Bone Marrow Transplantation (2012) 47, 1489 -- 1498
& 2012 Macmillan Publishers Limited All rights reserved 0268-3369/12
www.nature.com/bmt
REVIEW
Allogeneic cellular and autologous stem cell therapy for sickle
cell disease: ‘whom, when and how’
J Freed1,5, J Talano2,5, T Small3, A Ricci3 and MS Cairo4
Sickle cell disease (SCD) is an autosomal recessive inherited hematological disorder characterized by chronic hemolysis and
vaso-occlusion, resulting in multiorgan dysfunction and premature death. The only known curative therapy for patients with
severe SCD is myeloablative conditioning and allo-SCT from HLA-matched sibling donors. In this state of the art review, we
discuss current and future considerations including patient selection/eligibility, intensity of conditioning regimens, allogeneic
graft sources, graft manipulation, mixed donor chimerism, organ function and stability and autologous gene correction stem
cell strategies. Recent novel approaches to promote mixed donor chimerism have included the use of matched unrelated adult
donors, umbilical cord blood donors, haploidentical familial donors and the utilization of nonmyeloablative, such as reduced
intensity and reduced toxicity conditioning regimens. Future strategies will include gene therapy and autologous gene
correction stem cell designs. Prospects are bright for novel stem and cellular approaches for patients with severe SCD, and we
are currently at the end of the beginning for utilizing cellular therapeutics for the curative treatment of this chronic and
debilitating condition.
Bone Marrow Transplantation (2012) 47, 1489 -- 1498; doi:10.1038/bmt.2011.245; published online 19 December 2011
Keywords: allo-SCT; sickle cell disease; pediatrics
GENETICS, EPIDEMIOLOGY AND PATHOPHYSIOLOGY OF
SICKLE CELL DISEASE (SCD)
SCD is a rare recessive inherited disorder, secondary to a point
mutation that results in the replacement of valine for glutamic
acid at the sixth position of the b chain of human Hb (Figure 1).
Worldwide, there are an estimated 270 000 patients affected with
SCD.1,2 The polymerization of the deoxygenated form of Hb S
induces a major distortion of the shape of the human sickle cell
RBC resulting in a decrease in sickle cell RBC deformability, a
change in the rheology and consequential vaso-occlusion.3 The
most challenging aspect of the disease is the episodic and
unpredictable nature of the vaso-occlusive events.4
There are a significant number of clinical complications that can
occur in patients with SCD. Sickle cell-induced vaso-occlusive
events can occur anywhere in the circulation but most often occur
in bones within the chest, back, abdomen or extremities. These
episodes can last for days to weeks. Acute chest syndrome affects
about 40% of patients with SCD. Acute chest syndrome is more
common in children, and when it recurs it can cause chronic
respiratory disease.4 Stroke can occur in 10% of SCD patients
during childhood with silent central nervous system damage
occurring in 5 -- 9 times as many patients, and both can lead to
cognitive impairment. Priapism can occur in 10 -- 40% of men with
SCD, which can result in permanent erectile dysfunction. Patients
with SCD are predisposed to severe infections.5 This can be
attributed to a variety of immunological causes.6 - 12 The dominant
defect is attributed to poor splenic function. Other defects include
depletion of IgM memory B cells, reduced levels of T cell subsets
CD4 þ and CD8 þ , defects in the C0 , impaired opsonization and
1
phagocytosis, deficiency of factor B and skewed TH2 phenotype,
resulting in decreased TH1 function.6 - 12 This leads to the patient’s
vulnerability to encapsulated organisms, which can include
pneumococcus sepsis, salmonella osteomyelitis and E. coli
urosepsis. Despite this year being the 100th anniversary of the
original clinical description of SCD and that over the last 30 -- 40
years there has been extensive genetic characterization and
elucidation of the biochemical properties of sickle Hb, long-term
cure for most patients with SCD still remains elusive.13,14
STANDARD OF CARE (NON-TRANSPLANT) THERAPY FOR
PATIENTS WITH SCD
In industrialized countries, patients with SCD now survive into
their fifth and sixth decades (Figure 2) in large part due to many
advances in supportive care in the management of SCD, such as
penicillin prophylaxis, chronic transfusion regimens and screening
examinations, as detailed in Table 1.15
Hydroxyurea has emerged as an important therapeutic option
for children and adolescents with SCD. The percent of Hb F is
known to be protective against clinical severity in patients with
SCD.15,16 In comparison, increased leukocyte counts have been
associated with a poor clinical outcome because of the increased
viscosity caused by hyperleukocytosis.17,18 Hydroxyurea not only
increases the percent of Hb F, presumably by suppressing the
marrow and allowing for new marrow recovery and new RBC
production with early Hb F-containing RBC precursors, but also
decreases the leukocyte count, and thus has many characteristics
Department of Pediatrics, Hackensack University Medical Center, Hackensack, NJ, USA; 2Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI, USA;
Department of Pediatrics, Columbia University, New York, NY, USA and 4Department of Pediatrics, Medicine, Pathology, Microbiology and Immunology, Cell Biology and
Anatomy, New York Medical College, Valhalla, NY, USA; 5These authors contributed equally to this work. Correspondence: Dr MS Cairo, Pediatrics, Medicine, Pathology,
Microbiology & Immunology and Cell Biology & Anatomy, New York Medical College, Munger Pavilion Room 110A, Valhalla, NY 10595, USA. E-mail: [email protected]
This manuscript has been supported in part by a grant from the Pediatric Cancer Research Foundation.
Received and accepted 10 October 2011; published online 19 December 2011
3
Allogeneic and autologous therapy for SCD
J Freed et al
1490
Hemoglobin genes
α
Chromosome 16
α-globin
gene cluster
5’
Chromosome 11
β-globin
gene cluster
5’
α
3’
γ
γ
β
3’
Point mutation (βς)
in sickle cell disease
Hemoglobin development
Fetal hemoglobin
Birth/adult hemoglobin
α2γ2
α2γ2
α2β2
and
Normal
BCL11A
HBS1L-Myb
α2γ2
and
α2βς2
Severe sickle cell
Regulators of the
γ- to β-chain switch
during the fetal to
α2γ2
and
α2βς2
Figure 1. The Hb switch. The fetal (g) and adult (b) globin chains are expressed from genes on chromosome 11. SCD is caused by mutation of
the b chain to the sickle (bS) chain. Genome-wide association studies have identified loci on chromosome 2 (BCL11A) and chromosome 6
(HBS1LMyb) that modify HbF expression. These modifiers affect the expression switch from g to either b or bS globin. They may affect HbF
levels either directly or indirectly. Targeted therapy could reverse the fetal-to-adult switch, and hence reduce disease severity. Orkin SH et al.14
Table 1. Standard therapies and associated risks in the treatment of
patients with sickle cell disease
1.0
0.9
Probability of survival
0.8
0.7
Complication
Standard therapy
Risks of standard therapy
Infection85 - 89
Penicillin
prophylaxis
pneumococcal
vaccination
Chronic red cell
transfusion
Resistant infection,
medication noncompliance, sepsis-related
mortality
Iron overload,
alloimmunization,
hepatitis, increased risk of
graft failure
Progressive hypoxia,
pulmonary hypertension
0.6
Stroke (overt or
silent)90 - 92
0.5
0.4
Restrictive or
obstructive
pulmonary
disease93 - 97
Pulmonary
hypertension31,98 - 104
0.3
Females with SS
Males with SS
Black females
Black Males
0.2
0.1
0.0
0
10
20
30
40
Age (years)
50
60
70
Figure 2. Probability of OS stratified by sex in patients with SCD and
normal African Americans. Platt OS et al.5
that make it an ideal drug for patients with SCD. Initial studies in
adults have suggested that hydroxyurea significantly reduces the
number of veno-occlusive events.19,20 Subsequent studies demonstrated similar results in both infants and children with SCD.21,22
In the recently completed multicenter randomized Baby Hug
study by Wang et al.,23 hydroxyurea was shown to be safe and
effective in infants aged 9 -- 18 months of age with SCD.
Hydroxyurea as an alternative therapy for secondary prevention
of stroke coupled with phlebotomy for treatment of iron overload
has been proposed and recently tested in a multicenter
randomized clinical trial (SWITCH), stroke with transfusions
Bone Marrow Transplantation (2012) 1489 - 1498
Inhaled
bronchodilators
RBC transfusion
RBC transfusion
Congestive heart failure,
Oxygen
pulmonary edema
supplementation
changing to hydroxyurea, but was recently terminated for safety
and futility reasons (National Heart, Lung, and Blood Institute
(NHLBI) 3 June, 2010 press release), as the patients switched from
chronic transfusion regimens to hydroxyurea had a significantly
higher rate of second strokes. Although case reports of cancer
occurring in SCD patients on hydroxyurea exist,24 - 26 these are
isolated cases, and in order to truly determine whether the risk is
increased in SCD patients on hydroxyurea a registry of SCD
patients on hydroxyurea would be helpful.
MYELOABLATIVE CONDITIONING (MAC) AND ALLO-HSCT
FROM HLA-MATCHED SIBLING DONORS IN PATIENTS
WITH SCD
Allo-HSCT has been shown to be effective in other hemoglobinopathies such as thalessemia as the only curative option for the
& 2012 Macmillan Publishers Limited
Allogeneic and autologous therapy for SCD
J Freed et al
1491
Table 2.
HLA-matched sibling alloHSCTs after myeloablative conditioning in patients with SCD
Author
29
Panepinto
Walters28
Bernaudin30
Country or registry
N
OS (%)
EFS (%)
Graft rejection (%)
AGVHD (%)
CGVHD (%)
CIBMTR
USA
France
67
22
87
97
91
93
85
73
86
15 (N ¼ 9)
18 (N ¼ 4)
7 (N ¼ 6)
10
1
13
22
1
20
Abbreviations: AGVHD ¼ acute GVHD; allo-HSCTs ¼ allogeneic hematopoietic SCT; CIBMTR ¼ Center for International Blood and Marrow Transplant Research;
CGVHD ¼ chronic GVHD; EFS ¼ event-free survival; OS ¼ overall survival.
specific underlying disease. Johnson et al.27 first demonstrated the
cure of SCD by a myeloablative and HLA-matched sibling alloHSCT in a patient with SCD who was receiving an allo-HSCT for
acute leukemia. Walters et al.28 subsequently reported on the
successful use of HLA-matched sibling MAC allo-HSCT in a larger
series of patients with SCD. A total of 22 patients with severe
symptoms of SCD received allo-HSCT from a fully HLA-matched
sibling donor, after receiving a MAC regimen consisting of BU, CY
and anti-thymocyte globulin (ATG) (Table 2).28 The EFS and the
disease-free survival (DFS) rates at 4 years were 91% and 73%,
respectively.28 Panepinto et al.29 subsequently reported on 67
patients in the Center for International Blood and Marrow
Transplantation registry, and demonstrated that the 5-year overall
survival (OS) and DFS rates were 97% and 85%, respectively
(Table 2). Bernaudin et al.30 reporting results from France
demonstrated similar results in 87 allo-HSCT SCD recipients who
received allo-HSCTs from HLA-matched sibling donors, after MAC
with BU and CY. The 6-year OS was 93.1% and the EFS was 86.1%,
respectively (Table 2). These studies demonstrate that HLAmatched sibling allo-HSCTs after MAC offer very high survival
rates with few-transplant-related complications (Table 2).
In addition to symptom alleviation, HSCT has also been shown to
stabilize or reverse the organ damage due to SCD. In a long-term
follow-up study of patients who received HLA-matched related alloHSCT, pulmonary function tests were stable in 22 of 26 patients,
worse in 2 and not studied in 2.31,32 Linear growth measured by
median height s.d. score improved from 0.7 before HSCT to 0.2
after HSCT.31,32 Radiological improvement of a patient with
avascular necrosis of the humeral head has been reported,33 as
well as correction of splenic reticuloendothelial dysfunction.29,34
The effect of HSCT on reversal of cerebral vasculopathy has
been variable. Many studies have found that patients who
successfully engraft do not experience any sickle-related central
nervous system complications, and have evidence of stabilization
of central nervous system disease on magnetic resonance
imaging.31,35,36 In addition to stabilization, a few studies have
found improvement in areas of previous abnormality; however,
this was in an extremely small subset of patients.31,32,37 One study
compared the vessel diameter on magnetic resonance imaging of
patients with SCD treated with HSCT vs those treated with either
chronic transfusion or hydroxyurea.38 They found a 12% increase
in the lumen of 22 vessels in patients who underwent HSCT vs an
8% increase in the lumen of 42 vessels in the transfusion/
hydroxyurea patients. However, worsening of cerebral large vessel
disease and stroke has also been reported after HSCT.39
ALTERNATIVE ALLOGENEIC DONOR SOURCES FOR ALLO-HSCT
IN PATIENTS WITH SCD
We and others have demonstrated that unrelated umbilical cord
blood (UCB) is an excellent alternative allogeneic donor source for
some childhood malignant and nonmalignant conditions.28,40 - 43
However, the preliminary results of unrelated UCB transplantation
(UCBT) as an alternative allogeneic source for children and
adolescents with SCD, although limited in scope, have been
& 2012 Macmillan Publishers Limited
disappointing.44 - 46 Furthermore, the NHLBI BMT Clinical Trials
Network Trial 0601 of reduced intensity conditioning (RIC) before
UCBT in symptomatic patients with SCD recently closed to accrual
in the arm, utilizing unrelated cord blood grafts, secondary to
increased graft rejection (Memorandum BMT Clinical Trials
Network, 6/21/2010).
Ruggeri et al.47 examined the efficacy of unrelated UCBT in
children with SCD (n ¼ 16). OS and DFS were 94% and 50%,
respectively. The 2-year probability of DFS was 45% in patients
who received grafts with nucleated cell dose 45 107/kg and
13% with lower cell doses. Primary graft failure was the
predominant cause of treatment failure occurring in seven
patients with SCD. These results suggest that only UCB units
containing an expected infused nucleated cell dose 45 107/kg
should be considered for transplantation for hemoglobinopathies,
which further limits the available UCB units for this population of
patients. Additional studies are required to enhance improvement
in engraftment of UCB in patients with SCD, including double
UCBT, ex-vivo UCB expansion, and the combination of UCB and
other stem cell sources.
LESSONS LEARNED IN PATIENTS WITH THALASSEMIA
Allo-HSCT from multiple allogeneic donor sources has been
proven to be successful. Thalassemia is a hemoglobinopathy that
like SCD can lead to elimination of the chronic signs and
symptoms of that disease. Bernardo et al.48 transplanted 17
thalassemia patients with a conditioning regimen of treosulfan/
thiotepa/fludarabine/ATG followed by unrelated adult donor BMT.
A 2-year probability of survival and thalassemia-free survival was
95% (95% confidence interval, 85 -- 100%) and 85% (95%
confidence interval, 66 -- 100%), respectively.
Familial haploidentical (FHI) T-cell depletion allo-HSCT in patients
with thalassemia is another approach of overcoming the paucity of
well-matched unrelated donors. Lucarelli et al.49 and colleagues
originally designed a conditioning regimen consisting of hydroxyurea (30 mg/kg per day) and azathioprine (3 mg/kg per day)
between days 45 to 11, before fludarabine 20 mg/m2 per
day 6 days, BU 14 mg/kg per total dose and CY 60 mg/kg per total
dose in 33 poor-risk class 3 thalassemia patients prior to allo-HSCT.
Graft rejection was reduced to only 8% compared with the previous
30% reported from the same group without hydroxyurea,
azathioprine and fludarabine.50 Further improvements were
achieved in 22 poor-risk thalassemia patients by modifying the
conditioning regimen to add thiotepa 10 mg/kg per day, rabbit ATG
12.5 mg/kg per total dose, expanding the use of hydroxyurea and
azathioprine to day 59 and increasing CY to 200 mg/kg per total
dose. Grafts primarily from a maternal donor (N ¼ 20) were
depleted of T cells using the CliniMACS system (Miltenyi Biotec,
Auburn, CA, USA) to achieve a median of 14.2 106 CD34 þ cells/
kg, with a controlled add-back of 2x105 CD3 þ /kg and CY added as
acute GVHD prophylaxis. Engraftment was achieved in 16/22
patients without acute GVHD and with OS of 90%. These results
suggest that a similar approach could be investigated in high-risk
patients with SCD.
Bone Marrow Transplantation (2012) 1489 - 1498
Allogeneic and autologous therapy for SCD
J Freed et al
1492
RIC AND ALLO-HSCT IN PEDIATRIC RECIPIENTS
A major limitation of MAC and allo-HSCT is the risk of TRM or
treatment-related toxicities associated with MAC regimens. Organ
toxicities are more likely to occur and be more severe in
symptomatic patients with SCD who have impaired organ
function or have been exposed to multiple RBC transfusions
before MAC and allo-HSCT.51 - 53 MAC facilitates durable engraftment of donor cells, but is limited by toxicities of the conditioning
as well as allogeneic transplant-related complications.54,55 RIC
regimens have been examined in patients with malignant diseases
who could not tolerate MAC regimens. Pulsipher et al.56 examined
the use of a RIC regimen consisting of BU, fludarabine and ATG in
47 pediatric patients with hematological malignancies. With a
variety of graft sources including matched sibling donor, matched
unrelated adult donor (MUD) and UCB sustained engraftment
occurred in 79 -- 98% and full donor chimerism occurred in 76 -88% of patients. TRM was only 11%, although relapse was 43%.
Roman et al.23 and Del Toro et al.57 reported some success with
RIC regimen in children with hematological malignancies. RIC
regimens have also been examined in patients with nonmalignant
disorders.58 The use of a RIC regimen has also been examined
prior to UCBT. Bradley et al.59 investigated the use of a RIC
regimen before UCBT in 21 patients; 7 with nonmalignant
conditions. The 5-year OS for all patients was 60%. TRM was only
14%, and the incidence of acute GVHD and chronic GVHD was
28% and 17%, respectively.
The ongoing NHLBI BMT Clinical Trials Network 0601 trial
utilizes an RIC regimen, which includes alemtuzumab, fludarabine
and melphalan as a conditioning regimen. GVHD prophylaxis
consists of CYA/FK506, MTX and steroids. However, the major
difficulty with this approach is identifying an 8/8 HLA MUD, as
there are lower percentages of African-American and HispanicAmerican donors in the international BM registries. Approximately,
only 20 -- 25% of patients identified as a potential BMT candidate
have an 8/8 HLA MUD available. Clearly, other strategies of
increasing the donor pool or other alternatives are desperately
needed for identifying allogeneic donors for SCD.
DONOR CHIMERISM AFTER ALLO-HSCT IN PATIENTS WITH SCD
Walters et al.60 demonstrated that stable mixed chimerism after
MAC and allo-HSCT in patients with SCD may be sufficient to cure
the disease and prevent further SCD-related symptoms or
complications. In all, 50 patients survived free of SCD after
receiving MAC and HLA-identical sibling marrow transplants, and
of these 50, 13 developed stable mixed donor -- host chimerism.
Eight patients had a chimerism between 90-- 99%, but five patients
had a lower proportion of donor chimerism, ranging from 11-- 74%.
All five of these patients had Hb levels 411. In the three patients
whose donors had a normal Hb phenotype, the Hb S percentage
ranged from 0 -- 7%, and in the two patients whose donors had
sickle cell trait, the Hb S percentage was 36 and 37%. None of the
patients experienced painful events or other clinical complications
related to SCD. These results strongly support that a persistent
mixed donor -- host chimerism is sufficient to alleviate clinical and
laboratory manifestations of SCD. Andreani et al.61 investigated the
donor origin of mature erythrocytes in four patients with persistent
mixed chimerism after transplantation for hemoglobinopathies.
The percentage of donor-derived nucleated cells ranged from
15-- 71%; however, the percentage of donor-derived erythrocytes
ranged from 73 -- 100%. These results suggest that the majority of
the erythrocytes were donor-derived even in the patients with
minimal total donor-derived nucleated cells. This suggests that
perhaps only a small proportion of donor-engrafted cells may be
needed to prevent further symptoms or complications of SCD.
As stable mixed chimerism appears sufficient to eliminate all of
the symptoms of SCD,60,61 it is possible that RIC might be an effective
Bone Marrow Transplantation (2012) 1489 - 1498
alternate method of conditioning, even if it only results in mixed
donor chimerism. Krishnamurti et al.62 reported on stable donor
engraftment after RIC with BU, fludarabine, equine ATG, and TLI. Six
of seven patients with SCD demonstrated long-term engraftment.
However, because of the risk of graft rejection, particularly in the
heavily RBC-transfused patients with SCD, a high level of
immunoablation is important for successful donor engraftment.
The regimen of fludarabine and melphalan in combination with
alemtuzumab would provide a RIC regimen while potentially
preserving immunoablation. This conditioning regimen was used
in 44 patients with malignant disorders.63 Only two patients
experienced graft failure, and the incidence of acute GVHD and
chronic GVHD were 6.5% and 0%, respectively. In this study, the
alemtuzumab was given 4-- 8 days before SCT, which may account
for the low risk of acute GVHD and chronic GVHD. However, in an
immunocompetent host, such as a patient with SCD, this could
increase the risk of graft rejection. Owing to this theoretical
possibility, Shenoy et al.64 modified the regimen to dose
alemtuzumab on days 21 to 19, prior to allo-HSCT in patients
with nonmalignant disease. A total of 16 patients, 7 of whom had
SCD, between the ages of 2 and 20 were treated with this regimen,
all receiving either unrelated donors matched at 8 --10/10 loci, HLAidentical sibling donors or UCB matched at 4 --6/6 loci. Graft failure
occurred in 5% of patients, although two of these patients had
received a lower dose of melphalan in an attempt to further reduce
the intensity of the conditioning. The OS and DFS are 100% and
71%, respectively. Bhatia et al.65 also demonstrated the successful
use of an RIC in patients with SCD. A total of 18 patients received
BU, fludarabine and alemtuzumab prior to receiving either sibling or
unrelated allo-HSCT. The OS and EFS were 83% and 78%,
respectively. The median one and two year donor chimerism for
whole blood and CD71 were 93/90% and 94/95%, respectively.
Hsieh et al.66 performed RIC allo-HSCT in ten adult patients
(ages 16 -- 45 years) with high risk SCD. The conditioning regimen
consisted of alemtuzumab with low dose TBI (3 Gy) followed by
sirolimus for GVHD prevention. Nine patients showed stable
lymphohematopoietic engraftment at levels that sufficed to
reverse the SCD phenotype. The mean donor -- recipient chimerism
for T cells (CD3 þ ) and myeloid cells (CD14 þ 15 þ ) was 53.3%
and 83.3%, respectively. J Bolaños-Meade et al.67 reported on ten
adult patients with SCD who underwent a RIC haploidentical BMT
with post-transplant high-dose cytoxan. The ages ranged from
16 -- 33 years. The conditioning regimen included ATG, fludarabine,
cytoxan (14.5 mg/kg on days 6 and 5) and 2 Gy TBI. GVHD
prophylaxis consisted of post-transplant CY (50 mg/kg on days
þ 3 and þ 4), tacrolimus and mycophenolate mofetil. Four
patients rejected their graft (one primary and three secondary).
At a follow-up of 406 days, all patients were alive and six patients
were off immunosuppresion. This confirms that RIC regimens can
allow for sufficient donor whole blood and RBC engraftment to
ameliorate SCD. However, rejection remains a major hurdle to
overcome. These studies support the use of RIC regimens in
selected patient populations with symptomatic SCD (Table 3).
FHI T-CELL DEPLETED (TCD) FAMILY DONOR ALLO-HSCT IN
PATIENTS WITH MALIGNANT DISEASE
FHI TCD allo-HSCT has been an excellent allogeneic stem cell
source for children and adults with malignant disease.68,69 Aversa
et al.70 reported 100% engraftment and no acute GVHD/chronic
GVHD in 43 patients with hematologic malignancies, and utilizing
CD34 selection of FHI PBSCs. Aversa et al.70 also has reproduced
these results in a recent phase II trial and demonstrated high rates
of engraftment and low rates of acute and chronic GHVD.71 Also,
Evans et al.72 recently demonstrated long-term fetal microchimerism in PBMCs in healthy women, suggesting a potential selective
advantage to utilizing maternal FHI donors to promote tolerance
and/or decrease severe acute GVHD.73
& 2012 Macmillan Publishers Limited
Allogeneic and autologous therapy for SCD
J Freed et al
FHI TCD ALLO-HSCT IN PATIENTS WITH SCD AND INDICATIONS FOR ALLO-HSCT
We have created a multicenter, multidisciplinary consortium of
pediatric SCT centers, each having a substantial SCD patient
population with the intent of investigating FHI TCD allo-HSCT in
high-risk patients with SCD. We have adopted the conditioning
regimen that Lucarelli et al.49 piloted in the FHI TCD allo-HSCT
study in a high-risk thalassemia population (Figure 3). We have
included the addition of TLI in order to potentially reduce the
rejection rate in this SCD population. Selected high-risk patients
with SCD defined in the eligibility criteria will be enrolled on this
study. The eligibility criteria includes: (1) clinically significant
neurologic event (stroke) or any neurological deficit lasting 424 h
that is accompanied by an infarct on cerebral magnetic resonance
imaging; (2) minimum of two episodes of acute chest syndrome
(defined as new pulmonary alveolar consolidation involving at
least one complete lung segment associated with acute symptoms including fever 438.5, chest pain, tachypnea, intercostal
retractions, nasal flaring, use of accessory muscles of respiration,
wheezing, rales, or cough not attributable to asthma or
bronchiolitis) in the preceding 2 year period prior to enrollment
that have failed, been noncompliant or declined hydroxyurea
treatment; (3) recurrent painful events (at least 3 in the 2 years
prior to enrollment). Pain occurred in typical sites associated with
vaso-occlusive painful events, and cannot be explained by causes
other than SCD and (4) two consecutive abnormal transcranial
Doppler studies (2 months apart) with mean velocities in the
Table 3.
middle carotid artery, internal carotid artery or anterior carotid
artery of 4200 cm/s requiring chronic transfusion therapy.
Patients will receive a myeloimmunosuppressive conditioning
regimen (Figure 4) and will receive FHI TCD (CD34 selected) PBSC
transplantation using the CliniMACS device (IND no. 14359).
AUTOLOGOUS GENE REPLACEMENT/CORRECTION- A STEM
CELL APPROACH FOR PATIENTS WITH SEVERE SCD
Despite some success in allo-HSCT for patients with poor-risk SCD,
as mentioned above, there continues to be limitations with this
approach. These complications have stimulated interest in the use
of gene replacement/correction stem cell therapy. There have
been many attempts to correct the sickling globin gene via the
transfer of a regulated globin gene in autologous hematopoietic
stem cells using viral vectors. To date, remarkable success has
occurred in treating patients with X-linked SCIDS and adenosine
deaminase deficiency.74 Over 30 patients with SCIDS have been
treated with gene therapy, although there remains a problem with
insertional mutagenesis and oncogenesis. In 2000, a lentivirus was
used as the vector to cure a mouse model of thalassemia
intermedia.34 This breakthrough vector allowed high levels of
gene transfer without rearrangement of gene sequences35 and
increased b gene expression.75 Since this discovery, human trials
are underway. The major hurdles in using gene addition therapy
in treating SCD remain in obtaining therapeutic levels of b globin
gene expression and preventing insertional mutagenesis.76
Reduced intensity/toxicity conditioning regimens prior to allo-HSCT in patients with symptomatic SCD
Author
Country
N
Age
(years)
Regimen
Krishnamurti62
Shenoy64
Bhatia65
Hsieh66
J Bolaños-Meade67
USA
USA
USA
USA
USA
7
16
20
10
10
6 - 18
2 - 20
1 - 19
16 - 45
16 - 33
BU/Flu/ATG/TLI
Alemtuzumab/Flu/melphalan
Bu/Flu/alemtuzumab
Alemtuzumab/3GyTBI/sirolimus
ATG/CY/Flu/2GyTBI/ post-transplant CY/
Graft sources
Related BM
Related/MUD
Related BM, related/UCBT
Related PBSC
Related haploidentical BM
EFS (%)
Graft
rejection (%)
86
71
75
90
60
4
5
25
1
40
Abbreviations: Allo-HSCT ¼ allogeneic hematopoietic SCT; ATG ¼ anti-thymocyte globulin; Flu ¼ fludarabine; MUD ¼ matched unrelated donor; SCD ¼ sickle
cell disease; UCBT ¼ unrelated cord blood transplantation.
Tufts
Medical
medical center
college
New
york
(TMC)
of wisconsin
medical
(MCW)
college
(NYMC)
Columbia
university
(CUMC)
University of
California, San
Francisco
(UCSF)
Children’s hospital &
research center at
Oakl and
(CHRCO)
Washington
university
(Wash U)
Lead institution
Figure 3.
Clinical research centers
Cell processing cores
Health related quality of life core
Immunology cores
Admin/biostat/financial core
Neuroimaging and neurocognitive cores
Radiation therapy core
Donor chimerism core
Pulmonary function and vascular cores
Childhood and adolescent and young adult FHI allo-SCT SCD consortium.
& 2012 Macmillan Publishers Limited
Bone Marrow Transplantation (2012) 1489 - 1498
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J Freed et al
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Day –59 to –11
Hydroxyurea 60 mg/kg/day
X
Azathioprine 3 mg/kg/day
X
Keppra 5-10 mg/kg/day
Day –17 to –13
Day –12 to –9
Day –8
Day –7 to –4
Day –5 to –2
Day 0
Start day –18
Fludarabine 30 mg/m
X
Busulfan 3.2 mg/kg/day (4.0 ≤ 4 yrs)
X
Thiotepa 10 mg/kg IV
X
Cyclophophamide 50 mg/kg w/Mesna
X
Rabbit ATG 2.0 mg/kg/day
X
Total Lymphoid irradiation 500cGy
Day –2 only
SCT
X
Figure 4. Myeloimmunosuppressive conditioning regimen (hydroxyurea and azathioprine day 59 to day 11, fludarabine day 17 through
day 13, BU day 12 through day 9, thiotepa day 8, CY day 7 through day 4, ATG day 5 through day 2, TLI day 2) followed by FHI
T cell depleted (CD34 selected) PBSCT in patients with SCD.
(7)
Transplant corrected
hematopoetic progenitors
back into irradiated mice
(6)
Differentiate into
hematopoetic progenitors
hβS/hβS
tall tip
fibroblasts
hβS/hβS
Clone 3 picked
on day 16
hβS/hβS iPS line #3
established
after 2 passages
Humanized sickle cell
anemia mouse model
(hβS/hβS)
(1)
Harvest tail
tip fibroblasts
(5)
Differentiate into
embryoid bodies
ES
control
iPS
#3
iPS
#3.3
hβS/hβS
fibroblasts
hβA/hβS
iPS cells
(4)
Correct β sickle
mutation in iPS cells
by specific gene targeting
(3)
hβS/hβS
iPS cells
*
(2)
Infect with
Oct4, Sox2, Klf4
and loxP c-Myc
viruses
hβS/hβS mouse derived
iPS clones
3.8 Kb
5’ PCR
(Primers 1+2)
260 bp
on
Cl
tly
ES targ
e
Co
r
re
c
Cl
340 bp
(Primers 3+4
+ Bsu361 digest)
Cl
β globin allele
PCR
te
d
7.7 Kb
e
3’ PCR
(Primers 5+6)
on #3
e
#
on 10
e
Cl #1
on 1
e
#2
5
2.8 Kb
Figure 5. Derivation of autologous iPS cells from HbS/HbS mice and correction of the sickle allele by gene targeting.84 (a) Scheme for in vitro
reprogramming of skin fibroblasts with defined transcription factors combined with gene and cell therapy to correct sickle cell anemia in
mice. (b) Representative images of various steps of deriving hbs/hbs iPS line no. 3. (c) Southern blot for c-Myc viral integrations in (i) ES cells,
(ii) hbs/hbs iPS line no. 3 and (iii) its derived subclone hbs/hbs iPS no. 3.3 obtained after infection with adeno-Cre virus and deletion of the
viral c-Myc copies. *indicates endogenous c-Myc band. Arrows point to transgenic copies of c-Myc. (d) hbs/hbs iPS no. 3.3 displayed normal
karyotype 40xy (upper left), was able to generate viable chimeras (upper right), and formed teratomas (bottom). (e) Replacement of the hbs
gene with a hbA globin gene in sickle iPS cell line no. 3.3. Homologous recombinants were identified by PCR to identify correct 50 and 30 end
replacement. PCR with primers 3 and 4 followed by Bsu36I digestion was used to distinguish hbs and hbA alleles. The correctly targeted clone
no. 11 displayed a pattern identical to that previously obtained for the correctly targeted ES cell clone.
GENE REPLACEMENT THERAPY/HOMOLOGOUS RECOMBINATION (HR)
A different approach to preventing insertional mutagenesis and
oncogenesis is that of gene replacement therapy of the sickle
Bone Marrow Transplantation (2012) 1489 - 1498
gene with normal Hb A via HR. This method allows one to create a
break of the ds DNA upstream and downstream to the genetically
mutated site dissociating it from the DNA, and then replacing this
previously mutated site with the genetically corrected DNA.
& 2012 Macmillan Publishers Limited
Allogeneic and autologous therapy for SCD
J Freed et al
Townes et al.77 reported that by replacing one allele of sickled b
embryonic stem cells, then transplanting these cells into
blastocysts, hematopoietic cells are able to produce corrected
RBCs. However, the low yield of HR in mammals (1 per 106) makes
this technique very difficult. By using customized zinc-finger
nucleases, hypothetically, there is a possibility of increasing the
likelihood of HR at the mutation site by 10 000-fold.
INDUCED PLURIPOTENT STEM CELLS (IPS) CELLS
The main limitation after correcting patient-derived somatic
hematopoietic cells is the difficulty in expanding these cells while
maintaining multipotency. This decrease in self-renewal ability
affects long-term engraftment.
In recent years, there has been accelerated interest in the study
of pluripotent stem cells because of their limitless proliferation
potential and possible contribution in the study of genetic
diseases and gene manipulation.78 However, obtaining embryonic
stem cells presents with an ethical conundrum. In 2006,
Takahashi/Yamanaka et al.79 evinced that somatic cells could be
reprogrammed into pluripotent stem cells called iPS cells, which
had similar characteristics to embryonic stem cells. These iPS cells
had the capability of proliferating in vitro, differentiating into all
three germ layers and forming teratomas and embryoid bodies.
They expressed similar cell surface Ag-ic markers, and had a
similar morphology and chromatin methylation patterns as
embryonic stem cells. iPS cell production was accomplished by
the transduction of only four transcription factors Oct4, Sox2, Klf4
and c-Myc via a retroviral vector.79 As then, multiple different
vectors have been used, including adenoviral and lentiviral
vectors, plasmids and piggyback transposon system with and
without a non-viral vector system.76 Challenges in deriving iPS
cells using viral vectors include the potential for insertional
mutagenesis or oncogene reaction from viral integration.80
Transcription factors cMyc and KLF4 are also well-known
oncogenes. Alternative methods currently investigated encompass reprogramming with less factors, protein reprogramming,
mRNA reprogramming,81 small molecule reprogramming and
choosing cell types with high plasticity to generate iPS cells. The
inception of iPS cells paves the way for science to find new ways
of replacing damaged tissues, studying genetic diseases and
correcting their genetic mutation, autologous transplantation and
tissue engineering.78
By taking advantage of the regenerative features of pluripotent
stem cells, investigators are now able to derive an unlimited
amount of iPS cells obtained from sickled somatic cells, then
attempt to correct the mutated gene by HR and then eventuate
these genetically corrected iPS cells into hematopoietic stem cells.
This allows for an unlimited supply of gene-corrected hematopoietic stem cells, which can be used for autologous transplantation.82,83 In 2007, by using a humanized sickle cell knock-in mouse,
Hanna et al.84 derived hematopoietic progenitors from mouse
fibroblast iPS cells, which were able to reconstitute the
hematopoietic system of sickle cell mice and correct their disease
phenotype (Figure 5). These mice demonstrated stable engraftment up to 12 weeks after transplant. Compared with untreated
mice, the transplanted mice showed decreased markers of
hemolysis. This technique produced cells that are fully immune
compatible to the host, thus avoiding immunosuppressive
therapy. Currently there are many studies deriving gene-corrected
hematopoietic stem cells using human cell lines obtained from
patients with SCD to generate iPS cells, with the ultimate goal of
autologous transplantation (Figure 6).
Regenerative medicine is an emerging field with the increased
potential to cure genetic diseases in the near future. However,
there remain many complications, most notable being the
increased risk of oncogenesis. As this field continues to grow,
we are imminently approaching the possibility of auto-SCT to cure
patients with SCD with complications.
SUMMARY/FUTURE
MAC and HLA-matched sibling donor allo-HSCT is still the gold
standard and only known curative therapy in patients with SCD.
More novel approaches are being investigated to promote
permanent mixed donor chimerism in these patients, including
RIC and the use of alternative allogeneic donors (MUDs, UCBT and
haploidentical) and alternatively autologous gene correction/
Correct the β globin
gene via HR
3
Derive iPS cells
2
HbS
iPS cells
HbA
HβS MNC
Isolate mononuclear
cells
4
HβA iPS cells
Generate HSC
1
5
HβA HSC
Transplant gene
corrected cells
HβS HSC derived from cord blood,
bone marrow or peripheral blood
Figure 6. Studies deriving gene-corrected hematopoietic stem cells using human cell lines obtained from patients with SCD to generate iPS
cells, with the ultimate goal of autologous transplantation.
& 2012 Macmillan Publishers Limited
Bone Marrow Transplantation (2012) 1489 - 1498
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replacement stem cell therapies. Over the next 5 years, pilot
studies utilizing RTC and alternate allogeneic grafts including
MUDs, UCBT and haploidentical should be completed. Randomized phase III trials should be in development within 5 years to
compare some of these approaches with standard supportive
care, with end points of quality of life, organ function/stability and
neurocognitive stability or improvement. The future is bright for
the use of HPCT allogeneic and autologous stem cell therapy for
patients with severe manifestations of SCD. We are at the end of
the beginning of a new era for cellular curative therapies for this
chronic and debilitating genetic condition.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
ACKNOWLEDGEMENTS
We thank Erin Morris, RN, for her outstanding contribution to the preparation of this
manuscript, and to all the brave children and adults with SCD who suffer from this
chronic condition.
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