Martina Rossi Science Reviews - Biology, 2023, 2(3), 18 - 24

Advancements and Challenges in Gene Therapy Ap-

proaches for Sickle Cell Disease: A Comprehensive

Review

Martina Rossi, PhD

Independent Researcher, Strasbourg, France; martina.rossi108@gmail.com

https://orcid.org/0000-0002-8866-1844

https://doi.org/10.57098/SciRevs.Biology.2.3.3

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

Introduction

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

BioRender.

Martina Rossi Science Reviews - Biology, 2023, 2(3), 18 - 24

Gene Therapy approach to cure Sickle Cell Dis-

ease

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-

ments.

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-

der.

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

Strategies

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

Conclusion

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

worldwide.

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Conflict of Interest statement

The author declares no conflict of interest.