................................... 9 
TABLE OF CONTENTS ........................................................................................................ 11 
LIST OF TABLES IN THE TEXT ......................................................................................... 13 
LIST OF FIGURES IN THE TEXT ....................................................................................... 13 
1 
INTRODUCTION .............................................................................................................. 14 
1.1 
Potential Risks ............................................................................................................... 16 
1.1.1. 
Oncogenesis by Insertional Mutagenesis .............................................................. 16 
1.1.2. 
Vector Integration within the HMGA2 Gene ........................................................ 17 
1.1.3. 
Use of a Lentiviral Vector Derived from HIV-1 ................................................... 17 
1.1.4. 
Other Risks ............................................................................................................ 18 
2 
STUDY OBJECTIVES AND ENDPOINTS .................................................................... 18 
2.1 
Study Objectives ............................................................................................................ 18 
2.2 
Study Endpoints ............................................................................................................. 18 
2.2.1 
Efficacy Endpoints ................................................................................................ 18 
2.2.2 
Pharmacodynamic Endpoints ................................................................................ 18 
2.2.3 
Safety Endpoints .................................................................................................... 18 
3 
INVESTIGATIONAL PLAN ............................................................................................ 19 
3.1 
Overall Design and Plan of the Study ........................................................................... 19 
3.1.1 
Data Monitoring Committee .................................................................................. 20 
3.2 
Rationale for the Study .................................................................................................. 21 
3.2.1 
History of LentiGlobin Lentiviral Vectors HPV569 and BB305 .......................... 22 
3.2.2 
Rationale for the β-globin Gene Used ................................................................... 24 
3.3 
Rationale for the Study Design ...................................................................................... 24 
3.4 
Rationale for the Dose Selected .................................................................................... 24 
3.5 
Treatment Discontinuation and Enrollment Suspension Criteria .................................. 25 
3.5.1 
Stopping Rules for Busulfan.................................................................................. 25 
3.5.2 
Enrollment Suspension Criteria ............................................................................. 25 
4 
Study Population ................................................................................................................. 26 
4.1 
Number of Subjects ....................................................................................................... 26 
4.2 
Inclusion Criteria ........................................................................................................... 26 
4.3 
Exclusion Criteria .......................................................................................................... 26 
4.4 
Subject Identification and Registration ......................................................................... 28 
4.5 
Subject Withdrawal from the Study .............................................................................. 28 
5 
Study Treatments ................................................................................................................ 29 
5.1 
Description of LentiGlobin BB305 ............................................................................... 29 
5.2 
Summary of Treatments to be Performed or Administered .......................................... 30 
5.2.1 
Mobilization and Apheresis Procedure .................................................................. 30 
5.2.2 
Bone Marrow Harvest Procedure .......................................................................... 32 
5.2.3 
Conditioning .......................................................................................................... 33 
5.2.4 
Infusion Procedures, Dose, and Administration .................................................... 33 
5.3 
Transduction Process ..................................................................................................... 34 
5.4 
Storage and Stability of LentiGlobin BB305 Drug Product .......................................... 34 
5.5 
Product Accountability .................................................................................................. 34 

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CCI

Protocol HGB-204 Version 5.0 
Page 14 of 67 
29 June 2015  
 
bluebird bio, Inc. 
 
1 
INTRODUCTION 
β-Thalassemia is a rare hereditary blood disorder found most commonly in persons of 
Mediterranean, Middle Eastern, Indian, and South Asian descent (Colah et al., 2010). Due to the 
rarity of this disease in North America, published research on the prevalence of β-thalassemia in 
the United States (US) is limited. In California, it is estimated that 1.8 in 100,000 births are 
affected by β-thalassemia (Michlitsch et al., 2009), based on the mandatory screening for 
hemoglobinopathies in this state. Adjusting from this state-wide rate and assuming the incidence 
of milder forms, it is estimated that the prevalence of β-thalassemia in the US population is 
~5500 patients. However, this is likely an over-estimate as it assumes a normal life expectancy, 
which is not the case for the majority of patients (Delea et al., 2007), and immigration from Asia 
to California has likely led to a local increase in the state. The Thalassemia Clinical Research 
Network of the National Heart, Lung, and Blood Institute developed a registry and surveyed 
non-Network sites resulting in an estimate of ~1000 patients in the US and Canada with 
β-thalassemia. In brief, it appears that the estimated prevalence of β-thalassemia in the US is 
approximately 1,000 patients.  
β-Thalassemia results from a biochemical imbalance in the monomeric globin chains of the 
hemoglobin tetramer. Normally existing at an approximate 1:1 ratio, causative mutations impair 
the production of β-globin and lead to a relative excess of α-globin. Unable to form hemoglobin 
(Hb) tetramers on their own, the excess α-globin chains become insoluble and form a precipitate 
that damages the developing red blood cell (RBC). The clinical implications of the 
α-globin/β-globin imbalance are 2-fold: 1) patients lack sufficient RBCs and Hb to effectively 
transport oxygen throughout the body; and 2) RBCs suffer an elevated rate of hemolysis that 
leads to morbidity through chronic vasculature damage. 
The clinical course of β-thalassemia correlates with the degree of globin chain imbalance and is 
most severe in patients with β-thalassemia major. These patients require >100 mL/kg/year of 
packed RBCs (pRBC) or ≥8 transfusions per year, and produce little to no β-globin. In terms of 
genotype, most patients with the β0/β0 genotype and many with the βE/β0 genotype suffer from 
β-thalassemia major. β-Thalassemia major is diagnosed in infancy, and affected patients have 
profound hypochromic, microcytic anemia, producing as little as 1 to 7 g/dL of total Hb 
(Rachmilewitz and Giardina, 2011; Olivieri, 1999). In many developing countries where 
necessary chronic blood transfusions are not readily available, children with β-thalassemia 
major have a poor prognosis and experience growth retardation, hepatosplenomegaly, and 
skeletal deformities resulting from extra-medullary hematopoiesis. Ultimately, most die in 
childhood (Adams, III and Coleman, 1990). 
Where adequate treatment is available, patients with β-thalassemia major receive chronic blood 
hypertransfusion regimens aimed at maintaining steady-state Hb levels of about 9-10 g/dL. This 
regimen consists of infusions with 3 units of pRBCs every 3 to 5 weeks, and is highly effective 
at preventing the hallmark symptoms of childhood β-thalassemia major. However, chronic 
transfusions introduce a large amount of elemental iron into the body (Cao et al., 1996), and 
over time lead to mortality through iron overload-associated heart and liver toxicity (Vichinsky 
et al., 2005; Cao et al., 1996). In order to prevent iron overload associated risks, patients must 
adhere to iron chelation regimens administered orally (Hoffbrand et al., 2003; Cappellini et al., 
2006) or subcutaneously (Olivieri and Brittenham, 1997). However, compliance remains a key 

Protocol HGB-204 Version 5.0 
Page 15 of 67 
29 June 2015  
 
bluebird bio, Inc. 
 
challenge, and even with current therapies, overall survival until the age of 30 is only 55% 
(Modell et al., 2000; Delea et al., 2007). 
The only cure for β-thalassemia major is allogeneic hematopoietic stem cell transplant (HSCT). 
For pediatric subjects with available human leukocyte antigen (HLA)-identical sibling donors 
(< 25% of cases), outcomes from this potentially curative procedure are promising. A recent 
large-scale allogeneic HCT study in 179 pediatric subjects (Sabloff et al., 2011) reported overall 
and disease-free survival rates of 91% and 88%, respectively. Approximately 9.5% of subjects 
suffered graft failure, and 38% and 13% experienced acute or chronic graft-versus-host-disease 
(GVHD), respectively. Unfortunately, the experience with allogeneic HSCT in adult subjects is 
considerably worse, with a 27% transplant-related mortality and only a 67% rate of thalassemia-
free survival (Gaziev et al., 2005). In another study in 68 adults with thalassemia receiving 
HSCT using a matched, unrelated donor, overall rates of survival, disease-free survival, 
rejection, and transplant-related mortality were 79%, 66%, 14%, and 21%, respectively 
(Lucarelli et al., 2012). Therefore, in adults, HSCT is not a commonly prescribed approach to 
treatment and these subjects have a limited life-expectancy. 
Based on the pioneering work of Philippe Leboulch, MD, PhD to develop therapeutic 
applications of gene therapy for β-globin disorders (Cavazzana-Calvo et al., 2010), and in order 
to provide an effective and safer alternative to allogeneic HSCT, bluebird bio is developing 
LentiGlobin BB305 Drug Product (autologous CD34+ hematopoietic stem cells transduced with 
LentiGlobin BB305 lentiviral vector encoding the human βA-T87Q-globin for the treatment of 
β-thalassemia major.  
The LentiGlobin BB305 lentiviral vector is a replication defective, self-inactivating (SIN), 
human immunodeficiency virus type 1 (HIV-1) based lentiviral vector encoding a single codon 
variant of the β-globin gene, βA-T87Q, which conserves the protein’s function while allowing for 
quantification relative to other globin species. Expression of the βA-T87Q gene is driven by the 
erythroid lineage specific globin locus control region (LCR) to correct the β-globin/α-globin 
imbalance in erythrocytes.  
Gene therapy with LentiGlobin BB305 Drug Product involves the isolation of autologous 
mobilized hematopoietic CD34+ cells followed by transduction ex-vivo to introduce the 
βA-T87Q-globin therapeutic gene. After undergoing myeloablation, subjects receive LentiGlobin 
BB305 Drug Product via intravenous (IV) infusion, leading to peripheral reconstitution with 
corrected erythrocytes. As LentiGlobin BB305 Drug Product consists of genetically-modified 
autologous cells, the risks of GVHD and graft rejection are eliminated. 
A previous LentiGlobin lentiviral vector (LentiGlobin HPV569 lentiviral vector) was utilized in 
a clinical study, LG001, in which 3 subjects with β-thalassemia major were treated (Cavazzana-
Calvo et al., 2010). bluebird bio has designed LentiGlobin BB305 lentiviral vector to improve 
the transduction efficiency, integrated provirus stability, and safety of this earlier LentiGlobin 
lentiviral vector  (NC-11-001-R; NC-11-004-R). (See Section 3.2.1 for details.)  
Nonclinical studies have been performed to support the use of LentiGlobin BB305 lentiviral 
vector in clinical studies. Results of such studies showed transduction efficiency of CD34+ cells 
to be higher with LentiGlobin BB305 lentiviral vector than with HPV569. The improved 
transduction efficiency resulted in a greater amount of therapeutic globin produced in erythroid 
colonies with LentiGlobin BB305 lentiviral vector  than with HPV569, while the level of 
expression per copy remained unchanged (NC-11-001-R; NC-11-004-R). 

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Protocol HGB-204 Version 5.0 
Page 19 of 67 
29 June 2015  
 
bluebird bio, Inc. 
 
 incidence of transplant-related mortality through 100 days post-transplant, 
 overall survival, 
 detection of vector-derived RCL in any subject, 
 characterization of events of insertional mutagenesis leading to clonal dominance or 
leukemia, and 
 monitoring of laboratory parameters and frequency and severity of clinical adverse 
events (AEs). 
3 
INVESTIGATIONAL PLAN 
3.1 
Overall Design and Plan of the Study 
This is a non-randomized, open label, multi-site, single dose, Phase 1/2 study in up to 
18 subjects (including at least 3 adolescents ≥ 12 < 18 years of age) with β-thalassemia major 
who receive at least 100 mL/kg/year of packed red blood cells (pRBCs) or ≥8 transfusions of 
pRBCs per year in each of the 2 years preceding enrollment. The study will evaluate the safety 
and efficacy of autologous hematopoietic stem cell (HSC) transplantation (HSCT) using 
LentiGlobin BB305 Drug Product (autologous CD34+ HSCs transduced with LentiGlobin 
BB305 lentiviral vector encoding the human βA-T87Q-globin gene and resuspended in 
cryopreservative solution in the final immediate container for the intended medical use).  
Initially, treatment will be staggered. The second subject will begin myeloablative conditioning 
only after the first subject 1) engrafts (defined as an absolute neutrophil count [ANC] ≥ 
0.5 × 109/L for 3 consecutive days); and 2) has no LentiGlobin BB305 Drug Product 
treatment-related serious adverse event (SAE) unexpected to occur with autologous HSCT. 
After Subject 2 meets these same criteria, parallel drug product treatment can occur with 
additional subjects.  
The study has 4 distinct stages, as follows. 
Stage 1: Screening to determine eligibility 
Stage 2: Autologous CD34+ cell collection, LentiGlobin BB305 Drug Product manufacture and 
disposition  
Within the period of 30 days prior to HSC collection, the subject’s transfusion regimen may be 
adjusted to maintain a minimum of 10 g/dL of hemoglobin (Hb) in order to suppress 
dyserythropoiesis, which can impede the isolation of CD34+ cells enriched for undifferentiated 
HSC. 
Each subject will undergo HSC mobilization with filgrastim and plerixafor. Peripheral blood 
mononuclear cells (PBMCs) will be collected by apheresis using standard methods. A total of 2 
mobilization cycles may be performed if needed. Each mobilization cycle may include up to 5 
apheresis procedure days, but only 2 consecutive apheresis procedure products may be sent for 
transduction from any mobilization cycle. The other apheresis procedure products should be 
used for rescue cells, if they meet minimum requirements as a rescue product. If 2 mobilization 
cycles are needed to collect sufficient HSCs to meet the requirement of a total dose of 
≥3.0 × 106 CD34+ cells/kg, then 2 transductions will be performed: one on the cells collected 
during Mobilization Cycle 1 and one on the cells collected during Mobilization Cycle 2. Each 
transduced product from each Mobilization Cycles 1 and 2 is an independent drug product. 

Protocol HGB-204 Version 5.0 
Page 20 of 67 
29 June 2015  
 
bluebird bio, Inc. 
 
Thus, subjects who require 2 mobilization cycles to produce the minimum cell dose will be 
treated with 2 drug products in total. Mobilization cycles must be separated by at least 2 weeks. 
A bone marrow harvest is also allowed, but only to procure cells for rescue.  
The harvested cells to be used for transduction will be selected for the CD34+ marker to enrich 
for HSCs, transduced with LentiGlobin BB305 Lentiviral Vector, and stored under the vapor 
phase of liquid nitrogen while testing is ongoing. 
Stage 3: Myeloablative conditioning and infusion of LentiGlobin BB305 Drug Product 
After the transduced cells are dispositioned for clinical use, the subject will undergo 
myeloablative conditioning with busulfan. Busulfan will be administered intravenously (IV) at a 
starting dose of 3.2 mg/kg/day or 0.8 mg/kg every 6 hours for 4 consecutive days. The dose of 
busulfan will be adjusted based upon first dose busulfan pharmacokinetics in order to maintain 
appropriate levels for myeloablation (area under the curve [AUC] goal of 1000 [range 900 to 
1200] µM*min for an every 6 hours [q6h] dosing regimen, or 4000 [range 3600 to 
5000] µM*min for a once daily [qd] dosing regimen). Clinical sites that use a test dose of 
busulfan several days before beginning myeloablation to pre-determine busulfan dose may also 
do so in this protocol. After completion of the 4-day course of busulfan, there must be a 
minimum of 72 hours of busulfan washout before drug product infusion.   
On Day 0, after thawing, the LentiGlobin BB305 Drug Product(s) will be administered via IV 
infusion at a dose of ≥ 3.0 × 106 CD34+ cells/kg.  Subjects who undergo 2 mobilizations (and 
subsequent transduction of those cells) to achieve a total dose of ≥ 3.0 × 106 CD34+ cells/kg 
will have 2 LentiGlobin BB305 Drug Products, which should be administered in sequence, with 
the second administered immediately after the first. 
Stage 4: Follow-up, through engraftment and 24 months after drug product infusion 
Subjects will be followed daily in the transplant unit for adverse events (AEs), and laboratory 
parameters will be followed to monitor bone marrow engraftment. The subject may be 
discharged from the transplant unit once: 
1) engraftment occurs (defined as an ANC ≥ 0.5 × 109/L for 3 consecutive days); and 
2) the subject is considered medically stable.  
After discharge, subjects will be followed in this protocol for a minimum of 24 months after 
LentiGlobin BB305 Drug Product infusion. 
3.1.1 
Data Monitoring Committee 
A DMC comprised of members with appropriate scientific and medical expertise to monitor the 
study will be convened before the study is opened. A charter describing the composition and 
conduct of the DMC will be issued by the Sponsor and agreed to by all DMC members prior to 
the DMC’s initial meeting. 
A DMC chairperson will be appointed who will be responsible for the overall operation of the 
DMC. Minutes from each meeting will be recorded and archived. 
The DMC will meet by teleconference at regular intervals, approximately once every 3 months, 
or more or less frequently if needed, and depending on speed of subject enrollment and amount 
of new data generated. The DMC will be charged with review of all unexpected LentiGlobin 
Drug Product treatment-related SAEs following notification by the Sponsor. 

Protocol HGB-204 Version 5.0 
Page 21 of 67 
29 June 2015  
 
bluebird bio, Inc. 
 
It will be the responsibility of the Sponsor or Sponsor’s designee to promptly contact the 
chairperson of the DMC about all unexpected AEs (i.e., unexpected in nature and / or in 
severity). 
The DMC will have the right to recommend halting the study at any time due to concerns for the 
safety of the subjects. (Refer to Section 3.5.2 for the enrollment suspension criteria). 
3.2 
Rationale for the Study 
Allogeneic HSCT is associated with a high rate of transplant-related mortality, particularly in 
adults, where it is not considered a standard of care for β-thalassemia major. In order to provide 
a potentially curative treatment to adults with β-thalassemia major, bluebird bio is developing 
LentiGlobin BB305 Drug Product (autologous CD34+ HSCs transduced with LentiGlobin 
BB305 lentiviral vector encoding the human βA-T87Q-globin gene) for the treatment of 
β-thalassemia major.  
Nonclinical study findings have shown that the therapeutic βA-T87Q-globin gene is under the 
transcriptional control of the erythroid-specific human β-globin promoter and β-globin Locus 
Control Region elements (DNase I hypersensitive sites HS2, HS3, and HS4) (Pawliuk et al., 
2001; Oh et al., 2004; Imren et al., 2002). Similarly effective ex vivo globin gene transfer into 
human CD34+ HSCs was observed (Imren et al., 2002). The A-T87Q-globin protein level 
expressed can be quantified by high performance liquid chromatography (HPLC) and 
distinguished from that of wild-type -globin expressed from the endogenous genes or derived 
from transfused RBCs (Pawliuk et al., 2001). This property is important for demonstrating a 
biological effect in subjects with -thalassemia major by detection of engraftment with cells 
expressing the transferred gene, since subjects may be transfused with normal hemoglobin 
2A2 (HbA)-containing blood during the study. 
Nonclinical studies have been performed to support the use of LentiGlobin BB305 lentiviral 
vector in clinical studies; refer to the Investigator’s Brochure for summaries of study results. 
Treatment of -hemoglobinopathies with gene therapy is also expected to eliminate the major 
risk of GVHD associated with allogeneic HSCT, since the CD34+ cells transduced with the 
lentiviral vector are autologous. In addition, in order to avoid complications of extensive 
immunosuppression associated with the conditioning regimen necessary for allogeneic HSCT, 
the proposed protocol is designed to use the single agent busulfan for myeloablative bone 
marrow conditioning, and does not rely on immunosuppressive agents such as 
cyclophosphamide or fludarabine that are required for allogeneic transplant. 
Data from nonclinical studies and from clinical studies in subjects with hemoglobinopathies, 
including -thalassemia, indicate that partial chimerism with the transduced HSCs (animal 
studies) or allogeneic HSCs (human studies) results in quasi-complete peripheral reconstitution 
with corrected RBCs, because of their extended life-span (Vermylen, 2003; Pawliuk et al., 2001; 
Oh et al., 2004; Lucarelli et al., 1993; Imren et al., 2002). This is an important observation, 
which underscores the feasibility of the proposed clinical study. Indeed, bone marrow 
reconstitution with as low as 11% of HSCs containing the therapeutic gene is likely to result in 
clinical benefit to the patients (Walters et al., 2001; Lucarelli et al., 2001). 
There was no clinical data with LentiGlobin BB305 Drug Product when this study initiated. 
However, as stated previously, 3 subjects were treated using LentiGlobin HPV569 Drug Product 
under Protocol LG001. All 3 subjects treated were adults with β-thalassemia major who 

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Protocol HGB-204 Version 5.0 
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3.2.2 
Rationale for the β-globin Gene Used 
The -globin gene used in this study is the wild-type human -globin gene with 1 single 
modification at amino-acid position 87 [A87 Thr:Gln (A-T87Q)]. This change encodes 
1 amino-acid residue from the normal human - and -globin genes within a stretch of amino 
acids highly conserved in both human A-, - and - globin chains.  
This gene has an advantage over the normal -globin gene for the gene therapy of -thalassemia 
in that both A-T87Q-globin and Hb containing this chain can be distinguished from wild-type 
human -globin and HbA by HPLC (Pawliuk et al., 2001; Imren et al., 2002). This is especially 
important in a clinical study setting, as the ability to accurately detect the engraftment of 
transduced cells will not be impaired in subjects who require transfusion with normal 
HbA-containing blood post-transplant. 
Immunogenicity with A-T87Q is not anticipated. β-globin proteins are notoriously poorly 
immunogenic, as evidenced by the fact that patients with β0/β0 thalassemia do not develop anti-
hemoglobin antibodies despite receiving multiple transfusions (Olivieri, 1999). In addition, 
efforts as published by Reichlin to identify HbA antibody in multiply transfused patients with 
sickle cell disease yielded only negative results (Reichlin, 1975). 
3.3 
Rationale for the Study Design 
Because of the potential toxicity of busulfan, no treatment control group is included in this 
Phase 1/2 open label study. In addition, since 2 years of retrospective hematological data will be 
collected for each subject in the study, each subject may serve as his/her own control for 
evaluation of transfusion requirements and the evaluation of changes in the nature or frequency 
of the subject-specific main inclusion criterion. 
3.4 
Rationale for the Dose Selected 
Cellular doses of ≥ 0.75 × 106 CD34+ cells/kg are reported to provide acceptable engraftment, 
although the rate and response of engraftment are related to the dose (Perez-Simon et al., 
1998).  For apheresis-procured cells, the dose of ≥ 3 x 106 cells/kg was chosen because this 
number of cells is often obtainable with one mobilization cycle and two collection procedures 
(which are typically well tolerated by subjects) and are anticipated to provide the desired 
engraftment kinetics.  For bone marrow harvest procured cells, the dose of ≥ 1.5 x 106 cells/kg 
was chosen based on consideration of the lower yield expected from this collection method and 
the risk associated with the collection procedures.  In particular, the risks associated with a bone 
marrow harvest are significantly greater than the risks associated with a peripheral mobilization 
and apheresis, primarily due to the inherent toxicities of general anesthesia. The potential risk 
associated with a cell dose between 1.5 and 3.0 × 106 CD34+ cells/kg, as compared to a cell 
dose ≥ 3.0 × 106 CD34+ cells/kg, is a delay in engraftment which is likely to be less than 2 days 
(Miyamoto et al., 2004). Given these data, and the fact that rescue cells are non-transduced and 
therefore should have the best engraftment characteristics, Table 3-1 outlines the source of 
subject cells, usage, and minimal dose to be received. 

Protocol HGB-204 Version 5.0 
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Table 3-1 
Dose of LentiGlobin BB305 Drug Product or Rescue for -Thalassemia 
Major 
Usage 
Dose 
Drug Product 
≥3.0 × 106 CD34+ cells/kga 
Rescue Cells (obtained by apheresis) 
≥2.0 × 106 CD34+ cells/kg 
Rescue Cells (obtained by bone marrow harvest) 
≥1.5 × 106 CD34+ cells/kg or >1.0 × 108 TNC/kg 
TNC, total nucleated cells 
a  If 2 transductions are performed, the total dose of the 2 drug products together must meet this criterion. 
If LentiGlobin BB305 Drug Product fails to be dispositioned for clinical use, collection does not 
yield adequate cells for drug product manufacture, or manufacturing does not yield sufficient 
cells to meet the minimum dose criteria, then mobilization, apheresis, and additional 
transduction may be repeated.  
Myeloablation should not begin on a subject until it is confirmed that Drug Product for that 
subject has been dispositioned for clinical use and rescue cells are available. 
3.5 
Treatment Discontinuation and Enrollment Suspension Criteria 
See Section 4.5 for “subject withdrawal from the study”. 
3.5.1 
Stopping Rules for Busulfan  
Once myeloablation with busulfan has begun, there are no stopping rules for busulfan. In the 
anticipated very rare event of consent withdrawal during conditioning or the development of a 
new medical condition that, in the investigator’s opinion, puts the subject at risk with continued 
busulfan treatment, the Medical Monitor should be contacted immediately. In such situations in 
which busulfan conditioning has not been completed per protocol, LentiGlobin BB305 Drug 
Product should not be given, and it is likely that rescue therapy will be required. 
3.5.2 
Enrollment Suspension Criteria 
Enrollment in this study may be stopped at any time for safety reasons. It will be the 
responsibility of the DMC to determine if there is reasonable cause for suspending enrollment. 
The Sponsor will inform the regulatory authorities and the investigators, and each site’s 
Institutional Review Board (IRB) / Ethics Committee (EC) and other appropriate institutional 
regulatory bodies will be promptly notified if a decision to suspend enrollment is made. In the 
event enrollment is suspended, no new mobilization/conditioning/ or drug product infusion of 
subjects will be initiated, but subjects who have already been treated with LentiGlobin BB305 
Drug Product will continue in the study. If mobilization has been initiated, cell collection will be 
completed at investigator’s discretion. Likewise, if the study is halted while a subject is 
undergoing conditioning, conditioning will be completed at investigator’s discretion, and every 
effort will be taken to restart the study prior to their scheduled infusion. However, a subject may 
be infused with their rescue cells following conditioning if the study cannot be restarted in time.  
Enrollment and treatment with drug product will be temporarily suspended for any of the 
following reasons pending review and recommendations from the DMC and appropriate 
communication with the relevant regulatory agency(ies): 
 Death, until the cause of the death is determined* 

Protocol HGB-204 Version 5.0 
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 Detection of leukemia/lymphoma due to vector-mediated insertional oncogenesis** 
 Detection of vector-derived RCL in any subject  
 Failure to achieve reconstitution with transduced cells in 1 subject, requiring use of 
backup cells  
 Determination of unexpected, clinically significant, or unacceptable risk to subjects 
(e.g., development of study treatment-related Grade 3 or 4 toxicities in at least 
3 subjects)  
*Any death of a subject with β-thalassemia major on Studies HGB-204 or HGB-205 after receiving LentiGlobin 
BB305 Drug Product will result in a hold of further enrollment and treatment with drug product until an 
investigation into the cause of death is performed. If it is determined that the death was not related to the drug 
product, then enrollment/treatment with drug product may restart. If the relationship between the death and the drug 
product is not clear, or it appears that the death may be related to the study drug, enrollment will be held until the 
DMC assessment and recommendations as described above. In cases in which the cause of death is still under 
investigation, subjects that have already received conditioning may receive either back-up cells or BB305 Drug 
Product on a case-by-case basis in consultation with the DMC. 
**If a subject is diagnosed with leukemia or lymphoma after receiving LentiGlobin BB305 in any clinical study, 
enrollment will be held until determination is made as to whether the malignancy was related to a vector-mediated 
insertion. Once this assessment occurs, if the malignancy is not related to a vector-mediated insertion, enrollment 
may resume. If the relationship between the malignancy and an insertion is not clear or it appears they may be 
related, enrollment will be held until the DMC assessment and recommendations as described above. 
4 
STUDY POPULATION 
4.1 
Number of Subjects 
Up to 18 subjects, including at least 3 adolescents (≥ 12 < 18 years of age, inclusive) will be 
treated with drug product. Replacement subjects may be added if subjects withdraw or are 
withdrawn prior to conditioning.  
4.2 
Inclusion Criteria 
Subjects must meet all of the following criteria to be considered eligible for enrollment in the 
study. 
1. Subjects between 12 and 35 years of age, inclusive, at the time of consent or assent (as 
applicable), and able to provide written consent (adults, or legal guardians, as applicable) or 
assent (adolescents). 
2. Diagnosis of β-thalassemia major and a history of at least 100 mL/kg/year of pRBCs or 
≥8 transfusions of pRBCs per year for the prior 2 years. 
3. Documented baseline, or pretransfusion, hemoglobin level of ≤ 7 g/dL. 
4. Clinically stable, have a Karnofsky performance status of ≥ 60, and eligible to undergo 
HSCT. 
5. Treated and followed for at least the past 2 years in a specialized center that maintained 
detailed medical records, including transfusion history.  
4.3 
Exclusion Criteria 
Subjects meeting any of the following criteria will be excluded from the study. 

Protocol HGB-204 Version 5.0 
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1. Positive for presence of human immunodeficiency virus type 1 or 2 (HIV-1 or HIV-2), 
hepatitis B virus (HBV), or hepatitis C virus (HCV). (Note that subjects who are positive for 
anti-HBV antibody [to either core or envelope proteins] or for anti-HCV antibody are 
eligible as long as they have a negative HBV or HCV viral load by quantitative polymerase 
chain reaction [qPCR]. Where clinically and/or regionally indicated, one or more of the 
following tests may be performed, in which case positive results would exclude the subject 
from participating: human T-lymphotrophic virus-1 (HTLV-1) or HTLV-2, syphilis (RPR), 
toxoplasmosis, Trypanosoma cruzi, or West Nile Virus).  
2. Active bacterial, viral, fungal, or parasitic infection. 
3. A WBC count <3 × 109/L, and / or platelet count <100 × 109/L not related to hypersplenism. 
4. Uncorrected bleeding disorder.  
5. Any prior or current malignancy or myeloproliferative or immunodeficiency disorder. 
6. Immediate family member with a known or suspected Familial Cancer Syndrome (including 
but not limited to hereditary breast and ovarian cancer syndrome, hereditary non-polyposis 
colorectal cancer syndrome and familial adenomatous polyposis). 
7. Prior HSCT. 
8. Advanced liver disease, defined as: 
a. Baseline alanine transaminase or direct bilirubin value > 3 × the upper limit of normal 
(ULN), or 
b. Liver biopsy demonstrating cirrhosis, any evidence of bridging fibrosis, or active 
hepatitis.  
9. Baseline estimated glomerular filtration rate (eGFR) < 70 mL/min/1.73 m2, as determined 
using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) creatinine 
equation for ≥ 18 years of age, and Bedside Schwartz equation calculator < 18 years of age 
(see http://www.kidney.org/professionals/kdoqi/gfr_calculator.cfm). 
10. Uncontrolled seizure disorder. 
11. Diffusion capacity of carbon monoxide (DLco) < 50% of predicted (corrected for 
hemoglobin and/or alveolar volume, as clinically indicated). 
12. A cardiac T2* < 10 ms by magnetic resonance imaging (MRI) 
13. Any other evidence of severe iron overload that, in the investigator’s opinion, warrants 
exclusion. 
14. Clinically significant pulmonary hypertension, as defined by the requirement for ongoing 
pharmacologic treatment or the consistent or intermittent use of supplemental home oxygen. 
15. Participation in another clinical study with an investigational drug within 30 days of 
Screening. 
16. Failure to obtain appropriate informed consent. 
17. Any other condition that would render the subject ineligible for HSCT, as determined by the 
attending transplant physician or investigator.  

Protocol HGB-204 Version 5.0 
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18. Contraindications to the conditioning regimen. 
19. Prior receipt of gene therapy. 
20. Diagnosis of significant psychiatric disorder of the subject that could seriously impede the 
ability to participate in the study. 
21. Pregnancy or breastfeeding in a postpartum female or absence of adequate contraception for 
fertile subjects. Females of child-bearing potential are required to use effective 
contraception from Screening through at least 6 months after drug product infusion. Male 
subjects are required to use effective contraception (including condoms) from Screening 
through at least 6 months after drug product infusion. 
22. An assessment by the investigator that the subject would not comply with the study 
procedures outlined in the protocol. 
4.4 
Subject Identification and Registration 
Prior to the Screening Phase, the investigator will identify candidates potentially meeting the 
study eligibility criteria, based on review of medical records and clinical test findings performed 
routinely as standard of care for the treatment of the subject. Subjects who are determined by the 
investigator to be potentially eligible will be informed of the potential to participate in the study.  
If the subject is willing to participate in the study, then the site will begin the consent process as 
per institutional practices, according to Good Clinical Practice (GCP). Written informed consent 
must be obtained before the conduct of any Screening tests not performed routinely in the 
treatment of the subject.  
Upon signing the informed consent, the potential subject will be registered and assigned a 
unique subject number. Once a subject number has been assigned, it cannot be reused, and the 
number stays with the subject even if the subject is subsequently determined to be ineligible for 
the study; a new subject number will be assigned if the subject re-enrolls into the trial. 
4.5 
Subject Withdrawal from the Study 
Subjects have the right to withdraw from the study at any time for any reason. After giving 
informed consent, subjects may withdraw or be withdrawn from study-related procedures and 
treatments (e.g., apheresis, busulfan conditioning) under the following conditions: 
 withdrawal of consent, 
 the subject is unable to comply with protocol-defined visits or other requirements of the 
protocol,  
 any medical condition which, in the opinion of the investigators, would put the subject at 
risk for continuing treatment or follow-up studies, or 
 adequate cells are not collected during harvests, or failure of transduced cells to be 
dispositioned for clinical use.  
In addition, after subjects have been treated with LentiGlobin BB305 Drug Product, they may 
be withdrawn from the study under the following additional condition: 

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5
STUDY TREATMENTS 
5.1
Description of LentiGlobin BB305 
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5.2 
Summary of Treatments to be Performed or Administered 
After confirmation of eligibility, HSCs must be collected from the subject for 2 purposes – 
transduction for LentiGlobin BB305 Drug Product and for rescue cells (to be used if the subject 
fails to engraft with transduced cells). Table 3-1 outlines the source of the subject cells, usage, 
and the dose of LentiGlobin BB305 Drug Product or rescue.  
Additional details are provided in the following subsections. 
5.2.1 
Mobilization and Apheresis Procedure 
Within the period of 30 days prior to and during the period of mobilization and apheresis, the 
subject’s transfusion regimen may be adjusted to maintain a minimum of 10 g/dL of Hb in order 
to suppress dyserythropoiesis, which can impede the isolation of CD34+ cells enriched for 
undifferentiated HSC. 
A total of 2 mobilization cycles may be performed if needed. Each mobilization cycle may 
include up to 5 apheresis procedure days, but only 2 consecutive apheresis procedure products 
may be sent for transduction for each drug product. Apheresis procedure products are also to be 
used for rescue cells. Mobilization cycles must be separated by at least 2 weeks. A bone marrow 
harvest is also allowed, but only to procure cells for rescue. If 2 mobilization cycles are needed 
to collect enough HSCs to meet the requirement of a total dose of ≥ 3.0 × 106 CD34+ cells/kg, 
then 2 transductions will be performed, 1 on the cells collected during Mobilization Cycle 1 and 
another on the cells collected during Mobilization Cycle 2. Each transduced product from each 
Mobilization Cycles 1 and 2 is an independent drug product. Thus, subjects who require 2 
mobilization cycles to produce the minimum cell dose will be treated with 2 drug products in 
total. 
Potential toxicities of filgrastim, plerixafor, and apheresis are included in the Informed Consent 
form. 
5.2.1.1 
Mobilization 
Recent published data (Yannaki et al., 2013) has indicated that the combination of plerixafor 
and filgrastim may be the most appropriate mobilization strategy for subjects with β-
thalassemia. The mobilization approach below follows this recent data. Modifications to this 
approach are allowed only after consultation with the medical monitor. 
Table 5-1 
Mobilization for subjects who have NOT undergone splenectomy 
Mobilization 
day 
Filgrastim dosea 
Plerixafor dose 
(evening) 
Complete Blood 
Count (CBC) c 
Peripheral 
CD34+ count 
Apheresis 
1 
10 ug/kg 
 
 
 
 
2 
10 ug/kg 
 
X 
 
 
3 
10 ug/kg 
 
X 
 
 
4 
10 ug/kg 
0.24 mg/kg 
X 
 
 
5 
10 ug/kg 
0.24 mg/kg 
X 
Xd 
X 
6 (if needed) 
10 ug/kg 
b 
X 
X 
X 
7 (if needed) 
b 
b 
X 
X 
X 
8 (if needed) 
b 
b 
X 
X 
X 
9 (if needed) 
 
 
X 
X 
X 
a Dosage of filgrastim should be decreased if WBC > 100 × 109/L prior to initiation of apheresis 
b Mobilization regimen after 2 days of apheresis should be discussed with the Medical Monitor. 

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c Recommended but can be deferred in case of difficulty in obtaining CBC (e.g., weekend) at the discretion of the 
site PI. 
d CD34+ count is done prior to apheresis. 
 
Table 5-2 
Mobilization for subjects who have undergone a splenectomy 
Mobilization 
day 
Filgrastim dosea 
Plerixafor dose 
(evening) 
Complete Blood 
Count (CBC) c 
Peripheral 
CD34+ count 
Apheresis 
1 
5 ug/kg 
 
 
 
 
2 
5 ug/kg 
 
X  
 
 
3 
5 ug/kg 
 
X 
 
 
4 
5 ug/kg 
0.24 mg/kg 
X 
 
 
5 
5 ug/kg 
0.24 mg/kg 
X 
Xd 
X 
6 (if needed) 
5 ug/kg 
b 
X 
X 
X  
7 (if needed) 
b 
b 
X 
X 
X 
8 (if needed) 
b 
b 
X 
X 
X 
9 (if needed) 
 
 
X 
X 
X 
a Dosage of filgrastim should be decreased if WBC > 100 × 109/L prior to initiation of apheresis 
b Mobilization regimen after 2 days of apheresis should be discussed with the Medical Monitor. 
c Recommended but can be deferred in case of difficulty in obtaining CBC (e.g., weekend) at the discretion of the 
site PI. 
d CD34+ count is done prior to apheresis. 
 
After completion of Mobilization Cycle 1, the clinical site will be informed by the sponsor as to 
whether the total dose of drug product had been attained. This notification should come within 
approximately 7 days of the completion of Mobilization Cycle 1. Only if additional HSCs are 
needed either for drug product manufacture, or for rescue cells (i.e., if < 2.0 × 106 CD34+ 
cells/kg have been collected and stored at the site for rescue cells), subject should begin 
Mobilization Cycle 2 no sooner than 2 weeks after the completion of Mobilization Cycle 1. 
Mobilization Cycle 2 should follow the same procedures as Mobilization Cycle 1, although the 
dose of plerixafor and filgrastim and the apheresis schedule may be modified based on the 
subject’s experience from Mobilization Cycle 1. 
5.2.1.2 
Apheresis 
As described above, typically a subject’s first apheresis will occur on Mobilization Day 5. 
However, after discussion with the Medical Monitor, it may occur on Mobilization Day 4 or 6 if 
the subject has a particularly robust or sluggish response to mobilization, respectively. On each 
day of apheresis, the subject should have a physical examination and vital signs performed prior 
to beginning apheresis and again after completion of apheresis. Apheresis will be performed per 
SOP at the clinical site. Recommendations for apheresis are also provided in Section 10.2. 
The following examples are given as possible scenarios to show how cells for both drug product 
manufacture and rescue could be obtained from Mobilization Cycle 1. There should be close 
consultation between the Medical Monitor and the investigator during apheresis. 
Apheresis product from Mobilization Cycle 1: recommended evaluation and management: 
 If the Apheresis Procedure Day 1 collection is ≥ 10×106 CD34+ cells/kg, this collection 
should be sent to the transduction facility (as per the Study Operations Manual) no later 
than the next business day. The subject should return for as many additional apheresis 
procedures required (up to 5 total collections may be performed as part of any 1 

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mobilization cycle) to collect CD34+ cells for rescue, which should be stored indefinitely 
at the clinical site or per local procedures.  
 If the Apheresis Procedure Day 1 collection is between 1×106  and 10×106  CD34+ 
cells/kg, this collection should be held at an overnight, controlled storage facility at the 
clinical site (should be shipped immediately to the transduction facility if storage is not 
possible), and the subject should return for Apheresis Procedure Day 2. If Day 1 
collection was stored overnight, the collection from Apheresis Procedure Day 1 and 
Apheresis Procedure Day 2 should be sent together to the transduction facility 
immediately following the completion of the Apheresis Day 2 procedure (as per the 
Study Operations Manual). The subject should then return for as many additional 
apheresis procedures required (up to 5 total collections may be performed as part of any 
one mobilization cycle) to collect CD34+ cells for rescue, which should be stored 
indefinitely at the clinical site or per local procedures. 
 If the Apheresis Procedure Day 1 collection is < 1×106  CD34+ cells/kg, this collection 
should be stored towards collecting sufficient rescue cells and the subject should return 
for Apheresis Procedure Day 2.  
o If the Apheresis Procedure Day 2 collection is ≥ 2 × 106 CD34+ cells/kg, consult 
with Medical Monitor to determine how to proceed. In this scenario, it is possible 
that the subject will return for Apheresis Procedure Day 3, in which case the 
collections from Apheresis Procedure Days 2 and 3 would be sent together to the 
transduction facility.  
o If the Apheresis Procedure Day 2 collection is < 2 × 106 CD34+ cells/kg, then it 
should be stored towards collecting sufficient rescue cells, to be stored indefinitely at 
the clinical site or per local procedures. Plans for an additional mobilization cycle 
should be discussed with the Medical Monitor. 
Apheresis product from Mobilization Cycle 2: recommended evaluation and management: 
 If Mobilization Cycle 2 is for the procurement of rescue cells only, the subject should 
undergo as many apheresis procedures (up to 5) needed to collect the minimum dose of 
rescue cells required, which should be stored indefinitely at the clinical site or per local 
procedures. 
 If Mobilization Cycle 2 is for the procurement of additional HSCs for drug product, the 
management of the collection from each Apheresis Procedure day will be discussed with 
the Medical Monitor on a case-by-case basis prior to Apheresis Procedure Day 1, as it is 
dependent upon how many cells were collected and transduced from Mobilization 
Cycle 1.  
5.2.2 
Bone Marrow Harvest Procedure 
If sufficient cells for rescue are not procured after 2 Mobilization Cycles, the investigator can 
proceed with a bone marrow harvest. Bone marrow harvest will be performed according to 
standard operating procedures at the clinical site.  

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5.2.3 
Conditioning 
5.2.3.1 
General considerations 
Myeloablative conditioning of the subject will be performed using busulfan. 
Conditioning will only begin after the following criteria are met: 
1. All LentiGlobin BB305 Drug Product to be used for a particular subject has been release 
tested, dispositioned for clinical use, and is stored at the clinical site;  
2. The subject has undergone an interim medical history, AE and concomitant medications 
assessments, physical examination, vital signs, and laboratory tests as per the SOE and 
continues to meet the eligibility criteria based on these results. 
3.  Iron chelation therapy will be stopped 7 days prior to start of the conditioning. 
Busulfan will be administered at a starting dose of 3.2 mg/kg/day for 4 consecutive days via IV 
infusion on Days -7 through -4. The preferred schedule is busulfan via a central venous line 
(CVL) as a 3-hour infusion once daily for 4 consecutive days for a total of 4 doses. Divided 
dosing is permitted. In this case, busulfan should be administered at 0.8 mg/kg as a 2-hour 
infusion every 6 hours for 4 consecutive days for a total of 16 doses. Please note that the timing 
of busulfan pharmacokinetics (PK) sample collection is based on whether a 2-hour or 3-hour 
infusion is used. 
Clinical sites that use a test dose of busulfan several days before myeloablation to pre-determine 
the busulfan dose may also do so in this protocol.  
Anti-seizure prophylaxis should begin at least 12 hours before initiating busulfan, and should 
continue for at least 24 hours after completion of the four days of busulfan administration. All 
drugs other than phenytoin are allowed for anti-seizure prophylaxis.  Additional supportive 
medications are permitted as per site standard practices. 
5.2.3.2 
Pharmacokinetics for Busulfan Dose Adjustments 
Target first dose PK should be performed in all subjects regardless of busulfan dosing schedule. 
Daily dosing should target an AUC of 4000 (range 3600 to 5000) µM*min. Every 6 hour dosing 
should target an AUC of 1000 (range 900 to 1200) µM*min. 
Recommendations for Q24 hr dosing: Samples for busulfan PK analysis should be collected at 
the end of the first 3 hour infusion, 195 minutes after start of infusion, and 4, 5, 6, and 8 hours 
after the start of the first infusion. Samples should not be drawn from the lumen used to infuse 
busulfan. 
Recommendations for Q6hr dosing: Samples for busulfan PK analysis should be collected at the 
end of the first 2 hour infusion, 135 minutes after start of infusion, 150 minutes after start of 
infusion, and 3, 4, 5, and 6 hours after the start of the first infusion. Samples should not be 
drawn from the lumen used to infuse busulfan. 
5.2.4 
Infusion Procedures, Dose, and Administration 
LentiGlobin BB305 Drug Product is to be given after a minimum of 72 hours after completion 
of the busulfan conditioning regimen in order to achieve complete washout of busulfan.  
Pre-hydration and pre-medication of the subject are not required but sites should follow local 
procedures for HSC infusion.  

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5.3
Transduction Process 
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5.4
Storage and Stability of LentiGlobin BB305 Drug Product 
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5.5
Product Accountability 
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5.6 
Method of Assigning Subjects to Treatment 
All subjects entered into the study will be assigned to the single treatment group in this 
open-label study. 
5.7 
Blinding, Packaging, and Labeling 
5.7.1 
Blinding and Breaking the Blind 
This is an unblinded, open-label study. 
5.7.2 
Packaging and Labeling 
LentiGlobin BB305 Drug Product consists of autologous CD34+ HSCs transduced ex vivo with 
the LentiGlobin BB305 lentiviral vector and resuspended in cryopreservative solution in the 
final infusion bag.  
LentiGlobin BB305 Drug Product will be labeled by the transduction facility according to Good 
Manufacturing Practices.  
5.8 
Duration of Subject Participation 
Time between Screening and HSC-infusion will be variable, and is estimated to be between 2 to 
3 months; thereafter subject is planned to remain on study for approximately 24 months after 
HSC infusion. Eligible subjects will then be enrolled in a long-term follow-up study for another 
13 years, for a total of approximately 15 years. 
5.9 
Assessment of Treatment Compliance 
Eligible subjects will receive LentiGlobin BB305 Drug Product by IV administration on a single 
day as in-patients and thus will be monitored by hospital personnel. 
5.10 
Prior and Concomitant Medications 
5.10.1 
Prior Medications 
All medications taken within 30 days prior to the Screening Visit are to be recorded in the Prior 
and Concomitant Medication CRF.  
5.10.2 
Concomitant Medications and Therapies: General 
All concomitant treatments, including transfusions, will be recorded in the Prior and 
Concomitant Medication CRF. 
Iron chelation therapy should be discontinued at least 7 days prior to conditioning. Iron 
chelation therapy may be restarted at the discretion of the treating physicians no sooner than 30 
days after bone marrow engraftment (defined as an ANC ≥0.5 × 109/L for 3 consecutive days).  
5.10.3 
Concomitant Treatments: During Conditioning 
Permitted concomitant treatments during may include: 
 Hyperdiuresis, beginning 12 hours before initiating conditioning and continuing through 
24 hours thereafter 
 Prevention or treatment of nausea and vomiting 
o Prophylactic administration of ondansetron 

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o Metoclopramide for the treatment of nausea and vomiting 
 Seizure prophylaxis. All drugs other than phenytoin are allowed for anti-seizure 
prophylaxis during conditioning.   
 Ursodeoxycholic acid for prevention of hepatic veno-occlusive disease/ hepatic 
sinusoidal obstruction syndrome. 
 Transfusions should be given according to Hb and platelet counts. All blood products 
will be filtered and irradiated. 
 Subjects will be isolated in single rooms and appropriate precautions will be taken if the 
polynuclear leukocyte count is <0.5 × 109/L. Broad-spectrum antibiotic treatment will be 
administered according to the usual procedures for febrile neutropenia. 
5.10.4 
Concomitant Medications: Infection Surveillance and Prophylaxis 
The LentiGlobin BB305 Drug Product consists of CD34+ cells and is functionally T cell-
depleted. Thus, opportunistic infection surveillance and prophylaxis is recommended. Below is 
a suggested approach to infection surveillance and prophylaxis. Alternatives that follow 
institutional practices may be employed. 
 In subjects with past exposure to HBV, regular monitoring of HBV DNA by PCR for up 
to 3 months after drug product infusion, with pre-emptive lavimudine advised for any 
increase in HBV DNA titer (HBV reactivation). 
 Herpes simplex virus (HSV) prophylaxis: Acyclovir prophylaxis is recommended for at 
least 3 months after drug product infusion for patients who are sero-positive for HSV or 
VZV. 
 Pneumocystis pneumonia prophylaxis:  Trimethoprim-sulphamethoxazole or an 
equivalent drug is recommended for at least 3 months after drug product infusion.  
 Fungal prophylaxis:  Anti-fungal prophylaxis is recommended with agents such as 
Fluconazole for at least 3 months after drug product infusion.  
 Cytomegalovirus (CMV) surveillance:  CMV should be tested using the PCR based 
methods or CMV antigenemia for at least 3 months after drug product infusion. Antiviral 
therapy for CMV reactivation should be commenced pre-emptively if CMV testing 
reveals a high or rising viral load. If CMV reactivation occurs at or before engraftment, 
foscarnet may be considered to prevent marrow suppression. 
 Adenovirus surveillance:  Testing for adenovirus infection is recommended in the event 
of symptoms suspicious for infection such as diarrhea, hepatic dysfunction, respiratory 
symptoms, etc. If diagnosed with an active systemic infection, therapy should be 
instituted with cidofovir or other active agents. 
 Epstein-Barr virus (EBV) surveillance:  EBV monitoring by PCR is recommended for at 
least 3 months after drug product infusion. In the event of persistent EBV viremia or 
signs/symptoms consistent with EBV-related post-transplant lymphoproliferative 
disorder (adenopathy, fever, etc.) therapy such as Rituximab is recommended. 
 Intravenous Immune Globulin (IgG) may be administered for 3 months to maintain IgG 
levels >500 per institutional guidelines. 

Protocol HGB-204 Version 5.0 
Page 37 of 67 
29 June 2015  
 
bluebird bio, Inc. 
 
 Follow institutional post-transplantation guidelines for re-immunization of subjects. 
5.10.5 
Concomitant Medications: Iron Reduction guidelines 
Chelation should stop 7 days prior to starting busulfan.  
Post-transplant chelation guidelines are provided in Appendix 10.4. 
5.10.6 
Concomitant Medication: Transfusion Guidelines 
Recommendations for transfusions are made for 3 separate periods during treatment: 
1. Within the period of 30 days prior to and during mobilization and apheresis, the subject’s 
transfusion regimen may be adjusted to maintain a minimum of 10 g/dL of Hb in order to 
suppress dyserythropoiesis, which can impede the isolation of CD34+ cells enriched for 
undifferentiated HSC. 
2. During conditioning and hospitalization supporting engraftment: follow institutional 
guidelines.  
3. During follow-up: the goal is to maintain Hb ≥ 9 g/dL. Transfusions should be avoided for 
Hb ≥ 9 g/dL, unless the need is medically justified (e.g. as a pre-requirement for surgery). It 
is recommended that subjects should receive pRBC transfusions for any Hb < 7.0 g/dL, and 
for clinically symptomatic anemia, irrespective of Hb level. 
 
6 
STUDY ASSESSMENTS 
6.1 
Schedule of Events 
The study has 4 distinct stages, as follows. 
 Stage 1: 
Screening and eligibility assessments 
 Stage 2: 
Autologous CD34+ cell collection and LentiGlobin BB305 Drug Product 
manufacture and disposition 
 Stage 3: 
Myeloablative conditioning and infusion of LentiGlobin BB305 Drug Product 
 Stage 4: 
Follow-up through Month 24 
The SOE to be performed is outlined in Table 6-1 for Stages 1 and 2, Table 6-2 for Stage 3, and 
Table 6-3 for Stage 4.  
Detailed descriptions of the efficacy, pharmacodynamics, and safety procedures to be conducted 
during this study are provided in the following subsections. Additional details, including 
administrative information, will be provided in the Study Operations Manual. 
Evaluations and procedures identified in the SOE may be performed at unscheduled visits, as 
clinically indicated, at the investigator’s discretion in consultation with the Sponsor. 

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Schedule of Events: Screening and CD34+ Cell Harvest 
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Protocol HGB-204 Version 5.0 
Page 39 of 67 
29 June 2015  
 
bluebird bio, Inc. 
 
Table 6-2 
Schedule of Events:  Conditioning and Drug Product Infusion 
 
Preconditioning Conditioning 
Infusion 
Post-infusion  
Up to 7 days 
before 
busulfan 
Day -7 to -4 
inclusive 
Day 0 
Day 1 until 
discharge 
Interval medical history 
X 
 
 
 
Physical examination  
X 
X 
X12 
X13 
Vital Signs 
X 
X 
X14 
X15 
Stop iron chelation 
X16 
 
 
 
Local lab: Blood for clinical laboratory tests 
X17 
X17 
X17 
X13,17 
Local lab: Blood for serum β-human chorionic 
gonadotropin for women of child-bearing 
potential (WOCBP; serum pregnancy test) 
X 
 
 
 
Re-confirmation of eligibility 
X18 
 
 
 
Anti-seizure prophylaxis 
 
X19 
 
 
Busulfan chemotherapy (daily or q6h regimen, 
for 4 days) 
 
X 
 
 
Local laboratory: Blood for busulfan 
pharmacokinetics20 
X 
X 
 
 
Infusion of LentiGlobin BB305 Drug Product 
 
 
X 
 
Adverse event collection 
Continuous from ICF signing 
Concomitant medication collection 
Continuous from ICF signing 
                                                 
12 To be performed on Day 0 before drug product administration. For adolescents, include height, and Tanner test to monitor 
puberty if relevant. 
13 At least twice a week during hospitalization 
14 To be measured on Day 0 no less frequently than at the start, once during, and upon completion of the infusion 
15 To be performed daily until discharge 
16 Iron chelation must be stopped at least 7 days before initiating busulfan 
17 CBC; serum chemistry; liver function tests; and any other tests required by institutional guidelines, including for example 
testing for CMV, EBV, HSV and VZV IgG. CBC and clinical chemistry should be performed at least every second day until 
neutrophil engraftment is obtained, and frequency should increase if any abnormality of clinical significance is detected.  
18 Review CBC, serum chemistry, liver function tests, physical examination, and interval medical history; verification that 
LentiGlobin BB305 Drug Product has been clinically dispositioned, is available on site and that rescue cells are available.  
19 Anti-seizure prophylaxis to start at least 12 hours before initiating busulfan and to continue at least 24 hours after completion 
of 4 day busulfan course  
20 A test dose of busulfan several days before beginning myeloablation to pre-determine busulfan dose is also permitted.  

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Protocol HGB-204 Version 5.0 
Page 42 of 67 
29 June 2015  
 
bluebird bio, Inc. 
 
6.2.6 
Samples for Storage and Potential Biomarker Analysis 
Optional blood samples will be collected per the SOE for future research. These samples may be 
used for biomarker analyses of proteins, DNA, RNA and other molecules to study thalassemia 
and/or gene therapy.  Such samples may be stored until the samples are exhausted or until the 
repository is discontinued. The Sponsor will be the custodian of the samples in the repository 
and any unused samples will be destroyed at the Sponsor’s discretion. If possible, optional 
blood, bone marrow, and tissue samples also are to be collected in the event of a subject’s death 
if an autopsy is performed. Leftover samples from protocol procedures (e.g., blood draw for 
integration site analysis) may also be stored (optional) for potential future analyses as described 
above. 
Note that samples routinely collected as part of the manufacture of the drug product may be 
used to study the manufacturing process. In particular these samples may be used to understand 
how the process may be improved or made more robust. These potential studies are not optional. 
Other potential uses of these samples for non-manufacturing improvement research, such as 
biomarker analyses of proteins, DNA, RNA and other molecules to study thalassemia and/or 
gene therapy, are optional. 
Collection and storage of the samples described above will be subject to discretionary approval 
from each center’s IRB/IEC and the subject’s specific written consent. Samples will be labeled 
with a unique identification number that includes no subject identifying information. 
6.2.7 
Demographics and Medical History 
Subject demographic data such as gender, age, race, ethnicity, and country of birth will be 
obtained during Screening. A complete medical history also will be obtained during Screening. 
The medical history is to include all prior and current medical history, including age of first 
transfusion, age of starting regular transfusions (i.e., age at which transfusion needs stabilized to 
between once every 4 to 8 weeks), age of starting iron chelation treatment, and spleen size (cm 
below coastal margin) at enrollment. Transfusions details (mL/kg of RBC, along with the 
associated reticulocyte and Hb values taken prior to the transfusion where available), as well as 
relevant blood bank details (including average volume [mL] per unit), should be collected for 
the 2 years prior to enrollment. 
6.2.8 
Clinical/Physical Examination 
A complete physical examination (including Karnofsky performance status, general appearance; 
head eyes, ears, nose, and throat; cardiovascular; dermatologic, abdominal; genitourinary; 
lymph nodes; hepatic; musculoskeletal; respiratory; and neurological in addition to 
constitutional symptoms, GI, immunology/allergy, stomatology and psychiatric) is to be 
conducted as per the SOE. The subject’s weight is to be measured at every visit that includes a 
physical examination. The subject’s height is to be measured at the Screening visit. 
For adolescents, Tanner staging for assessment of puberty will be done at baseline and 
monitored throughout the study as applicable. Height will be collected for adolescents as per the 
SOE. 
See Section 10.3 for more information on Karnofsky performance status. 

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Vital Signs 
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Electrocardiogram 
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Imaging Studies 
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6.2.13
Hormonal Testing 
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6.2.14
Immunological Testing 
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Sperm / Testicular Tissue or Oocyte Banking 
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Liver Biopsy and Liver Imaging 
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Pregnancy Testing 
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Clinical Laboratory Tests 
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Hematology and Clinical Chemistry 
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Urinalysis 
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Assessment of Clonal Dominance and/or Suspicion of 
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Protocol HGB-204 Version 5.0 
Page 46 of 67 
29 June 2015  
 
bluebird bio, Inc. 
 
Figure 6-1 
Schematic for Assessment of Clonal Dominance  
 
 
Note that for the purposes of this protocol, the term “oligoclonality” is used to describe 
situations in which the above clonal dominance criteria apply. Therefore, if a subject meets the 
above criteria and further work-up is required, a regulatory submission will be performed as per 
FDA Guidance for Industry: Gene Therapy Clinical Trials - Observing Subjects for Delayed 
Adverse Events (November 2006). 
6.2.21.2 
Other Criteria that can Trigger Clinical Work-up for Malignancy 
 Any clinical suspicion of malignancy including leukemia or lymphoma 
 Unexplained WBC count > 30,000 (cells/μL) on two consecutive measurements  
 After achievement of a WBC count within the normal range post-drug product infusion 
and engraftment of gene-modified cells, the development of a WBC < 1000 (cells/μL) 
on two consecutive measurements  
6.2.21.3 
Clinical Work-up for Malignancy 
If any of the above criteria is met, the Medical Monitor will be notified, and a work-up will be 
performed that may include the following:  
 Physical exam  
 Complete blood count (CBC) with differential and lymphocyte subsets  
 Studies to rule out infectious cause 
 Studies to rule out autoimmune disease 
 Imaging studies, as appropriate                                    
 Bone marrow analysis   

Protocol HGB-204 Version 5.0 
Page 47 of 67 
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bluebird bio, Inc. 
 
If clinical results indicate a diagnosis of a malignancy or myelodysplasia, enrollment into this 
study will be suspended, and further analyses will be determined by the Sponsor, in consultation 
with the DMC (see also Section ). It should be noted it may not be possible to distinguish the 
source of malignancy, e.g. arising from transplant-related medications or procedures, or from 
expansion of gene-modified cells due to insertional mutagenesis, and all efforts should be made 
to confirm the source of malignancy before determining to halt or alternatively to resume the 
study.  
If there is no evidence of malignancy or myelodysplasia, subject will continue to be monitored 
as per the protocol-defined SOE, or more frequently at discretion of the investigator and 
sponsor. 
6.2.22 
Adverse Events 
6.2.22.1 
Assessments During the Study 
Monitoring of AEs will be conducted throughout the study. AEs, as defined in the next section, 
will be monitored and recorded in the CRFs from the time informed consent is signed through 
the following timepoints: 
 ≥Grade 1 AEs: through 30 days after LentiGlobin BB305 Drug Product infusion. 
 ≥Grade 2 AEs:  through 12 months after LentiGlobin BB305 Drug Product infusion. 
 SAEs and LentiGlobin BB305 Drug Product-related AEs: through 24 months after 
LentiGlobin BB305 Drug Product infusion. 
All AEs should be monitored until they are resolved or are clearly determined to be due to a 
subject’s stable or chronic condition or inter-current illness(es). See Section 4.5 for AEs 
monitoring of subjects who withdraw from the study. 
Note that at the completion of this study, subjects will be asked to enroll into a long-term 
follow-up study (LTF-303), that will monitor the safety of subjects (including drug product-
related AEs) through a total of 15 years after LentiGlobin BB305 Drug Product infusion. 
6.2.22.2 
Definitions, Documentation, and Reporting 
Adverse Events 
An AE is any change in physical signs, symptoms, and/or clinically significant laboratory 
change occurring in any phase of a clinical study regardless of its relationship to study drug. An 
AE can therefore be any unfavorable and unintended sign (including abnormal laboratory 
findings), symptom, or disease temporally associated with the use of a medicinal product, 
whether or not considered related to the medicinal product. This definition includes intercurrent 
illnesses or injuries, and exacerbation of pre-existing conditions. A pre-existing condition is a 
clinical condition (including a condition being treated) that is diagnosed before the subject signs 
the informed consent form and is documented as part of the subject’s medical history. 
For the purposes of this study, engraftment failure is defined as an AE and is to be reported as 
such. 
Unexpected Adverse Events 
An AE is considered unexpected with LentiGlobin BB305 Drug Product if it is not consistent in 
nature or severity with information contained in the written information or current Investigator’s 
Brochure provided to the investigator by the Sponsor.  

Protocol HGB-204 Version 5.0 
Page 48 of 67 
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bluebird bio, Inc. 
 
Serious Adverse Events 
An SAE is any AE, occurring at any dose and regardless of causality that: 
 Results in death. 
 Is life-threatening. Life-threatening means that the subject was at immediate risk of 
death from the reaction as it occurred, ie, it does not include a reaction which 
hypothetically might have caused death had it occurred in a more severe form. 
 Requires in-patient hospitalization or prolongation of existing hospitalization. 
Hospitalization admissions and/or surgical operations scheduled to occur during the 
study period, but planned prior to study entry are not considered AEs if the illness or 
disease existed before the subject was enrolled in the study, provided that it did not 
deteriorate in an unexpected manner during the study (eg, surgery performed earlier than 
planned). 
 Results in persistent or significant disability/incapacity. Disability is defined as a 
substantial disruption of a subject’s ability to conduct normal life functions. 
 Is a congenital anomaly/birth defect. 
 Is an important medical event. An important medical event is an event that may not 
result in death, be life threatening, or require hospitalization but may be considered an 
SAE when, based upon appropriate medical judgment, it may jeopardize the subject and 
may require medical or surgical intervention to prevent 1 of the outcomes listed in the 
definitions for SAEs. 
 For the purposes of this study, any new malignancy or new diagnosis of a neurologic, 
rheumatologic, or hematologic disorder that, in the investigator’s opinion, is clinically 
significant and requires medical intervention will be considered medically important and 
therefore serious. 
6.2.22.3 
Procedures for AE and SAE Reporting 
Each subject must be carefully monitored for the development of any AEs. This information 
should be obtained in the form of non-leading questions (eg, “How are you feeling?”) and from 
signs and symptoms detected during each examination, observations of study personnel, and 
spontaneous reports from subjects. 
All AEs (serious and non-serious) spontaneously reported by the subject and/or in response to 
an open question from study personnel or revealed by observation, physical examination or 
other diagnostic procedures will be recorded on the appropriate page of the CRF. Any clinically 
relevant deterioration in laboratory assessments or other clinical findings is considered an 
adverse event and must be recorded on the appropriate pages of the CRF. When possible, signs 
and symptoms indicating a common underlying pathology should be noted as 1 comprehensive 
event. 
All SAEs that occur during the course of the study must be promptly reported by the 
investigator to the medical monitor (see below) within 24 hours from the point in time when the 
investigator becomes aware of the SAE. All SAEs must be reported whether or not they are 
considered causally related to LentiGlobin BB305 Drug Product. SAE forms, created 
specifically by bluebird bio, Inc., will be provided to each clinical site. The information 

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 Death (Grade 5) 
If the intensity (grade) changes within a day, the maximum intensity (grade) should be recorded. 
If the intensity (grade) changes over a longer period of time, the changes should be recorded as 
separate events (having separate onset and stop dates for each grade). 
Relationship will be determined by the investigator according to the criteria that follow. 
Relationship of AEs to LentiGlobin BB305 Drug Product will be determined after the start of 
LentiGlobin BB305 Drug Product infusion on Day 0; prior to that time, relationship to study 
treatment other than LentiGlobin BB305 Drug Product will be assessed: 
 Not Related:  Exposure to the study treatment did not occur, or the occurrence of the AE 
is not reasonably related in time, or the AE is considered unlikely to be related to the 
study treatment. 
 Unlikely Related:  The study treatment and the AE were not closely related in time, 
and/or the AE could be explained more consistently by causes other than exposure to the 
study treatment product. 
 Possibly Related:  The study treatment and the AE were reasonably related in time, and 
the AE could be explained equally well by causes other than exposure to the study 
treatment product. 
 Related:  The study treatment and the AE were reasonably related in time, and the AE 
was more likely explained by exposure to the study product than by other causes, or the 
study treatment was the most likely cause of the AE. 
For the purpose of safety analyses, all AEs that are classified as possible or probable will be 
considered treatment-related events. 
6.2.22.4 
Pregnancy 
Pregnancy is neither an AE nor an SAE, unless a complication relating to the pregnancy occurs 
(eg, spontaneous abortion, which may qualify as an SAE). However, all pregnancies occurring 
during this study (in subjects or female partners of subjects) are to be reported in the same time 
frame as SAEs using the Pregnancy Form. The course of all pregnancies, including perinatal 
and neonatal outcome, regardless of whether the subject has discontinued participation in the 
study, will be followed until resolution, including follow-up of the health status of the newborn 
to 6 weeks of age. 
Busulfan has been shown in animal studies to be teratogenic. The effects of administration of 
LentiGlobin BB305 Drug Product on the pregnant female or the developing fetus are unknown. 
Female subjects of child-bearing potential are required to use effective contraception from 
Screening through at least 6 months after drug product infusion. Male subjects are required to 
use effective contraception (including condoms) from Screening through at least 6 months after 
drug product infusion. Beyond 6 months, subjects should discuss with their physician prior to 
resuming unprotected intercourse. 
6.2.23 
Unscheduled Visits 
Unscheduled visits may be performed at any time during the study whenever necessary to assess 
for or to follow-up on AEs or as deemed necessary by the investigator. Evaluations and 

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procedures to be performed at unscheduled visits will be at the investigator’s discretion in 
consultation with the sponsor, and may be based on those listed in the SOE. 
6.2.24 
Long-Term Follow-Up Protocol 
All subjects will be followed for 24 months post-transplant under this protocol. Then, if 
appropriate consent is obtained, subjects will be followed for an additional 13 years under a 
separate long-term follow-up protocol (LTF-303), which will focus on long-term safety, with an 
emphasis on integration site analysis, insertional oncogenesis, and long-term efficacy. This 
follow-up includes recording of SAEs, and archiving of peripheral blood leukocyte cell samples 
for RCL and clonality testing.  
7 
STATISTICAL PROCEDURES 
7.1 
Sample Size Estimation 
The sample size for this study was not determined by formal statistical methods, but is sufficient 
to demonstrate a robust effect on the binary response endpoint, where a responder is defined as a 
subject with production of ≥2.0 g/dL of HbA containing βA-T87Q-globin for the 6-month period 
between Month 18 and Month 24 post-transplant. For example, a one-sided lower 90% 
confidence bound on the rate of response would exceed 22%, should the true response rate be 
50% or more, with at least 80% power. 
7.2 
Populations for Analysis 
The following subject populations will be evaluated and used for presentation and analysis of 
the data: 
 Intent-to-Treat (ITT) population:  All subjects who initiate any study procedures, 
beginning with mobilization by filgrastim and/or plerixafor. 
 Transplant Population (TP):  All subjects who undergo LentiGlobin BB305 Drug 
Product treatment, should this be a smaller number of subjects than ITT. It is anticipated 
that the ITT population and TP will be identical. 
The ITT population is the primary population for the analysis of efficacy parameters and safety 
parameters. The TP is the primary population for transplant parameter endpoints in the case that 
TP and ITT are not identical.  
Since it is of interest to evaluate the efficacy of the gene therapy in subjects who are 
successfully transplanted and are followed for sufficient time to evaluate efficacy, an evaluable 
population will be defined as those subjects who 1) receive LentiGlobin BB305 Drug Product; 
2) engraft, defined as an ANC ≥0.5 × 109/L for 3 consecutive days; and 3) have sufficient study 
visit compliance to acquire clinical laboratory and transfusion data for a minimum of 24 months 
after drug product infusion. Subjects in this population must be compliant with the visit window 
for the Month 24 evaluation.  
7.3 
Procedures for Handling Missing, Unused, and Spurious Data 
No imputation will be performed for missing data elements. Subjects in the ITT population or 
TP will be considered treatment failures in the primary endpoint analysis, should they have less 
than 6 months post-transplant follow-up. 

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When tabulating AE data, partial dates will be handled as follows. If the day of the month is 
missing, the onset day will be set to the first day of the month unless it is the same month and 
year as study treatment. In this case, in order to conservatively report the event as 
treatment-emergent, the onset date will be assumed to be the date of treatment. If the onset day 
and month are both missing, the day and month will be assumed to be January 1, unless the 
event occurred in the same year as the study treatment. In this case, the event onset will be 
coded to the day of treatment in order to conservatively report the event as treatment-emergent. 
A missing onset date will be coded as the day of treatment. 
7.4 
Statistical Methods 
7.4.1 
General Methods 
Tabulations will be produced for appropriate demographic, baseline, efficacy, and safety 
parameters. For categorical variables, summary tabulations of the number and percentage within 
each category (with a category for missing data) of the parameter will be presented. For 
continuous variables, the mean, median, standard deviation, minimum and maximum values will 
be presented. Two-sided, 90% confidence intervals will be calculated as appropriate; a one-
sided 90% confidence interval will be calculated for the primary response endpoint.  
Descriptive summary statistics as well as 2-sided, 90% confidence intervals will be presented on 
selected parameters, as described in the sections below. By-subject listings of data for all 
completed and discontinued subjects will be provided.  
For disease-specific biological parameters and clinical events, including RBC transfusion 
requirements, baseline will be defined as the average of these parameters over the 2 years prior 
to study entry. For other change from baseline analyses, baseline will be defined as the value 
closest to, but prior to transplant. Longitudinal data (collected serially over time on study and 
follow-up) will be presented by appropriate time intervals, such as monthly, quarterly and so 
forth, depending on the nature of the data. 
7.4.2 
Disposition of Subjects 
A tabulation of the disposition of subjects will be presented, including the number enrolled, the 
number with any post-transplant data available for analysis, and the extent of data available. 
Tables and listings will be provided for subjects in each analysis data set, including the 
distribution of subjects according to the β-hemoglobinopathy type. Subject data will also be 
displayed by site. The number of subjects completing the study through 2 years post-transplant 
and reasons for study discontinuation will be reported. Deviations from protocol treatment and 
assessment specifications will be tabulated and listed.  
7.4.3 
Demographic and Baseline Characteristics 
The following demographic and baseline characteristic factors will be summarized: age (current 
and age at diagnosis), country of birth, race and ethnicity, time from diagnosis of 
β-hemoglobinopathy type to confirmation for inclusion in the study, method of diagnosis of 
disease, the presence of any significant co-morbid conditions, the time from diagnosis of 
β-hemoglobinopathy type to treatment, and amount of pRBCs transfused per year (volume per 
year and volume per month) in the 2 years prior to study enrollment. 

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Efficacy and Pharmacodynamic Analysis 
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7.4.5
Safety Analysis 
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7.5
Procedures for Reporting Deviations to Original Statistical Analysis Plan 
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8
ADMINISTRATIVE REQUIREMENTS 
8.1
Good Clinical Practice 
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CCI
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be established at the beginning of the study, maintained for the duration of the study and 
retained according to the appropriate regulations. 
8.2 
Ethical Considerations 
The study will be conducted in accordance with ethical principles founded in the Declaration of 
Helsinki. The IRB / EC and other appropriate institutional regulatory bodies will review all 
appropriate study documentation in order to safeguard the rights, safety, and well-being of the 
subjects. The study will only be conducted at sites where IRB / EC and other appropriate 
institutional regulatory body approval has been obtained. The protocol, Investigator’s Brochure, 
informed consent, advertisements (if applicable), written information given to the subjects 
(including diary cards), safety updates, annual progress reports, and any revisions to these 
documents will be provided to the IRB / EC and other appropriate institutional regulatory bodies 
by the investigator. 
8.3 
Subject Information and Informed Consent 
After the study has been fully explained, written informed consent will be obtained from either 
the subject or his/her guardian or legal representative prior to study participation. The method of 
obtaining and documenting the informed consent and the contents of the consent will comply 
with ICH-GCP and all applicable regulatory requirement(s). 
8.4 
Subject Confidentiality 
In order to maintain subject privacy, all CRFs, accountability records, study reports, and 
communications will identify the subject by initials and the assigned subject number. The 
investigator will grant monitor(s) and auditor(s) from bluebird bio or its designee and regulatory 
authority(ies) access to the subject’s original medical records for verification of data gathered on 
the CRFs and to audit the data collection process. The subject’s confidentiality will be 
maintained and will not be made publicly available to the extent permitted by the applicable 
laws and regulations. 
8.5 
Protocol Compliance 
The investigator will conduct the study in compliance with the protocol provided by bluebird 
bio, and given approval/favorable opinion by the IRB / EC and other appropriate institutional 
regulatory bodies. Modifications to the protocol should not be made without agreement of both 
the investigator and bluebird bio. Changes to the protocol will require written IRB / EC and 
other appropriate institutional regulatory body approval/favorable opinion prior to 
implementation, except when the modification is needed to eliminate an immediate hazard(s) to 
subjects. The IRB / EC may provide, if applicable regulatory authority(ies) permit, expedited 
review and approval/favorable opinion for minor change(s) in ongoing studies that have the 
approval /favorable opinion of the IRB / EC and other appropriate institutional regulatory 
bodies. bluebird bio, Inc., will submit all protocol modifications to the regulatory authority(ies) 
in accordance with the governing regulations.  
When immediate deviation from the protocol is required to eliminate an immediate hazard(s) to 
subjects, the investigator will contact bluebird bio, if circumstances permit, to discuss the 
planned course of action. Any departures from the protocol must be fully documented in the 
CRF and source documentation. 

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8.6 
Direct Access to Source Data 
Monitoring and auditing procedures developed by bluebird bio will be followed, in order to 
comply with GCP guidelines. 
The study will be monitored by bluebird bio or its designee. Monitoring will be done by 
personal visits from a representative of the Sponsor (site monitor) and will include on-site 
review of the CRFs for completeness and clarity, cross-checking with source documents, and 
clarification of administrative matters will be performed. The review of medical records will be 
performed in a manner to ensure that subject confidentiality is maintained. 
The site monitor will ensure that the investigation is conducted according to protocol design and 
regulatory requirements by frequent communications (letter, e-mail, telephone, and fax). 
Regulatory authorities, the IRB / EC and other appropriate institutional regulatory bodies, 
and/or bluebird bio’s clinical quality assurance group may request access to all source 
documents, CRFs, and other study documentation for on-site audit or inspection. Direct access 
to these documents must be guaranteed by the investigator, who must provide support at all 
times for these activities. 
8.7 
Case Report Form Completion 
bluebird bio will provide the clinical sites with a CRF for each subject. 
CRFs will be completed for each study subject. It is the investigator’s responsibility to ensure 
the accuracy, completeness, and timeliness of the data reported in the subject’s CRF. Source 
documentation supporting the CRF data should indicate the subject’s participation in the study 
and should document the dates and details of study procedures, AEs, and subject status.  
The investigator or designated representative should complete the CRF screening pages as soon 
as possible after information is collected, preferably on the same day that a study subject is seen 
for an examination, treatment, or any other study procedure. Any outstanding entries must be 
completed immediately after the final examination. An explanation should be given for all 
missing data. 
The investigator must sign and date the Investigator’s Statement at the end of the CRF to 
endorse the recorded data.  
8.8 
Record Retention 
The investigator will maintain all study records according to ICH-GCP and applicable 
regulatory requirement(s). Records will be retained for at least 2 years after the last marketing 
application approval or 2 years after formal discontinuation of the clinical development of the 
investigational product or according to applicable regulatory requirement(s). If the investigator 
withdraws from the responsibility of keeping the study records, custody must be transferred to a 
person willing to accept the responsibility. bluebird bio, Inc., must be notified in writing if a 
custodial change occurs. 
The Sponsor has full rights over any invention, discovery, or innovation, patentable or not, that 
may occur when performing the study. 

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8.9 
Liability and Insurance 
bluebird bio, Inc., has subscribed to an insurance policy covering, in its terms and provisions, its 
legal liability for injuries caused to participating persons and arising out of this research 
performed strictly in accordance with the scientific protocol as well as with applicable law and 
professional standards. 
8.10 
Publication and Presentation of Study Findings and Use of Information 
All information regarding LentiGlobin BB305 lentiviral vector and Drug Product supplied by 
bluebird bio to the investigator is privileged and confidential information. The investigator 
agrees to use this information to accomplish the study and will not use it for other purposes 
without consent from bluebird bio. It is understood that there is an obligation to provide 
bluebird bio with complete data obtained during the study. The information obtained from the 
clinical study will be used towards the development of LentiGlobin BB305 lentiviral vector and 
Drug Product and may be disclosed to regulatory authority(ies), other investigators, corporate 
partners, or consultants as required. 
It is anticipated that the results of this study will be presented at scientific meetings and/or 
published in a peer reviewed scientific or medical journal. A Publications Committee comprised 
of investigators participating in the study and representatives from bluebird bio, as appropriate, 
will be formed to oversee the publication and presentation of the study results, which will reflect 
the experience of all participating clinical sites.  
No publication or disclosure of study results will be permitted except under the terms and 
conditions of a separate written agreement between Sponsor and the investigator and/or the 
investigator's institution. 
 

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9 
REFERENCES 
 
Adams,J.G., III and Coleman,M.B. (1990). Structural hemoglobin variants that produce the 
phenotype of thalassemia. Semin. Hematol. 27, 229-238. 
Biffi,A., Bartolomae,C.C., Cesana,D., Cartier,N., Aubourg,P., Ranzani,M., Cesani,M., 
Benedicenti,F., Plati,T., Rubagotti,E., Merella,S., Capotondo,A., Sgualdino,J., Zanetti,G., 
Von,K.C., Schmidt,M., Naldini,L., and Montini,E. (2011). Lentiviral vector common integration 
sites in preclinical models and a clinical trial reflect a benign integration bias and not oncogenic 
selection. Blood 117, 5332-5339. 
bluebird bio France, Technical Report NC-11-001-R Comparison of LentiGlobin vectors BB305 
and HPV 569. Assessment of transduction efficiency in Sickle Cell Anemia (S/E) CD34+ cells 
as measured in colony forming cells and long term culture colony forming cells, and assessment 
of globin chain expression in BFUe colonies, March 2012. 
bluebird bio France, Technical Report NC-11-004-R Comparison of LentiGlobin vectors BB305, 
HPV524 and HPV 569. Assessment of transduction efficiency in Sickle Cell Disease (S/+thal) 
CD34+ cells as measured in colony forming cells and long term culture colony initiating cells, 
and assessment of globin chain expression in erythroid colonies, 4 April 2012. 
bluebird bio, Inc., Technical Report NC-12-016-R, Amendment 1, Medizinische Hochschule 
Hannover, 06 November, 2012.  
Boztug,K., Schmidt,M., Schwarzer,A., Banerjee,P.P., Diez,I.A., Dewey,R.A., Bohm,M., 
Nowrouzi,A., Ball,C.R., Glimm,H., Naundorf,S., Kuhlcke,K., Blasczyk,R., Kondratenko,I., 
Marodi,L., Orange,J.S., Von,K.C., and Klein,C. (2010). Stem-cell gene therapy for the Wiskott-
Aldrich syndrome. N. Engl. J. Med. 363, 1918-1927. 
Cao,A., Galanello,R., Rosatelli,M.C., Argiolu,F., and De,V.S. (1996). Clinical experience of 
management of thalassemia: the Sardinian experience. Semin. Hematol. 33, 66-75. 
Cappellini,M.D., Cohen,A., Piga,A., Bejaoui,M., Perrotta,S., Agaoglu,L., Aydinok,Y., 
Kattamis,A., Kilinc,Y., Porter,J., Capra,M., Galanello,R., Fattoum,S., Drelichman,G., 
Magnano,C., Verissimo,M., Athanassiou-Metaxa,M., Giardina,P., Kourakli-Symeonidis,A., 
Janka-Schaub,G., Coates,T., Vermylen,C., Olivieri,N., Thuret,I., Opitz,H., Ressayre-Djaffer,C., 
Marks,P., and Alberti,D. (2006). A phase 3 study of deferasirox (ICL670), a once-daily oral iron 
chelator, in patients with beta-thalassemia. Blood 107, 3455-3462. 
Cartier, N., Hacein-Bey, Abina S., Bartolomae, C. C., Schmidt, M., Kutschera, I., and Vidaud, 
M. Hematopoietic Stem Cell Gene Therapy for X-linked Adrenoluekodystrophy.  10-29-2012.  
Ref Type: Conference Proceeding 

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Cavazzana-Calvo,M., Payen,E., Negre,O., Wang,G., Hehir,K., Fusil,F., Down,J., Denaro,M., 
Brady,T., Westerman,K., Cavallesco,R., Gillet-Legrand,B., Caccavelli,L., Sgarra,R., Maouche-
Chretien,L., Bernaudin,F., Girot,R., Dorazio,R., Mulder,G.J., Polack,A., Bank,A., Soulier,J., 
Larghero,J., Kabbara,N., Dalle,B., Gourmel,B., Socie,G., Chretien,S., Cartier,N., Aubourg,P., 
Fischer,A., Cornetta,K., Galacteros,F., Beuzard,Y., Gluckman,E., Bushman,F., Hacein-Bey-
Abina,S., and Leboulch,P. (2010). Transfusion independence and HMGA2 activation after gene 
therapy of human beta-thalassaemia. Nature 467, 318-322. 
Colah,R., Gorakshakar,A., and Nadkarni,A. (2010). Global burden, distribution and prevention 
of beta-thalassemias and hemoglobin E disorders. Expert. Rev. Hematol. 3, 103-117. 
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(2007). Consequences and costs of noncompliance with iron chelation therapy in patients with 
transfusion-dependent thalassemia: a literature review. Transfusion 47, 1919-1929. 
Gaziev,J., Sodani,P., Polchi,P., Andreani,M., and Lucarelli,G. (2005). Bone marrow 
transplantation in adults with thalassemia: Treatment and long-term follow-up. Ann. N. Y. Acad. 
Sci. 1054, 196-205. 
Hacein-Bey-Abina,S., Garrigue,A., Wang,G.P., Soulier,J., Lim,A., Morillon,E., Clappier,E., 
Caccavelli,L., Delabesse,E., Beldjord,K., Asnafi,V., Macintyre,E., Dal,C.L., Radford,I., 
Brousse,N., Sigaux,F., Moshous,D., Hauer,J., Borkhardt,A., Belohradsky,B.H., Wintergerst,U., 
Velez,M.C., Leiva,L., Sorensen,R., Wulffraat,N., Blanche,S., Bushman,F.D., Fischer,A., and 
Cavazzana-Calvo,M. (2008). Insertional oncogenesis in 4 patients after retrovirus-mediated gene 
therapy of SCID-X1. J. Clin. Invest 118, 3132-3142. 
Hoffbrand,A.V., Cohen,A., and Hershko,C. (2003). Role of deferiprone in chelation therapy for 
transfusional iron overload. Blood 102, 17-24. 
Howe,S.J., Mansour,M.R., Schwarzwaelder,K., Bartholomae,C., Hubank,M., Kempski,H., 
Brugman,M.H., Pike-Overzet,K., Chatters,S.J., de,R.D., Gilmour,K.C., Adams,S., Thornhill,S.I., 
Parsley,K.L., Staal,F.J., Gale,R.E., Linch,D.C., Bayford,J., Brown,L., Quaye,M., Kinnon,C., 
Ancliff,P., Webb,D.K., Schmidt,M., Von,K.C., Gaspar,H.B., and Thrasher,A.J. (2008). 
Insertional mutagenesis combined with acquired somatic mutations causes leukemogenesis 
following gene therapy of SCID-X1 patients. J. Clin. Invest 118, 3143-3150. 
Imren,S., Payen,E., Westerman,K.A., Pawliuk,R., Fabry,M.E., Eaves,C.J., Cavilla,B., 
Wadsworth,L.D., Beuzard,Y., Bouhassira,E.E., Russell,R., London,I.M., Nagel,R.L., 
Leboulch,P., and Humphries,R.K. (2002). Permanent and panerythroid correction of murine beta 
thalassemia by multiple lentiviral integration in hematopoietic stem cells. Proc. Natl. Acad. Sci. 
U. S. A 99, 14380-14385. 
Inoue,N., Izui-Sarumaru,T., Murakami,Y., Endo,Y., Nishimura,J., Kurokawa,K., Kuwayama,M., 
Shime,H., Machii,T., Kanakura,Y., Meyers,G., Wittwer,C., Chen,Z., Babcock,W., Frei-Lahr,D., 
Parker,C.J., and Kinoshita,T. (2006). Molecular basis of clonal expansion of hematopoiesis in 2 
patients with paroxysmal nocturnal hemoglobinuria (PNH). Blood 108, 4232-4236. 

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Lucarelli,G., Andreani,M., and Angelucci,E. (2001). The cure of the thalassemia with bone 
marrow transplantation. Bone Marrow Transplant. 28 Suppl 1, S11-S13. 
Lucarelli,G., Galimberti,M., Polchi,P., Angelucci,E., Baronciani,D., Giardini,C., Andreani,M., 
Agostinelli,F., Albertini,F., and Clift,R.A. (1993). Marrow transplantation in patients with 
thalassemia responsive to iron chelation therapy. N. Engl. J. Med. 329, 840-844. 
Lucarelli,G., Isgro,A., Sodani,P., and Gaziev,J. (2012). Hematopoietic stem cell transplantation 
in thalassemia and sickle cell anemia. Cold Spring Harb. Perspect. Med. 2, a011825. 
Michlitsch,J., Azimi,M., Hoppe,C., Walters,M.C., Lubin,B., Lorey,F., and Vichinsky,E. (2009). 
Newborn screening for hemoglobinopathies in California. Pediatr. Blood Cancer 52, 486-490. 
Miyamoto,T., Shinozuka,T., Maeda,H., Hirasawa,T., Muramatsu,T., Murakami,M., Makino,T., 
Itagaki,H., and Nakamura,Y. (2004). Effect of peripheral blood progenitor cell dose on 
hematopoietic recovery: identification of minimal progenitor cell requirements for rapid 
engraftment. Bone Marrow Transplant. 33, 589-595. 
Modell,B., Khan,M., and Darlison,M. (2000). Survival in beta-thalassaemia major in the UK: 
data from the UK Thalassaemia Register. Lancet 355, 2051-2052. 
Montini E, Biffi A, Calabria A, Cesani M, Benedicenti F, and Lorioli L. Integration site analysis 
in a clinical trial of lentiviral vector-based HSC gene therapy for MLD shows high levels of 
polyclonal hematopoietic reconstitution and no signs of genotoxicity at one year after transplant. 
American Society of Gene & Cell Therapy 15th Annual Meeting.  2012. 5-16-2012.  
Ref Type: Conference Proceeding 
Montini,E., Cesana,D., Schmidt,M., Sanvito,F., Ponzoni,M., Bartholomae,C., Sergi,S.L., 
Benedicenti,F., Ambrosi,A., Di,S.C., Doglioni,C., Von,K.C., and Naldini,L. (2006). 
Hematopoietic stem cell gene transfer in a tumor-prone mouse model uncovers low genotoxicity 
of lentiviral vector integration. Nat. Biotechnol. 24, 687-696. 
Murakami,Y., Inoue,N., Shichishima,T., Ohta,R., Noji,H., Maeda,Y., Nishimura,J., Kanakura,Y., 
and Kinoshita,T. (2012). Deregulated expression of HMGA2 is implicated in clonal expansion of 
PIGA deficient cells in paroxysmal nocturnal haemoglobinuria. Br. J. Haematol. 156, 383-387. 
Oh,I.H., Fabry,M.E., Humphries,R.K., Pawliuk,R., Leboulch,P., Hoffman,R., Nagel,R.L., and 
Eaves,C. (2004). Expression of an anti-sickling beta-globin in human erythroblasts derived from 
retrovirally transduced primitive normal and sickle cell disease hematopoietic cells. Exp. 
Hematol. 32, 461-469. 
Olivieri,N.F. (1999). The beta-thalassemias. N. Engl. J. Med. 341, 99-109. 
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thalassemia. Blood 89, 739-761. 
Paruzynski A, Boztug K Braun CJ Pyl PT Ball C Nowrouzi A et al. Molecular Therapy 20, 
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Ellis,J., London,I.M., Eaves,C.J., Humphries,R.K., Beuzard,Y., Nagel,R.L., and Leboulch,P. 
(2001). Correction of sickle cell disease in transgenic mouse models by gene therapy. Science 
294, 2368-2371. 
Perez-Simon,J.A., Caballero,M.D., Corral,M., Nieto,M.J., Orfao,A., Vazquez,L., Amigo,M.L., 
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CD34+ cells to ensure successful leukapheresis and engraftment in autologous peripheral blood 
progenitor cell transplantation. Transfusion 38, 385-391. 
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crossroads. Blood 119, 1107-1116. 
Ronen,K., Negre,O., Roth,S., Colomb,C., Malani,N., Denaro,M., Brady,T., Fusil,F., Gillet-
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treat beta-thalassemia. Mol. Ther. 19, 1273-1286. 
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Bredeson,C.N., Cowan,M.J., Gale,R.P., Hale,G.A., Horan,J., Hongeng,S., Eapen,M., and 
Walters,M.C. (2011). HLA-matched sibling bone marrow transplantation for beta-thalassemia 
major. Blood 117, 1745-1750. 
Scaramuzza, S., Ferrua, F., Castiello, M. C., Giannelli, S, Cicalese, M. P, Evangelino, C., 
Assanelli, A., Biasco, L., Casiraghi, M., Bosticardo, M, Finocchi, A, Metin, A, Banerjee, P. P., 
Orange, J. S., Biffi, A., Ciceri, F., Roncarolo, M. G., Villa, A., Naldini, L., and Aiuti, A. 
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Stein,S., Ott,M.G., Schultze-Strasser,S., Jauch,A., Burwinkel,B., Kinner,A., Schmidt,M., 
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(2010). Genomic instability and myelodysplasia with monosomy 7 consequent to EVI1 
activation after gene therapy for chronic granulomatous disease. Nat. Med. 16, 198-204. 
Uchida,N., Washington,K.N., Lap,C.J., Hsieh,M.M., and Tisdale,J.F. (2011). Chicken HS4 
insulators have minimal barrier function among progeny of human hematopoietic cells 
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Jonlin,E., Papayannopoulou,T., Anagnostopoulos,A., and Stamatoyannopoulos,G. (2013). 
Hematopoietic stem cell mobilization for gene therapy: superior mobilization by the combination 
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Hum. Gene Ther. 24, 852-860. 
 
 

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10 
APPENDICES 
10.1 
Creatinine Clearance Calculation 
 
Calculate creatinine clearance for males and females as follows: 
In males: 









dL
mg
creatinine
kg
weight
age
/
72
140



 
In females: 









85
.0
/
72
140




dL
mg
creatinine
kg
weight
age
 
 

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10.2 
Peripheral Blood Stem Cell Collection Recommendations 
Product 
Parameter 
Criteria 
Apheresis 
machine 
COBE Spectra (Terumo BCT), Optia (Terumo BCT), Amicus (Fenwal) 
Venous access 
Bilateral peripheral venous catheters or central venous access catheter 
Collection 
Large volume processed daily (>15L of recirculated blood volume). 
ACD-A to whole blood ratio of 1:12; use of heparin is allowed. 
 
For Spectra manual program: 
 Enter patient’s weight, height, total blood volume, Hct 
 Collect line monitored frequently to ensure correct interface position 
 
Target Hct to be in 3% ± 1% on the collect line colorgrama.  Change plasma 
pump 0.1 – 0.3 mls/min to maintain desired Hct. 
 
Collect line rate 0.9 – 1.5 mls/min 
 
For Optia: 
 Enter patient’s weight, height, total blood volume, Hct, WBC, Platelet, to 
establish flow rates (~ 30 to 40 ml/min) appropriately.  
 Start with collection preference at default.  If RBCs are seen exiting the chamber, 
decrease collection preference immediately to 20.b 
 Faster centrifugation rate also aids in adequate collectiona. 
 
For Amicus: 
 
Enter patient’s weight, height, total blood volume, Hct to establish flow 
rates. 
 
Set MNC offset at 1.5 and RBC offset at 6.0. 
 
Monitor the MNC chain collection during each cycle.  Increase MNC offset 
as needed to collect as many MNCs as possible. 
 
Process at least 7 cycles with cycle volume 1000 mls. 
Recommended 
Monitoring  
CD34+ count from the collection bag after 1 to 2 hours of collection  
Appearance 
Container intact with no leakage 
No gross clumps or clots 
Label 
Proper label affixed 
Product Seal 
Hermetically seal product with three (3) seals.  Cut third seal away from the product 
leaving 2 seals with the product and as much tubing as possible.  Heat seal or 
grommets are acceptable seals. 
Abbrev. : ACD-A, Anticoagulant Citrate Dextrose Solution, Solution A, USP (2.13% free citrate ion); Hct, 
hematocrit; MNC, mononuclear cells; RBC, red blood cells; WBC, white blood cells 
a   Due to increased number of nucleated RBCs and reticulocytes in the peripheral blood, the color of the buffy coat 
is darker than compared to healthy donors/oncology patients. Therefore, following standard collection 
recommendations (low Hct, colorgram in ~2% to 3% range) will erroneously target the platelet layer in 
thalassemia patients, especially in patients with prior splenectomy. 
b   Using the Optia and observing the Cell Separation connector, one may notice a second RBC layer above the buffy 
coat.  If seen, quickly go deeper to collect MNCs vs. RBCs by changing the collection preference. 
 
Target collection CD34+ is ≥ 10×106 CD34+ cells/kg. 

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10.3 
Karnofsky Performance Status Scale 
 
The following table presents the Karnofsky performance status scale. 
Points 
Description 
100 
Normal, no complaints, no evidence of disease 
90 
Able to carry on normal activity 
80 
Normal activity with effort; some signs or symptoms of disease 
70 
Cares for self; unable to carry on normal activity or to do active work 
60 
Required occasional assistance but is able to care for most of his/her needs 
50 
Required considerable assistance and frequent medical care 
40 
Disabled; required special care and assistance 
30 
Severely disabled; hospitalization indicated. Death not imminent 
20 
Very sick; hospitalization necessary; active support treatment necessary 
10 
Moribund; fatal processed progressing rapidly 
0 
Dead 
Source:  Mor V, Laliberte L, Morris JN, Wiemann M. The Karnofsky Performance Status Scale:  an examination of its reliability 
and validity in a research setting. Cancer 1984;53:2002-2007. 
 

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10.4 
Iron Reduction Guidelines 
10.4.1 
For subjects who achieve transfusion independent (TI) status within 
3 months post-drug product infusion (“post-HSCT”) 
Assessing chelation needs  
Screening cardiac MRI and liver LIC will be used to evaluate the need for starting iron reduction 
methods at Day +90 post-HSCT based on the table below. 
 
Table 10-1 
Iron reduction guidelines post-drug product infusion 
Liver LIC (mg/g) 
Cardiac T2* (ms) 
>20 ms 
20-10 ms 
< 8 mg/g 
No chelation is required  
Chelation is indicated 
8-15 mg/g 
Consider based on serum ferritin and 
hepatitis serologya 
Chelation is indicated 
>15 mg/g 
Chelation is indicated 
Chelation is indicated 
a Chelation is recommended only if serum ferritin is >2000 ng/ml at Day +90 or hepatitis B/C serology was positive pre-transplant. 
 
 
If iron reduction methods need to be started based on guidelines in Table 10-1, they should be 
initiated after Day +90, once subjects are medically stable. Earlier initiation of iron reduction 
(<90 days post-HSCT) should be discussed with the medical monitor.  
Subjects who have achieved the TI status but do not start iron reduction methods on Day +90 
(based on Table 10-1), should still be continually reassessed for chelation needs post-HSCT 
based on the SOE of the relevant protocol. 
Iron Reduction Recommendations 
Chelation is recommended using oral deferasirox (Exjade) or deferoxamine (Desferal) infusion, 
depending on institutional choice and subjects’ pre-transplant experience.  
Deferiprone is not recommended at Day +90 because of its effect on the bone marrow, but its use 
can be evaluated > 6 months post-transplant.  
Starting dose of chelator is recommended based on institutional protocols. Doses may need to be 
adjusted continuously based on serum ferritin levels to prevent toxicity.  
Phlebotomy can also be used in subjects who have Hb consistently ≥ 11 g/dl.  
Reducing and Stopping Iron Reduction Treatment 
To avoid toxicity, once serum ferritin is ≤ 1000 ng/ml, downward adjustment of dose of chelator 
and decreasing the frequency of phlebotomy is advised, as per institutional protocols. 
Iron reduction methods should be stopped once liver LIC is ≤ 5 mg/g dry wt. and serum ferritin 
is <500 ng/ml. 

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10.4.2 
For subjects who are not TI at 3 months post-HSCT 
For subjects who contain to require transfusions, chelation should be re-started at Day +90 post-
HSCT following guidelines in Section 10.4.1 
To avoid toxicity, once serum ferritin is ≤ 1000 ng/ml, downward adjustment of dose of chelator 
and decreasing the frequency of phlebotomy is advised, as per institutional protocols. 
Chelation should be discontinued if they become TI, and their Liver LIC is <5mg/g and serum 
ferritin is <500 ng/ml.  
Subjects who are not taking chelators should be continually reassessed for chelation needs post-
HSCT based on the SOE of the relevant protocol. 
 

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Protocol Title: 
A Phase 1/2, Open Label Study Evaluating the Safety and Efficacy of Gene 
Therapy in Subjects with -Thalassemia Major by Transplantation of 
Autologous CD34+ Stem Cells Transduced Ex Vivo with a Lentiviral 
A-T87Q-Globin Vector (LentiGlobin BB305 Drug Product) 
Protocol Number: HGB-204 Version 5.0 (29 June 2015)  
INVESTIGATOR STATEMENT 
I have read, understood, and agree to abide by all the conditions and instructions contained in this protocol. 
I understand that all documentation provided to me by bluebird bio or its designated representative(s) concerning 
this study that has not been published previously will be kept in the strictest confidence. This documentation 
includes the study protocol, investigator brochure, case report forms, and other scientific data. 
I agree to personally conduct or supervise the described investigation(s). 
I agree to inform any subjects, or any persons used as controls, that the Drug Product is being used for 
investigational purposes and I will ensure that the requirements relating to obtaining informed consent, as per local 
regulations and under Good Clinical Practice (GCP), are met. 
I agree to report to the Sponsor adverse events that occur in the course of the investigation(s) in accordance with this 
protocol and as required by local regulations and under GCP.  
I have read and understand the information in the Investigator’s Brochure, including the potential risks and side 
effects of the Drug Product. 
I agree to maintain adequate and accurate records and to make those records available for inspection in accordance 
with local regulations and under GCP. 
I will ensure that an ethics committee that complies with all local regulations and GCP requirements will be 
responsible for the initial and continuing review and approval of the clinical investigation.  
I also agree to promptly report to the ethics committee all changes in the research activity and all unanticipated 
problems involving risks to human subjects or others.  
I agree that this study will not commence without the prior approval of the appropriate national health authorities 
together with a properly constituted ethics committee. I agree that no changes will be made to the study protocol 
without the prior written approval of bluebird bio and the aforementioned regulatory bodies, as applicable in the 
relevant laws and regulations. 
I agree to ensure that all associates, colleagues, and employees assisting in the conduct of the study(ies) are 
informed about their obligations in meeting the above commitments. 
 
Investigator Name 
 
Investigator Signature 
 
Date 
 
Investigational site or name of institution and location (printed)