Gene Therapy of the β- Hemoglobinopathies by Lentiviral ...

Gene Therapy of the β-Hemoglobinopathies by Lentiviral

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Citation Negre, O., A. Eggimann, Y. Beuzard, J. Ribeil, P. Bourget, S.Borwornpinyo, S. Hongeng, et al. 2016. “Gene Therapy of theβ-Hemoglobinopathies by Lentiviral Transfer of the βA(T87Q)-Globin Gene.” Human Gene Therapy 27 (2): 148-165. doi:10.1089/hum.2016.007. http://dx.doi.org/10.1089/hum.2016.007.

Published Version doi:10.1089/hum.2016.007

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Gene Therapy of the b-Hemoglobinopathies by LentiviralTransfer of the bA(T87Q)-Globin Gene

Olivier Negre,1,2,* Anne-Virginie Eggimann,1 Yves Beuzard,2,3 Jean-Antoine Ribeil,4

Philippe Bourget,4 Suparerk Borwornpinyo,5 Suradej Hongeng,5 Salima Hacein-Bey,6

Marina Cavazzana,4 Philippe Leboulch,2,3,5,7 and Emmanuel Payen2,3,8,*1bluebird bio, Cambridge, Massachusetts; 2CEA, Institute of Emerging Disease and Innovative Therapies (iMETI), Fontenay aux Roses, France; 3UMR 007, University

of Paris 11 and CEA, CEA-iMETI, Fontenay aux Roses, France; 4Necker Hospital, Assistance Publique-Hopitaux de Paris, Paris, France; 5Mahidol University, Bangkok,

Thailand; 6Immunology Laboratory, Groupe Hospitalier Universitaire Paris-Sud, Assistance Publique–Hopitaux de Paris, Paris, France; 7Harvard Medical School and

Genetics Division, Department of Medicine, Brigham & Women’s Hospital, Boston, Massachusetts; 8INSERM, Paris, France.

b-globin gene disorders are the most prevalent inherited diseases worldwide and result from abnormal b-globin synthesis or structure. Novel therapeutic approaches are being developed in an effort to movebeyond palliative management. Gene therapy, by ex vivo lentiviral transfer of a therapeutic b-globin genederivative (bAT87Q-globin) to hematopoietic stem cells, driven by cis-regulatory elements that confer high,erythroid-specific expression, has been evaluated in human clinical trials over the past 8 years. bAT87Q-globin is used both as a strong inhibitor of HbS polymerization and as a biomarker. While long-termstudies are underway in multiple centers in Europe and in the United States, proof-of-principle of efficacyand safety has already been obtained in multiple patients with b-thalassemia and sickle cell disease.

INTRODUCTION

SICKLE CELL DISEASE (SCD) and b-thalassemia major(b-TM), the latter defined clinically as transfusion-dependent cases regardless of the underlying geno-type, are the most common monogenic disordersworldwide with approximately 400,000 affected con-ceptions or births each year.1,2 These disorders fallinto two large groups of b-globin gene mutations thatresult in either abnormal hemoglobin structure (SCD)or massively reduced/absent production of b-globinchains (b-TM). The clinical manifestations of theseinherited disorders typically appear several monthsafter birth, when gene expression switches from thefetal c-globin chain, which forms fetal hemoglobin(HbF), to the adult bA-globin chain forming hemo-globin (HbA).3 Note that adult bA-globin is also simplyreferred to as b-globin when no confusion with otherb-like globin chains is possible. In low-income coun-tries, most of the affected children succumb in earlychildhood, whereas in developed countries, neonataldiagnosis and supportive care have greatly improved

survival. However, even with modern and specializedcare, life expectancy is still reduced by several de-cades,4–6 and quality of life greatly suffers.7–9

Allogenic hematopoietic stem cell transplanta-tion (AHSCT) as a curative option is currentlyrecommended for b-TM if a human leukocyte an-tigen (HLA)-matched sibling donor is available.10,11

Disease-free survival after AHSCT is approxima-tely 88% in pediatric subjects12 and 65% in adults.13

Recent transplantation trials conducted duringthe last 15 years for young SCD patients reporteda disease free-survival rate of 85–90%.11 However,fewer than 25% of patients have a suitable in-trafamilial donor.14 In the absence of matchedsibling donors, AHSCT from HLA-matched unre-lated or haploidentical donors or minimally mis-matched cord blood products may be used as thesource of donor cells, although these approachesexhibit a lower benefit/risk ratio and thus remainexperimental.15 Although outcomes are improv-ing, AHSCT continues to carry a substantial risk

*Correspondence: Dr. Emmanuel Payen, CEA/iMETI, 18 route du Panorama, 92260 Fontenay aux Roses, France. E-mail: [email protected] or Dr. Olivier Negre,bluebird bio, 150 Second Street, Cambridge, MA 02141. E-mail: [email protected]

ª Olivier Negre et al. 2016; Published by Mary Ann Liebert, Inc. This Open Access article is distributed under the terms of the CreativeCommons Attribution Noncommercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits any noncommercial use,distribution, and reproduction in any medium, provided the original author(s) and the source are credited.

148 j HUMAN GENE THERAPY, VOLUME 27 NUMBER 2 DOI: 10.1089/hum.2016.007Mary Ann Liebert, Inc.

of severe adverse events and mortality,16,17 bothincreasing with recipient age and disease severi-ty.18,19 Severe adverse events include graft failure,graft-versus-host disease (GVHD), early or late sideeffects from conditioning regimens that are bothmyeloablative and immunosuppressive (infections,hemorrhages, secondary malignancies), and aggra-vation of preexisting organ damage.20–22

For patients who lack a suitable HLA-matcheddonor, ex vivo gene therapy using autologous HSCsbrings hope as a potential curative treatment option.If proven safe and effective, gene therapy may thenbe extended to most b-TM and severe SCD patients,as there is no concern here for histocompatibility-related complications and the conditioning regimendoes not need to include immunosuppressive drugs.However, gene therapy shares with AHSCT therisks associated with the transplant procedureand the toxicity of the myeloablative agent. Globingene addition to HSCs by means of lentiviral vectors(LVs) is a promising approach under investigation(Fig. 1). Several clinical trials of gene therapy forb-TM and severe SCD are ongoing in France and inthe United States (Tables 1 and 2). Other recentapproaches under study for the gene therapy of theb-hemoglobinopathies include pharmacological23 orgenetic induction of c-globin production throughinterference with the BCL11A pathway24,25 or dis-ruption of the BCL11A erythroid enhancer byCRISPR/CAS9 technology as well as zinc finger ortranscription activator-like effector nuclease,26,27

or even attempts at repairing the defective bA-globin gene in HSCs by genome editing.28–30 These

approaches are at the stage of collecting evidenceof efficacy in relevant cell and animal models andscoring potentially untoward off-target events. Evenif these approaches are ultimately successful, geneaddition has the advantage of making use of a singleproduct applicable to all cases of b-TM and SCDregardless of the genotype, whereas gene repair willhave to tackle separately the hundreds of mutationsknown to cause b-thalassemia in humans.

While we recognize the important contribu-tions from other laboratories, this review willlargely focus on our own experience with the de-velopment of the HPV569 and BB305 vectors, self-inactivating (SIN) LVs bearing a human bA-globinmini-gene encoding an ‘‘antisickling’’ b-globin withamino-acid substitution (T87Q) and driven by cis-regulatory elements of the human b-globin genelocus (promoter, locus control region [LCR] ele-ments), designed in the Leboulch laboratory incollaboration with bluebird bio (formerly GenetixPharmaceuticals).

THE b-HEMOGLOBINOPATHIESThe b-thalassemias

b-TM is a microcytic hemolytic anemia that israpidly fatal in the absence of palliative life-longred blood cell (RBC) transfusions and iron chela-tion.31 The disease results from absence (b0) ormassive reduction (e.g., b+, bE/b0) in bA-globin geneexpression. While complete absence of bA-globinexpression is not compatible with RBC production,even a severe diminution in its expression also re-

Figure 1. The milestones of ex vivo gene therapy research and development for hemoglobin disorders. LG001, HGB204, HGB205, and HGB206 clinical studiesare conducted with our lentiviral vectors (Table 1). Gene therapy trials using other lentiviral vectors are summarized in Table 2.

GENE THERAPY OF b-HEMOGLOBINOPATHIES 149

Tab

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ber

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sor

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date

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prim

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plet

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udy15

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tical

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sults in b-TM. This is because the protein compo-nent of adult hemoglobin comprises two a- and twob-globin chains, and massive decrease in b-globinexpression results in a relative excess of unpairedand toxic free a-chains. Free a-chains precipitate,damage the cell membrane,32 and sequester thechaperone heat shock protein 70 (HSP70), which isno longer available to protect the GATA-1 ery-throid maturation transcription factor fromcaspase-3 cleavage.33 a/b-chain imbalance resultsin cell death and ineffective erythropoiesis (dyser-ythropoiesis)34 in bone marrow as well as reducederythrocyte lifespan and hemolysis.35

Among the severe b-thalassemias, the bE/b0 ge-notype presents interesting features and is highlyfrequent. In this disease, one b-globin allele iscompletely silent (b0) while the other encodes themissense mutation (26AAG>GAG) that results bothin an amino-acid substitution (GLU26LYS) and inabnormal RNA splicing.36 Part of the transcribedbE mRNA is not translated because of abnormalsplicing, whereas the small amount of mutated(GLU26LYS) protein produced is functional butslightly unstable.37 Thus, when the bE allele iscompounded with a b0 allele, a profound decreasein b-like globin production is observed.38 Approxi-mately 50% of patients with bE/b0-thalassemiahave transfusion-dependent b-TM, depending onthe patient’s genetic makeup in modulators of dis-ease severity.39 Hemoglobin E is one of the world’smost common mutations40 and is especially preva-lent in Southeast Asia. The frequency can approach60% of the population in some parts of Thailand,Cambodia, and Laos,40 where it is estimated that100,000 new cases of bE/b0 are expected in the nextfew decades. It is also found at high frequency inother Asian nations (India, Sri Lanka, Malaysia,and southern China) and increasingly in Europeand North America through immigration.41,42

Sickle cell diseaseSCD is a multisystem disorder that results from

a single mutation in the bA-globin gene, changing aglutamic acid into a valine at the sixth position ofthe b-globin chain, known as the bS mutation.43 Inhomozygous patients (bS/bS), the most frequentgenotype, or in the compound heterozygous states(bS/bC, bS/bThalassemia) of SCD, the chronic hemo-lytic anemia is complicated by painful vaso-occlusive crises (VOC), acute chest syndrome (ACS),increased risk of infections, as well as organ vascu-lopathy and dysfunctions, particularly affectingbrain, kidney, lung, heart, bone, eye, and the skin.44

At the protein level, the single amino-acid substi-tution at codon 6 (bS chain) prompts the formation of

HbS polymers at low oxygen pressure,45 the rate ofwhich is proportional to the corpuscular concentra-tion of HbS, the extent of hemoglobin deoxygen-ation in the microcirculation,46 and the quantity ofHbF, which inhibits HbS polymerization.47 Vaso-occlusion, resulting from erythrocyte stiffness andthe adhesive interaction of abnormal erythrocytesto endothelial cells and leukocytes,48 leads to organinfarction and inflammation, which in turn en-hances adhesive interactions,49 microvascularocclusion, and ischemia. The increased number ofactivated white blood cells produces high levels ofreactive oxygen species (ROS) and other factorscontributing actively to micro-vessel clogging, in-creased local hypoxia, and increased proportionsof rigid RBCs containing HbS polymers.50 Thecycles of ischemia/reperfusion cause oxidativestress51 that contributes to the proinflammatoryphenotype,52 worsening the vicious circle. Further-more, acute and chronic intravascular hemolysisimpairs endothelial functions, causing progressivesystemic and pulmonary vasculopathy.53

CHALLENGES FOR EFFECTIVEGENE THERAPYThe therapeutic bA(T87Q)-globin gene

With regard to the gene therapy of SCD by geneaddition, wild-type human bA-globin is a relativelyweak inhibitor of HbS polymerization because itacts by mere dilution. When PO2 is relatively low,as in capillary vessels, phenylalanine 85 (bF85) andleucine 88 (bL88) form an acceptor hydrophobicpocket on one a2b

S2 tetramer that binds to the mu-

tated valine (bV6) of a close a2bS

2 complex.54 Thisphenomenon is responsible for HbS fiber polymeri-zation after further aggregation and fiber elonga-tion.46 HbA (a2b

A2) has these same hydrophobic

residues and thus does not inhibit polymer forma-tion when it is incorporated.54–56 In contrast, otherhuman ‘‘b-like’’-globin chains such as c-globin andd-globin are stronger inhibitors.57,58 This inhibitionof polymerization is best exemplified by cases ofhereditary persistence of HbF (HPFH), especiallyof the pan-cellular type (within all erythrocytes),where as little as 20% HbF is sufficient to inhibitHbS formation in vivo and to alleviate substantiallythe clinical manifestations of SCD in homozygousbS/bS patients.59,60 In these cases, the c-chainexcludes the heterotetramer a2cb

S from the HbSpolymer.61–63

However, neither c- or d-globin genes are highlyexpressed in the ‘‘adult’’ (postglobin switch) RBCenvironment. Because the bA-, c-, and d-globinchains are co-linear and comprise only a few dif-

152 NEGRE ET AL.

ferences in amino-acid residues between them, es-pecially between bA and d with only 10 differencesout of 146, biochemical studies were performed todetermine which positions were critical to inhibitHbS polymerization. Within the acceptor hydro-phobic pocket, a critical residue differs at position87 between bA (T87) and c or d (Q87), and has beendetermined to be responsible for most of the inhib-itory effect of c- or d-globins on HbS polymeriza-tion.55 A few other residues co-contribute to a lesserdegree, and in particular Ala-22.64 In an effort toexpress a strong antisickling b-globin derivative athigh levels in adult RBCs, a modified bA-globin genewas designed (Leboulch Laboratory) that comprisesa point mutation within a human b-globin mini-gene suitable for retroviral transfer, so that theexpressed therapeutic protein is bA(T87Q). The re-sulting bA(T87Q)-globin chain appears as efficientas c-globin to inhibit HbS polymerization in bio-chemical assays.65 The oxygen affinity of the mu-tated tetramer (HbAT87Q: a2b

T87Q2) is in the range

of that of HbA,65 whereas the oxygen affinity ofHBF is substantially higher than that of HbA.66

Furthermore, the b-globin T87Q is a valuablebiomarker of biological efficacy in human clinicaltrials. The bT87Q-globin chain can be distinguishedand quantified from bA- and c-globin chains byhigh-performance reverse-phase liquid chroma-tography (HPLC).67,68 In the case of b+ thalasse-mia or when RBC transfusions are still provided,there would otherwise be no possibility of quan-tifying vector-derived gene expression if wild-typebA-globin was used as the therapeutic gene.

Therapeutic levels of bA(T87Q)-globin

gene expressionThe threshold level of therapeutic bA(T87Q)-

globin expression required for effective gene ther-apy of b-TM will vary depending on residual levels,if any, of endogenous b-globin production. It willalso depend on genetic modulators that contributeto reducing the severity of the disease in each in-dividual patient. These modulators include asso-ciated a-thalassemia69,70 or the ability to producesubstantial amounts of c-globin after birth,71,72

both reducing the magnitude of free a-chains inerythroid cells and the severity of the disease.

With respect to SCD, one can surmise that theantisickling properties of bA(T87Q)-globin will becomparable to or slightly lower than that of c-globin.65 HPFH with HbF levels of 30% or evenlower are consistently associated with completeabsence of clinical and biological signs of SCD inotherwise homozygous bS/bS patients, when HbFexpression is well distributed among RBCs (pan-

cellular HPFH).73 When HbF distribution is moreheterogeneous, the antisickling effect is less pro-nounced, although SCD symptoms of homozygousbS/bS were alleviated in a few reported cases ofstable mixed chimerism as low as 11% afterAHSCT.74 It is thus likely that expression ofbA(T87Q)-globin between 20% and 30%, preferablywith moderate variegation in expression will meetthe minimum threshold to prevent most of theclinical manifestations and complications of SCDin homozygous bS/bS patients.

Cis-regulatory control elementsto be incorporated in vectors

Most of the genetic elements that control tissuespecificity and developmental switching of b-globin gene expression are located within or nearthe transcription unit in the promoter region,downstream of the polyadenylation signal, andwithin the second intron.75–78 Nevertheless, in-troducing these elements to control b-globin geneexpression in gamma-retroviral vectors (c-RV) hasresulted in very low levels of human b-globin geneexpression in erythroid cells of mice transplantedwith transduced HSCs.79,80 The discovery of chro-matin domains, referred to as DNase I hypersensi-tive sites (HS), several kilobases upstream of theb-globin gene,81,82 has shed light on the regulatoryorganization of the locus. The ability of the 15 kbLCR to confer physiological levels of human b-globin, when inserted immediately 5¢ to the b-globingene in transgenic mice,83,84 has yielded clues fordesigning effective expression systems. The mostimportant HS sites to cis-link to the human b-globingene are HS2, HS3, and HS4 that have enhanceractivity when tested individually in cell cultureand in transgenic mice.85,86 Several laboratorieshave undertaken to reduce the size of each of theHS sites while maintaining most of their en-hancer effect. However, while the so-called ‘‘coreelements’’ of 250–350 bp long show useful activitywhen combined in c-RV,87–89 expanded elementsappear necessary for near-optimal enhancer ef-fect.84,85,90,91

Gene transfer vectors suitable for the genetherapy of the b-hemoglobinopathies

Sustained gene therapy of inherited hemato-logical disorders by ex vivo gene addition requiresintegration of the vector in the chromosomes ofHSCs to obtain proper replication and segregationin the daughter cells for the lifespan of the recipi-ent. To date, only retroviral vectors have repro-ducibly shown this capability in animal models.Efforts have first made use of c-RV derived from


the Moloney murine leukemia virus (M-MuLV).The first M-MuLV vectors containing a human b-globin gene and its promoter were reported by theMulligan and Nienhuis laboratories.92,93 However,b-globin expression was erythroid specific but wellbelow a possible therapeutic effect.79,80,92,93

Incorporation of core LCR elements resulted inlow-titer c-RV that were also highly unstable withmultiple rearrangements of the transferred provi-ral structures.94,95 Reducing the size of the LCR tominimal elements is unsatisfactory as b-globinexpression levels are too low.96,97 Leboulch et al.88

identified untoward polyadenylation and splicingof the genomic viral RNA before packaging as themain mechanisms of both low titers and provirusrearrangements (internal splicing and poly-adenylation). LCR elements, the promoter, and theactual globin gene were placed in reverse orienta-tion to avoid splicing of the bona fide introns beforepackaging, which resulted in the unmasking ofconsensus polyadenylation and splicing signalsalong the vector genomic viral RNA. A small in-ternal deletion of sequence repeats within thesecond (minigene), together with site-directedmutagenesis eliminating those sequences, resultedin stable proviral transmission.88 Sadelain et al.89

achieved a similar result by applying the same in-ternal deletion within the second intron whilepermutating LCR elements, possibly resulting in aconformational change in the viral genomic RNAthat also inhibited untoward splicing. In spite ofthese advances, vector-encoded b-globin remainedbelow therapeutic levels, and transduction of HSCsby c-RV was suboptimal in vivo.98,99

Grants from the National Institutes of Health(NIH) were awarded in the mid-1990s to focus onmaking further progress in this area. Sadelain andLeboulch (together with R. Nagel, I. Mondon, C.Eaves, and K. Humphries) were awarded grants tofocus on the gene therapy of b-TM and SCD, re-spectively. With the discovery that the Rev re-sponsive element (RRE) system of lentiviruses,including the human immunodeficiency virus(HIV), allows for efficient nucleocytoplasmic exportand subsequent packaging of full-length, unsplicedgenomic viral RNA100 and the subsequent adventof lentiviral vectors (LVs),101,102 there appeared agreat opportunity to apply this class of vectors tothe complex genomic b-globin structures. An ad-ditional benefit is that LVs pseudotyped with theenvelope of the G protein of the vesicular stomatitisvirus (VSV-G) are amenable to concentration andare much more effective than c-RV at transducingcells arrested at the G1-S boundary of the cell cy-cle101–103 and in quiescent HSCs.104,105 Sadelain

and colleagues published the first correction ofmurine b-thalassemia by an LV containing the b-globin mini-gene described above,106,107 althoughwith substantially larger LCR elements than thoseincorporated in previous c-RV (LV referred to asTNS9). Leboulch and colleagues published the firstcorrection of transgenic mouse models of SCD,65 bymaking use of their own vector, also containing theb-globin mini-gene together with similarly largerLCR elements (644 bp for HS2, 845 bp for HS3, and1153 bp for HS4). Importantly, this LV contains theantisickling bA(T87Q)-globin gene. Human CD34+

cells transduced with a SIN version of this vectorgenerated long-term hematopoietic reconstitutionin immunodeficient mice,108 indicating effectivetransduction of human HSCs. This vector was mod-ified further in the Leboulch lab to yield HPV569109

and BB305110 vectors, which have become the basisfor our own clinical trials described in this review.We and other groups have subsequently publishedother b-globin- or c-globin-based LVs and reportedefficient transduction of mouse HSCs and high levelsof b-globin expression.111–114

REGULATORY APPROVALPROCESS IN THE UNITED STATESAND THE EUROPEAN UNION

In spite of existing regulatory frameworks forgene therapy projects,115,116 there are still manyunknowns, in part because of the lack of precedents.Innovative regulatory science can help foster asupportive ecosystem for gene therapy products andaccelerate their development in a sustainablemanner. An example of such a framework is theadaptive biomedical innovation framework.117

Frequent, science-based, transparent, and pro-active interactions with regulators can also con-tribute to the overall development strategy for genetherapy products by reducing regulatory uncer-tainty. The majority of the regulatory mechanismsthat exist to accelerate the development of medic-inal products, including gene therapy products,require (1) targeting a serious or life-threateningcondition with significant unmet medical need, (2)having the potential for outstanding efficacy withan acceptable safety profile, and (3) having a robustdevelopment plan. A clear mechanism of action andearly proof-of-concept studies are also helpful.

Typically, the first interaction with regulatoryagencies in the development of a gene therapyproduct is to discuss the nonclinical development(traditionally referred to as pharmacology andtoxicology) and the design of the planned first-in-human study. In the United States, this first in-

154 NEGRE ET AL.

teraction is a pre–Investigational New Drug (IND)meeting with the Food and Drug Administration(FDA). In the European Union (EU), this first in-teraction can be a presubmission meeting beforefiling a clinical trial application (CTA) with na-tional authorities and/or a scientific advice meetingwith the European Medicines Agency (EMA). Inaddition, ‘‘pre-pre-IND’’ informal discussions withFDA and meetings with the EMA Innovation TaskForce can be useful to discuss the early develop-ment of very innovative products, including inno-vative gene therapy products. Seeking orphandesignation for products developed for rare dis-eases (defined as fewer than 5/10,000 affected in-dividuals in the EU118 and fewer than 200,000prevalent cases in the United States),119 as well asseeking EU gene therapy,120 can also be helpfulearly on for sponsors to define their products andtheir active substance, clarify their mechanism ofaction, and outline their potential for significantclinical benefit.

Clinical trials are regulated under Directive2001/20/EC in the EU and under 21 CFR Part 312in the United States. In the EU, a new regulation121

will come into force in 2016 that will replace thecurrent directive. This regulation should stream-line the process and allow for centralized submis-sions via a new electronic portal system to onereference member state. For gene therapy prod-

ucts, seeking approval to initiate a clinical trial inthe EU currently requires filing a CTA including aninvestigational medicinal product dossier (IMPD)in each member state involved, and requires filing agenetically modified organism (‘‘GMO’’) submissionsometimes in advance of the CTA. GMO submis-sions for gene therapy product are cumbersome andoften unclear. In the future, specific applicationscustomized for gene therapy medicinal productsshould be created and these should be managed bythe same regulatory agencies receiving the CTAapplications. In the United States, filing of an INDapplication is required. INDs follow the commontechnical dossier (CTD) format of the internationalconference harmonization (ICH). IMPDs headingsare also consistent at a high level with the CTDorganization (Fig. 2).122 IMPDs and INDs mustcontain information on manufacturing and quality,on the nonclinical studies conducted, and on theplanned clinical trial, including a protocol and In-vestigator’s Brochure. For gene therapy products,filing a pediatric investigational plan (PIP) withEMA early in the development is advisable if thetargeted condition affects pediatric patients. Dur-ing this process, sponsors can receive advice ontheir preliminary long-term development plans.

Other key regulatory mechanisms that can beleveraged to accelerate the development of genetherapy products include seeking fast-track desig-

Figure 2. The common technical document, adapted from www.ich.org/products/ctd.html The common technical document is organized into five modules.Module 1 is region specific, and modules 2–5 are intended to be common for all regions.


nation or breakthrough therapy designation in theUnited States.123 In the EU, in addition to scientificadvice, applying for an EMA nonclinical and quali-ty/manufacturing certification procedure should beconsidered (for small and medium-size enterprises),as well as seeking the recently created prioritymedicines (PRIME) designation,91 and leveragingthe flexibility of the risk-based approach and of theadvanced therapy medicinal product (ATMP) Reg-ulation No. 1394/2007/EC, particularly for thequality/manufacturing development.124,125

InthespecificcasestudyofHPV569andBB305drugcandidates, a number of above-mentioned existingregulatory mechanisms were used to support and ac-celeratedevelopment,withanemphasisonevaluatingearly the entire life-span of the product in a creativeway, including leveraging the potential of real-worldevidence and ‘‘Safe Harbor’’ discussions with multiplestakeholders, including patients’ representatives.

Looking forward, in order to obtain marketingauthorization, a biological license application (BLA)must be submitted to the FDA in the United Statesin accordance with 21 CRF Part 601.126 In the EU, amarketing authorization application (MAA) mustbe submitted to the EMA in accordance with Direc-tive 2001/83/EC and the advanced therapy medici-nal product (ATMP) regulation 1394/2007/EC.127 Inboth regions, mechanisms exist to accelerate thereview of the BLA or MAA: priority review and ac-celerated assessment, respectively.123,128 Mechan-isms also exist to provide flexibility in the timing toobtain approval. In the United States, ‘‘acceleratedapproval’’ is the only mechanism based on the use ofa surrogate or intermediate endpoint.123 In the EU,centralized marketing authorizations that aremeant to accelerate access of medicines to patientsin need for serious conditions can be granted ‘‘underexceptional circumstances’’ or as a ‘‘conditional’’MAA.129,130

Gene therapy products provide unique opportu-nities to contribute to the evolution of regulatoryscience because of their transformative efficacypotential, their increasing complexity, and theirpending individualized nature.

HPV569 AND BB305 VECTOR DEVELOPMENTThe HPV569 lentiviral vector was assessedin the first human trial LG001

The original LVs we used to achieve long-termcorrection of SCD and b-thalassemia in mice65,112

comprised (1) HIV1’s RRE and central polypurinetract; (2) the human bA-globin gene, in reverse ori-entation, either wild-type or with the bT87Q muta-tion; (3) the human bA-globin promoter, and (4) amini-LCR composed of HS2, HS3, and HS4 of 644,845, and 1153 bp length, respectively.

To prepare for human clinical trials, safety mod-ifications were made to the original vector, as fol-lows, to yield the vector referred to as HPV569.109,112

These include mutating the GAG gene and deletionof the viral enhancer and promoter elements in theU3 region of the 3¢LTR to generate a SIN vector(Fig. 3). The SIN modification reduces the likeli-hood of propagation of replication-competent re-combinant LV in the vector producer and targetcells,105,131 decreases the risk of mobilization of thevector genome upon HIV secondary infection,132 andreduces the residual activation of cellular onco-genes by enhancer/promoter activities of integratedLTRs.133,134 Another modification has concerned theU5 region of the 3¢LTR, which was replaced by anartificial polyadenylation/termination signal de-rived from the rabbit b-globin gene.135 This lattermodification yielded higher viral titers for the SINversions of the HPV569 vector because of more effi-cient polyadenylation/termination of the viral tran-script.136 In an effort to protect transduced cells

Figure 3. Diagram of the HPV569 b-globin (bA-T87Q) lentiviral vector. The 3¢ b-globin enhancer, the 372 bp IVS2 deletion, the bA-T87Q mutation (ACA[Thr] toCAG[Gln]), and DNase I hypersensitive sites (HS) 2, HS3, and HS4 of the human b-globin locus control region (LCR) are indicated. Safety modifications,including the 2 stop codons in the w+ packaging signal, the 400 bp deletion in the U3 of the right HIV LTR, the rabbit b-globin polyA signal, and the 2 · 250 bpcHS4 chromatin insulators, are indicated. In the BB305 lentiviral vector, U3 promoter/enhancer has been replaced by cytomeglovirus (CMV) promoter/enhancerand the 2 · 250 bp cHS4 insulator elements have been removed. cPPT/flap, central polypurine tract; HIV LTR, human immunodeficiency type-1 virus longterminal repeat; ppt, polypurine tract; RRE, Rev-responsive element; bp, human b-globin promoter.

156 NEGRE ET AL.

against cis activation of adjacent genes by enhanceractivities present within the b-globin LCR,137 twocopies of the 250 bp core elements of the chicken b-globin chromatin insulator (chicken b-globin locusDNase I hypersensitive site 4: cHS4) were insertedin place of the deleted U3 region of the vector.

The HPV569 vector109 was produced at clinicalgrade68 and used to evaluate therapeutic efficacyand safety in a mouse model of b-thalassemia.138

The gene-corrected mice showed normalization oftheir phenotype. Consistent with other LV stud-ies,139,140 the HPV569 vector was shown to favortargeting of transcription units (&70%) and gene-dense regions. Importantly, no enrichment of in-tegration sites within proto-oncogenes was foundposttransplantation, and no relationship was seenbetween the proximity of integration sites to on-cogenes and site abundance in mice.138 A variety ofcomplementary efficacy and safety studies wereperformed in human CD34+ cells from patients andin several mouse models before filing a CTA dossierwith the French regulatory agency, as described inthe section Regulatory approval process in theUnited States and the European Union. The firstclinical trial with the HPV569 drug product can-didate vector (LG001) was initiated in 2006 inFrance. Three subjects with b-TM were treated andone (subject No. 1003) became transfusion inde-pendent (see details below).141

The BB305 lentiviral vectorAlthough the LG001 trial brought the proof-of-

principle of clinical efficacy in a human patient(subject No. 1003), partial clonal dominance, whichsubsequently resulted, was observed upon vectorintegration within the HMGA2 gene. This inte-gration triggered abnormal splicing of the endog-enous HMGA2 RNA using a cryptic acceptor sitewithin the cHS4 insulator core in the left LTR.Studies performed in mouse samples showed thatmost of the integrated provectors had lost one of thetwo 250 bp cHS4 insulator cores at each end.138

This phenomenon was observed in subject No. 1003as well.141 Data reported by several contributorsalso showed that inclusion of cHS4 chromatin in-sulator elements in the 3¢LTR of LVs reduced theirtiters and transduction efficacy for human CD34+

cells.142–144 Moreover, their presence had limitedefficacy on transgene expression in human hema-topoietic cells in vitro and in vivo,144 while theprotection provided was dependent on the locationof cHS4 integration in the genome.145 For all thesereasons (decreased titers and transduction effi-ciency, insulator loss, untoward splicing, and lim-ited enhancer blocking activity), removal of the

cHS4 insulator from our HPV569 vector was war-ranted.

In addition, we decided to replace the Tat-dependent U3 promoter/enhancer of the 5¢LTR bythat of the cytomegalovirus (CMV) (Fig. 3) in orderto further increase the titer of the vector.105 Over-all, the modified vector, referred to as the BB305vector,110 is identical to HPV569 after chromo-somal integration, except for the absence of thecHS4 insulator (Fig. 3). In plasmid form, however,as used in producer cells, the CMV promoter nowdrives transcription instead of the 5¢LTR.

The new-generation BB305 vector was pro-duced, purified, and tested on human CD34+ cellsas well as in a mouse model of b-thalassemia.110

Side-by-side comparison of HPV569 and BB305showed that the vector changes resulted inboth increased vector titers (3–4-fold) and in-creased transduction efficiency (2–3-fold). Com-prehensive toxicological mouse studies and vectorcomparison showed b-thalassemic phenotype cor-rection with no evidence of toxic effect related toany of the vectors, and similar integration patterns(mostly within RefSeq genes), without any sign ofclonal outgrowth or in vivo selection.110 In vitroimmortalization (IVIM) assays, developed by theBaum lab,146 were run in order to evaluate the riskof hematopoietic cell transformation upon vectorintegration. Both vectors (HPV569 and BB305)exhibited a strongly reduced risk of immortaliza-tion of murine hematopoietic cells as comparedwith control retroviral vectors, and LVs containingthe strong SFFV viral promoter.110

VECTOR AND DRUG PRODUCTMANUFACTURINGProduction of lentiviral vectors

Guidance on development and manufacturingof LVs, which are considered starting materials byboth U.S. and EU regulatory agencies when usedfor ex vivo transduction, is provided by FDA andEMA. This guidance provides recommendations forLV design, manufacturing, and characterization,including transducing activity, LV particle quan-tification, and testing for replication-competentlentiviruses (RCLs). bluebird bio has developeda GMP manufacturing process for large-scaleproduction (>40 liters) of third-generation VSV-G-pseudotyped HIV-1-based LVs. Clinical-grade ve-sicular stomatitis virus glycoprotein-pseudotypedlentiviral particles are produced by a plasmid-based co-transfection method. Purification is doneby chromatography and buffer is exchanged byultrafiltration before final filtration according to


published protocols.147,148 This method is used toproduce large-scale clinical-grade LV lots to sup-port our clinical trials.

Release tests performed on the manufacturedLV include potency and identity, safety, and pu-rity. The LV potency is determined using assaysthat measure both the concentration of viral par-ticles and infectious titer. The concentration ofvector particles is assessed by measuring the HIV-1p24 antigen. The titer is defined as the number offunctional transduction units per milliliter (TU/ml).149 Vector infectivity (or specific transducingactivity) is defined as the ratio between transduc-tion unit per milliliter and the concentration of p24[(TU/ml)/(ng/ml)]. This ratio gives a reliable pa-rameter to evaluate the quality of the vector prep-aration.150 High-quality vector preparations withan infectious titer >108 TU/ml and a particle/infectious ratio between 100 and 500 are obtained.151

The ability of transduced cells to produce thetherapeutic bT87Q-globin is verified after erythroiddifferentiation of transduced CD34+ hematopoieticcells. It is assessed by reverse-phase HPLC analy-sis of globin chains in differentiated erythroid cells.The therapeutic bT87Q-globin can be easily distin-guished from the normal b-chain, as well as fromthe bE and bS polypeptides.67,68

The generation of RCL is highly improbable be-cause of the multiplasmid packaging system andthe U3-deletion. Nevertheless, RCL testing is per-formed. It relies upon the permissive cell lineC8166-45, allowing the amplification and the de-tection of replicative-competent particles.152,153

Drug product (geneticallymodified CD34+ cells)

Once subjects have been screened and eligibil-ity has been determined, autologous hematopoi-etic CD34+ cells are procured either by apheresisof mobilized peripheral blood cells (for b-TM sub-jects) or by bone marrow harvest (for subjects withSCD). Multiple harvests may be undertaken ifneeded to meet the minimum cell dose required fordrug product manufacturing and untransducedbackup. Mobilization is performed with filgrastim,a recombinant form of granulocyte-colony stimu-lating factor (G-CSF), in combination with pler-ixafor. The combination of plerixafor and filgrastimmay be the most effective mobilization strategyfor subjects with b-thalassemia.154,155 Apheresisis performed on the fifth day of mobilization. Theobjective is to collect sufficient cells for bothmanufacturing and for rescue therapy. After pro-curement, the CD34+ cell population is enrichedvia purification. A portion of the product is cryo-

preserved for rescue therapy. CD34+ cells are grownin serum-free medium supplemented with recom-binant human cytokines stem cell factor (SCF),thrombopoietin (TPO), and FMS-like tyrosine ki-nase receptor-3 (Flt3-L) for approximately two days.The cells are then incubated for an additional dayfor the lentiviral transduction step. After trans-duction, a portion of the cells and supernatant areremoved for release testing. The reminder of thecells is cryopreserved. After the completion of re-lease testing and disposition, the drug product, de-fined as CD34+ hematopoietic stem cells transducedwith the BB305 LV, is infused after the patient hasundergone myeloablative conditioning.

CLINICAL TRIALS: INTERIM RESULTSFROM FRANCE

The HPV569 and BB305 vectors are designedto be used as a single product for the gene therapyof the b-hemoglobinopathies (SCD and b-TM).Vector design is intended to overcome the deficitof b-globin chains in b-thalassemia and to pro-vide anti-HbS polymerizing activity in SCD. TheHPV569 vector is the first vector to have been tes-ted worldwide in an approved human trial for thegene therapy of the b-hemoglobinopathies, withthe first patient transplanted with transduced cellsin 2006.

The French trials led the way in the testing ofthe HPV569 and BB305 drug products and are theprimary focus of this review article. The BB305program has now expanded internationally, andinitial results are summarized in Table 1 and in thesection Multicenter U.S. and international trialsfor b-TM (HGB-204) and SCD (HGB-206) with theBB305 drug product. Other ongoing trials of othergene therapies for hemoglobinopathies are sum-marized in Table 2.

Study design and pretransplant conditioningClinical studies of HPV569 and BB305 drug

products run in France are nonrandomized, open-label, single-dose, phase 1/2 studies and only enrollpatients with no sibling donor. The first trial per-formed, with regulatory approval obtained in 2006,was termed LG001 and aimed to assess the HPV569drug product, as described in the section, ‘‘TheHPV569 lentiviral vector was assessed in the firsthuman trial LG001’’ above, in 10 subjects with b-TMor severe SCD. After 3 subjects had been treated, theHPV569 vector was replaced with the improvedBB305 vector and manufacturing thereof, and a newclinical trial protocol, termed HGB-205, was im-plemented to treat the remaining 7 subjects. The

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HGB-205 study is currently ongoing in France for b-TM and SCD.

In addition to the characteristics of the clinical-grade vector (high-titer, high purity of the manu-factured lot, intrinsic therapeutic globin expres-sion properties), key parameters to success include(1) the dose of CD34+ cells infused, (2) the meanvector copy number (VCN) in CD34+ cells preinfu-sion, and (3) efficient myeloablative conditioning.Because there is little convincing evidence in ani-mal models and clinical trials that effective lenti-viral gene transfer to HSCs can be achieved with ahigh degree of sustained chimerism for vector-bearing cells in the absence of extensive myeloa-blation, when a CD34+ cell dose similar to thatapplicable to human autologous settings is used, wedecided to apply a full dose of myeloablative agentwith intravenous (IV) busulfan as pretransplantconditioning regimen. However, the additional useof immunosuppressive agent (e.g., cyclophospha-mide) is not required in this autologous setting.Monitoring of busulfan conditioning to optimizeHSC transplantation is an important componentof the success156 and is meticulously performed.When the drug product manufacture is complete,the subject receives myeloablative conditioningwith IV busulfan at a starting dose of 3.2 mg/kg/dayfor 4 days with pharmacokinetics analysis. The doseand schedule of busulfan is monitored daily andmay be adjusted based upon busulfan plasma levelsin order to maintain appropriate levels for myeloa-blation (AUC exposure of 4500–5000 [lM�min]/dayfor a daily dosing regimen, over 4 days). After bu-sulfan washout for several days after the end of IVbusulfan administration, the drug product is in-fused. The subject remains hospitalized until en-graftment occurs (absolute neutrophil count [ANC]‡0.5 · 109/liter for 3 consecutive days) and the pa-tient is medically stable. Subjects are followedmonthly for the first 6 months posttransplant, andthen every 3 months through 24 months post-transplant. Subjects are then enrolled in a long-term follow-up study for an additional 13 years.

Endpoints. The primary study objective is toassess the safety, tolerability, and success of en-graftment with autologous CD34+ HSCs transducedwith the vectors encoding the human bA-T87Q-globingene after conditioning with intravenous busulfanin subjects with b-TM and severe SCD. The primaryoutcome measures are success and kinetics of HSCengraftment, incidence of transplant-related mor-tality through 100 days posttreatment, overall sur-vival, detection of RCL, characterization of anyevents of insertional mutagenesis, and monitoring of

laboratory parameters, and frequency and severityof clinical adverse events (AEs).

Secondary endpoints include therapeutic globin(HbAT87Q) expression quantified by HPLC analy-sis, VCN levels in peripheral blood (and in bonemarrow, if collected), and RBC transfusion re-quirements posttransplant. They also include theassessment of dyserythropoiesis for b-TM subjectsand VOC and ACS frequencies for SCD subjects.

Inclusion criteria. All subjects must be between 5and 35 years old. They must have transfusion-dependent (‡100 ml/kg/year of packed RBCs forb-TM) or severe SCD, confirmed by Hb studies.Subjects must be eligible for AHSCT based on insti-tutional medical guidelines, but without a suitable,willing HLA-identical sibling donor. In addition,subjects with SCD must have failed to achieve ade-quate clinical benefit after hydroxyurea treatmentfor at least 4 months unless this treatment was notindicated or not well tolerated, and have a history of 1or more of the following poor prognostic risk factors:recurrent VOC, ACS (at least 2 episodes), significantcerebral abnormality on magnetic resonance imag-ing (MRI), stroke, antierythrocyte alloimmunization(>2 antibodies), presence of SCD cardiomyopathydocumented by Doppler echocardiography, or osteo-necrosis of 2 or more joints.

All subjects must have been treated and followedfor at least the past 2 years in a specialized centerthat maintained detailed medical records, includ-ing transfusion history. Sperm preservation ortestis or ovary biopsy is offered to subjects enrolledon the study. All subjects or their parents orguardians must provide written informed consent.

Exclusion criteria. Subjects meeting any of thefollowing criteria cannot be enrolled in the study:(1) availability of HLA-identical sibling hemato-poietic cell donor, (2) clinically significant, activebacterial, viral, parasitic, or fungal infection, (3)prior or current malignancy, myeloproliferativedisorder or immunodeficiency, (4) contraindicationto anesthesia for bone marrow harvesting, (5)white blood cell count lower than 3 · 109/liter and/or platelet count lower than 120 · 109/liter, and (6)history of major organ damage.

Trial results to dateThe LG001 and HGB-205 were the first gene

therapy studies worldwide to treat b-TM and SCDsubjects, respectively, achieving the first conver-sion to long-term transfusion independence of a b-TM subject15,141,157,158 and the first evidence ofclinical benefit in SCD.159


b-TM patients in the first French trial LG001 withHPV569 drug product. Four subjects with b-TMwere enrolled, 3 of whom were treated between2006 and 2011, under the LG001 protocol. Subject1002 was 29 years old with b+/b0 genotype and re-ceived a dose of 0.93 · 106 cells/kg with a mean VCNof 1.3. Subject No. 1003 was 18 years old with bE/b0

genotype and received a dose of 3.9 · 106 cells/kgwith a mean VCN of 0.6. Subject 1004 was 22 yearsold with bE/b0 genotype and received a dose of4.3 · 106 cells/kg and a VCN of 0.3. Transplantationwas uneventful in all subjects. Transduced cells didnot successfully engraft in subject 1002, althoughgene marking was detected for a few months; thissubject received backup hematopoietic cells forrescue and remains transfusion dependent. Neu-trophil engraftment was reported for subject No.1003 at day +27 and for subject 1004 at day +22postinfusion. The HPV569 drug product was welltolerated, with no nonhematologic serious AE andno drug product-related AEs reported. In addition,no subject has developed vector-derived RCL, leu-kemia, or lymphoma.

Clinical benefit of treatment with HPV569 drugproduct was observed in 1 of the 2 treated subjectswho did not receive backup cells. Subject No. 1003sustained clinical benefit as evidenced by long-term transfusion independence that was achievedapproximately 1 year posttransplant15,141 andsustained through approximately 8 years post-transplant. The hemoglobin level stabilized 1.5years posttreatment, and the mean VCN per mye-loid cell was close to 0.2. A detailed report on thissubject after 33 months of follow-up has beenpublished.15,141 After approximately 7 years oftransfusion independence, this subject received afew transfusions to address symptoms of anemia.Ongoing follow-up will determine whether or notperiodic transfusion support will again be required.His total hemoglobin and levels of HbAT87Q trans-genic hemoglobin have remained generally con-sistent from years 2 through 8 postinfusion ataround 8 g/dl total Hb of which 30% is HbAT87Q.

Integration-site analysis revealed the relativedominance of a clone bearing the vector inside in-tron 3 of the HMGA2 gene, the proportion of whichreached a maximum representation of *30% of thetransduced myeloid cells (i.e.,*4% of hematopoieticcells) 15 months posttransplantation. Molecularanalyses revealed that the integrated vector hadcaused transcriptional activation of the HMGA2promoter in erythroid cells. Furthermore, a crypticsplice acceptor site (GTAT(C)6AG), located withinthe cHS4 insulator core of the 5¢LTR, generated atruncated mRNA containing HMGA2 exons 1, 2,

and 3. Cleavage/polyadenylation occurred withinthe adjacent R region of the 5¢LTR that leads to in-creased stability of the RNA because of deletion ofthe Let7 microRNA binding sites (located in exon 5)and an excess of truncated HMGA2 mRNA. Theclone has remained under homeostatic control, witha peak at *4% of hematopoietic cells 4 years aftertransplantation, gradually decreasing to approxi-mately 1% of total nucleated blood cells at 5 yearsposttransplant.158 The clone is absent from lym-phocytes. Despite declining levels of this clone, theamount of bT87Q-globin has remained stable, indi-cating that the observed therapeutic benefit is notdependent on this specific clone. The insertion ofviral vectors in the HMGA2 gene has been observedin other gene therapy studies, with no case of leu-kemia or lymphoma related to this insertion site inany patient.158,160–162

Transduced cells successfully engrafted in sub-ject No. 1004, but this subject did not achieve sus-tained clinical benefit and remains transfusiondependent. Hemoglobin levels containing bT87Q-globin account for *5% of total hemoglobin.158

b-TM and SCD patients in French trial HGB-205with BB305 drug product. As of November 2015,four subjects with b-TM and one subject with SCDhave been treated with BB305 drug product underthe HGB-205 protocol. Initial results have beenpresented at scientific meetings but not yet pub-lished, and the trial is ongoing.157–159 All of the fourtreated b-TM subjects have so far become transfu-sion independent, and the treated SCD subjectshows early clinical benefit.

MULTICENTER U.S. AND INTERNATIONALTRIALS FOR b-TM (HGB-204) AND SCD(HGB-206) WITH THE BB305 DRUGPRODUCT

Additional subjects with b-TM and SCD arecurrently being treated under the HGB-204 andHGB-206 protocols, in two separate internationaland U.S. studies (Table 1).163,164 Early initial re-sults have been presented at scientific meetingsbut not yet published.

CONCLUSIONS

Gene therapy for hemoglobin disorders hasmade major progress, from early discovery of b-globin regulatory elements; development of LVs,including the HPV569 and BB305 vectors de-scribed here; efficient transduction of hematopoi-etic stem cells; proof-of-principle of efficacy in

160 NEGRE ET AL.

mouse models; the first conversion to multiyeartransfusion independence of a patient with b-TM;and early clinical benefit now observed in SCD.

Successfully implementing a gene therapystrategy for the b-hemoglobinopathies involves anintegrated approach of regulatory, manufacturing,and clinical trial design, and execution. Thepromise of bringing this therapeutic modality to alarge number of patients will require the successfulindustrialization and commercialization as well asregulatory product approval.

To date, no serious adverse effects have beenattributed to ex vivo LV-based HSC gene therapyfor hemoglobinopathies, but long-term data in alarger number of patients are needed to assess therisks fully. Current vectors are designed to mini-mize the possibility of genotoxicity, including on-cogenesis, but this risk cannot yet be conclusivelyquantified or excluded. Ongoing and future clinicalstudies will allow for a more complete under-standing of the benefit–risk profile of this thera-peutic approach. Implementation of gene therapyfor b-TM and SCD to a large extent will depend onthe benefit/risk/cost ratios.

ACKNOWLEDGMENTS

We thank Eliane Gluckman (Saint-Louis hospi-tal, Paris, France), the principal investigator at the

beginning of the LG001 study; Francoise Bernau-din (CHIC, Creteil, France) for contributions tostudy design and for clinical care; and OlivierHermine and Felipe Suarez (Necker Hospital,Paris), the transplanters for the HGB-205 study.We thank Christof von Kalle, Manfred Schmidt(National Center for Tumor Disease, Heidelberg,Germany) and Frederic Bushman (University ofPennsylvania, PA) for vector insertion site ana-lyses. We thank Laure Caccavelli (Necker hospital,Paris, France) and the bluebird bio France team(CEA, Fontenay aux Roses, France) for technicalassistance. We thank Philipp Gregory, SandeepSoni, Kate Lewis, Gabor Veres, Michael Paglia,John Pierciey, and Tim Douros (bluebird bio,Cambridge) for manuscript proofreading. Thework was supported by an Industrial Chair fromFrance’s Agence Nationale pour la Recherche(ANR) awarded to Philippe Leboulch. bluebird biois the sponsor of the clinical trials and providedfunds to Necker Hospital, Assistance Publique-Hopitaux de Paris.

AUTHOR DISCLOSURE

O.N. and A.-V.E. are employees of bluebird bio,Inc. Y.B., P.L., and E.P. have financial relation-ships with bluebird bio, Inc. All other authors haveno competing interests to disclose.

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Received for publication January 14, 2016;

accepted after revision January 21, 2016.

Published online: January 22, 2016.


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