| 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

Downloaded from https://ashpublications.org/hematology/article-pdf/2018/1/361/1254816/hem01850.pdf by guest on 24 April 2020

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 fective treatment modality when it has been efficiently delivered. The
• Understand the scope of options available now and in the near reliability with which safe blood, iron chelation, and complex
future to treat thalassemia disorders monitoring varies not only between countries but even within
• Be aware of ongoing clinical trials for new treatments and in countries. Since the introduction of iron chelation in the mid- to late
particular those in allogeneic transplantation, gene therapy, 1970s, life expectancy and comorbidities have markedly improved.1
and small molecules that modify erythropoiesis Deaths from cardiomyopathy have decreased and this improved
• Understand the mechanism of action, benefits, and risks of further after the introductions of oral chelation and cardiac magnetic
these novel therapies resonance monitoring. There is a strong cohort effect on survival
• Gain insight into how quality of life and economic issues may (predicated by the date of introduction of chelation to the population
also help to inform decisions about which approach is most in question) but patients with transfusion-dependent thalassemia
suitable (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
Achievements and limitations of transfusion chelation. For the best results, however, chelation needs to start
plus chelation before irreversible iron-mediated tissue damage has occurred and for
Thalassemias are a heterogeneous range of anemias, caused by globin patients now alive in their 50s, regular chelation with desferriox-
chain imbalance with ineffective erythropoiesis as its hallmark. This amine 5 to 7 nights a week by subcutaneous slow infusion only
therefore requires a range of measures, from simple observation, to became available after 10 years of age, when irreversible tissue
intensive transfusion with chelation and to potentially curative ther- damage had already typically occurred, particularly to the endocrine
apies. Unfortunately, geographic regions with the highest prevalence system. Now, with an aging thalassemia population, in centers
are often those with the lowest level of economic development, often where chelation has been established for 4 decades or more, liver
lacking in universal health care provision. With an estimated global disease and bone pain are increasing issues. For example, bone pain
birth rate of 50 000 to 60 000 per annum and a global prevalence of has been reported in .80% of patients older than age 40 in our clinics
288 000 for severe b-thalassemias, for economic reasons alone, at University College London Hospitals and Whittington. It remains
prevention is an important component of overall management. 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 statements now recommend that patients with a matched sibling
carcinomas than current patients .40 years of age. donor should be offered HSCT at an early age.9

This success comes at a price and, unfortunately, the challenge of Improving outcomes in higher-risk patents
providing blood, chelation therapy, adequate monitoring, and patient Modifications to conditioning regimes have improved outcomes in
support can threaten to overwhelm health care systems,2 even in high-risk class 3 patients (Table 1). Intensive pretransplant trans-
areas with relatively well-developed economies such as Cyprus and fusion, intensive chelation, and hydroxyurea combined with im-
Sardinia. Consequently, effective treatment is often not delivered: for proved myeloablative conditioning, improved outcomes (Table 1,
example, as recently as a decade ago, it was estimated that only 12% Sodani, 2004). Sinusoidal obstruction syndrome (veno-occlusive
of children with TDT globally receive adequate transfusion therapy, disease [VOD]) is another important complication, particularly in
and ,40% of those transfused receive adequate iron chelation. It has those with significant preexisting liver dysfunction and conditioning
been estimated in the United Kingdom that a lifetime of treatment to with treosulfan rather than busulfan may also improve outcomes.
50 years of age is about £483 454 ($646 934).3 Costs have increased Another approach is to use a “reduced intensity” regimen in the
because of newer chelator regimes and monitoring techniques such knowledge that partial mixed chimerism of donor- and recipient-
as magnetic resonance imaging of heart and liver iron: for example. derived stem cells can produce adequate and stable hematopoiesis
increasing 32% in the United Kingdom in the past 16 years.3 Indirect (Table 1, Andreani, 2008).

Downloaded from https://ashpublications.org/hematology/article-pdf/2018/1/361/1254816/hem01850.pdf by guest on 24 April 2020

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 Extending the donor pool
days per month and children 2.8 days of school per month, from Because HLA-matched related donors are available for only about
treatment and monitoring. A further challenge with chronic trans- one-quarter of patients (except in countries with larger families or
fusion and chelation is adherence to treatment.4 To minimize ad- communities with consanguineous marriages), a goal has been to
herence issues, long-term care also requires lifelong specialist, extend the donor pool. In principle, this can be achieved by hap-
multidisciplinary input, often not organizationally available, even in loidentical or by matched unrelated donor (MUD) transplantation.
highly developed economic regions. For all these reasons, curative MUD potentially extends the donor pool close to 50% of patients of
treatments or for treatments that decrease the requirements for blood Caucasian origin. High-resolution molecular typing for both HLA
transfusion have been eagerly sought. 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,
Allogeneic HSCT expert panel recommendations are more cautious than for sibling
Risks, benefits, and outcomes using matched sibling allografts,9 and suggest that if a well-matched MUD is available,
donors allogeneic HSCT with high-resolution molecular typing for both HLA
The effectiveness and risk factors of allogeneic transplantation with class I and II loci is a suitable option for a child with life-long control of
HLA-matched stem cells from a sibling were established in the 1980s iron overload and in the absence of iron-related tissue complications.
(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 Cord blood (CB) transplant) has been explored as a way to decrease
fibrosis, or poor previous chelation)5 with age older than age 14 is an acute and chronic GVHD. However, for matched sibling trans-
independent risk factor. Increased risk of graft rejection, probably plantations, thalassemia-free survival did not differ between cord
related to repeated transfusions5 as well as massive expansion of the blood transplant and blood marrow transplant.10 Unrelated CB
erythropoietic marrow required high-intensity conditioning initially transplantation has also been explored for patients lacking matched
using busulfan and cyclophosphamide. There has been gradual family or matched unrelated donors. Unfortunately, high rates of
improvement in outcome over the past 20 years, resulting from better graft failure and delayed hematopoietic recovery have been seen,
risk stratification, targeted dose adjustment of intravenous busulfan, mainly because of inadequate cell dose (Ruggeri, Table 1). This
modified conditioning regimens, and improvements in supportive limitation might be mitigated by using $ 1 CB donor unit or by
care. Today, if hematopoietic stem cell transplantation (HSCT) is giving CB together with T cell–depleted HLA-haploidentical CD34
performed from an HLA-matched sibling donor before 14 years of with cells. Current recommendations for CB transplant suggest using
age, procedure-related mortality is ,10% (Table 1); if performed units containing at least 3.5 3 107 total nucleated cell/kg recipient
before 5 years of age, mortality is reduced further to ~5%.6 Risk body weight before cryopreservation, and with less than 2 HLA
in adults (aged 18-45) remains higher than children, so that al- disparities.
though overall survival is 88%, only 69% to 81% of adults show
thalassemia-free survival.6 Remaining complications include about HLA-haploidentical family donors may be another way to extend the
a 10% risk of severe acute graft-versus-host disease (GVHD) (grade donor pool. Initial reports, using T-depleted CD34 donation, showed
3-4) and 2-year risks for limited or extended chronic GVHD of 15% 27% graft rejection a low thalassemia-free survival (Sodani, 2004,
and 6%, respectively. Graft rejection/failure is another important Table 1). There have been recent improvements in graft rejection
complication, which was reduced by the use of ATG (Table 1).7 and overall survival followed using T-cell receptor ab1 (TCRab1)/
GVHD may also be reduced by this maneuver.7 Health-related QOL CD19 1 –depleted grafts (Gaziev 2018, Table 1). However, there
(HRQOL) measures are generally good in children surviving HSCT: was delayed immune reconstitution with associated morbidity and
chronic GVHD was the most significant factor lowering HRQOL. mortality. Furthermore posttransplant lymphoproliferative disorders
Twenty years after HSCT, better HRQOL outcomes have been re- such as autoimmune hemolytic anemia or thrombocytopenia have
ported than in those having conventional noncurative therapy.8 The been seen. Procedure-related mortality, as well as morbidities,
long-term effects of HSCT on fertility are not well described, therefore remain greater than in matched sibling transplantation9; as
however, particularly in males and while fertility might occasionally a result, HLA-haploidentical family donation should currently be
be preserved after HSCT, this is a rare event. Overall consensus confined to clinical trials.11

362 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

Downloaded from https://ashpublications.org/hematology/article-pdf/2018/1/361/1254816/hem01850.pdf by guest on 24 April 2020

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 to efficacy and safety. The role of insulators, ostensibly to decrease
Although costs vary between countries, the estimated costs of an the risk of insertional mutagenesis is less clear: the first clinically
allogeneic HSCT from a sibling are almost invariably lower than of successful LV vector, HPV569, contained 2 insulator elements.
conventional therapy with lifelong blood transfusion and iron che- However, because of low engraftment in 3 of 4 patients and because
lation (CT).3,12 Cost analysis makes assumptions about life expec- of a transient clone in 1 patient, the insulators (cHS4) were omitted
tancy with transfusion plus chelation difficult and vary considerably, from the subsequent vector BB305, when Bluebird Bio (BBB)
depending on health care resources and geographic organizational became involved (Table 2). Insulators are included in the TNS9.3.55
issues. In the United Kingdom, the approximate cost of an allogeneic vector used at Memorial Sloan Kettering Cancer Center17 but not in
blood marrow transplant13 is equivalent to about 8.7 years of CT the vector developed in the Italian GLOBE study (Table 2).16 The
for a patient surviving 50 years.3 The advantages of transplan- potential for long-term correction of thalassemia, using LV-transduced
tation over CT will be greater when the latter cannot be provided HSC was first shown in a murine model of thalassemia intermedia18
to the necessary standards for financial or organizational reasons. and subsequently clinically in a transfusion-dependent adult Eb
Comparison of outcomes with either HSCT or CT, in Sardinia, thalassemia patient.19
where both are available to a high standard, showed that overall
survival of 83% in transplanted patients was similar to CT with Increasing fetal hemoglobin synthesis by gene editing. A
overall survival of 85%.14 Outside matched sibling allograft, risks second general approach is to promote g chain synthesis in the

Downloaded from https://ashpublications.org/hematology/article-pdf/2018/1/361/1254816/hem01850.pdf by guest on 24 April 2020

are still higher and there is less consensus about the optimal risk/ developing erythroblast to a point where the a/non-a ratio is suf-
benefit ratio. Long-term follow-up at least yearly is recommended ficiently balanced to prevent intramarrow apoptosis in developing
for after HSCT. 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.
Gene therapy
Editing methodologies include designer nucleases such as zinc
Gene therapy thalassemia has already become a clinical reality using
fingers or the repurposing of bacterial clustered regularly interspaced
lentiviral (LV) vectors to replace key elements of the b globin gene15
short palindromic repeats (CRISPR) to allow RNA-guided cleavage
and other approaches are in advanced preclinical stages. The use of
of complementary DNA. By using CRISPR/Cas9 and guide RNA in
autologous modified stem cells for engraftment has the obvious
human HSC, a 40% reduction in BCL11A mRNA resulted in
advantage of providing HSCT for all patients. Further theoretical
corresponding twofold increase in g-globin transcript ex vivo.20
advantages are the lower procedure-related mortality and the lack of
need for immune suppression, as well as no risk of acute or chronic
Another way to enhance fetal hemoglobin (HbF) is to disrupt g2d
GVHD or immune-mediated graft rejection. Some transplant-related
intergenic HbF silencers. This has been recently demonstrated in
issues will remain while myeloablative regimes are used, however;
human progenitor cells, using CRISPR/Cas9 to edit a 13.6-kb ge-
these include VOD and long-term effects on fertility. Overall, the
nomic region encompassing the d- and b-globin genes.21 At least 2
clinical benefit of gene therapy must outweigh the risks of mye-
such approaches are being initiated in clinical trials for thalassemia
loablative conditioning regimens; also, the long-term safety will need
(Table 2). The first uses zinc finger nuclease to boost HbF by dis-
to be demonstrated with respect to the oncogenic potential of gene
ruption of an erythroid specific enhancer of the BCL11A gene
therapy itself (see the following section).
(Sangamo, Bioverativ). The second is CTX001, a gene-edited cel-
lular product that uses CRISPR Cas9 technology to target an
Approaches to gene modification used in clinical trials erythroid-specific enhancer of the BCL11A gene (Table 2), which
In principle, correction of the abnormal or missing gene can be has been are approved for clinical trials in several countries.22 The
performed by gene addition, such as using LV vectors or by precisely latter demonstrates a high ex vivo editing rate and no identified off-
correcting single-point mutations using designer nucleases. Be- target sites in preclinical models. The safety and specificity of the
cause there are .200 b-thalassemia mutations, a strategy that can CRISPR approach to gene editing is currently debated. A key issue is
be applied irrespective of the individual b mutation is preferable. the so called “off target” effects (OTEs). These occur under con-
Early studies with murine thalassemia models used recombinant ditions in which, although cleavage at sites with perfect comple-
g-retroviral vectors, derived from the Moloney leukemia virus, as mentarity to the guide RNA (gRNA) is highly efficient, this can also
carriers to introduce the human b-globin gene in HSC. Limitations occur at sites where 1 or more bases are mismatched with the gRNA.
included low vector titers, low expression of b-globin, and unstable In principle, OTE could lead to loss of a key stem cell function or
vectors with a tendency to rearrangements. b-globin expression was could have oncogenic potential by activating oncogenes or inacti-
then increased by incorporation of the core elements of the hyper- vating a tumor suppressor genes. Methods that result in higher
sensitive sites 2, 3, and 4 of the human b-globin locus control region. sustained levels of cellular Cas9 and gRNA tend to have higher OTE.
However vector rearrangements and position effects were still seen. At high editing efficiencies, double-strand DNA breaks are induced
Self-inactivating LV vectors were then evaluated for which over- by Cas9 and, although these usually lead to cell death through a P53/
came some of these issues; it is these that trials are most advanced TP53-dependent mechanism, cells with mutated P53 may be re-
sistant, risking the emergence of mutated cells.23 It remains to be
Gene replacement of functional b gene(s) into HSC using LV seen whether these concerns are real in clinical use and/or whether
vectors. Here the intent is to insert a fully functional gene (or part of strategies to minimize OTE such as with high-fidelity Cas9 variants
it) that will result in sufficient correction of globin chain imbalance are effective.
that is the hallmark of thalassemia. Self-inactivating, LV vectors are
now used by several laboratories for clinical trials for b-thalassemia, Autologous transplantation of corrected stem cells. Irrespective
which have been reviewed in detail recently elsewhere (Table 2).16 of the gene modification, transplantation typically involves 4 steps:
These vectors have many shared features.17 For gene expression to mobilization of autologous CD341 stem cells, ex vivo correction of
be adequate, promoters are required, and the insertion site is critical the abnormal gene, myeloablative conditioning, and the reinfusion of

364 American Society of Hematology

sufficient corrected stem cells. Autologous CD341 cells can be ob- Pharmacological enhancers of effective erythropoiesis
tained by direct collection from bone marrow aspiration16 or from stem Enhancers of erythropoiesis for thalassemia patients have been known
cell mobilization.15,19 Mobilization of CD341 cells has been generally for .2 decades, working either by direct stimulation: erythropoiesis-
achieved using granulocyte colony stimulating factor with or without stimulating agents or by enhancing HbF synthesis such as with
plerixafor (Table 2). Myeloablation has been achieved using busulfan hydroxyurea (Hu) and butyrates. Unfortunately, responses have
monotherapy, for example, with total doses of 12.8 mg/kg used over generally been highly variable and unpredictable so that, unlike sickle
4 days in the BBB studies. The procedure has been generally well cell disease, their place in the management of TDT and non-TDT is
tolerated with adverse events and frequencies typical of an au- still unclear. For reasons of space, this section will focus only on agents
tologous transplant.15 A less intensive myeloablative regime would that have reached the stage of clinical use or clinical trials.
presumably lead to lower VOD risk, which although less frequent
than in allogeneic transplantation, is not 0.15 Reduced intensity Erythropoiesis-stimulating agents
busulfan conditioning has been used in the Memorial Sloane In non-TDT, erythropoietin (EPO) levels decrease with age, as does
Kettering Cancer Center study (Table 2). By contrast, in the Italian erythropoietic response to EPO, so that Hb values also tend to fall.
GLOBE study, treosulfan and thiotepa have been used for con- EPO acts by binding to the EPO receptor on the earliest erythroid
ditioning, which may reduce toxicity when compared with bu- progenitors, burst-forming unit-erythroid, with subsequent activation
sulfan.24 Experience thus far suggests that suppression of the bone of Jak2 kinase and downstream Stat5. EPO also mediates erythro-

Downloaded from https://ashpublications.org/hematology/article-pdf/2018/1/361/1254816/hem01850.pdf by guest on 24 April 2020

marrow before HSC by hypertransfusion may improve engraft- blast expansion though suppression of Fas-FasL coexpression,
ment. The duration of hypertransfusion and the optimal levels are a potentially undesirable effect in non-TDT, risking increased and
yet to be determined. Good chelation control before transplant is unwanted expansion of extramedullary erythropoiesis. EPO doses
advisable and is a requirement of some protocols to reduce the risk have typically been 1000 IU/kg weekly or twice weekly and mainly
of liver complications in the peritransplant period. Again, the short term in uncontrolled trials so that the long-term risks of
optimal liver iron concentration values for minimizing hepatic risk extramedullary expansion are not well defined. Highly variable
have not been formally evaluated. Infertility is a likely consequence responses have been reported: from no response up to 3 g/dL in some
of this regime and VOD is also occasionally seen. non-TDT patients, and with transfusion independence in some
hitherto transfused patients. Pegylated EPO is now in a phase 1
After myeloablation, the genetically modified autologous HSPC are clinical trial for b thalassemias (NCT02950857), but this may risk
returned, where they repopulate the hematopoietic compartment. In 1 increased extramedullary hematopoiesis if used alone. Surprisingly,
trial, infusion was into the posterior superior iliac crest (Table 2) with in the light of recent reports of iron restriction enhancing erythro-
the aim of enhancing engraftment and minimizing a first-pass in- poiesis (see the following section), some trials used iron supple-
travenous filter.16 The vector copy number (VCN) in the infused mentation to further increase the Hb response.
CD341 cells is important to levels of HbA or HbF obtained postgraft.
For example, improvement in manufacturing process of LentiGlobin Jak2 inhibition
has led to both increased peripheral blood VCN and also increased A secondary effect of ineffective erythropoiesis (IE) and hypoxia in
levels of posttransplant Hb (HbAT87Q). This has allowed current thalassemias is the EPO and Jak2 phosphorylation-dependent ex-
trials to be extended to more severe forms of thalassemia (b0/b0) pansion of immature erythroblasts that are unable to bypass apoptosis
(BBB study 212). at the polychromatophilic stage, to 5 to 6 times that of healthy controls.
Chronic anemia in thalassemia skews the erythropoietic response, so
Clinical outcomes reported to date. Interim combined data have that proliferation outpaces differentiation. In principle, inhibitors of
been reported from 2 phase 1-2 studies of autologous HSC trans- Jak2 kinase may decrease IE by improving the balance between
duced ex vivo with the LV BB305 (BBB) vector to 22 patients (aged proliferation and differentiation. Murine studies in a thalassemia
12-35) (TDT trials numbers NCT01745120 and NCT02151526).15 model showed improvement in IE and decrease in spleen size,
At 26 months (range, 15-42) after infusion, 12 of 13 patients with possibly at the cost of decreased overall erythropoietic activity.
a non-b(0)/b(0) genotype were transfusion free with HbA(T87Q) Furthermore, in humans with myelofibrosis, a Jak2 inhibitor
levels from 3.4 to 10.0 g/dL, and total hemoglobin ranged from 8.2 to (INCB018424) reduced spleen size. A phase 2A open label single
13.7 g/dL. Furthermore, in 9 patients with the more severe IVS1-110 arm study was therefore undertaken of ruxolitinib (INC424, Jakavi)
mutation, the annual transfusion volume was decreased by 73%. No (NCT02049450) in 30 TDT patients with splenic enlargement25 to
safety issues were attributed to the BB305 vector in either studies 204 determine whether transfusion requirement and/or spleen size could
or 205. Nine serious adverse events were reported, including 2 be decreased. Treatment was given for 30 weeks at a starting dose of
episodes of veno-occlusive liver disease attributed to busulfan 10 mg twice daily. Overall, transfusion requirement did not decrease
conditioning. No replication-competent lentivirus was detected in the (12 decreases, 7 increases, 8 did not change). However there was
patients in either study, and serial monitoring of vector integration a significant 26% reduction in splenic volume at 30 weeks. Thus,
sites in blood samples has consistently shown polyclonal profiles of although the lack of decrease in transfusion requirements are con-
unique integration sites without dominant. However, the overall sistent with murine studies, ruxolitinib may have a role in reducing
long-term risks remain unclear: in particular, it is unknown whether spleen size in TDT patients with advancing splenomegaly. In
there is increased oncogenic potential in humans over several de- principle, after reduction of spleen size and cessation of ruxolitinib,
cades. However, no clonal dominance related to vector integration transfusion requirement may then decrease, so that intermittent therapy
has been thus far observed in the LentiGlobin studies. This is may be applicable in selected TDT patients with splenomegaly.
a rapidly evolving field and other clinical studies also using self-
inactivating LV vectors or CRISPR both in thalassemia and sickle HbF induction
cell disease are less advanced but also promising (Table 2). Ulti- HbF-inducing agents increase g-globin, which binds to unpaired
mately, a conditioning regime that did not have the risk of infertility a-chains in b thalassemias, thereby decreasing globin imbalance,
would also be welcome. ineffective erythropoiesis, and hemolysis. Many drugs increase HbF

Hematology 2018 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

Downloaded from https://ashpublications.org/hematology/article-pdf/2018/1/361/1254816/hem01850.pdf by guest on 24 April 2020
 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

Downloaded from https://ashpublications.org/hematology/article-pdf/2018/1/361/1254816/hem01850.pdf by guest on 24 April 2020

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

Downloaded from https://ashpublications.org/hematology/article-pdf/2018/1/361/1254816/hem01850.pdf by guest on 24 April 2020
occurring at later stages of erythropoiesis31 and, unlike EPO re- Restricted iron delivery to the erythron
sponses, appear to require accessory cells. Modulation of GDF-11 Animal models suggest that by restricting iron delivery to the
has been implicated as the mechanism of action in thalassemia31: thalassemic erythron, the efficiency of erythropoiesis can be im-
GDF11 is upregulated in spleen and erythroid cells of thalassemic proved. Iron restriction, using intraperitoneal iron-free human
animals and inhibits murine erythroid maturation. GDF11 expression transferrin or by increasing plasma hepcidin levels, improved Hb in
and recombinant human homodimeric activin type IIB receptor in thalassemic mice. Both manipulations decrease iron delivery to
erythroid precursors decreases progressively with maturation.31 normoblasts, thereby decreasing heme synthesis, decreasing hemi-
Inactivation of GDF11, as with these drugs, decreases oxidative chrome formation, and hence reactive oxygen species–mediated
stress and a-globin membrane precipitates in red blood cells. GDF11 oxidative stress and apoptosis. In principle, plasma hepcidin can be
also corrected the abnormal ratio of immature/mature erythroblasts in increased by giving exogenous hepcidin(s) or by upregulating
thalassemic mice by inducing apoptosis of immature erythroblasts hepcidin synthesis. Administration of minihepcidins, short peptides
through the Fas–Fas ligand pathway.32 that mimic the activity of endogenous hepcidin, improved ineffective
erythropoiesis, anemia, and iron overload in thalassemic mice.36
Clinical trials. Phase 2 clinical trials in non-TDT with sotatercept Hepcidin synthesis can be increased by suppressing Tmprss6,
showed dose-dependent increments in Hb typically .1 g/dL with a transmembrane serine protease (matriptase-2) that normally sup-
improvements in red cell morphology (Table 3).33 Because of the presses hepcidin synthesis by deactivating hemojuvelin to form

Downloaded from https://ashpublications.org/hematology/article-pdf/2018/1/361/1254816/hem01850.pdf by guest on 24 April 2020

long plasma half-life of ~23 days, subcutaneous dosing was given soluble hemojuvelin. An antisense oligonucleotide approach has
every 3 weeks, with Hb improvement seen within the first 3 cycles in been shown to decrease Tmprss6 protein, thereby increasing hep-
non-TDT. In TDT, a reduction in transfusion burden was also seen. cidin and leading to improved anemia in murine thalassemia
Tolerability was generally good and reported adverse events in- models.37 Injection of siRNA formulated in lipid nanoparticles di-
cluded grade 3 bone pain in 1 TDT patient and grade 2 phlebitis in rected to the liver improved anemia by ameliorating of IE and
a non-TDT patient. There were similar findings in phase 2a trial of improving red cell survival38 in thalassemic mice. Iron chelation has
luspatercept (ACE-356; NCT01749540) in 31 non-TDT and 32 TDT a less clear effect, however, at least when given alone, suggesting that
patients at dose levels titrated up to 1.25 mg/kg every 3 weeks were restriction of iron delivery may need to be through the transferrin
presented at the European Hematology Association Congress in uptake. This may have a role when combined with hepcidin mi-
2018.34 In NTDT an increase of .1 g/dL over baseline was seen in metics, however. In humans, large doses of purified human apo-
71% of patients and of .1.5G in 55% at 3 months. In a patient- transferrin showed decrements in transferrin saturation or NTBI.39
reported outcome QOL questionnaire, a significant increase in score Several clinical trials are under way or imminently planned to in-
was seen in patients showing .1 g/dL Hb increase.34 The 6-minute vestigate the effect of hepcidin, mini-hepcidin or manipulation
walk test showed a significant improvement at 48 weeks of treatment through TMPRSS6 on transferrin saturation, NTBI and hence
and improvement correlated with the Hb increase at this time. In TDT erythropoiesis in non-TDT (Table 3). Secondary effects on iron
patients, a $20% reduction in transfusion requirement was seen in uptake by myocardium are also under investigation.
78% of patients and a $30% reduction in 69% of patients after
a median treatment duration of 14 months. In patients as a whole, Correspondence
tolerability was generally good with the majority of adverse effects John Porter, Department of Haematology, University College London,
being grade 1-2: bone pain was seen in 38%, headache in 28%, Paul O'Gorman Building, 72 Huntley St, London WC1E6HX, United
myalgia in 22%, and arthralgia in 19%. Other adverse effects Kingdom; e-mail: [email protected].
($10%) were asthenia, injection site pain, and back pain. This study
is now in a 5-year extension phase (NCT02268409). The detailed References
phase 2 findings with sotatercept and luspatercept are in press. 1. Ladis V, Chouliaras G, Berdoukas V, et al. Survival in a large cohort of
Greek patients with transfusion-dependent beta thalassaemia and mor-
Luspatercept was selected for phase 3 development, mainly because, tality ratios compared to the general population. Eur J Haematol. 2011;
unlike sotatercept, it binds only minimally to activin A and thus may 86(4):332-338.
have lower off-target effects. Randomized double-blind phase 3 2. de Silva S, Fisher CA, Premawardhena A, et al; Sri Lanka Thalassaemia
studies are now under way for TDT (NCT02604433: Efficacy and Study Group. Thalassaemia in Sri Lanka: implications for the future
health burden of Asian populations. Lancet. 2000;355(9206):786-791.
Safety Study of Luspatercept (ACE-536) Versus Placebo in Adults
3. Weidlich D, Kefalas P, Guest JF. Healthcare costs and outcomes of
Who Require Regular Red Blood Cell Transfusions Due to Beta [b]
managing b-thalassemia major over 50 years in the United Kingdom.
Thalassemia) and non-TDT (NCT03342404; A Study to Determine Transfusion. 2016;56(5):1038-1045.
the Efficacy and Safety of Luspatercept in Adults With Non 4. Porter JB, Evangeli M, El-Beshlawy A. Challenges of adherence and
Transfusion Dependent Beta [b]-Thalassemia). The predetermined persistence with iron chelation therapy. Int J Hematol. 2011;94(5):453-460.
criterion for efficacy in TDT is a 33% decrease in the number of 5. Lucarelli G, Galimberti M, Polchi P, et al. Bone marrow transplantation
transfused red blood cell units from baseline. In summary, both in patients with thalassemia. N Engl J Med. 1990;322(7):417-421.
sotatercept and luspatercept regularly increase Hb in non-TDT to 6. Baronciani D, Angelucci E, Potschger U, et al. Hemopoietic stem cell
levels associated with improved QOL. In TDT, the reduction in transplantation in thalassemia: a report from the European Society for
transfusion burden is likely to decrease the use of chelation and the Blood and Bone Marrow Transplantation Hemoglobinopathy Registry,
2000-2010. Bone Marrow Transplant. 2016;51(4):536-541.
frequency of hospital visits. It is reassuring that RAP-536 corrected
7. Goussetis E, Peristeri I, Kitra V, et al. HLA-matched sibling stem cell
the complications of ineffective erythropoiesis, splenomegaly, and
transplantation in children with b-thalassemia with anti-thymocyte
bone pathology in a mouse model of non-TDT.35 However, as globulin as part of the preparative regimen: the Greek experience.
transfusion decreases in TDT and autologous erythropoiesis pro- Bone Marrow Transplant. 2012;47(8):1061-1066.
portionately increases, it will be important to establish that the known 8. La Nasa G, Caocci G, Efficace F, et al. Long-term health-related quality
complications of non-TDT, such as thrombosis and extramedullary of life evaluated more than 20 years after hematopoietic stem cell
erythropoietic expansion, do not become problematic. transplantation for thalassemia. Blood. 2013;122(13):2262-2270.

Hematology 2018 369

 9. Angelucci E, Matthes-Martin S, Baronciani D, et al; EBMT Inborn Error conditioning regimen based on the use of treosulfan. Blood. 2012;120(2):
and EBMT Paediatric Working Parties. Hematopoietic stem cell trans- 473-476.
plantation in thalassemia major and sickle cell disease: indications and 25. Taher AT, Karakas Z, Cassinerio E, et al. Efficacy and safety of rux-
management recommendations from an international expert panel. olitinib in regularly transfused patients with thalassemia: results from
Haematologica. 2014;99(5):811-820. a phase 2a study. Blood. 2018;131(2):263-265.
10. Locatelli F, Kabbara N, Ruggeri A, et al; Eurocord and European Blood 26. Elalfy MS, Adly AA, Ismail EA, Elhenawy YI, Elghamry IR.
and Marrow Transplantation (EBMT) group. Outcome of patients with Therapeutic superiority and safety of combined hydroxyurea with
hemoglobinopathies given either cord blood or bone marrow trans- recombinant human erythropoietin over hydroxyurea in young b-
plantation from an HLA-identical sibling. Blood. 2013;122(6): thalassemia intermedia patients. Eur J Haematol. 2013;91(6):
1072-1078. 522-533.
11. de Montalembert M, Brousse V, Chakravorty S, et al. Are the risks of 27. Taher AT, Musallam KM, Karimi M, et al. Overview on practices in
treatment to cure a child with severe sickle cell disease too high? BMJ. thalassemia intermedia management aiming for lowering complication
2017;359:j5250. rates across a region of endemicity: the OPTIMAL CARE study. Blood.
12. Sruamsiri R, Chaiyakunapruk N, Pakakasama S, et al. Cost utility 2010;115(10):1886-1892.
analysis of reduced intensity hematopoietic stem cell transplantation in 28. Karimi M, Darzi H, Yavarian M. Hematologic and clinical responses of
adolescence and young adult with severe thalassemia compared to thalassemia intermedia patients to hydroxyurea during 6 years of therapy
hypertransfusion and iron chelation program. BMC Health Serv Res. in Iran. J Pediatr Hematol Oncol. 2005;27(7):380-385.

Downloaded from https://ashpublications.org/hematology/article-pdf/2018/1/361/1254816/hem01850.pdf by guest on 24 April 2020

2013;13(1):45. 29. Lohani N, Bhargava N, Munshi A, Ramalingam S. Pharmacological and
13. Weidlich D, Kefalas P, Guest JF. Healthcare costs and outcomes of molecular approaches for the treatment of b-hemoglobin disorders. J Cell
managing b-thalassemia major over 50 years in the United Kingdom. Physiol. 2018;233(6):4563-4577.
Transfusion. 2016;56(5):1038-1045. 30. Paulson RF. Targeting a new regulator of erythropoiesis to alleviate
14. Caocci G, Orofino MG, Vacca A, et al. Long-term survival of beta anemia. Nat Med. 2014;20(4):334-335.
thalassemia major patients treated with hematopoietic stem cell trans- 31. Suragani RN, Cadena SM, Cawley SM, et al. Transforming growth
plantation compared with survival with conventional treatment. Am J factor-b superfamily ligand trap ACE-536 corrects anemia by promoting
Hematol. 2017;92(12):1303-1310. late-stage erythropoiesis. Nat Med. 2014;20(4):408-414.
15. Thompson AA, Walters MC, Kwiatkowski J, et al. Gene therapy in 32. Dussiot M, Maciel TT, Fricot A, et al. An activin receptor IIA ligand trap
patients with transfusion-dependent b-thalassemia. N Engl J Med. 2018; corrects ineffective erythropoiesis in b-thalassemia. Nat Med. 2014;
378(16):1479-1493. 20(4):398-407.
16. Lidonnici MR, Ferrari G. Gene therapy and gene editing strategies for 33. Porter J, Cappellini M, Origa R, et al. Interim results from a phase 2a,
hemoglobinopathies. Blood Cells Mol Dis. 2018;70:87-101. open-label, dose-finding study to determine the safety, efficacy, and
17. Mansilla-Soto J, Riviere I, Boulad F, Sadelain M. Cell and gene therapy tolerability of sotatercept (ACE-011) in adults with beta-thalassemia
for the beta-thalassemias: advances and prospects. Hum Gene Ther. [abstract]. Haematologica. 2014;99(suppl 1):230. Abstract S662.
2016;27(4):295-304. 34. Piga A, Tartaglione I, Gamberini R, et al. Improvements in hemoglobin,
18. May C, Rivella S, Callegari J, et al. Therapeutic haemoglobin synthesis in quality of life, and six-minute-walk distance in adults with b-thalassemia
beta-thalassaemic mice expressing lentivirus-encoded human beta- treated with luspatercept: long-term phase 2 study [abstract]. Haema-
globin. Nature. 2000;406(6791):82-86. tologica. 2018;103(suppl 1). Abstract S844.
19. Cavazzana-Calvo M, Payen E, Negre O, et al. Transfusion independence 35. Suragani RN, Cawley SM, Li R, et al. Modified activin receptor
and HMGA2 activation after gene therapy of human b-thalassaemia. IIB ligand trap mitigates ineffective erythropoiesis and disease
Nature. 2010;467(7313):318-322. complications in murine b-thalassemia. Blood. 2014;123(25):
20. Yu VWC, Liu Y, Curran M, et al. CRISPR/Cas9 gene-edited hemato- 3864-3872.
poietic stem cell therapy for sickle cell disease [abstract]. Blood. 2017; 36. Casu C, Oikonomidou PR, Chen H, et al. Minihepcidin peptides as
130(suppl 1). Abstract 535. disease modifiers in mice affected by b-thalassemia and polycythemia
21. Antoniani C, Meneghini V, Lattanzi A, et al. Induction of fetal hemo- vera. Blood. 2016;128(2):265-276.
globin synthesis by CRISPR/Cas9-mediated editing of the human b- 37. Bauer DE, Kamran SC, Lessard S, et al. An erythroid enhancer of
globin locus. Blood. 2018;131(17):1960-1973. BCL11A subject to genetic variation determines fetal hemoglobin level.
22. Lin I, Paik E, Mishra B, et al. CRISPR/Cas9 genome editing to treat Science. 2013;342(6155):253-257.
sickle cell disease and B-thalassemia: re-creating genetic variants to 38. Schmidt PJ, Toudjarska I, Sendamarai AK, et al. An RNAi therapeutic
upregulate fetal hemoglobin appear well-tolerated, effective and durable targeting Tmprss6 decreases iron overload in Hfe(-/-) mice and ame-
[abstract]. Blood. 2017;130(suppl 1). Abstract 284. liorates anemia and iron overload in murine b-thalassemia intermedia.
23. Ihry RJ, Worringer KA, Salick MR, et al. p53 inhibits CRISPR-Cas9 Blood. 2013;121(7):1200-1208.
engineering in human pluripotent stem cells. Nat Med. 2018;24(7): 39. de Vries B, Snoeijs MG, von Bonsdorff L, Ernest van Heurn LW,
939-946. Parkkinen J, Buurman WA. Redox-active iron released during machine
24. Bernardo ME, Piras E, Vacca A, et al. Allogeneic hematopoietic stem perfusion predicts viability of ischemically injured deceased donor
cell transplantation in thalassemia major: results of a reduced-toxicity kidneys. Am J Transplant. 2006;6(11):2686-2693.

370 American Society of Hematology