Life Sciences 178 (2017) 100–108

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Life Sciences

journal homepage: www.elsevier.com/locate/lifescie

Review article

Therapeutic strategies in Sickle Cell Anemia: The past present and future
Queenie Fernandes ⁎,1
University of Greenwich, United Kingdom

a r t i c l e i n f o a b s t r a c t

Article history: Sickle Cell Anemia (SCA) was one of the first hemoglobinopathies to be discovered. It is distinguished by the mu-
Received 25 January 2017 tation-induced expression of a sickle cell variant of hemoglobin (HbS) that triggers erythrocytes to take a char-
Received in revised form 22 March 2017 acteristic sickled conformation. The complex physiopathology of the disease and its associated clinical
Accepted 22 March 2017
complications has initiated multi-disciplinary research within its field. This review attempts to lay emphasis
Available online 20 April 2017
on the evolution, current standpoint and future scope of therapeutic strategies in SCA.
Keywords:
© 2017 Elsevier Inc. All rights reserved.
Sickle Cell Anemia
Sickle Cell Therapeutics
Sickle Cell Research
Hemoglobinopathies

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
2. Evolution of therapeutic strategies targeting SCA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
3. Existing therapies for the management of SCA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
3.1. Hydroxyurea therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
3.2. Blood transfusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
3.3. Bone marrow transplantation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
4. Current research standpoint and prospects on future treatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
4.1. Basic research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
4.2. Genetic-based strategies in research and gene therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
4.2.1. Vector based genetic approaches. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
4.2.2. Vector-less genetic approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
5. Conclusion-scientific prospects & future scope of SCA targeted research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
Conflict of interest statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106

1. Introduction Under deoxygenated conditions, HbS molecules tend to aggregate into

Sickle Cell Anemia (SCA) arises out of a point mutation in the bases
coding for the sixth amino-acid of the β-chain of hemoglobin. A single
nucleotide substitution from GTG to GAG causes the amino acid valine
to replace glutamic acid in the growing protein chain. This altered trans-
lational event gives rise to a mutant variant of hemoglobin (HbS) with
different properties than the wildtype hemoglobin (HbA) molecule.
long rigid chains that distort the shape of the erythrocytes causing it
to take up its characteristic sickle-shaped conformation.
This sickling event remains to be the underlying cause for a trail of
associated clinical complications. Its principal & mechanism of occur-
rence has inspired multi-disciplinary research, commencing right from
the origin and discovery of the SCA mutation.

2. Evolution of therapeutic strategies targeting SCA

⁎ Corresponding author.
E-mail address: Queenie.Fernandez@gmail.com.
1
Current Address: National Center for Cancer Care and Research (NCCCR), Hamad SCA has been a common genetic disorder that has prevailed over
Medical Corporation, PO Box; 4572 Doha, Qatar. centuries with the highest incidence in Sub-Saharan Africa and the

http://dx.doi.org/10.1016/j.lfs.2017.03.025
0024-3205/© 2017 Elsevier Inc. All rights reserved.
 Q. Fernandes / Life Sciences 178 (2017) 100–108
Caribbean. It is said to have originated among African and Afro-Ameri-
can populations.
The co-occurance of the disease among generalized African popula-
tions during a period of endemic malaria sparked an interest to deter-
mine whether the two diseases were linked by a selective advantage.
In 1949, J.S.B. Haldane popularized a theory stating that infectious
diseases may be a driving force of natural selection [1]. In accordance
with this theory, Haldane suggested that patients with the β-thalasse-
mic mutation showed an increased fitness or resistance to malaria.
Later in 1954 these findings were confirmed when A. C. Allison stated
that Sickle cell heterozygotes have considerable protection against ma-
laria [2].This protective effect thus brought about an equilibrium in
which the homozygotic hemoglobinopathic disadvantage was balanced
by a heterozygotic advantage offering protection against malaria [3].
Currently this hypothesis has been accepted and supported by a number
of studies [4–9]. Certain other, studies state that the SCA mutation does
not offer direct protection from the malarial parasite (Plasmodium
falciparum) but instead deters progression to clinical malaria by improv-
ing clearance of infected erythrocytes thereby interrupting parasite
growth [10].
Moreover, certain ancient African beliefs associated with the disease
emphasized that the disease was a curse from tribal Gods. No later, a
common myth originated in Nigeria; according to which the disease af-
fected only children that are reincarnated [11].
However, the onset of medical research and the development of
novel diagnostic techniques, led researchers to unveil the true cause
and nature of the disease through a series of milestones (Table 1).
Sickle cell hemoglobin (HbS) was first detected in the blood stream
by James Herrick in 1910. Subsequent research initiatives over an exten-
sive period of time were inefficient in identifying factors that caused the
disease. A major breakthrough along these lines was brought about by
Ingram (1957) [12], who unraveled the mutational mechanics underly-
ing the disease and its resultant expression of the mutated protein
(HbS).
Early investigations began with a study [13] in 1948 where it was
first noted that the symptoms of SCA appeared in infants only after
levels of fetal hemoglobin (HbF) began to fall and be replaced by adult
hemoglobin. This substantiated a theory that fetal hemoglobin proved
advantageous over the sickle cell trait, which paved the way for a series
of experiments targeting the increase of fetal hemoglobin in the blood.
Hydroxyurea was one such drug that was found to trigger the expres-
sion of fetal hemoglobin in adults. However, it was only in 1998 that
the Food & Drug Administration (FDA) approved the use of hydroxyurea
Table 1
Showing the timeline of events in the history of SCA.
Timeline Events
1910 Sickle Cell Hemoglobin (HbS) detected in the blood stream by James
Herrick.
1948 SCA identified in infants only after levels of fetal hemoglobin (HbF)
dropped; thus initiating a theory that HbF showed a beneficial effect
over the disease [13].
1957 The mutational basis (point mutation) underlying the disease was
determined [12].
1980 Clinical trials undertaken with 33 SCA patients. Erythrocyte
adherence to the endothelium was identified to be a key aspect
contributing to the severity of the disease [18].
1998 FDA approves the use of Hydroxyurea (an inducer of HbF) for the
treatment of adult SCA patients.
2000–2002 Studies on Ion-channel blockers that control cation homeostasis
initiate in order to control increased permeability of erythrocytes,
detected to initiate polymerization of HbS that eventually leads to
sickling [16,17].
Research focuses on compounds regulating the expression of
pro-adhesive molecules (aiding erythrocyte adherence to the
endothelium) at the transcriptional level [19].
Present Treatment with Hydrea, blood transfusion and haematopoietic
transplantation/CBT is currently practiced in the treatment of SCA.
101
in the treatment of adults with Sickle Cell Anemia. However its applica-
tion in therapy was still controversial due to its reported toxicity in a
number of cases [14,15].
Further reports stated that polymerization of hemoglobin initiates
due to an increased permeability of the erythrocyte membrane [16].
This permits the entry of calcium ions and the loss of potassium ions
and water leading to severe dehydration and rigidity of the red blood
cells. Relevant therapeutic measures that could control cation homeo-
stasis began to surface in the form of ion-channel blockers like Clotrima-
zole and ICA-17043 [17]. Although these received success in terms of
decreasing membrane-permeability, they were unable to curb the pain-
ful crises associated with the disease and showed numerous fatal side-
effects.
In another effort to conceptualise therapy, Hebbel, et al. [18] under-
took a clinical trial with 33 patients diagnosed with Sickle Cell Anemia.
Their work established a firm relationship between erythrocyte adher-
ence to the endothelium and severity of the disease stating that higher
the adherence greater the severity. This theory triggered the reasoning
of an anti-adhesion treatment. Nearly two decades later, transcription
inhibitors that facilitate down regulation of pro-adhesive molecules
aiding erythrocyte adhesion began to surface. Solovey et al. [19] empha-
sized on the use of sulfasalazine as inhibitors of cell adhesion. Although
sulfasalazine led the way to new dimensions in therapeutic advances, it
was reported that patients regained symptoms once the steroid was
discontinued.
Blood transfusion was also evaluated for possible corrective mea-
sures in SCA [20]. Apart from it being laborious, it is not generally prac-
ticed due to a high risk of exposing patients to other infections.
Currently, Haematopoietic (bone-marrow) transplantation is prac-
tised in the treatment of SCA [21]. Although it has proved successful in
a number of cases, it is still not widely practiced due to the difficulties
faced in finding an appropriate donor-match.
3. Existing therapies for the management of SCA
3.1. Hydroxyurea therapy
The use of Hydroxyurea or Hydrea in the treatment of SCA was ap-
proved by the Food and Drug Administration in 1988.
Multiple mechanisms of action have been attributed to the use of
Hydrea; one such mechanism being the re-synthesis of HbF in adults.
Under normal circumstances, a general shift from HbF to HbA/HbS (in
the case of SCA patients) is observed within a few months after birth
(6–12 months) [22]. Clinical symptoms of SCA are known to arise only
after levels of HbF drop. This is usually because the increasing synthesis
of HbF brings about a decrease in the overall levels of HbS [14].
Hydroxyurea is also known to function by causing a reduction in the
number of white blood cells and also by acting as a nitric oxide donor in
blood cells. Other mechanisms of function include a reduced expression
of certain cell adhesion molecules that promote vaso-occlusive crisis.
One of the major underlying drawbacks of the use of Hydrea is its re-
ported toxicity in a number of cases. It was observed that an increased
dosage brought about an increased therapeutic effect, however greater
was the subsequent toxicity [23]. Further, being a cell-cycle specific
agent, long-term adverse effects were also speculated upon the
prolonged usage of the drug [15].
A particular randomized controlled trial was carried out for nearly
18 years to assess the benefits versus risks of Hydrea treatment
among adults [24]. According to this study, treatment initiated after
40 years of age proved to be associated with lower risks, while also
showing a considerable reduction in morbidity.
Since Hydrea has been known to be cytotoxic, its use was approved
only for adult patients. Early evidences supporting the use of the drug
for the treatment of children were inadequate. Clinical trials and subse-
quent follow-up analysis did not provide satisfactory results to support
its use among children.
102 Q. Fernandes / Life Sciences 178 (2017) 100–108
In 1984, the first report of the use of the drug in children was pub-
lished [25]. This led to a rise of a number of paediatric studies (BABY
HUG, Toddler HUG, HUG - KIDS etc.) that mainly focussed on evaluating
the risk of treatment. Through these randomized control trials it was
proven that 85–90% of child patients were capable of tolerating a dosage
of approximately 20 mg per day.
After a series of strenuous research on the risk assessment of the
drug, the use of Hydrea in the treatment of children was finally support-
ed by the National Heart, Lung, and Blood Institute (NHLBI) in 2002.
Hydroxyurea is now also available in liquid formulations to aid ad-
ministration in child patients [26]. Usually, the effect of the treatment
begins to take place within weeks; however, certain specific cellular
mechanisms like decreased leucocyte counts and the synthesis of HbF
may take up to 6 months [27].
A number of reports have proved the success of Hydrea treatment. It
is also known that if HbF levels have been considerably raised through
Hydrea treatment during early life, the progression of the disease, its as-
sociated complications and even mortality can be considerably delayed
or prevented.
3.2. Blood transfusion
SCA is known to be associated with a number of clinical complica-
tions ranging from frequent episodes of acute vaso-occlusive crisis to
splenic sequestration and stroke. Under the severity of such settings,
transfusions of blood components are often life-saving and hence be-
come the primary mode of managing the disease.
Exchange transfusions have been a traditional method of disease
management since decades. However, improved understanding of indi-
cations is now evident; especially those that include the dangers of hyp-
oxemia or adidosis for which partial exchange may be recommended in
adults or children [28].
Regular transfusions have been carried out in adult, child, as well as
pregnant patients of SCA. Additionally, the management of crisis in
pregnant patients through the practise of exchange transfusion has led
to spontaneous and dramatic improvements [29].
Studies have also proven that frequent blood transfusions may also
alleviate the burden of certain complications associated with SCA. Re-
current cerebral infarcts or Strokes are a common vascular complication
in both adult and child patients of SCA. A certain study was carried out
to compare the recurrence of strokes between children on regular trans-
fusions and children who received standard care. Participants were
within the age group of 1–15 years and were followed for a median of
3 years. This study was able to prove that regular blood transfusions sig-
nificantly reduced the incidence of stroke in children with SCA as com-
pared to other standard approaches of treatment [30].
Moreover, Steinberg [31] also highlights importance of blood trans-
fusions in preventing recurrent strokes. Apparently, a stroke affects
nearly 10% children who do not undergo long-term blood transfusion
after transcranial Doppler screening. A suggested mechanism by
which transfusions may help prevent a stroke is by reducing the velocity
of cerebral blood flow, thereby reducing endothelial damage and cere-
bral vascular occlusion [31].
Although transfusions have proven to be life-saving under condi-
tions of acute crisis, they are also known to tag along certain short or
long-term risks and/adverse effects.
These dangers have been identified as early as 1950 [32], in a partic-
ular study that reported 17 adverse reactions and 3 deaths from 44
transfusions. Moreover hemorrhagic complications with thrombocyto-
penia and other coagulation disorders are also frequently observed.
Another major issue facing blood transfusion is iron overload that
poses elevated risks to patients of SCA undergoing frequent transfu-
sions. This problem arises due to an excessive release of free heme into
the plasma; also known to be the major cause of tissue damage [33].
In these patients, iron-induced complications are known to appear dur-
ing the course of treatment and patients eventually develop liver
cirrhosis and cardiac or pancreatic iron deposition [34,35]. Iron storing
complexes like Ferritin and hemosiderin are found to be present in
large numbers in SCA patients mainly in the reticulocytes [36], thus fur-
ther highlighting the burden of iron overload.
Red blood cells received through transfusion are ultimately phago-
cytoses by the liver, spleen or bone marrow macrophages at the end
of their life cycle. The protein is digested causing the release of free
iron or heme into the cytosol. However prolonged transfusion limits
the capacity of these macrophages leading to the release of excess iron
into the plasma [37]. Once in the plasma, free heme is bound by transfer-
rin; however the increase and saturation of transferring causes the re-
cruitment of hepatocytes to act as storage sites for the excess iron.
Any further increase in the iron concentration then causes it to be re-
leased into the plasma in quantities that exceed the transport capacity
of transferrin. This non- transferrin bound iron found in the plasma is
known to be the major cause of tissue damage [33].
The non-transferrin bound heme is capable of entering a variety of
cells like hepatocytes, anterior pituitary or pancreatic cells. Further,
iron accumulation in these cells causes the release of activated oxygen
species that are known to cause molecular and cellular damage that
may also lead to cellular malfunctioning and necrosis.
However, despite the risk of iron overload, nowadays there seems to
be an increased tendency to encourage recurrent blood transfusions.
This may be attributed to the options currently available to manage
overload like the use of iron chelators.
Currently, three iron-chelating agents are available for commercial
use. Deferoxamine is one such iron-binding compound that is obtained
from bacteria (Streptomyces pilosus). A single molecule is able to bind a
single atom of heme leading to the formation of a biochemically and
metabolically inert complex that is then excreted by the kidneys. Oral
doses are proven to show poor absorption; hence intravenous dosage
is preferred.
A synthetic compound called Deferasirox is also known for its iron-
chelating properties. It is known to be absorbed through the gastro in-
testinal tract and is gradually disposed of the blood vasculature [38].
In this case, deferasirox-iron complexes are solely discarded by the
liver into the bile.
Deferiprone is another compound that is widely used an iron chela-
tor. It is administered through oral doses and has also been used to ease
the burden of iron overload in children with transfusion dependent he-
moglobinopathies. In a particular study children (mean age 6.5 years)
were administered oral doses of deferiprone for a mean duration of
21.5 months. It was reported that the drug was well tolerated and did
not show association with any major safety concerns [39]. Moreover,
certain other studies also identified that deferiprone may also possess
an ability to protect the heart, thereby increasing survival [40,41].
Iron-chelating therapy is essential for patients on long term blood
transfusion. However, the use of chelators in pregnant or breast-feeding
patients needs to be exercised with caution.
Iron-chelators have also been associated with toxicity and adverse
effects in certain cases. A particular study reported visual and auditory
toxicity through the use of deferoxamine [42]. It has also been proved
to interfere with natural growth [43] due to skeletal dysplasia [44].
In other trials, Deferasirox was found to be associated with rash, gas-
trointestinal disturbances, and an increase in serum-creatinine levels
[45–47].
Another complication found associated with Blood transfusion is
“Thromboembolism”. It is a phenomenon that arises out of the obstruc-
tion of a blood vessel by a blood clot. Venous thromboembolism (VTE)
[48] is commonly associated with SCA patients that have received trans-
fusions. Thromboembolism can be particularly fatal when it occurs in
the brain or the lungs [49] leading to conditions of hypoxia. Moreover,
thromboembolic events in SCA have been reported under both steady
state and acute crisis [50].
Anticoagulant therapy may be used in SCA patients for the preven-
tion of VTE [50]. However, there is no medical evidence that the use of
 Q. Fernandes / Life Sciences 178 (2017) 100–108
anticoagulants for the primary or secondary prevention of VTE may be
any different between SCA patients and other medically ill patients.
SCA specific studies targeting the management of VTE are much needed
in order to rule out possible therapies.
It is not surprising that coagulation abnormalities are commonly as-
sociated with SCA. This is due to the fact that SCA is a hypercoagulable
state [50–54]. SCA patients although at steady-state are at an elevated
risk to coagulation abnormalities in comparison to healthy controls
with normal hemoglobin [55].
A number of factors are known to promote this phenomenon. For
example, in SCA patients, higher levels of in-vivo markers of thrombin
and fibrin generation were noted in the plasma at steady-state [56–
63]. Moreover, a marked increase of circulating Tissue Factor (TF) was
also noted in the endothelial cells [62,64–66]. TF is a common activator
of coagulation and is therefore isolated from contact with the plasma
protein by a layer of endothelial cells. As endothelial and vascular injury
is common in SCA it is not surprising that TF exposure at these sites trig-
ger coagulation. In addition, heme, released during hemolysis is also
known to induce the expression of TF, thereby further elevating the
risk of coagulation abnormalities in SCA patients.
Similarly, platelets are also seemingly activated in SCA patients at
the steady-state and are known to undergo further activation during
vaso-occlusive crisis [67–76].
A sub-population of RBCs in SCA patients are known to be marked by
an increased expression of phosphatidylserine (PS) due to the loss of
membrane phospholipid asymmetry [77]. Due to impaired spleen-func-
tion in SCA patients, these PS marked RBC are not removed from circu-
lation thereby serving as docking sites for enzyme complexes involved
in coagulation pathways [78].
Further, in SCA patients at steady-state the concentration of circulat-
ing microparticles is noted to be higher as compared to subjects with
normal hemoglobin [66,79,80] thus exposing them to an increased
risk of procoagulant activity [81,82].
Additionally, high plasma levels of coagulation factor VIII (FVIII)
have been noted in SCA patients at steady-state [56,83–86], while cer-
tain other naturally present anti-coagulant proteins like protein C and
protein S were found to be decreasingly expressed at steady-state as
compared to normal subjects [56–59,63,84,87,88].
Recently, research has been focused on the development of certain
artificially engineered biomolecules like (HB)-based oxygen carriers
(HBOCs) that can act as substitutes to blood transfusion particularly in
critical situations like trauma [89]. HBOCs may be chemically or geneti-
cally engineered and are designed to have a higher oxygen affinity and a
smaller size that makes it capable of reaching the microvasculature to
reverse the sickling event, thereby alleviating vaso-occlusion. However,
upon hemolysis, infused HBOCs are also known to interfere in the func-
tioning of certain signalling pathways thus negating its potential bene-
fits [89]. Studies have also reported HBOCs to be toxic; stating that they
may also be associated with serious adverse events like hypertension,
myocardial infraction, renal injury or even mortality [90].
3.3. Bone marrow transplantation
Currently, haematopoietic stem cell transplantation appears to be
the only potential cure for SCA. The first case of a successful Bone Mar-
row Transplant (BMT) was reported in 1984 in a child with SCA who
had developed acute myeloid leukemia [91]. However, despite a series
of promising results proven by a series of successful reports [92,93],
high mortality rates in patients older than 16 years of age and the diffi-
culty in obtaining HLA-identical donors have limited the potential of
BMT.
However, the use of BMT in the treatment of SCA is now regaining
focus due to a variety of factors like improved conditioning regimes, var-
iants of stem cell transplantation like Cord Blood Transplantation (CBT)
as well as the emergence of global registries to detect donor availability.
103
Recently, the use of reduced-intensity regimens before BMT is
gaining attention. It is known to reduce toxicity in adults with SCA.
Such forms of preparatory therapy are able to discard the patients'
bone marrow cells thereby creating space for donor engraftment and
haematopoiesis. This creates a state of chimerism between the patient
and donor cells. Under such conditions, sickling of erythrocytes is still
evident; however is known to occur at a much lowered ratio. A particu-
lar study employed the use of reduced-intensity BMT in adults between
16 and 45 years of age before BMT through matched donors [94]. The
study did not identify any cases of mortality or Graft Versus Host Dis-
ease (GVHD).
BMT involving haploidentical donors is another novel approach that
can enable a larger proportion of SCA patients to qualify for BMT. Pa-
tients are more likely to have a donor who is half-matched at HLA anti-
gens. In a particular study, 14 patients received a BMT from a
haploidentical donor [95]. Although no deaths were reported, 7% of
the study sample were reported to have rejected their grafts and
reverted to SCA.
Advancements and improvements in conditioning regimes of BMTs
for SCA have caused it to become a more convenient option for treat-
ment. Most patients with SCA receive conditioning with oral busulphan
in combination with intravenous cyclophosphamide [96]. Moreover,
the use of antilymphocyte treatment (antilymphocyte globulin or
campath) before transplantation has also been reported by the same
group to reduce the chances of graft rejection. It was observed that
the graft rejection rate dropped from 22.6% to 3% when antilymhocyte
treatment was added to the conditioning regime [97]. A particular
study also reported the used of thoracoabdominal radiations in a patient
population above 12 years of age [98].
Certain studies have also researched the use of Reduced-Intensity
Conditioning (RIC) regimes. Early results remained unsuccessful with
a high rejection rate. However, more recently a trial conducted with
10 SCA patients reported better results through the use of alemtuzumab
as an anti-thymophocyte treatment along with a total body radiation of
3 Gy which was then followed by HLA-identical peripheral blood stem
cell transplantation [99]. Other studies have also reported the use of dif-
ferent conditioning regimes comprising of low dosage of busulfan or
total body radiation with fludarabine, alemtuzumab or melphalan. In
order to reach a common conditioning protocol, it is important to deter-
mine which regime is able to achieve full or mixed chimerism and
which may cause short or long-term toxicity [100].
An alternative approach to BMT is transplantation involving the use
of cord blood stem cells. Results obtained through the use of Cord Blood
Transplantation (CBT) have been motivating in the field of
hemaglobinopathies [101]. In a particular study carried out to compare
the results of BMT with CBT it was found that patients who had received
CBT had lowered chances of GVHD and also showed slower neutrophil
recovery [102]. Moreover, diagnostic analysis has been performed
through pre-implantation genetics and embryo selection in order to se-
lect a healthy HLA-identical sibling [103,104]. Studies involving the use
of CBT from unrelated donors have also shown favourable results that
are evident by a high survival and disease-free status at the age of 2
[105].
BMT and CBT in SCA patients are now being increasingly per-
formed mainly due to the improving ease in finding a suitable
donor. With the increasing numbers of unrelated donor listings in
BM registries worldwide, it is now comparatively simpler to identify
a suitable donor.
4. Current research standpoint and prospects on future treatments
Due to the complex pathology of the disease, it is natural that multi-
disciplinary research has been initiated globally. Here focus is laid on
some of the cutting-edge aspects in research and future prospects of
therapeutic approaches towards SCA.
104 Q. Fernandes / Life Sciences 178 (2017) 100–108

4.1. Basic research prescribe zinc supplements for child patients in order to treat growth
impairment.
A few of the earliest attempts to elucidate the complex pathophysi- On the basis of these supporting evidences, this study [106] was
ology of SCA and target therapeutic research are summarized in Table 2. targeted at determining whether the supplementation of
Recently, a classic study was carried out on the effects of micronutrients (Minerals: Magnesium Folate, Zinc, Vitamin A, B6, B12,
micronutrients on the symptoms of the disease [106]. SCA is often C, D & E, folic acid, omega-3 fatty acids) could improve the conditions
found associated with poor growth [107], high susceptibility to infec- of the disease or reduce the frequency of the occurrence of painful epi-
tions [108], recurring episodes of pain and irreversible organ damage sodes. The study included participants who were either homozygous for
[109]. It was observed that a deficiency in micronutrients increases sus- SCA or were diagnosed for thalassemia. Upon analysis it was found that
ceptibility to these events. This study was focussed on utilizing a nutri- the supplementation of micronutrients did show a positive response in
tional approach in order to bring down the effects of these improving the conditions associated with the disease to a certain extent.
complications. As part of this study, six randomized controlled trials In a particular report, it was demonstrated that children who re-
were carried out in order to identify key knowledge gaps for nutritional ceived zinc supplementation of 10 mg/day showed a significant in-
interventions. The study implied that SCA patients may suffer from a crease in mean height in a period of approximately 1 year, while on
limited nutritional supply. This could possibly be due to a decrease in the other hand, supplementation with folic acid did not yield any signif-
the intake, assimilation and absorption [110], or due to the intestinal icant difference in terms of growth in children [107].
mucosa being damaged [111]. Other possibilities include a hyper-dy- In another report it was observed that when adult patients of SCA
namic circulation [109] or an increased demand for folate and other vi- were given 50 mg supplements of zinc for a period of about 3 months,
tamins due to a rapid destruction of red blood cells. Moreover most SCA there was a considerable fall in the number of infections for the given
patients also suffer from an increased rate of renal excretion that also period of time [118]. On an average the number of infections was
contributes to a lowered nutritional state due to excessive loss of nutri- brought down from seven to one per patient. Another trial with similar
ents [112]. Many studies have also reported a deficiency of zinc supplementations doses reported an overall reduction in respirato-
micronutrients like omega-3 fatty acids [113], minerals like magnesium ry infection [119]. However in another randomized controlled trial, in-
[114] and zinc [107] and vitamins like B6, C [115], D [116,117] and E volving the supplementation of folic acid, no difference in number of
[117]. Many screening campaigns for the detection of SCA in children infections was noted among patients and control groups [120].
The effects of zinc supplementation on the frequency of painful
vaso-occlusive episodes was evaluated in a particular study carried
Table 2 out over a period of 1 year [121]. However there was no significant dif-
Showing some of the earliest basic research strategies employed in the curative treatment ference in the number of hospital admissions for vaso-occlusive crisis
of SCA. between treated and control groups. Similarly another study reported
Year Analysis Results no significant difference in the frequency of vaso-occlusive crisis in chil-
dren upon supplementation with folic acid [38]. In another study based
Reversal and blocking of erythrocyte sickling character
1971 In-vitro treatment of erythrocytes Treatment prevented the formation
on the supplementation of omega-3 fatty acids in a study conducted
with Urea in Invert Sugar (UIS) in of hydrophobic bonds between the with 10 adult patients of SCA for over 1 year, it was reported that the
order to reverse and block sickling. adjacent hemoglobin molecules number of painful episodes reduced to nearly 50% [122]. However,
thereby preventing polymerization since the study group was small (n b 30), the results may not be conclu-
and subsequent sickling of
sive due to a possibility of sampling bias.
erythrocytes [135].
Evidently, zinc supplementation holds a promising potential in initi-
Hemoglobin modifications ating a nutritional-based therapy as it has been associated with a reduc-
1973 Incubation of HbS or HbA with Hoglobin was acetylated showing
tion of infections and an improvement of growth in children.
aspirin lowered hydrophobic character and
increased oxygen affinity [136]. Since the evidences are few, further analysis over varying treatment
1980 Removal of amino acids from the Both α & β chains of hemoglobin periods and doses of micronutrient supplements becomes necessary be-
hemoglobin tetramer by incubation was modified displaying an fore any conclusions are drawn. However it is evident that the
with ninhydrin. improved affinity towards oxygen micronutrients status may possibly show a positive impact over the
[137].
morbidity and mortality of the disease.
Micronutrient therapy
1983 Evaluation of Folic acid No difference in number of 4.2. Genetic-based strategies in research and gene therapy
supplementation on the frequency infections was noted among patients
of painful vaso-occlusive episodes. and control groups [117].
1995 Evaluation of Zn supplementation No significant differences were Although bone marrow transplantation has shown huge success in
on the frequency of painful observed in the number of hospital the treatment of SCA, its application is still limited, as a majority of
vaso-occlusive episodes. admission for vaso-occlusive crisis adult patients lack a human HLA matched sibling donor. Despite this,
between treated and control groups
it was able to provide a huge platform for the development of genetic
[118].
1999 Evaluation of the effect of Zn An overall reduction in respiratory based curative strategies.
supplementation on the incidence of infection was reported [116]. The two main targets for most genetic based approaches are to firstly
infection in sickle cell patients. bring about a reduction in the overall concentration of HbS in the eryth-
2001 Evaluation of supplementation of Nearly 50% reduction in the number rocytes in order to prevent polymerization of hemoglobin and secondly
omega-3 fatty acids on the of painful episodes was observed
occurrence of painful episodes in ten [119].
to bring about modification in a fraction of erythrocytes in order to pre-
patients. vent vaso-occlusive crises associated with the disease. This approach
2008 Evaluation of the effect of Zn A considerable fall was observed in could possibly involve the efficient transfer and integration of a trans-
supplementation on the incidence of the number of infections for the gene into the haematopoietic stem cells followed by its subsequent
infection in sickle cell patients. given period of 3 months [115].
transplantation into the bone marrow tissue.
2012 Evaluation of supplementation of Micronutrient supplementation
micronutrients (Magnesium, Folate, improved conditions of the disease
Zn, Vitamin A, B6, B12, C, D & E, folic to a certain extent [103]. 4.2.1. Vector based genetic approaches
acid, omega-3 fatty acids)to improve One of the major challenges in gene therapy is the appropriate con-
conditions of the disease and reduce struction and use of a gene transfer vehicle. Haematopoietic cells are
the occurrence of painful episodes.
usually available in very small quantities and their ex-vivo culture is
 Q. Fernandes / Life Sciences 178 (2017) 100–108 105

laborious. In such cases a highly efficient gene transfer methodology be- major drawback of using SSOs is that they are often prone to attack by nu-
comes necessary in order to ensure that the target gene gets incorporat- cleases in the cells. This led to the development of Peptide Nucleic Acids
ed into the chromosome while also maintaining its viability. Usually (PNAs). PNAs are DNA analogs in which a polyamide polymer is used to
viral vectors have been the method of choice till date. Retroviruses, len- replace the entire sugar-phosphate backbone in nucleic acids. These syn-
tiviruses and spumaviruses have often been used in gene transfer thetic DNA strands bind to the target site via complementary base pairing.
experiments as they offer high gene accuracy. An important characteris- In a particular study, Wang et al. [134] designed PNAs with properties that
tic of lentivirus vectors is their high genomic stability as compared to could enable it to selectively bind to a particular sequence of the human
other viral vectors. Hence lentiviruses have been established as reliable beta-globin gene in human K562 erythroleukaemia cells. Since then,
globin vectors and are of relevance in correcting the hemoglobin gene in this strategy has opened newer lines for gene therapy.
order to treat SCA. However, all three viral vectors have been successful- Another genetic based approach involves the use of ribozymes and
ly used in the transduction of haematopoietic stem cells [123–125]. All trans-splicing mechanisms. One of the earliest studies carried out in
three vector systems differ in their integration mechanisms. Retrovirus order to analyse the activity of ribozymes showed that a certain class
generally tend to get incorporated upstream of transcribed genes. On of ribozymes (group I ribozymes) could perform trans-splicing to repair
the other hand lentiviral vector systems generally target the complete human β-globin gene transcripts in-vitro [135]. This system functions
transcribed gene sequence [126]. Relatively weak promoters are also by allowing the ribozyme to first recognise the target site on the HBB
sufficient for the alteration of hemoglobin specific genes. In a particular transcript followed by binding through complementary base in the re-
study it was identified that erythroid-specific globin gene transfers gion of interest. The ribozyme then induces cleavage of the sickle cell
could be particularly targeted through the engineering of lentiviral vec- HBB transcripts leading to the release of the cleavage product contain-
tor viruses harbouring transcriptional elements in combinations of ing the mutation. Thus the expression of the diseased protein (HbS) is
proximal and distal hemoglobin genes [127]. Apart from this system, withheld or inhibited at the post-transcriptional level. Subsequent stud-
several vectors encoding a chimeric hemoglobin gene have also been ies have been able to identify newer target sites with improved accessi-
reported and tested for their potential in the treatment of Sickle Cell bility resulting in an enhanced RNA-cleaving capacity of ribozymes
Anemia [128,129]. [136]. However adequate research is still required in order to develop
Among the various hemoglobinopathies, the first mice models were a stable, efficient and safe expression system synthesizing catalytically
generated for SCA & β-thalessemia in order to treat severe genetically active ribozymes in-vivo.
associated Anemia. In a particular study the efficiency of a certain lenti- Targeted gene integration through homologous recombination in
virus vector in haematopoietic stem cell transplantation was evaluated regions of homology has also been attempted in an effort to develop
in transgenic mice models [130]. Three months after transplantation, it gene-based therapies for SCA. Moreover, it has proved advantageous
was observed that reticulocyte counts and red blood cells had returned due to being an error-free DNA repair mechanism that does not lead
to normal. Moreover the number of irreversibly sickled erythrocytes in to insertion or deletion at the site of cleavage. This technique has been
peripheral blood cells was reduced to a minimum. successfully used to correct or repair diseased or mutated genes at a
In genetic approaches based on the expression of the corrected globin specific site [137]. Thus it provides a wide future scope in offering a
gene, precise quantification of the proportions of γ or β chain expression cell mediated treatment of SCA.
that is required to inhibit the sickling of erythrocytes becomes essential. Evidently, the applications of genetic implements in research are as-
Inducing the production of HbF through altered gene expression has sociated with sets of benefits and drawbacks (Table 3). However, al-
been a main target since decades. Although humans naturally contain though these studies are at primitive stages, they have shown a
about 4 to 10% of HbF in their blood, it was identified that increasing
this proportion to a level of 20% could prove beneficial in improving con- Table 3
ditions of SCA [131]. Based on this theory, a certain study was conducted Showing the role of various genetic tools employed in SCA research.
in which a line of double-transgenic mice was generated. This line was
Genetic tools Typical characteristic Advantages/drawbacks
obtained as a result of a cross between sickle cell anaemic mice model
and other mice models expressing different levels of HbF [130]. As a result Triplex Forming Aims at blocking or TFOs can be destabilized by
Oligonucleotides selectively knocking out the charge repulsions within the
the transgenic mice showed an improved pathology, haematological pro-
(TFOs) expression of the mutated nucleic acid strands [129].
file and an overall increased life span. Through this study it was also HBS gene.
established that even HbF levels of nearly 16% were sufficient to trigger Single Stranded Used to cause a single base Often prone to attack by
a therapeutic response. Oligonucleotides substitution from adenine to nucleases in the cells.
However, one of the biggest threats faced by viral vector-driven (SSOs) thymine at the site of the SCA
mutation (sixth codon of the
treatments is the occurrence of insertational mutagenesis. It is a phe- β-hemoglobin gene) [130].
nomenon arising out of the inactivation of a functional gene when a Peptide Nucleic Synthetic DNA strands PNAs show stronger binding
transgene is incorporated within its sequence. Thus vector-less tech- Acids (PNAs) capable of binding to the affinities TFOs [131].
niques may often be a preferred choice. target site via complementary Capable of binding to both
base pairing. single & double-stranded
DNA targets [131].
4.2.2. Vector-less genetic approaches Ribozymes Perform trans-splicing to Ribozymes are said to
Triplex Forming Oligonucleotides (TFOs) have been evaluated since repair human β-globin gene assume multiple
many years in order to target modified gene expression in in-vitro and transcripts in-vitro [132]. conformations in-vivo,
in-vivo studies. This technique aims at blocking or selectively knocking thereby complicating target
recognition [133].
out the expression of the diseased gene, in this case the HbS gene. In a par- The efficiency of ribozymes is
ticular study, an attempt was made to target cytosines through the use of dependent on the
TFOs within the second intron of the human HBB gene in human accessibility of the target
erythroleukaemia K562 cells [132]. The TFOs were reported to have suc- substrate [133].
Vectors
cessfully bound to the target; however mutation of the locus was not ob-
Lentiviruses Generally applied when High genomic stability.
served. Moreover a number of attempts were also made to induce targeting the complete Established as reliable globin
mutations in the HBB gene through using short Single Stranded Oligonu- transcribed gene sequence vectors.
cleotides (SSOs). It was reported that SSOs were used to cause a single [123].
base substitution from adenine to thymine at the site of the mutation Retroviruses Gets incorporated upstream -
of transcribed genes
which is at the sixth codon of the β-hemoglobin gene [133]. However a
106 Q. Fernandes / Life Sciences 178 (2017) 100–108
Fig. 1. Gene Therapy Strategies in Sickle Cell Anemia.
promising scope in tackling the scientific and clinical challenges pre-
sented. Fig. 1 graphically summarizes the genetic strategies employed
towards therapeutic research in SCA.
5. Conclusion-scientific prospects & future scope of SCA targeted
research
The medley of strategies targeting treatment of SCA that have been ac-
tively sought after reflects the dire need for improved curative measures.
Although the complexity of the disease complications and treatment out-
comes has been fathomed, there is still an urgent need for scientific re-
search in order to conquer the clinical conditions of the disease.
Considerable progress has been made in terms of the use of hy-
droxyurea, and other novel drugs to manage the disease. The discovery
of micronutrients therapy has indeed opened newer lines into the ther-
apeutic venture; however extensive research still needs to be exercised.
Moreover, gene therapy approaches have also displayed a wide poten-
tial through the safe and effective modification of pluripotent stem
cells. This has led to the conceptualization of possibilities for a perma-
nent cure. The engineering of vector-less techniques have eliminated
the concerns of insertational inactivation. The only identifiable concerns
remaining are its genotoxicity related issues that require further de-
tailed analysis and scrutiny.
Conflict of interest statement
The author has no competing interests.
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