Martina Rossi Science Reviews - Biology, 2023, 2(3), 18 - 24
Advancements and Challenges in Gene Therapy Ap-
proaches for Sickle Cell Disease: A Comprehensive
Martina Rossi, PhD
Independent Researcher, Strasbourg, France;
Received October 6, 2023. Revised October 24, 2023. Accepted October 24, 2023. Published online October 31, 2023.
Abstract: Sickle cell disease (SCD) is an autosomal recessive genetic blood disorder that occurs when both
alleles of the HBB gene have mutations, leading to the production of abnormal haemoglobin (HbS). The
presence of HbS causes red blood cells (RBCs) to take on the distinctive sickle-shaped form associated with the
disease. This, in turn, leads to blockages in blood vessels, decreased blood circulation, and organs’ damage.
Traditional treatments such as blood transfusions and hydroxyurea offer relief but come with their own
limitations and associated risks. Gene therapy has emerged as a promising paradigm shift in the quest to cure
SCD, offering personalised solutions by targeting the genetic root of the disease.
This review article explores the principles and recent advancements in gene therapy for SCD. However,
before gene therapy can become the main curative strategy for this disease, several challenges need to be
overcome including the need for long-term safety and efficacy evaluations. Ongoing research and innovation
hold the promise of enhanced treatments and the potential for a widely available gene therapy, ultimately
improving the quality of life for individuals living with SCD.
Keywords: Sickle cell disease (SCD), HBB gene, haemoglobin S (HbS), gene therapy autologous
haematopoietic stem cells (HSCs), gene addition, lentiviral vectors, gene editing, CRISPR/Cas9
Molecular Basis of Sickle Cell Disease
Beyond Sickle cell disease, also known as sickle
cell anemia, is the most prevalent autosomal reces-
sive genetic blood disorder. It results from an in-
herited mutation in both alleles of the HBB gene
(1). This monogenic disease is caused by a specific
single base pair mutation (A=T) located within the
sixth codon of the HBB gene (2). The HBB gene is
responsible for encoding the β-globin subunit of
haemoglobin A (HbA), which is the primary oxy-
gen-carrying protein in adult red blood cells
(RBCs). The missense mutation in the HBB gene
results in the substitution of the hydrophilic amino
acid glutamic acid with the hydrophobic amino
acid valine. This change in amino acids leads to the
misshaping of the β-globin chains within the HbA.
Figure 1: Haemoglobin A (HbA) protein is made up
of four subunits: two α-globin subunits (pink) and
two β-globin subunits (blue) each bound to a heme
group (yellow). Image created using BioRender
Science Reviews - Biology, 2023, 2(3), 18 - 24 Martina Rossi
Haemoglobin is composed of four subunits: two α-
subunits and two β-subunits each bound to a heme
group (Figure 1). Heme groups enable haemoglo-
bin to bind to oxygen molecules by forming a sta-
ble and reversible association. Sickle cell hae-
moglobin (HbS) can perform the function of carry-
ing out oxygen (which will be distributed
throughout the body) perfectly well. However,
when de-oxygenated, HbS molecules undergo
anomalous hydrophobic interactions, leading to
their aggregation into long, rigid structures within
the RBCs (2). This eventually gives rise to the char-
acteristic deformation of RBCs, causing them to
assume a sickle-like shape (2) (Figure 2).
The sickle shape of these cells is problematic as it
reduces their flexibility, making them more prone
to getting stuck in small blood vessels. This, in turn,
results in vascular blockages, decreased blood cir-
culation, and premature cell death (1,3). Moreover,
reduced blood circulation can lead to chronic or-
gan damage, potentially culminating in conditions
such as strokes, kidney failure, lung-related com-
plications, and bone necrosis, ultimately contrib-
uting to premature mortality (4).
Heterozygote Advantage in Sickle Cell Disease
Sickle cell disease is the most prevalent monogenic
disorder in the United States, affecting approxi-
mately 1 in every 500 African Americans. It is even
more common in malaria-endemic regions, a phe-
nomenon known as "heterozygote advantage" (5).
Individuals with only one copy of the mutated
HBB gene typically experience mild or no symp-
toms. In turn, research has shown that those with
this heterozygous mutation are more resistant to
severe malaria (5-7). This resistance occurs because
HbS interferes with the Plasmodium parasite's life
cycle within RBCs, providing a survival advantage
for individuals carrying the heterozygous muta-
tion in malaria-prone areas. Remarkably, approxi-
mately 1 in every 12 African Americans carries the
autosomal recessive mutation in the HBB gene,
resulting in roughly 300,000 infants being born
with the condition each year (2,8).
Traditional Treatment Approaches
To date, the main therapeutic treatments for SCD
remain blood transfusion and hydroxyurea (1).
Blood transfusions aim to enhance oxygen-
carrying capacity and reduce the ratio of HbS to
HbA, thereby alleviating the complications associ-
ated with vascular occlusion (9). However, in
many regions around the world, patients lack ac-
cess to a secure and sustainable blood source. Even
in countries where blood is accessible and econom-
ically viable, long-term transfusion therapy carries
inherent risks, including alloimmunisation (an
immune response to foreign cell antigens), iron
overload (due to the body's inability to naturally
break down excess iron), and potential risks of in-
fections from contaminated blood (9). Overall,
long-term transfusion therapy is associated with a
significant burden on the patient, including the
need for regular hospital attendance and the use of
iron chelators to eliminate excess metal. Therefore,
the approach to transfusion must balance these
risks with the benefits, both in decisions regarding
when to transfuse and in the practical aspects of
how transfusions are administered.
Another common therapeutic approach for SCD is
hydroxyurea, an oral medication renowned for its
effectiveness in reducing the frequency and severi-
ty of various complications associated with the
disease by elevating fetal haemoglobin levels (HbF)
(10). Fetal haemoglobin differs from the adult
haemoglobin by featuring two γ-globin subunits in
place of β-globin subunits, thus effectively side-
stepping the challenges stemming from the genetic
mutation in the β-globin subunits. However, de-
spite its proven efficiency in reducing the frequen-
cy of disease-related complications, its use requires
careful monitoring to ensure that the dosage is tai-
lored to the patient, as it can lead to side effects
such as bone marrow suppression and a tempo-
rary decrease in blood counts.
Figure 2: Healthy mature red blood cell (left) vs sickled
de-oxygenated red blood cell (right). Image created using
Martina Rossi Science Reviews - Biology, 2023, 2(3), 18 - 24
Gene Therapy approach to cure Sickle Cell Dis-
More recently, besides blood transfusions and hy-
droxyurea, other FDA-approved therapies like
voxelotor, crizanlizumab, and L-glutamine have
been used to diminish the frequency and severity
of vaso-occlusive crises (1). However, the potential
adverse effects associated with these therapies
highlight the need for innovative interventions
that can address the limitations of current treat-
To date, the only cure for SCD is bone marrow
transplant, which consists of the transplantation of
healthy hematopoietic stem cells (HSCs) from a
donor to the patient. However, this technique is
associated with significant issues, including organ
injury, infection, and graft-versus-host disease,
which could eventually lead to death. Only about
10% of patients affected by the disease have a his-
tocompatible sibling donor (11). Although it repre-
sents a viable curative option, before performing a
bone marrow transplantation, the patient needs to
undergo extensive testing and evaluation to de-
termine eligibility and find a suitable donor (11,12).
Gene therapy offers a potential solution to the is-
sues raised by bone marrow transplantation. By
using autologous HSCs, scientists can either insert
the functional gene or correct the genetic mutation
and re-insert the cells back into the patient, thus
overcoming potential immune complications and
eradicating the disease at its core.
Gene Therapy: A Promising Paradigm Shift
Overview of Gene Therapy Principles
With a focus on patient-tailored medication, gene
therapy approach holds an immense promise in
revolutionising medical treatments by addressing
the disease at the genetic level. This approach is
grounded on the simple principle of replacing or
correcting faulty genetic sequences causing a speci-
fic disease, to restore their functionality.
Autologous HSC therapy has been actively pur-
sued and holds a great promise for curing SCD
(11). This technique consists of isolating the HSCs
cells from either the bone marrow or the periphe-
Figure 3: Schematic representation of gene therapy strategies for SCD. (1) Haematopoietic stem cells are collected
from the patient affected by SCD. (2) Gene therapy strategies are implemented to either (2A) insert the functional HBB
into a lentiviral vector or (2B) engineer CRISPR/Cas9 technology to correct the underlined gene mutation. (3)
Haematopoietic stem cells are then transfected and (4) re-inserted back into the patient. Image created using BioRen-
Science Reviews - Biology, 2023, 2(3), 18 - 24 Martina Rossi
ral blood and manipulating them ex vivo to either (i)
insert the functional gene, (ii) correct the SCD mu-
tation, or (iii) induce HbF expression. Once HSCs
have been successfully transfected, the patient re-
ceives bone marrow conditioning with myeloabla-
tive agents, followed by infusion of the modified
HSCs (Figure 3).
Two Types of Gene Therapy for Sickle Cell Dis-
ease: Gene Addition and Gene Editing
Over the years, two different approaches have
emerged in the field of gene therapy for SCD: gene
addition and gene editing. Gene addition consists
of introducing a functional copy of the β-globin
gene into a patient's HSCs, usually using lentiviral
vectors (Figure 3 2A). This approach aims to re-
place defective haemoglobin with healthy haemo-
globin, ultimately improving the quality and func-
tionality of RBCs (11,13). On the other hand, gene
editing seeks to provide a one-time treatment ca-
pable of either correcting the genetic mutation res-
ponsible for SCD or inducing the expression of
HbF, using gene editing tools such as
CRISPR/Cas9 (Figure 3 2B) (11,13).
Both approaches offer promising avenues for de-
veloping effective treatments and potential cures
for SCD patients. However, there is a key distinc-
tion between the two methods. While gene addi-
tion does not integrate the functional gene into the
genome - resulting in a transient curative strategy -
gene editing has the potential to permanently cor-
rect the underlying genetic defect, offering a more
robust curative solution. Ongoing research and
clinical trials are continually enhancing our under-
standing of the effectiveness and safety of these
therapeutic strategies.
Gene Addition: Lentiviral-Based Strategies
The transplant of genetically engineered
autologous HSCs has emerged as a promising cu-
rative strategy for SCD. One approach consists of
introducing the functional HBB gene inside the
HSCs using viral vectors. Over the years, ad-
vancements in viral vectors manipulation have led
to a transition from the use of γ-retroviral vectors
to lentiviral vectors as the preferred approach (14).
Initially, γ-retroviruses offered advantages such as
stable integration, versatility in target cell types,
and ease of vector manipulation. However, γ-
retroviruses have been shown to come with limita-
tions including limited transgene expression and
risks of insertional mutagenesis (14,15). Lentiviral
vectors have emerged as a promising alternative
due to their ability to accommodate more complex
DNA cassettes, a crucial factor for achieving high-
level of β-globin expression (14). Unlike γ-
retroviruses, lentiviral vectors can integrate into
non-dividing HSCs, ensuring a safer and more
stable integration profile. Safety modifications,
including self-inactivation and removal of viral
enhancer and promoter sequences, have been im-
plemented to address concerns about insertional
mutagenesis. Additionally, transgene expression
has been improved by incorporating into the lenti-
viral vectors key transcriptional regulatory ele-
ments from the β-globin locus control region (16).
While gene addition strategies have marked signif-
icant progress in the development of gene therapy
for SCD, they exhibit only partial effectiveness in
alleviating the clinical manifestations of the disease
(17). To tackle this challenge, a gene silencing ap-
proach has been employed. This method utilises a
lentiviral vector that expresses a microRNA to si-
lence the expression of the BCL11A gene, a crucial
regulator of the gene encoding the γ-globin subu-
nit in adulthood (18,19). Ongoing clinical trials are
currently assessing the potential therapeutic bene-
fits of reactivating HbF using lentivirus-based
strategies (20). Nevertheless, it still remains the
issue that even when combining gene addition
with gene silencing, the formation of HbS cannot
be completely prevented, ultimately leading to the
premature degradation of RBCs (17,21).
A recent development involves the generation of a
bifunctional lentiviral vector designed to express
functional β-globin while concurrently employing
a microRNA to specifically down-regulate sickling
β-globin expression. This technique allows for the
reduction of HbS levels and promotes the incorpo-
ration of functional β-globin into the haemoglobin
molecule (21). The efficient transduction of autolo-
gous HSCs by this bifunctional lentiviral vector
results in a significant expression of the functional
β-globin and a reduction of the sickling β-globin
transcripts within the erythroid progenitors and
RBCs, ultimately resulting in the successful correc-
tion of the sickling phenotype (21). Overall, the
integration of both gene addition and gene silenc-
ing strategies holds great promise for enhancing
the effectiveness of existing lentiviral-based thera-
peutic methods. In particular, this approach pre-
Martina Rossi Science Reviews - Biology, 2023, 2(3), 18 - 24
sents an innovative treatment option ready for pre-
clinical and clinical testing.
Gene Editing: Prime Editing and Base Editing
Genome editing allows for precise alterations to
the human genome, with the goal of rectifying mu-
tations that underlie genetic disorders (22). This
process usually involves inducing DNA double-
strand breaks (DSBs) using engineered designer
nucleases, such as zinc finger nucleases (ZFNs),
transcription activator-like effector nucleases
(TALENs), or the CRISPR/Cas9 system. When
DSBs occur under physiological conditions, they
activate two primary repair pathways: non-
homologous end joining (NHEJ) and homology-
directed repair (HDR) (23). NHEJ is a rapid but
error-prone mechanism, often resulting in inser-
tions and deletions at the break site. Conversely,
HDR is a slower but highly accurate DNA repair
pathway that employs an introduced DNA frag-
ment as a template to precisely rectify the occurred
error. Because HDR requires a template strand, it
is largely restricted to the S and G2 phases of the
cell cycle; therefore, achieving gene targeting rates
higher than 20% in predominantly inactive HSCs
remains a challenge (24). Moreover, inducing DSBs
is known to hold genotoxic potential, and muta-
tions, loss of heterozygosity, and chromosome re-
arrangements can occur during DNA repair (25).
To overcome this issue, alternative techniques
where only one strand of DNA is cleaved have
been developed, and they are mostly based on the
CRISPR/Cas9 system (26).
One such technique is base editing, which allows
for the introduction of single-nucleotide variants in
the DNA or RNA of living cells. Base editors are
composed of a Cas9 fused with a deaminase en-
zyme capable of precisely converting A to G or C
to T at specific sites directed by single-guide RNA
(sgRNA). In a 2021 study, successful base editing
was implemented to rectify the SCD mutation in
both patient blood-forming cells and a mouse
model (27). While base editors cannot generate the
required T-to-A change to restore the SCD muta-
tion to its normal sequence, converting A to G - or
T to C on the complementary DNA strand - gener-
ates a "haemoglobin Makassar," which consists of a
rare benign haemoglobin variant found in healthy
individuals (27).
Another emerging gene editing tool that allows for
targeted small insertions, deletions, and base
swapping - without the need for DSB - is the prime
editing tool. In a recent study, prime editing has
been used effectively to correct the HBB gene in
HSCs collected from SCD patients (28). Correction
rates ranged from 15% to 41% and exhibited suc-
cessful engraftment, differentiation, and lineage
maturation. Importantly, a genome-wide analysis
revealed minimal off-target effects. These studies
suggest the potential for a one-time treatment for
SCD that mitigates the undesirable effects associat-
ed with DSB.
Challenges to overcome and future directions
Overall, lentiviral vectors have shown effective
results and flexibility in incorporating various anti-
sickling genes. Importantly, no vector-related clin-
ical adverse events have been noted. Nonetheless,
lentiviral vectors do carry a potential risk of inser-
tional mutagenesis, although current data suggest
this risk is relatively low in the treated patient
population (14). Alternative approaches to gene
adding, such as the CRISPR/Cas9 system, are in
the early stages of clinical data collection, while
others, like base editing and prime editing are just
beginning clinical evaluation. A major concern po-
sed by the use of CRISPR/Cas9 system, is the
generation of off-target effects and potential geno-
toxicity. Evaluating the long-term effectiveness
and safety of all these therapeutic strategies will
require several decades of ongoing observation. In
the meantime, it is likely that new approaches will
continue to emerge, potentially offering even
greater promise.
The ultimate approach for HSC-based gene editing
to treat SCD would involve direct genome editing
(in vivo), rather than the current method of isolat-
ing HSCs outside the body (ex vivo) with chemo-
therapy conditioning. If it becomes feasible to sys-
tematically administer gene editing agents and
achieve efficient editing within HSCs in vivo, it
would significantly broaden the application of this
treatment and drastically reduce costs. The ulti-
mate goal is to make gene therapy globally acces-
sible, particularly for the majority of SCD patients
who have limited resources (14).
Science Reviews - Biology, 2023, 2(3), 18 - 24 Martina Rossi
The pursuit of a cure for SCD has witnessed re-
markable advancements over the years, transition-
ing from traditional therapies like blood transfu-
sion and hydroxyurea to innovative gene therapy
approaches. While traditional treatments provide
some relief, they come with limitations and risks,
highlighting the need for more advanced interven-
tions. Gene therapy has emerged as a promising
paradigm shift in the quest to cure SCD. By target-
ing the genetic root of the disease, gene therapy
aims to offer a patient-tailored solution. Ongoing
research and clinical trials are shedding light on
the effectiveness and safety of these innovative
therapeutic strategies. However, despite the pro-
gress made in gene therapy, challenges persist, and
long-term observations are required to assess the
safety and efficacy of these approaches fully. The
ultimate goal would be to transition from ex vivo
gene editing, involving HSC isolation with chemo-
therapy conditioning, to in vivo editing, which
could make gene therapy more accessible and cost-
effective. With continued research and innovation,
the vision of a globally accessible gene therapy for
SCD may become a reality, providing lasting relief
and improving the lives of countless individuals
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Conflict of Interest statement
The author declares no conflict of interest.