Villamizar 2014

Gene Therapy for Severe
Haemoglobin Disorders
Olga Villamizar, Department of Medical Microbiology, Immunology and Cell Biology,
Southern Illinois University School of Medicine, Springfield, Illinois, USA
Christopher B Chambers, Department of Medical Microbiology, Immunology and Cell
Biology, Southern Illinois University School of Medicine, Springfield, Illinois, USA
Andrew Wilber, Department of Medical Microbiology, Immunology and Cell Biology,
Southern Illinois University School of Medicine, Springfield, Illinois, USA
Sickle cell disease (SCD) and b-thalassaemia result from
inherited mutations that cause structural abnormality or
deficient synthesis of adult haemoglobin. Palliative
therapies improve the quality/duration of life for many,
but side effects result from long-term use. Bone marrow
transplantation can be curative, but is limited to indivi-
duals with a matched donor. Thus, gene delivery into the
patient’s haematopoietic stem cells is a desirable therapy.
Lentiviral vectors encoding for erythroid-specific expres-
sion of either g- or b-globin genes have been developed for
this purpose. These vectors have been used to cure SCD
and b-thalassaemia in mouse models with positive results
emerging from clinical trials. Concurrently, innovative
strategies intending to reactivate endogenous g-globin
expression or correct b-globin mutations at the genome
level are showing promise for the future. Ultimately,
clinical utility of all these approaches depend on safety
and efficacy so that a cure can be consistently achieved.
Introduction
Red blood cells (RBCs; erythrocytes) are responsible for
gas exchange in the lungs and delivery of oxygen to cells
and tissues. In humans, mature RBCs lack a nucleus and
are filled with the metalloprotein haemoglobin. All hae-
moglobin molecules have an iron-containing haem group
and are tetramers of two different types of globin chains,
which undergo two major ‘switches’ during development
to ensure adequate oxygen binding (Stamatoyannopoulos,
2005; Figure 1). At the earliest stages of life, embryonic
haemoglobins (HbE) are produced by RBCs generated in
the yolk sac. The first switch (primitive to definitive) occurs
during foetal development, when HbE is replaced by foetal
haemoglobin (HbF; a2,g2) synthesised by erythroid cells of
the liver. The second switch (foetal to adult) begins shortly
before birth and continues throughout the first year of life
as HbF is progressively replaced by adult haemoglobin
(HbA; a2,b2). In adults, HbF is produced at low levels
(52%) with variation subject to individual genetics (Thein
and Menzel, 2009). See also: Haemoglobin: Cooperativity
in Protein–Ligand Interactions
The principal haemoglobin disorders (sickle cell disease
(SCD) and b-thalassaemia) are prevalent, autosomal
recessive diseases in which coinheritance of two mutated b-
globin genes results in pathology. In the b-globin protein of
SCD, valine replaces glutamic acid at the sixth amino acid
resulting in an altered charge (Bunn, 1997). This substitu-
tion creates haemoglobin S (HbS; a2,bS
2) which poly-
merises when deoxygenated conferring a rigid, sickle shape
to RBCs. From birth, these sickled cells can clog small
blood vessels and cause severe pain, progressive organ
damage and increased risk for strokes (Bunn, 1997).
Alternatively, b-thalassaemia results from poor to no
production of b-globin due to various mutations. Indivi-
duals that lack b-globin expression have a b0
genotype,
while those that have some level of expression are con-
sidered b+
. The severity of b-globin deficiency determines
how much a-globin remains unpaired, thus forming inso-
luble aggregates that cause premature death of RBCs and
with that severe anaemia (Weatherall, 2001). See also:
Globin Genes: Polymorphic Variants and Mutations;
Sickle Cell Anaemia; Thalassemias
The clinical relevance of persistent production of HbF
is evidenced by its role as a potent modifier of both SCD
and b-thalassaemia (Stamatoyannopoulos, 2005; Thein
Advanced article
Article Contents
. Introduction
. Premise of Gene Therapy
. Retroviral Vectors: Designed to Deliver
. Retroviral Vector Gene Therapy Trials: Success with
Safety Concerns
. Lentiviral Vectors: A Retrovirus with Benefits
. Lentiviral Vectors for Globin Gene Expression
. Correction of Mouse Models of SCD and b-Thalassaemia
. Correction of Human SCD and b-Thalassaemia RBCs
. Haemoglobin Gene Therapy in the Clinic
. Alternative Approaches
. Conclusion and Outlook
. Acknowledgements
Online posting date: 15th
October 2014
eLS subject area: Genetics & Disease
How to cite:
Villamizar, Olga; Chambers, Christopher B; and Wilber, Andrew
(October 2014) Gene Therapy for Severe Haemoglobin Disorders.
In: eLS. John Wiley & Sons, Ltd: Chichester.
DOI: 10.1002/9780470015902.a0025829
eLS & 2014, John Wiley & Sons, Ltd. www.els.net 1

and Menzel, 2009). Individuals homozygous for muta-
tions in their b-globin genes who also have enhanced HbF
production have less severe disease. The benefit of per-
sistent HbF production has fuelled a long-standing
interest in understanding the mechanisms responsible for
the switch from foetal to adult haemoglobin production
(Bunn, 1997; Weatherall, 2001; Perrine, 2008). Now
spanning nearly 50 years, these studies have led to the
identification of several agents which enhance HbF
synthesis (Perrine, 2008).
Hydroxyurea has emerged as the most widely used
drug. This antimetabolite medication was approved for
the treatment of adult patients with severe SCD following
a clinical trial which demonstrated its ability to reduce
pain crisis, acute chest syndrome and transfusion
requirements (Charache et al., 1995). Additional trials
conducted in children, teenagers and adults confirmed the
generalised benefit of hydroxyurea for patients with SCD
and b-thalassaemia (Testa, 2009). Although a variety of
mechanisms have been suggested, the beneficial effects of
hydroxyurea are most often attributed to increases in
levels of HbF observed in responding patients. Although
hydroxyurea and related medicines can improve the
quality and duration of life for some individuals, overall
cures for the severe haemoglobin disorders remain
unsatisfactory.
Bone marrow (BM) transplantation from human leu-
kocyte antigen (HLA)-matched sibling donors is a highly
effective treatment with cure rates approaching 85% for
both diseases (Bhatia and Walters, 2008). Approximately
200 SCD patients and 2000 b-thalassaemia patients have
been cured by this procedure. However, limitations in the
numbers of matched donors limit widespread use of this
therapy. Furthermore, mortality rates up to 10% still exist
due to treatment-associated infection and graft-versus-
host disease (Bhatia and Walters, 2008). As few patients
have a HLA-matched sibling donor, alternative donor
sources such as HLA matched unrelated donors (La Nasa
et al., 2005), HLA mismatched family members (Gaziev
et al.,2013) and unrelated umbilical cord blood units (Jaing
et al., 2010) are being explored but conclusive recommen-
dations are currently lacking. See also: Transplantation of
Haematopoietic Stem Cells
Premise of Gene Therapy
Reversal of genetic disease by introduction of corrective
sequences into a patient’s own haematopoietic stem cells
(HSCs) is one goal of gene therapy, and represents a valid
alternative to all forms of BM transplant. Because this
approach utilises the patient’s own stem cells, it has uni-
versal application with no need for immunosuppression or
risk of graft-versus-host disease. Initial studies have
focused on rare genetic disorders of the immune system for
which existing treatments did not exist or were unsuccess-
ful. Viruses have served as the prototype gene delivery
vehicle as they are naturally adapted for transferring their
genetic information into human cells. Therefore, much
effort has been devoted to the development of viral vectors
capable of delivering and expressing corrective genes in
HSCs to restore normal function to their progeny. See also:
Gene Delivery by Viruses; Viral Vectors for Gene Therapy
Chr. 16
Gower I/II HbF
Birth
HbA
1
ζ 2
HBZ
HBE HBG2 HBG1 HBD HBB
HBA2 HBA1
Stage: Embryonic Fetal Adult
Yolk sac
Age (weeks)
Chr. 11
LCR
1 2 3 4 5 ε
6 12 18 24
Post-conception
30 36 6 12 18 24
Post-natal
30 36 42 48
Site:
Gγ A
γ
Bone marrowFetal liver
Figure 1 Genomic structural organisation of the human a-globin and b-globin loci and developmental expression patterns of the various types of
haemoglobin. Diagram of the a-globin locus on chromosome 16 and the b-globin locus on chromosome 11 and the types of haemoglobin tetramers
produced during the indicated stages of human development with respect to the timeline. HbA, adult haemoglobin; HbE, embryonic haemoglobin; HbF,
foetal haemoglobin; LCR, locus control region. Reproduced with permission from Wilber et al. (2011b). & American Society of Hematology.
eLS & 2014, John Wiley & Sons, Ltd. www.els.net2
Gene Therapy for Severe Haemoglobin Disorders

Retroviral Vectors: Designed to
Deliver
Retroviruses are relatively simple pathogens that have a
single-stranded ribonucleic acid (RNA) genome between 7
and 12 kb. Upon entering a cell, the RNA is converted into
double-stranded deoxyribonucleic acid (DNA) and inser-
ted into a host-cell chromosome. Because of this property,
retroviruses were considered candidates for HSC gene
therapy applications requiring life-long expression. The
first vectors were based on Murine Leukaemia Virus
(MLV) because in-depth information about genome
structure and life cycle was available. Upon integration,
MLV DNA is flanked by the long terminal repeat (LTR)
sequences U3, R and U5 (Figure 2a). Viral DNA tran-
scription is controlled by enhancer/promoter elements in
the 5’ U3 region, the genomic transcript starts with R, and
is followed by U5, the primer binding site (PBS) for reverse
transcription, and the packaging signal (c). Beyond these
regulatory sequences are coding genes for essential viral
proteins: gag (structural), pol (enzymes) and env (surface or
envelope). The 3’ end includes a polypurine tract and a
polyadenylation site. See also: Retroviral Vectors in Gene
Therapy; Retroviruses in Human Gene Therapy
To create a gene therapy vector, the general strategy is to
separate sequences for Gag/Pol and Env onto independent
vectors, thus removing most of the coding information
from the viral genome (Figure 2b). The resulting retroviral
transfervectorcontainsc,thePBSandboth5’and3’LTRs,
but has a therapeutic gene that replaces gag/pol and env.
Vector particles are produced by introducing the transfer
vector into cells that either have transient or stable
expression of Gag/Pol and Env proteins (packaging cells),
and typically yield around 106
infectious units (IU) per mL
(Markowitz et al., 1990). While Gag/Pol proteins are highly
conserved, the Env proteins are subject to change (pseu-
dotyping). The flexibility of the Env proteins is important
for gene therapy as it permits selection of the optimal
envelope for each application. Expression of these collec-
tive genes within cells is necessary and sufficient to generate
virus particles capable of delivering a genetic payload.
Retroviral Vector Gene Therapy Trials:
Success with Safety Concerns
In the 1980s, MLV-based vectors were successfully used to
introduce marker genes into HSCs of mice (Williams et al.,
1984; Dick et al., 1985). As the delivered gene was inte-
grated into the cell genome, virus-modified cells and their
progeny could be identified by sensitive molecular methods
(i.e. polymerase chain reaction), even if they only repre-
sented a very small fraction of the total population. Lym-
phocytes isolated from tumours of five patients with
advanced malignant melanoma were marked using this
approach and their distribution and survival monitored
after re-administration to each patient (Rosenberg et al.,
1990). Gene-modified lymphocytes were detected in the
blood and tumour tissue of all five patients, albeit at low
frequency, and there was no incidence of infectious MLV
virus. This seminal study demonstrated the feasibility and
safety of MLV gene delivery, and suggested that this
technology could be applied to patients with heritable,
single gene disorders.
Candidate diseases for initial trials included the severe
combined immune deficiencies (SCID). As a class SCID is a
rare, recessive disorder in which patients lack a functional
immune system. These disorders are unique in that there is a
strong selective advantage for corrected cells. This char-
acteristic reduces the requirement for highly efficient gene
delivery into HSC, evidenced by clinical findings that rare
patients have undergone spontaneous reversions of their
original mutation to correct their immunodeficiency. One of
the more common versions of SCID is caused by deficiency
of adenosine deaminase (ADA), an enzyme required for
proper lymphocyte development. In 1992, MLV vectors
were used to introduce a functional ADA gene into the HSC
of two patients with SCID-ADA (Bordignon et al., 1995).
Unfortunately, clinical benefit was limited and did not to
confer sufficient development of mature lymphocytes nee-
ded to cure the disease. Still, these results indicated that a
patient’s own HSCs could be collected, modified and
returned safely and effectively. See also: Severe Combined
Immune Deficiency (SCID): Genetics
Where early studies suffered from low gene transfer effi-
ciencies, recent trials have demonstrated remarkable suc-
cess. SCID-ADA patients showed improved engraftment
of MLV-modified HSCs when they received mild (non-
myeloablative) preconditioning alone (Aiuti et al., 2002) or
when this regimen was combined with discontinuation of
enzyme replacement therapy (Gaspar et al., 2006). Nearly
10 years later, all participants receiving a successful treat-
ment showed immunological improvement and were free of
severe adverse effects. Another immunodeficiency disease is
X-SCID1, where boys have dysfunctional B-cells and lack
T- and Natural killer (NK)-cells due to deficiency of the
common gamma chain of interleukin 2 (IL2gc). The
majority of X-SCID1 patients in an MLV-based gene
transfertrialdemonstratedsignificantclinicalimprovement
of their immune deficiency (Hacein-Bey-Abina et al., 2002),
with the exception of two older ones (Thrasher et al., 2005).
However, five X-SCID1 patients developed T cell acute
lymphoblastic leukaemia due to vector integration near
proto-oncogenes (Howe et al., 2008; Hacein-Bey-Abina
etal.,2008).Similarproliferativedisorderswereobservedin
HSC gene therapy trials for X-chromosome-linked chronic
granulomatous disease (Ott et al., 2006) and Wiskott–
Aldrich syndrome (Braun et al., 2014), again the result of
vector-dependent integration and activation of proto-
oncogene(s). The MLV LTR was used in these trials to
express the therapeutic gene, but it is still unknown why this
LTR was safe in the context of SCID-ADA. Still, these
adverse events have underscored vector design as an
important safety issue for gene therapy. See also: Gene
Therapy for Primary Immunodeficiency

Lentiviral Vectors: A Retrovirus with
Benefits
Lentiviruses (LV), including human immunodeficiency
virus (HIV), are members of the retrovirus family; in
contrast to MLV, HIV has increased genetic complexity
and can infect nondividing cells. The HIV genome encodes
Gag, Pol and Env proteins, typical of retroviruses,
but includes additional accessory proteins (Figure 3a).
Multiple refinements in LV vector design have led to
improved safety by reducing the amount of viral genome
present, such that current gene delivery vectors encode for
less than one quarter (packaging vectors) and less than 5%
(transfer vector) of the viral genome (Zufferey et al., 1997;
Figure 3b). Additional modifications include removal of
enhancer/promoter sequences from the 3’ LTR to render
the vectors self-inactivating (SIN), a feature that makes
transgene expression dependent on an internal promoter.
If selected properly and appropriately tested, SIN LV
vectors are less prone to proto-oncogene activa-
tion following integration (Ryu et al., 2008). See also:
Lentiviral Vectors in Gene Therapy; Viral Vectors for
Gene Therapy
Lentiviral vector preparations are created by transiently
transfecting human cells with a transfer vector and at
least three separate packaging plasmids that typically yield
about 107
IU permL (Figure 3c). Lentiviral particles
are advantageous over retroviral particles as they are cap-
able of transferring complex globin expression cassettes into
cells without rearrangement. For the first time, preclinical
studies could be performed to establish parameters
needed to achieve erythroid-restricted expression of globin
genes at levels sufficient to ameliorate or cure SCD and b-
thalassaemia. See also: Haemoglobin Disorders: Gene
Therapy
Lentiviral Vectors for Globin Gene
Expression
Early experiments performed using retroviral vectors
provided critical information about globin coding
sequences, transcriptional regulatory elements and pro-
duction conditions. Building on this evidence, a number of
lentiviral vectors have been developed to introduce func-
tional globin genes into HSCs. The most effective designs
contain the human b-globin locus control region (LCR)
and promoter, a genomic globin sequence and b-globin 3’
untranslated (UTR) sequences, all in reverse orientation
(Arumugam and Malik, 2010). The LCR contains DNase I
hypersensitive sites (HS) that are critical for high-level,
long-term and erythroid-specific expression from the b-
globin promoter. Therapeutic globin sequences include
g-globin, b-globin or derivatives engineered with specific
mutations for enhanced activity. The b-globin UTR is used
because, unlike g-globin, it contains a stem-loop structure
that stabilizes b-globin transcripts in maturing adult RBCs
(Jiang et al., 2006). Additional modifications include the
insertion of chromatin insulator elements in the 3’ LTR to
prevent gene silencing (barrier activity) and activation
(enhancer blocking activity). All of these iterations have
demonstrated successful therapeutic outcomes in pre-
clinical mouse and human cell models (Figure 4).
Correction of Mouse Models of SCD
and b-Thalassaemia
Michel Sadelain’s group was the first to demonstrate that
a human b-globin LV vector with LCR fragments
(TNS9) could cure b-thalassaemia in mouse models
5’LTR
5’LTR
3’LTR
3’LTR
U3 R U5 gag pol
pol pA
U3 R U5
env
SA
PPT
PPT
PBS SD Ψ
PBS SD Ψ
gag
env
Transgene
pA
Promoter
Promoter
(a)
(b)
Figure 2 General structure of a retroviral genome and recombinant vector systems. (a) Graphical representation of a typical MLV pro-viral genome.
Indicated are the 5’ and 3’ LTR sequences, regions for U3, R and U5 and coding regions for gag, pol and envelope (env) proteins. (b) Diagram of
vectors encoding for expression of the essential viral proteins (packaging vectors, top two) or recombinant vector genome (transfer vector, bottom) that
can be used to make a recombinant retrovirus. The sequences for Gag/Pol and Env are placed on separate vectors to reduce recombination and creation
of infectious retrovirus that can replicate in the target cell. This strategy removes most of the coding information from the viral genome to incorporate
the therapeutic gene (Transgene). c, packaging signal; pA, polyA signal; PBS, primer binding site; PPT, polypurine tract; SA, splice acceptor; SD, splice
donor.

(May et al., 2000, 2002). In these studies, expression of the
human b-globin gene from a 3.2 kb LCR was sufficient to
produce mixed haemoglobin tetramers (mouse a2, human
b2) representing about 20% of the total haemoglobin in the
RBCs. Molecular analysis revealed that this was achieved
with nearly every HSC harbouring the vector. An inde-
pendent b-globin LV vector with shortened LCR sequen-
ces (bA
) was also able to cure b-thalassaemia mice, but this
time haematologic and pathologic improvement depended
on a vector copy number of at least 3 in each HSC (Imren
et al., 2002). The benefit of the TNS9 vector was also tested
in a mouse model of the most severe form of b-thalassae-
mia, b8-thalassaemia (Rivella et al., 2003). Even in this
model, all mice engrafted with vector transduced cells
showed some level haematological improvement with one
animal, having an average vector copy per cell 42, being
cured.
Building on the successful use of LV vectors in b-tha-
lassaemia models, other groups pursued studies in mice
modelling SCD. Leboulch and colleagues used a LV vector
similar to TNS9 to express a variant human b-globin gene
(Pawliuk et al., 2001). Where the aforementioned studies
utilised a wild-type b-globin gene, this version has a
threonine to glutamine substitution at amino acid 87
(bT87Q
); glutamine is present in g-globin and thought to
promote anti-sickling activity. A smaller 2.7 kb LCR con-
ferred expression of bT87Q
sufficient to resolve anaemia and
reduce organ damage in mice with three vector copies per
HSC. Using a more severe model of SCD, Townes and
colleagues evaluated another b-globin variant that inclu-
ded the T87Q change and two additional g-globin-based
substitutions (bAS3
) (Levasseur et al., 2003). Using a 3.4 kb
LCR, they were able to express bAS3
to levels about
20–25% that of endogenous bS
at a copy number of 2.2 per
HSC; this level reduced organ pathology but failed to
completely resolve anaemia.
As sustained production of HbF can improve the clinical
severity of b-thalassaemia and SCD, other groups have
used LV vectors to introduce the g-globin gene into HSC.
Persons and colleagues obtained significant correction of
severe b-thalassaemia intermedia mice using a g-globin
vector containing a 1.7 kb LCR and minimal b-globin
vif
pol
gag
LTR
SIN
Promoter
Promoter
Promoter
Transfect Collect Concentrate Titer
gag pol
rev
env
pA
pA
pA
RREΨ PPT
Promoter Transgene SIN
env
nef
vpu
vpr LTR
tat
rev
(a)
(b)
(c)
Figure 3 General structure of a HIV genome and recombinant vector systems used to produce lentivirus particles. (a) Graphical representation of a typical
HIV pro-viral genome. Indicated are the 5’ and 3’ LTR sequences, coding regions for gag, pol and envelope (env) proteins and additional accessory proteins
Tat, Rev, Nef, Vif, Vpu and Vpr. The tat protein enhances viral gene transcription, the rev protein facilitates nuclear to cytoplasmic transport of viral mRNA
and the nef, vif, vpu and vpr proteins are virulence factors. (b) Diagram of a SIN vector genome for an integrated version of the provirus (transfer vector,
top). This SIN vector lacks the enhancer sequences in the 3’-LTR that are duplicated on integration and requires an internal promoter to regulate
transcription of the therapeutic gene (Transgene). Also shown are three independent vectors used to express a fusion of gag/pol, rev or env (packaging
vectors, bottom three). (c) Recombinant lentivirus particles are produced by transient transfection of human embryonic kidney (HEK-293 T) cells with a
transfer vector and three packaging plasmids. The following day medium is refreshed and cells cultured for an additional 24 h before medium is collected,
cleared of debris and filter sterilised. This product can be concentrated to increase particle numbers per mL and applied to cultured cells to determine
infectious titre and sterility. c, packaging signal; RRE, rev response element; pA, polyA signal; RRE, rev response element.

promoter (D432bDg) (Persons et al., 2003). Expression of
g-globin produced chimeric HbF tetramers (mouse a2,
human g2) averaging approximately 20% at 2.4 vector
copies per HSC. A follow-up study performed using
a version of this vector with a more substantial LCR
(3.2-kb) and extended b-globin promoter sequences
(mLARbDgV5) achieved more consistent g-globin expres-
sion and improved therapeutic efficacy (Hanawa et al.,
2004). Most recently, this group replaced the g-globin 3’
UTR in mLARbDgV5 with its b-globin counterpart
(mLARbDgV5m3) and demonstrated improved efficacy of
disease correction in a SCD mouse model, with g-globin
levels sufficient to cure the majority of animals at only one
vector copy per HSC (Pestina et al., 2009).
Correction of Human SCD and
b-Thalassaemia RBCs
Cell culture models of human erythropoiesis have been
equally useful for testing the performance of globin vec-
tors. In this assay, undifferentiated CD34+ cells isolated
from BM or peripheral blood of patients with SCD or
TNS9
A
T87Q
AS3
AS3
-FB
BG-I
AnkT9w
LentiGlobinTM
d432 Δ
mLAR Δ V5
V5m3
V5m3-400
3’e
615
266
Pr
Pr
840
644
HS2 HS3 HS4
845 1153
266
T87Q
T87Q
T87Q
G16D
E22A
644 845 1153
266 1203 1213 954
254 700 1000 1400
615 840 1308 1069
266 644 845 1153
130 379 869 756
130 1095 851 1254
130 1097 869 1254
HS2 HS3 HS4 SIN
SIN
Pr HS2 HS3 HS4 SIN
Pr HS2 HS3 HS4
Pr HS2 HS3 HS4
Pr HS2 HS3 HS4
Pr HS2 HS3 HS4
Pr HS2 HS3 HS4 SIN
Pr HS2 HS3 HS4 SIN
Pr HS2 HS3 HS4 SIN
400
77
SIN
1308 1069
3’
3’e
3’e
3’e
3’e
3’e
3’e
3’e
3’e
3’e
SIN
SIN
SIN
400
1200
190
2X250
5’
SIN
SIN
SIN
SIN
77
190
2x250
1200
Figure 4 Globin lentiviral vectors used to correct murine and human models of b-thalassaemia and sickle cell disease. Schematics of the integrated provirus
genome for lentiviral vectors used by different groups. All vectors are SIN. Highlighted are the constellation of the DNase I HS2, HS3 and HS4 for each LCR
and b-globin promoter (bPr, black box) sequences that are critical for high-level, erythroid-specific expression; the genomic globin sequences (orange or
green); 3’ UTR sequences (g: turquoise, b: pink); 3’ enhancer (3’e: purple box) and insulator elements (white boxes). Therapeutic globin sequences are in
reverse orientation and include b-globin (orange arrows) or g-globin (green arrows) with amino acid mutations indicated. The length (in base pairs) of each
HS, b-globin promoter and insulator element is indicated.

b-thalassaemia are used as a seed population that is first
transduced with vector and then cultured to promote
erythroid differentiation. These models recapitulate the
composition and levels of haemoglobin associated with
disease, and allow for quantitative assessment of transgene
expression and haemoglobin synthesis per vector copy.
These are important criteria as corrected RBCs in hemo-
globinopathy patients will likely have a limited selective
advantage, as evidenced from clinical data from SCD
patients that have partial chimerism after allogenic trans-
plant (Walters et al., 2005). In fact, it is estimated that
between 15% and 20% of all engrafted stem cells would be
required to be transduced with a therapeutic globin vector
in order to achieve clinical benefit (Persons et al., 2001).
Thus, these culture models permitted testing and optimi-
sation of alternative vector designs in maturing erythroid
cells from patients with a range of variability in hae-
moglobin production.
Malik and colleagues showed that a b-globin LV vector
with 3.1 kb of LCR sequences coupled to a b-globin pro-
moter (BG-I) corrected the b-thalassaemia major pheno-
type at a vector copy of 2.2 per HSC (Puthenveetil et al.,
2004). This vector also included a 1.2 kb cHS4 insulator in
the 3’-LTR which was confirmed to permit more consistent
b-globin expression in a follow-up study (Arumugam et al.,
2007). Likewise, transduction of b-thalassaemia major
CD34+ cells with a g-globin vector modified to include
400 bp of core cHS4 sequences (mLARbDgV5m3–400)
demonstrated therapeutic levels of HbF (up to 50% total
haemoglobin) at an average of one vector copy per cell
(Wilber et al., 2011a). A modified version of the TNS9
vector that included an ankyrin insulator (AnkT9W) was
evaluated in erythroid cells derived from transduced
CD34+ cells isolated from blood of patients with varying
degrees of b-thalassaemia or SCD (Breda et al., 2012).
Curative levels of total haemoglobin were achieved at a
single vector copy per cell unless patients had the most
severe form of b-thalassaemia (b0/0
). SCD patient CD34+
cells have also been transduced with a bAS3
globin vector
insulated with a 77 bp FB-element (bAS3
-FB) (Romero
et al., 2013). This vector produced chimeric haemoglobin
tetramers to about 25% total haemoglobin at one vector
copy per cell, suggesting that this design could achieve
therapeutic levels of the anti-sickling haemoglobin in SCD
patients. These studies demonstrated that insulated LV
vectors encoding either g- or b-globin sequences were able
to meet or exceed the therapeutic threshold requirements of
the severe haemoglobin disorders and may be applied in
clinical trials.
Haemoglobin Gene Therapy in the
Clinic
In 2007, Leboulch and colleagues initiated a Phase I/II
clinical trial (LG001) in France for patients with b-tha-
lassaemia major or severe SCD (Cavazzana-Calvo et al.,
2010). BM CD34+ cells were isolated, prestimulated and
transduced with the bT87Q
vector further modified to
include two copies of a 250 bp cHS4 insulator in the 3’ LTR
(LentiGlobinTM
). Patients received a myeloablative dose
of busulfan before autologous transplant of transduced
cells. Two patients were enroled with one demonstrating
haematopoietic reconstitution after transplant. This
patient, an 18-year-old male with severe, transfusion-
dependent HbE/b0
-thalassaemia was treated in June 2007.
One year after gene therapy, he had an average vector copy
of 0.6 per cell with haemoglobin of about 10 g dL21
(nor-
mal range is 13–17 g dL21
) which resulted in him becoming
blood transfusion independent. The therapeutic bT87Q
globin contributed one-third of the total haemoglobin,
while HbE and HbF accounted equally for the remaining
portion. In this case, it appears that bT87Q
haemoglobin
tetramers and HbF both contributed to the therapeutic
efficacy. An initial point of concern was the appearance of a
partially dominant population of myeloid cells in which the
integrated vector caused alternative splicing and activation
of high mobility group AT-hook 2 (HMGA2). Uncon-
trolled expression of HMGA2 can support cellular growth,
however, this particular population is no longer dominant
and the patient remains blood transfusion independent 6
years after gene therapy (Leboulch, 2013).
For this patient, a detailed molecular analysis of vector
integrants revealed few instances where both copies of the
250 bp insulator remained intact in flanking LTR sequen-
ces (Cavazzana-Calvo et al., 2010). This led to several
improvements in the vector design and production condi-
tions in route to a new therapeutic product and follow-up
clinical study (HGB-205). A press release dated 14 June
2014 summarised early study results for two subjects, both
with b-thalassaemia major and the HbE/b0
genotype
(Bluebird Bio, Inc., 2014). This time both subjects quickly
transitioned to blood transfusion independence on day 10
(patient 1) and 12 (patient 2) after gene therapy. Although
only a few months have passed since treatment (patient 1:
4.5 months; patient 2: 2 months), total haemoglobin levels
have improved to 10.1 and 11.6 g dL21
with average vector
copy numbers 1.5 and 2.1 per cell, respectively. At this
point, both patients are in good health with no product-
related adverse events. The rapid onset of transfusion
independence for these two patients (52 weeks versus
1 year for the first trial) is a remarkable achievement and we
look forward to additional updates on this trial.
Alternative Approaches
After decades of studying factors responsible for the switch
from foetal to adult haemoglobin production, a number of
regulators have emerged, the majority of which function to
silence g-globin expression (Wilber et al., 2011b). Many of
these have been tested using lentiviral vectors encoding for
shRNA sequences designed to downregulate these repres-
sors in maturing adult erythroid cells with ‘reactivation’ of
endogenous g-globin and HbF expression serving as a

quantifiable products. One of the most compelling candi-
dates is the zinc finger transcription factor BCL11A, a
major regulator of HbF silencing in humans (Sankaran
et al., 2008). Knockdown of BCL11A increased HbF
expression from baseline levels (52%) to more than 30%
in erythroblasts derived from CD34+ cells of healthy adult
donors (Sankaran et al., 2008). Similar studies performed
using CD34+ cells of patients with b-thalassaemia major
revealed that BCL11A knockdown increased HbF and
total haemoglobin to potentially therapeutic levels (Wilber
et al., 2011a).
All of the aforementioned gene therapy approaches rely
on LV-mediated insertion of corrective sequences and cell-
type specific regulatory elements into host cell chromo-
somes. Although LV vectors have shown improved safety
profiles, the risk of insertional mutagenesis and genotoxi-
city remains a lingering concern. Furthermore, the mutant
genes are still present and in some cases expressed in the
treated cell population. Sequence specific nucleases, such
as zinc finger nucleases, transcription activator-like effec-
tor nucleases, and RNA-guided nucleases (CRISPR/Cas9)
are capable of direct genome editing. These technologies
introduce double-stranded breaks in the genome and can
stimulate repair of mutations when a functional template is
provided. Genome editing is an attractive approach to
treating b-globin disorders because the corrected gene
would be physiologically regulated by endogenous pro-
moter/enhancer sequences. With the first evidence of gene
correction in SCID-X1 HSCs reported (Genovese et al.,
2014), steps can be taken to apply these methods to the
severe haemoglobin disorders. However, significant
improvements in correction efficiency will be required to
achieve therapeutic benefit. See also: Gene Targeting by
Homologous Recombination
Conclusion and Outlook
Nearly 15 years have passed since lentiviral vectors were
first used to introduce complex globin expression cassettes
into HSCs. During this time, vectors have been optimised
to confer high-level expression that is restricted to matur-
ing RBCs. The most optimised versions have been used to
correct a variety of mouse and human cell models of SCD
and b-thalassaemia. These advances have culminated in a
successful gene therapy treatment of one b-thalassaemia
patient with encouraging results emerging for two more.
This trial indicates that current technology in HSC gene
transfer with globin lentiviral vectors may also benefit
patients with SCD, however, none have been treated. The
first US-based clinical trial using a LV vector of similar
design is enroling patients (NCT01639690; Boulad et al.,
2014) with others seeking approval from the FDA. Inno-
vative strategies designed to reactivate endogenous g-glo-
bin expression have been effective in human cell culture
models and may be integrated into globin vectors to aug-
ment therapeutic potential. Gene correction efforts are
showing potential with efficiency being the rate-limiting
variable in their application. These collective successes
coupled with an ever-increasing knowledge of stem cell
biology and transplantation provides good reason for
optimism about the future of gene therapy for the severe
haemoglobin disorders.
Acknowledgements
This work was supported by the Doris Duke Charitable
Foundation (2010037, A.W.) and National Heart, Lung
and Blood Institute (PO1HL053749, A.W.).
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