| THALASSEMIA SYNDROMES: DIAGNOSIS AND MANAGEMENT IN A CHANGING WORLD |

Beyond transfusion therapy: new therapies in thalassemia

including drugs, alternate donor transplant, and gene therapy
John Porter

University College London, London, United Kingdom

Transfusion combined with chelation therapy for severe b thalassemia syndromes (transfusion-dependent thalassemia
[TDT]) has been successful in extending life expectancy, decreasing comorbidities and improving quality of life. However,
this puts lifelong demands not only on the patients but also on the health care systems that are tasked with delivering
long-term treatment and comprehensive support. Prevention programs and curative approaches are therefore an
important part of overall strategy. Curative treatments alter the dynamic of a patient’s health care costs, from financial

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commitment over 50 years, into a potential “one-off” investment. Since the 1980s, this has usually been available only to
the 30% or so of young children with matched sibling donors. By improving the safety of matched related donors and
haploidentical hematopoietic stem cell transplants, the potential size of the donor pool for curative therapies may be
increased. Recent advances in gene therapy demonstrate that even patients lacking a matched donor can be rendered
transfusion independent with an autograft of genetically modified autologous stem cells, with a low short-term risk.
Noncurative treatments are also of potential value by decreasing use of blood and chelators and decreasing hospital
visits. An example is luspatercept, an activin-receptor trap that modifies transforming growth factor-b signaling, thereby
increasing the efficiency of erythropoiesis. This has entered phase 3 clinical trials for TDT and non-TDT and, usefully
increases in both Hb and quality of life in non-TDT as well as decreasing transfusion requirements in TDT. Other novel
noncurative treatments are entering clinical trials such improvement of erythropoiesis through pharmacological ma-
nipulation of hepcidin and iron metabolism.

Transfusion combined with chelation has undoubtedly been an ef-

Learning Objectives
• Understand the scope of options available now and in the near
future to treat thalassemia disorders
• Be aware of ongoing clinical trials for new treatments and in
particular those in allogeneic transplantation, gene therapy,
and small molecules that modify erythropoiesis
• Understand the mechanism of action, benefits, and risks of
these novel therapies
• Gain insight into how quality of life and economic issues may
also help to inform decisions about which approach is most
suitable
Achievements and limitations of transfusion
plus chelation
Thalassemias are a heterogeneous range of anemias, caused by globin
chain imbalance with ineffective erythropoiesis as its hallmark. This
therefore requires a range of measures, from simple observation, to
intensive transfusion with chelation and to potentially curative ther-
apies. Unfortunately, geographic regions with the highest prevalence
are often those with the lowest level of economic development, often
lacking in universal health care provision. With an estimated global
birth rate of 50 000 to 60 000 per annum and a global prevalence of
288 000 for severe b-thalassemias, for economic reasons alone,
prevention is an important component of overall management.
fective treatment modality when it has been efficiently delivered. The
reliability with which safe blood, iron chelation, and complex
monitoring varies not only between countries but even within
countries. Since the introduction of iron chelation in the mid- to late
1970s, life expectancy and comorbidities have markedly improved.1
Deaths from cardiomyopathy have decreased and this improved
further after the introductions of oral chelation and cardiac magnetic
resonance monitoring. There is a strong cohort effect on survival
(predicated by the date of introduction of chelation to the population
in question) but patients with transfusion-dependent thalassemia
(TDT) should now expect to live for 50 years or more, provided
treatment is not sporadic or interrupted.1 Quality of life (QOL) has
also been improved further by the introduction of orally active
chelation. For the best results, however, chelation needs to start
before irreversible iron-mediated tissue damage has occurred and for
patients now alive in their 50s, regular chelation with desferriox-
amine 5 to 7 nights a week by subcutaneous slow infusion only
became available after 10 years of age, when irreversible tissue
damage had already typically occurred, particularly to the endocrine
system. Now, with an aging thalassemia population, in centers
where chelation has been established for 4 decades or more, liver
disease and bone pain are increasing issues. For example, bone pain
has been reported in .80% of patients older than age 40 in our clinics
at University College London Hospitals and Whittington. It remains
to be seen whether cohorts that have been optimally chelated from

Conflict-of-interest disclosure: J.B.P. is on the board of directors or has served on an advisory committee for BBB, Vifor, Protagonist, La Lolla, Celgene, and Novartis.
Off-label drug use: None disclosed.

Hematology 2018 361

a young age will experience less pain, osteoporosis, cirrhosis, and
carcinomas than current patients .40 years of age.
This success comes at a price and, unfortunately, the challenge of
providing blood, chelation therapy, adequate monitoring, and patient
support can threaten to overwhelm health care systems,2 even in
areas with relatively well-developed economies such as Cyprus and
Sardinia. Consequently, effective treatment is often not delivered: for
example, as recently as a decade ago, it was estimated that only 12%
of children with TDT globally receive adequate transfusion therapy,
and ,40% of those transfused receive adequate iron chelation. It has
been estimated in the United Kingdom that a lifetime of treatment to
50 years of age is about £483 454 ($646 934).3 Costs have increased
because of newer chelator regimes and monitoring techniques such
as magnetic resonance imaging of heart and liver iron: for example.
increasing 32% in the United Kingdom in the past 16 years.3 Indirect
costs also need to be added: a large international study showed that
adult TDT patients in paid employment lose a mean of 2.1 working
days per month and children 2.8 days of school per month, from
treatment and monitoring. A further challenge with chronic trans-
fusion and chelation is adherence to treatment.4 To minimize ad-
herence issues, long-term care also requires lifelong specialist,
multidisciplinary input, often not organizationally available, even in
highly developed economic regions. For all these reasons, curative
treatments or for treatments that decrease the requirements for blood
transfusion have been eagerly sought.
Allogeneic HSCT
Risks, benefits, and outcomes using matched sibling
donors
The effectiveness and risk factors of allogeneic transplantation with
HLA-matched stem cells from a sibling were established in the 1980s
(Table 1). These remain largely unchanged as: class 1, 2, or 3, based
on the presence of 1 to 3 risk factors (out of hepatomegaly, liver
fibrosis, or poor previous chelation)5 with age older than age 14 is an
independent risk factor. Increased risk of graft rejection, probably
related to repeated transfusions5 as well as massive expansion of the
erythropoietic marrow required high-intensity conditioning initially
using busulfan and cyclophosphamide. There has been gradual
improvement in outcome over the past 20 years, resulting from better
risk stratification, targeted dose adjustment of intravenous busulfan,
modified conditioning regimens, and improvements in supportive
care. Today, if hematopoietic stem cell transplantation (HSCT) is
performed from an HLA-matched sibling donor before 14 years of
age, procedure-related mortality is ,10% (Table 1); if performed
before 5 years of age, mortality is reduced further to ~5%.6 Risk
in adults (aged 18-45) remains higher than children, so that al-
though overall survival is 88%, only 69% to 81% of adults show
thalassemia-free survival.6 Remaining complications include about
a 10% risk of severe acute graft-versus-host disease (GVHD) (grade
3-4) and 2-year risks for limited or extended chronic GVHD of 15%
and 6%, respectively. Graft rejection/failure is another important
complication, which was reduced by the use of ATG (Table 1).7
GVHD may also be reduced by this maneuver.7 Health-related QOL
(HRQOL) measures are generally good in children surviving HSCT:
chronic GVHD was the most significant factor lowering HRQOL.
Twenty years after HSCT, better HRQOL outcomes have been re-
ported than in those having conventional noncurative therapy.8 The
long-term effects of HSCT on fertility are not well described,
however, particularly in males and while fertility might occasionally
be preserved after HSCT, this is a rare event. Overall consensus
362
statements now recommend that patients with a matched sibling
donor should be offered HSCT at an early age.9
Improving outcomes in higher-risk patents
Modifications to conditioning regimes have improved outcomes in
high-risk class 3 patients (Table 1). Intensive pretransplant trans-
fusion, intensive chelation, and hydroxyurea combined with im-
proved myeloablative conditioning, improved outcomes (Table 1,
Sodani, 2004). Sinusoidal obstruction syndrome (veno-occlusive
disease [VOD]) is another important complication, particularly in
those with significant preexisting liver dysfunction and conditioning
with treosulfan rather than busulfan may also improve outcomes.
Another approach is to use a “reduced intensity” regimen in the
knowledge that partial mixed chimerism of donor- and recipient-
derived stem cells can produce adequate and stable hematopoiesis
(Table 1, Andreani, 2008).
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Extending the donor pool
Because HLA-matched related donors are available for only about
one-quarter of patients (except in countries with larger families or
communities with consanguineous marriages), a goal has been to
extend the donor pool. In principle, this can be achieved by hap-
loidentical or by matched unrelated donor (MUD) transplantation.
MUD potentially extends the donor pool close to 50% of patients of
Caucasian origin. High-resolution molecular typing for both HLA
class I and II loci has been important. Improved conditioning regimes
including ATG and methotrexate for GVHD prophylaxis also im-
proved outcome (Table 1). Because of the greater risks with MUD,
expert panel recommendations are more cautious than for sibling
allografts,9 and suggest that if a well-matched MUD is available,
allogeneic HSCT with high-resolution molecular typing for both HLA
class I and II loci is a suitable option for a child with life-long control of
iron overload and in the absence of iron-related tissue complications.
Cord blood (CB) transplant) has been explored as a way to decrease
acute and chronic GVHD. However, for matched sibling trans-
plantations, thalassemia-free survival did not differ between cord
blood transplant and blood marrow transplant.10 Unrelated CB
transplantation has also been explored for patients lacking matched
family or matched unrelated donors. Unfortunately, high rates of
graft failure and delayed hematopoietic recovery have been seen,
mainly because of inadequate cell dose (Ruggeri, Table 1). This
limitation might be mitigated by using $ 1 CB donor unit or by
giving CB together with T cell–depleted HLA-haploidentical CD34
with cells. Current recommendations for CB transplant suggest using
units containing at least 3.5 3 107 total nucleated cell/kg recipient
body weight before cryopreservation, and with less than 2 HLA
disparities.
HLA-haploidentical family donors may be another way to extend the
donor pool. Initial reports, using T-depleted CD34 donation, showed
27% graft rejection a low thalassemia-free survival (Sodani, 2004,
Table 1). There have been recent improvements in graft rejection
and overall survival followed using T-cell receptor ab1 (TCRab1)/
CD19 1 –depleted grafts (Gaziev 2018, Table 1). However, there
was delayed immune reconstitution with associated morbidity and
mortality. Furthermore posttransplant lymphoproliferative disorders
such as autoimmune hemolytic anemia or thrombocytopenia have
been seen. Procedure-related mortality, as well as morbidities,
therefore remain greater than in matched sibling transplantation9; as
a result, HLA-haploidentical family donation should currently be
confined to clinical trials.11
American Society of Hematology
Table 1. Landmarks in development of allogeneic transplantation for thalassemia

Main donor
Reference Year Center population Recipients Innovation Key outcome

5 1990 Pesaro, Italy HLA-identical 222 TM First large series. Class 1: mortality 6%,
family donors age ,16 y Identification of class 1, rejection 0%
2, 3 risk conditioning: Class 3: mortality 24%,
busulfan (3.5 mg/kg rejection 35%
per d for 4 d) 1
cyclophosphamide (50
mg/kg per d for 4 d)
Sodani, Blood 2004 Rome, Italy HLA-identical 33 TM: all class Reduced C 1 add All class 3: mortality 7%,
family donors 3, age ,17 y fludarabine rejection 8%
Immunosuppression
with azathioprine
chelation. Hydroxyurea
to suppress BM (from

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day 45)
Andreani, 2008 Rome, Italy HLA-identical 93 TM median Stable mixed chimerism in Stable mixed chimerism in
Blood sibling donor age 9.2 y mature erythrocytes 46% of patients. Residual
Transf Standard Bu, Cy host cells increase
conditioning plus: rejection risk
6fludarabine,
hydroxyurea, and
azathioprine
Gaziev, Blood 2010 Rome, Italy HLA-identical 71 children with Pharmacokinetic 7% mortality, 5% rejection;
family donor liver damage monitoring of Bu pharmacokinetic
monitoring improves
outcome
7 2012 Athens, HLA-matched 75 pediatric TM ATG with conditioning to Mortality 4%, rejection 4%,
Greece siblings reduce graft rejection overall mortality 6%,
class 3
Galambrun, 2013 France, Mainly matched 108: TM ATG to reduce graft 35% rejection without ATG
Biol Blood multicenter sibling 96/108 rejection 10% rejection with ATG
Marrow
Transplant
Mathews, 2013 Vellore, India Matched related 50 high-risk TM Treosulfan-based Mortality 13%, rejection 8%
Blood donors (class 3) conditioning
8 2005 Cagliari, Italy MUD 27 TM adults; High-resolution HLA Mortality 21%, rejection 14%
BMT Group median age, molecular type
22 y conditioning: addition of
thiotepa to Bu, Cy
Li, Blood 2012 Guangzhou, 52 MUD, 30 82 TM children Conditioning Mortality 9%, rejection 4%
China matched sibling and adolescents modifications: Bu with MUD novel
donors adjusted to PK conditioning improves
fludarabine 200 mg/kg, survival in MUD
thiotepa, azathioprine 1
Hu on days 245 to 211
24 2012 Rome, Italy MUD (n 5 40) Children and Conditioning with Mortality 7%, rejection 9%;
HLA-identical adults median treosulfan 1 thiotepa treosulfan-based regimen
sibling (n 5 20) 7 y, classes 1-3 and fludarabine to effective and safe
reduce toxicity
Ruggeri, Biol 2011 Paris, France, Cord umbilical 35 children Unrelated umbilical cord Mortality 38%, rejection
Blood Eurocord unrelated with TM 57%; low cell dose
Marrow office associated with graft failure
Transplant
Sodani, Blood 2010 Rome, Italy HLA-haploidentical 22 TM children T depleted CD341 Mortality 10%, rejection
maternal donors and adults donation 27%; no GVHD in those
with full chimerism (n 5 14)
Gaziev, Blood 2018 Rome, Italy HLA-haploidentical 14 TM (TCR) 40 TCRab1/CD191- Mortality 16% in TCR group,
Adv family donors TM CD341 depleted grafts (TCR) rejection 14%;
comparators ATG containing lymphoproliferative
preparative regimen complications,
thrombocytopenia,
hemolytic anemia

Bu, busulfan; Cy, cyclophosphamide; TM, thalassemia major.

Hematology 2018 363

Cost analysis and conclusion
Although costs vary between countries, the estimated costs of an
allogeneic HSCT from a sibling are almost invariably lower than of
conventional therapy with lifelong blood transfusion and iron che-
lation (CT).3,12 Cost analysis makes assumptions about life expec-
tancy with transfusion plus chelation difficult and vary considerably,
depending on health care resources and geographic organizational
issues. In the United Kingdom, the approximate cost of an allogeneic
blood marrow transplant13 is equivalent to about 8.7 years of CT
for a patient surviving 50 years.3 The advantages of transplan-
tation over CT will be greater when the latter cannot be provided
to the necessary standards for financial or organizational reasons.
Comparison of outcomes with either HSCT or CT, in Sardinia,
where both are available to a high standard, showed that overall
survival of 83% in transplanted patients was similar to CT with
overall survival of 85%.14 Outside matched sibling allograft, risks
are still higher and there is less consensus about the optimal risk/
benefit ratio. Long-term follow-up at least yearly is recommended
for after HSCT.
Gene therapy
Gene therapy thalassemia has already become a clinical reality using
lentiviral (LV) vectors to replace key elements of the b globin gene15
and other approaches are in advanced preclinical stages. The use of
autologous modified stem cells for engraftment has the obvious
advantage of providing HSCT for all patients. Further theoretical
advantages are the lower procedure-related mortality and the lack of
need for immune suppression, as well as no risk of acute or chronic
GVHD or immune-mediated graft rejection. Some transplant-related
issues will remain while myeloablative regimes are used, however;
these include VOD and long-term effects on fertility. Overall, the
clinical benefit of gene therapy must outweigh the risks of mye-
loablative conditioning regimens; also, the long-term safety will need
to be demonstrated with respect to the oncogenic potential of gene
therapy itself (see the following section).
Approaches to gene modification used in clinical trials
In principle, correction of the abnormal or missing gene can be
performed by gene addition, such as using LV vectors or by precisely
correcting single-point mutations using designer nucleases. Be-
cause there are .200 b-thalassemia mutations, a strategy that can
be applied irrespective of the individual b mutation is preferable.
Early studies with murine thalassemia models used recombinant
g-retroviral vectors, derived from the Moloney leukemia virus, as
carriers to introduce the human b-globin gene in HSC. Limitations
included low vector titers, low expression of b-globin, and unstable
vectors with a tendency to rearrangements. b-globin expression was
then increased by incorporation of the core elements of the hyper-
sensitive sites 2, 3, and 4 of the human b-globin locus control region.
However vector rearrangements and position effects were still seen.
Self-inactivating LV vectors were then evaluated for which over-
came some of these issues; it is these that trials are most advanced
Gene replacement of functional b gene(s) into HSC using LV
vectors. Here the intent is to insert a fully functional gene (or part of
it) that will result in sufficient correction of globin chain imbalance
that is the hallmark of thalassemia. Self-inactivating, LV vectors are
now used by several laboratories for clinical trials for b-thalassemia,
which have been reviewed in detail recently elsewhere (Table 2).16
These vectors have many shared features.17 For gene expression to
be adequate, promoters are required, and the insertion site is critical
364
to efficacy and safety. The role of insulators, ostensibly to decrease
the risk of insertional mutagenesis is less clear: the first clinically
successful LV vector, HPV569, contained 2 insulator elements.
However, because of low engraftment in 3 of 4 patients and because
of a transient clone in 1 patient, the insulators (cHS4) were omitted
from the subsequent vector BB305, when Bluebird Bio (BBB)
became involved (Table 2). Insulators are included in the TNS9.3.55
vector used at Memorial Sloan Kettering Cancer Center17 but not in
the vector developed in the Italian GLOBE study (Table 2).16 The
potential for long-term correction of thalassemia, using LV-transduced
HSC was first shown in a murine model of thalassemia intermedia18
and subsequently clinically in a transfusion-dependent adult Eb
thalassemia patient.19
Increasing fetal hemoglobin synthesis by gene editing. A
second general approach is to promote g chain synthesis in the
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developing erythroblast to a point where the a/non-a ratio is suf-
ficiently balanced to prevent intramarrow apoptosis in developing
red cells. This has been achieved in human hematopoietic cells by
editing genes that suppress g globin synthesis, such as the BCL11A
transcription factor, without affecting other hematopoietic lineages.
Editing methodologies include designer nucleases such as zinc
fingers or the repurposing of bacterial clustered regularly interspaced
short palindromic repeats (CRISPR) to allow RNA-guided cleavage
of complementary DNA. By using CRISPR/Cas9 and guide RNA in
human HSC, a 40% reduction in BCL11A mRNA resulted in
corresponding twofold increase in g-globin transcript ex vivo.20
Another way to enhance fetal hemoglobin (HbF) is to disrupt g2d
intergenic HbF silencers. This has been recently demonstrated in
human progenitor cells, using CRISPR/Cas9 to edit a 13.6-kb ge-
nomic region encompassing the d- and b-globin genes.21 At least 2
such approaches are being initiated in clinical trials for thalassemia
(Table 2). The first uses zinc finger nuclease to boost HbF by dis-
ruption of an erythroid specific enhancer of the BCL11A gene
(Sangamo, Bioverativ). The second is CTX001, a gene-edited cel-
lular product that uses CRISPR Cas9 technology to target an
erythroid-specific enhancer of the BCL11A gene (Table 2), which
has been are approved for clinical trials in several countries.22 The
latter demonstrates a high ex vivo editing rate and no identified off-
target sites in preclinical models. The safety and specificity of the
CRISPR approach to gene editing is currently debated. A key issue is
the so called “off target” effects (OTEs). These occur under con-
ditions in which, although cleavage at sites with perfect comple-
mentarity to the guide RNA (gRNA) is highly efficient, this can also
occur at sites where 1 or more bases are mismatched with the gRNA.
In principle, OTE could lead to loss of a key stem cell function or
could have oncogenic potential by activating oncogenes or inacti-
vating a tumor suppressor genes. Methods that result in higher
sustained levels of cellular Cas9 and gRNA tend to have higher OTE.
At high editing efficiencies, double-strand DNA breaks are induced
by Cas9 and, although these usually lead to cell death through a P53/
TP53-dependent mechanism, cells with mutated P53 may be re-
sistant, risking the emergence of mutated cells.23 It remains to be
seen whether these concerns are real in clinical use and/or whether
strategies to minimize OTE such as with high-fidelity Cas9 variants
are effective.
Autologous transplantation of corrected stem cells. Irrespective
of the gene modification, transplantation typically involves 4 steps:
mobilization of autologous CD341 stem cells, ex vivo correction of
the abnormal gene, myeloablative conditioning, and the reinfusion of
American Society of Hematology
sufficient corrected stem cells. Autologous CD341 cells can be ob-
tained by direct collection from bone marrow aspiration16 or from stem
cell mobilization.15,19 Mobilization of CD341 cells has been generally
achieved using granulocyte colony stimulating factor with or without
plerixafor (Table 2). Myeloablation has been achieved using busulfan
monotherapy, for example, with total doses of 12.8 mg/kg used over
4 days in the BBB studies. The procedure has been generally well
tolerated with adverse events and frequencies typical of an au-
tologous transplant.15 A less intensive myeloablative regime would
presumably lead to lower VOD risk, which although less frequent
than in allogeneic transplantation, is not 0.15 Reduced intensity
busulfan conditioning has been used in the Memorial Sloane
Kettering Cancer Center study (Table 2). By contrast, in the Italian
GLOBE study, treosulfan and thiotepa have been used for con-
ditioning, which may reduce toxicity when compared with bu-
sulfan.24 Experience thus far suggests that suppression of the bone
marrow before HSC by hypertransfusion may improve engraft-
ment. The duration of hypertransfusion and the optimal levels are
yet to be determined. Good chelation control before transplant is
advisable and is a requirement of some protocols to reduce the risk
of liver complications in the peritransplant period. Again, the
optimal liver iron concentration values for minimizing hepatic risk
have not been formally evaluated. Infertility is a likely consequence
of this regime and VOD is also occasionally seen.
After myeloablation, the genetically modified autologous HSPC are
returned, where they repopulate the hematopoietic compartment. In 1
trial, infusion was into the posterior superior iliac crest (Table 2) with
the aim of enhancing engraftment and minimizing a first-pass in-
travenous filter.16 The vector copy number (VCN) in the infused
CD341 cells is important to levels of HbA or HbF obtained postgraft.
For example, improvement in manufacturing process of LentiGlobin
has led to both increased peripheral blood VCN and also increased
levels of posttransplant Hb (HbAT87Q). This has allowed current
trials to be extended to more severe forms of thalassemia (b0/b0)
(BBB study 212).
Clinical outcomes reported to date. Interim combined data have
been reported from 2 phase 1-2 studies of autologous HSC trans-
duced ex vivo with the LV BB305 (BBB) vector to 22 patients (aged
12-35) (TDT trials numbers NCT01745120 and NCT02151526).15
At 26 months (range, 15-42) after infusion, 12 of 13 patients with
a non-b(0)/b(0) genotype were transfusion free with HbA(T87Q)
levels from 3.4 to 10.0 g/dL, and total hemoglobin ranged from 8.2 to
13.7 g/dL. Furthermore, in 9 patients with the more severe IVS1-110
mutation, the annual transfusion volume was decreased by 73%. No
safety issues were attributed to the BB305 vector in either studies 204
or 205. Nine serious adverse events were reported, including 2
episodes of veno-occlusive liver disease attributed to busulfan
conditioning. No replication-competent lentivirus was detected in the
patients in either study, and serial monitoring of vector integration
sites in blood samples has consistently shown polyclonal profiles of
unique integration sites without dominant. However, the overall
long-term risks remain unclear: in particular, it is unknown whether
there is increased oncogenic potential in humans over several de-
cades. However, no clonal dominance related to vector integration
has been thus far observed in the LentiGlobin studies. This is
a rapidly evolving field and other clinical studies also using self-
inactivating LV vectors or CRISPR both in thalassemia and sickle
cell disease are less advanced but also promising (Table 2). Ulti-
mately, a conditioning regime that did not have the risk of infertility
would also be welcome.
Hematology 2018
Pharmacological enhancers of effective erythropoiesis
Enhancers of erythropoiesis for thalassemia patients have been known
for .2 decades, working either by direct stimulation: erythropoiesis-
stimulating agents or by enhancing HbF synthesis such as with
hydroxyurea (Hu) and butyrates. Unfortunately, responses have
generally been highly variable and unpredictable so that, unlike sickle
cell disease, their place in the management of TDT and non-TDT is
still unclear. For reasons of space, this section will focus only on agents
that have reached the stage of clinical use or clinical trials.
Erythropoiesis-stimulating agents
In non-TDT, erythropoietin (EPO) levels decrease with age, as does
erythropoietic response to EPO, so that Hb values also tend to fall.
EPO acts by binding to the EPO receptor on the earliest erythroid
progenitors, burst-forming unit-erythroid, with subsequent activation
of Jak2 kinase and downstream Stat5. EPO also mediates erythro-
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blast expansion though suppression of Fas-FasL coexpression,
a potentially undesirable effect in non-TDT, risking increased and
unwanted expansion of extramedullary erythropoiesis. EPO doses
have typically been 1000 IU/kg weekly or twice weekly and mainly
short term in uncontrolled trials so that the long-term risks of
extramedullary expansion are not well defined. Highly variable
responses have been reported: from no response up to 3 g/dL in some
non-TDT patients, and with transfusion independence in some
hitherto transfused patients. Pegylated EPO is now in a phase 1
clinical trial for b thalassemias (NCT02950857), but this may risk
increased extramedullary hematopoiesis if used alone. Surprisingly,
in the light of recent reports of iron restriction enhancing erythro-
poiesis (see the following section), some trials used iron supple-
mentation to further increase the Hb response.
Jak2 inhibition
A secondary effect of ineffective erythropoiesis (IE) and hypoxia in
thalassemias is the EPO and Jak2 phosphorylation-dependent ex-
pansion of immature erythroblasts that are unable to bypass apoptosis
at the polychromatophilic stage, to 5 to 6 times that of healthy controls.
Chronic anemia in thalassemia skews the erythropoietic response, so
that proliferation outpaces differentiation. In principle, inhibitors of
Jak2 kinase may decrease IE by improving the balance between
proliferation and differentiation. Murine studies in a thalassemia
model showed improvement in IE and decrease in spleen size,
possibly at the cost of decreased overall erythropoietic activity.
Furthermore, in humans with myelofibrosis, a Jak2 inhibitor
(INCB018424) reduced spleen size. A phase 2A open label single
arm study was therefore undertaken of ruxolitinib (INC424, Jakavi)
(NCT02049450) in 30 TDT patients with splenic enlargement25 to
determine whether transfusion requirement and/or spleen size could
be decreased. Treatment was given for 30 weeks at a starting dose of
10 mg twice daily. Overall, transfusion requirement did not decrease
(12 decreases, 7 increases, 8 did not change). However there was
a significant 26% reduction in splenic volume at 30 weeks. Thus,
although the lack of decrease in transfusion requirements are con-
sistent with murine studies, ruxolitinib may have a role in reducing
spleen size in TDT patients with advancing splenomegaly. In
principle, after reduction of spleen size and cessation of ruxolitinib,
transfusion requirement may then decrease, so that intermittent therapy
may be applicable in selected TDT patients with splenomegaly.
HbF induction
HbF-inducing agents increase g-globin, which binds to unpaired
a-chains in b thalassemias, thereby decreasing globin imbalance,
ineffective erythropoiesis, and hemolysis. Many drugs increase HbF
365
366
Table 2. Gene therapy for thalassemia

Patient target BMT conditioning VCN of

Product and no. and age Cell source CD341 infusion infused Stage of trial
study name Sponsor Principle of therapy Transgene vector eligibility mobilization dose CD341 Outcomes development

HVP569, LG001 Genetix France Autologous b A2.T 87Q 4 TDT 5-35 y Bone marrow Busulfan 12.8 mg/ 0.6 1 case of Phase 1 started
transplant of encoding or peripheral kg over 4 d; dose transfusion July 2006
transduced human b-globin blood adjusted to PK independence in
CD341 HSC (T87Q) IV 3.9 3 106 Eb thalassemia
LV vector CD34/kg BM with stable clone
HVP569 4.3 3 106 cells/ at 7 y FU; low
kg from PB engraftment in 3
other patients
LlentigGlobin BBB As above As above, with 18 TDT PB GCSF 1 Busulfan 12.8 mg/ 0.3-1.5 Interim published; Phase 1/2
BB305 HGB removal 12-35 y plerixafor kg 6-18 3 106 see text started 2013
2204 of insulator LV CD34/kg b0/b0
vector BB305 5-13 3 106
CD34/kg others
LlentigGlobin BBB As above As above 7 TDT 5-35 y As above Busulfan 12.8 mg/ 0.8-2.1 Interim published; Phase 1/2
HGB-205 kg IV 9-14 3 106 see text started
NCT02151526 CD34/kg August 2014
LlentigGlobin BBB As above As above non b0/b0 n 5 As above Busulfan 12.8 mg/ 2.4-3.2 Interim results Phase 3
HGB 2207 15 12-50 y kg IV 7-8 3 106 presented at
CD34/kg ASH 2017
Transfusion-free
3/3 at .6 mo
LlentigGlobin BBB As above As above 1 b0/b0 n 5 4, As above Busulfan 12.8 mg/ Ongoing Ongoing Phase 3
HGB 2212 improved 3-7 y n 5 3, kg 3 4 d ongoing
manufacturing to 8-17 y
increase VCN
Was TNS9.3.55 Memorial Transplant of Lentiviral vector 10 TDT .18 y As above Reduced intensity 0.39-0.21 Stable Phase 1 started
NCT01639690; Sloan Kettering autologous conditioning 2 d; engraftment July 2012;
now CD341 HSC busulfan 8 g/kg; without clonal completion
TNS9.3.55.A1 transduced interval 2 d IV dominance17 July 2018?
with TNS9 3.55 11.8-8.4 3 106
CD34/kg
ST-400 Sangamo, Transplant of Zinc finger 6 TDT 18-40 y As above Standard NA Change in HbF; Phase 1/2
Bioverativ autologous nuclease to conditioning change in started May
modified boost HbF by anticipated annual volume of 2018; ends
CD341 HSC disruption packed RBC May 2020
of BCL11A gene
TIGET-BTHAL Telethon Transplant of b globin GLOBE TDT 3/$18 y As above Treosulfan 42 g/m2 0.7-1.5 Ongoing Phase 1/2
NCT02453477 Foundation autologous 3/8-17 y 1 thiotepa 8 mg/ Started May
modified 4/3-7 y kg 16-19.5 3 2015
CD341 HSC 106 CD341/kg
intramarrow

BM, bone marrow; GCSF, granulocyte colony stimulating factor; PB, peripheral blood; PK, pharmacokinetics; SCD, sickle cell disease.

American Society of Hematology

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 but few have undergone systematic clinical evaluation. g-Globin can

beginning in
Stage of trial
development
be increased by cytostatic agents that disturb the balance between

Europe
proliferation and differentiation: such as Hu. In non-TDT, responses

Phase 1
have been highly variable, from very little to .2 g/dL. QOL im-
provement has been reported when Hb increased with EPO plus
hydroxyurea by 1.6 g/dL or by 0.7 g/dL using Hu alone.26 Decreased
extramedullary hematopoiesis, pulmonary hypertension, leg ulcers,

annual RBC ex

mRNA to 39%
vivo; increases

patient sample
Change in HbF;

b-thalassemia
hypothyroidism, and osteoporosis were reported in a retrospective

(as a ratio of
Outcomes

in g-globin
analysis.27 In TDT, response has been particularly variable. Some
change in

g/a) in 1
reports have been remarkable: 78% of “transfusion-dependent”
patients becoming transfusion independent in an Iranian study.28 A
Cochrane review found no convincing evidence that Hu reduces
transfusion requirement, however. Some of this variability may
reflect the local clinical definitions TDT but underlying genetic
infused
VCN of

CD341

determinants cannot be excluded.

NA

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Short-chain fatty acids such a butyrates activate the Ag gene promoter
busulfan 8 mg/kg;
conditioning 2 d;

and possibly also inhibit histone deacetylase. Despite the occasional

Reduced intensity

11.8-8.4 3 106
BMT conditioning
CD341 infusion

dramatic individual responses, initially with intravenous arginine

interval 2 d

butyrate, Hb responses .1 g/dL have been rare. Responses with orally

CD34/kg
dose

bioavailable butyrates have been inconsistent and insufficient. Other

modulators of HbF have included DNA demethylating agents such as
5-azacytidine and decitabine to hypomethylate the g-globin genes. Hb
increments of about 1 g/dL were reported, but long-term safety and the
role in management of non-TDT is not established. Pharmacological
mobilization
Cell source

BM, bone marrow; GCSF, granulocyte colony stimulating factor; PB, peripheral blood; PK, pharmacokinetics; SCD, sickle cell disease.

enhancers of HbF are under preclinical investigation, such as LSD-1

As above

inhibitors, and may have application for non-TDT. Other targets for
HbF manipulation, such as SOX3, KLF1, HBSL1-MYB intergenic
region, DRED complex (TR2 and TR4), and Lim domain biding 1
have yet to be evaluated clinically.29
Patient target
no. and age

TDT (also
eligibility

SCD)

Modulation of GDF11 with activin receptor traps,

sotatercept and luspatercept

Background to development for thalassemia. These agents

to boost HbF by
Transgene vector

BCL11A gene

were originally conceived to improve bone mineral density in

disruption of

postmenopausal women, preventing osteoclast-dependent bone

nuclease
Zinc finger

resorption through inhibition of activin-dependent signaling, but

sotatercept unexpectedly increased Hb values. This was later
shown with luspatercept (ACE-356), but unlike in sotatercept,
increases in bone mineral density were not seen, possibly because
ACE-356 does not bind with high affinity to activin A. In murine
Principle of therapy

studies using murine equivalents of these drugs (RAP-536 or RAP-

CD341 HSC
autologous
Transplant of

011), Hb increases were seen in both healthy animals and models

modified

of anemia that included: thalassemia, chronic renal disease,

myelodysplastic syndromes, Diamond Blackfan anemia, and iron-
restricted anemia.

Structure and mechanisms of action. Sotatercept (ACE-011), is

a recombinant human homodimeric activin type IIa receptor fusion
Therapeutics
Sponsor

protein comprising the extracellular domain of the human activin

type IIA receptor, fused to the Fc domain of human immunoglobulin
CRISPR

Vertex
and

G1 with 2 disulfide-linked chains, dimerized through the Fc region.

Luspatercept (ACE-536) is a similar fusion protein, but for activin
Table 2. (continued)

receptor type IIB. These act as traps for a wide range of ligands of the
transforming growth factor-b superfamily and inhibit signaling by
binding ligands extracellularly, sequestering them away from their
Product and
study name

receptors. Linking clinical effects to inhibition of a single ligand can

be challenging, but studies have also provided insights into the
CTX001

regulation of hematopoiesis itself. Effects appear EPO-independent:

the kinetics of response are too rapid for an EPO-like effect,30

Hematology 2018 367

368
Table 3. Potential enhancers of erythropoiesis in ongoing or imminent clinical trials

Drug name Route and

and drug Molecular Potential applications frequency of Observed clinical
class target Secondary effect in thalassemias administration Clinical trial stage effects to date Company Ref

Sotatercept Trap for Decreased GDF11 Improved Hb and Subcutaneous, Phase 2a for Dose-dependent Hb Celgene, See text
(ACE-011) TGF-b signaling; increased QOL in non-TDT; 3 weekly thalassemia increase 1-2 g/dL Acceleron
activin ligands erythropoiesis reduced transfusion in non-TDT;
receptor IIa effectiveness in TDT decreased
transfusion in
some TDT
Luspatercept As above As above As above Subcutaneous, Phase 3 for Improved anemia Celgene, See text
(ACE-536) 3 weekly thalassemia in non-TDT as Acceleron
activin above; improved
receptor IIb QOL; improved 6-min
walk; decreased
transfusion in
some TDT
PTG-300 Ferroportin Hepcidin mimetic Improved Subcutaneous Phase 1: no Prolonged plasma Protagonist Presented EHA 2018
hepcidin increases erythropoiesis serious adverse half-life and serum Therapeutics (abstract S843)
derivative effectiveness events expected; iron decrease
of erythropoiesis hypoferremia
observed
LJPC-401 Ferroportin Hepcidin mimetic Secondary iron overload Subcutaneous Phase 1: no Expected hypoferremia La Jolla Presented EHA 2018
synthetic synthetic hepcidin distribution toxicity reported, observed Pharmaceutical (abstract S894)
hepcidin healthy volunteers Company
formulation
Tmprss6- Tmprss6 As above Improved Subcutaneous Validated in NYR Alnylam Animal model ASH
siRNA erythropoiesis preclinical studies Pharmaceuticals (2013)
Tmprss6- Tmprss6 Stimulates hepcidin Improved Subcutaneous Phase 1 ongoing NYR Ionis Ionis Pharma press
ASO production by erythropoiesis Pharmaceuticals release; in pipeline
suppressing
Tmprss6
SLN124 Tmprss6 As above Improved Subcutaneous Phase 1 NYR Silence Animal data
Tmprss6- erythropoiesis; planned for Therapeutics presented
siRNA iron overload late 2019 EHA (2018)
(abstract S893)
VIT-2763 Ferroportin Ferroportin Prevent iron Oral Phase 1 NYR Vifor Pharma Vifor press release
ferroportin inhibitors overload; improved planned for (2018)
inhibitor erythropoiesis late 2018
decrease
MHs (PR65, Ferroportin Hepcidin mimetic As above Subcutaneous Preclinically NYR University of 36
PR73, (mini-hepcidin) validated in early California,
M009, low MW clinical trials Los Angeles
M012)

ASH, American Society of Hematology; EHA, European Hematology Association; NYR, not yet reported.

American Society of Hematology

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occurring at later stages of erythropoiesis31 and, unlike EPO re-
sponses, appear to require accessory cells. Modulation of GDF-11
has been implicated as the mechanism of action in thalassemia31:
GDF11 is upregulated in spleen and erythroid cells of thalassemic
animals and inhibits murine erythroid maturation. GDF11 expression
and recombinant human homodimeric activin type IIB receptor in
erythroid precursors decreases progressively with maturation.31
Inactivation of GDF11, as with these drugs, decreases oxidative
stress and a-globin membrane precipitates in red blood cells. GDF11
also corrected the abnormal ratio of immature/mature erythroblasts in
thalassemic mice by inducing apoptosis of immature erythroblasts
through the Fas–Fas ligand pathway.32
Clinical trials. Phase 2 clinical trials in non-TDT with sotatercept
showed dose-dependent increments in Hb typically .1 g/dL with
improvements in red cell morphology (Table 3).33 Because of the
long plasma half-life of ~23 days, subcutaneous dosing was given
every 3 weeks, with Hb improvement seen within the first 3 cycles in
non-TDT. In TDT, a reduction in transfusion burden was also seen.
Tolerability was generally good and reported adverse events in-
cluded grade 3 bone pain in 1 TDT patient and grade 2 phlebitis in
a non-TDT patient. There were similar findings in phase 2a trial of
luspatercept (ACE-356; NCT01749540) in 31 non-TDT and 32 TDT
patients at dose levels titrated up to 1.25 mg/kg every 3 weeks were
presented at the European Hematology Association Congress in
2018.34 In NTDT an increase of .1 g/dL over baseline was seen in
71% of patients and of .1.5G in 55% at 3 months. In a patient-
reported outcome QOL questionnaire, a significant increase in score
was seen in patients showing .1 g/dL Hb increase.34 The 6-minute
walk test showed a significant improvement at 48 weeks of treatment
and improvement correlated with the Hb increase at this time. In TDT
patients, a $20% reduction in transfusion requirement was seen in
78% of patients and a $30% reduction in 69% of patients after
a median treatment duration of 14 months. In patients as a whole,
tolerability was generally good with the majority of adverse effects
being grade 1-2: bone pain was seen in 38%, headache in 28%,
myalgia in 22%, and arthralgia in 19%. Other adverse effects
($10%) were asthenia, injection site pain, and back pain. This study
is now in a 5-year extension phase (NCT02268409). The detailed
phase 2 findings with sotatercept and luspatercept are in press.
Luspatercept was selected for phase 3 development, mainly because,
unlike sotatercept, it binds only minimally to activin A and thus may
have lower off-target effects. Randomized double-blind phase 3
studies are now under way for TDT (NCT02604433: Efficacy and
Safety Study of Luspatercept (ACE-536) Versus Placebo in Adults
Who Require Regular Red Blood Cell Transfusions Due to Beta [b]
Thalassemia) and non-TDT (NCT03342404; A Study to Determine
the Efficacy and Safety of Luspatercept in Adults With Non
Transfusion Dependent Beta [b]-Thalassemia). The predetermined
criterion for efficacy in TDT is a 33% decrease in the number of
transfused red blood cell units from baseline. In summary, both
sotatercept and luspatercept regularly increase Hb in non-TDT to
levels associated with improved QOL. In TDT, the reduction in
transfusion burden is likely to decrease the use of chelation and the
frequency of hospital visits. It is reassuring that RAP-536 corrected
the complications of ineffective erythropoiesis, splenomegaly, and
bone pathology in a mouse model of non-TDT.35 However, as
transfusion decreases in TDT and autologous erythropoiesis pro-
portionately increases, it will be important to establish that the known
complications of non-TDT, such as thrombosis and extramedullary
erythropoietic expansion, do not become problematic.
Hematology 2018
Restricted iron delivery to the erythron
Animal models suggest that by restricting iron delivery to the
thalassemic erythron, the efficiency of erythropoiesis can be im-
proved. Iron restriction, using intraperitoneal iron-free human
transferrin or by increasing plasma hepcidin levels, improved Hb in
thalassemic mice. Both manipulations decrease iron delivery to
normoblasts, thereby decreasing heme synthesis, decreasing hemi-
chrome formation, and hence reactive oxygen species–mediated
oxidative stress and apoptosis. In principle, plasma hepcidin can be
increased by giving exogenous hepcidin(s) or by upregulating
hepcidin synthesis. Administration of minihepcidins, short peptides
that mimic the activity of endogenous hepcidin, improved ineffective
erythropoiesis, anemia, and iron overload in thalassemic mice.36
Hepcidin synthesis can be increased by suppressing Tmprss6,
a transmembrane serine protease (matriptase-2) that normally sup-
presses hepcidin synthesis by deactivating hemojuvelin to form
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soluble hemojuvelin. An antisense oligonucleotide approach has
been shown to decrease Tmprss6 protein, thereby increasing hep-
cidin and leading to improved anemia in murine thalassemia
models.37 Injection of siRNA formulated in lipid nanoparticles di-
rected to the liver improved anemia by ameliorating of IE and
improving red cell survival38 in thalassemic mice. Iron chelation has
a less clear effect, however, at least when given alone, suggesting that
restriction of iron delivery may need to be through the transferrin
uptake. This may have a role when combined with hepcidin mi-
metics, however. In humans, large doses of purified human apo-
transferrin showed decrements in transferrin saturation or NTBI.39
Several clinical trials are under way or imminently planned to in-
vestigate the effect of hepcidin, mini-hepcidin or manipulation
through TMPRSS6 on transferrin saturation, NTBI and hence
erythropoiesis in non-TDT (Table 3). Secondary effects on iron
uptake by myocardium are also under investigation.
Correspondence
John Porter, Department of Haematology, University College London,
Paul O'Gorman Building, 72 Huntley St, London WC1E6HX, United
Kingdom; e-mail: j.porter@ucl.ac.uk.
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