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\begin{document}

\title{Novel therapies in \selectlanguage{greek}β- \selectlanguage{english}thalassemia\selectlanguage{english}}



\author[1]{Laura Grech}%
\author[2]{Karen Borg}%
\author[3]{Joseph Borg}%
\affil[1]{University of Malta Faculty of Medicine and Surgery}%
\affil[2]{Affiliation not available}%
\affil[3]{University of Malta Faculty of Health Sciences}%


\vspace{-1em}



  
  \date{May 05, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Beta-thalassaemia is one of the most significant haemoglobinopathies
worldwide resulting in the synthesis of little or no \selectlanguage{greek}β-\selectlanguage{english}globin chains.
Without treatment, \selectlanguage{greek}β-\selectlanguage{english}thalassaemia major is lethal within the first
decade of life due to the complex pathophysiology which leads to wide
clinical manifestations. Current clinical management for these patients
solely relies on repeated transfusions followed by iron chelating
therapy which can eventually results into multi-organ damage. A number
of novel approaches to correct the resulting \selectlanguage{greek}α/β \selectlanguage{english}globin chain imbalance
are currently being developed. These include reactivation of foetal
haemoglobin by pharmacological compounds, allogenic hematopoietic stem
cell transplantation (HSCT) and gene therapy. Up to now, the only
curative treatment for beta-thalassemia is HSCT, but this is a risky and
costly procedure. Gene therapy either by gene addition or gene editing
is emerging as a powerful approach to treat this disease. Gene addition
is currently based on transplantation of autologous hematopoietic stem
cells genetically modified with an integrating lentiviral vector
expression the globin gene while gene editing involves the use of
CRISPR/Cas9 to correct the causative mutation. Although the early
outcomes of the clinical trials in gene therapy are showing promising
results, they have also highlighted a number of limitations. In this
review we will discuss about the current management strategies used to
treat beta-thalassaemia and also focus on novel therapies.%
\end{abstract}%



\sloppy


Novel therapies in \selectlanguage{greek}β- \selectlanguage{english}thalassemia

Grech Laura\textsuperscript{1}, Borg Karen\textsuperscript{2}, Borg
Joseph\textsuperscript{1,3}

1 - Centre for Molecular Medicine and Biobanking, University of Malta

2 -- Department of Public Health Medicine, Ministry for Health Malta

3 -- Applied Biomedical Science, Faculty of Health Sciences, University
of Malta

Abstract

Beta-thalassaemia is one of the most significant haemoglobinopathies
worldwide resulting in the synthesis of little or no \selectlanguage{greek}β-\selectlanguage{english}globin chains.
Without treatment, \selectlanguage{greek}β-\selectlanguage{english}thalassaemia major is lethal within the first
decade of life due to the complex pathophysiology which leads to wide
clinical manifestations. Current clinical management for these patients
solely relies on repeated transfusions followed by iron chelating
therapy which can eventually results into multi-organ damage. A number
of novel approaches to correct the resulting \selectlanguage{greek}α/β \selectlanguage{english}globin chain imbalance
are currently being developed. These include reactivation of foetal
haemoglobin by pharmacological compounds, allogenic hematopoietic stem
cell transplantation (HSCT) and gene therapy. Up to now, the only
curative treatment for beta-thalassemia is HSCT, but this is a risky and
costly procedure. Gene therapy either by gene addition or gene editing
is emerging as a powerful approach to treat this disease. Gene addition
is currently based on transplantation of autologous hematopoietic stem
cells genetically modified with an integrating lentiviral vector
expression the globin gene while gene editing involves the use of
CRISPR/Cas9 to correct the causative mutation. Although the early
outcomes of the clinical trials in gene therapy are showing promising
results, they have also highlighted a number of limitations. In this
review we will discuss about the current management strategies used to
treat beta-thalassaemia and also focus on novel therapies.

\textbf{Introduction}

Beta-thalassaemia is one of the most common genetic blood disorders
worldwide, caused by a spectrum of mutations that results in a
quantitative reduction of structurally normal \selectlanguage{greek}β-\selectlanguage{english}globin chains. It is
most prevalent in the Mediterranean region, parts of North and
sub-Saharan Africa, the Middle East, Indian subcontinent and Southeast
Asia and it is estimated that 68,000 babies are born each year suffering
from this disease {[}1{]}, {[}2{]}.

\selectlanguage{greek}\textbf{Genetics and Pathophysiology of β-thalassaemia}\selectlanguage{english}

Downregulation of \selectlanguage{greek}β-\selectlanguage{english}globin gene (\emph{HBB} ) can be caused by a whole
spectrum of mutations. In fact, more than 400 mutations in the \selectlanguage{greek}β-\selectlanguage{english}globin
gene have now been described ranging from point mutations to small
deletions limited to the \emph{HBB} to extensive deletions of the whole
\selectlanguage{greek}β \selectlanguage{english}globin cluster
(\emph{https://ithanet.eu/db/ithagenes?action=list\&hem=2}). The
non-deletional mutations include single base substitutions, small
insertions or deletions of one to a few bases located within the gene or
its immediate flanking sequences. These mutations can affect every stage
of \selectlanguage{greek}β-\selectlanguage{english}globin expression, from transcription to RNA processing to
translation. Although \selectlanguage{greek}β-\selectlanguage{english}thalassaemia is rarely caused by deletions, a
number of deletions that are restricted to the \emph{HBB} gene itself
have been described. These deletions range from 25bp up to 6kb {[}3{]}.
Subjects with a single \selectlanguage{greek}β-\selectlanguage{english}thalassaemia allele are called \selectlanguage{greek}β-\selectlanguage{english}thalassaemia
traits or carriers and they are usually asymptomatic. Such individuals
present with a mild hypochromic, microcytic anaemia and elevated
HbA\textsubscript{2}. When both \selectlanguage{greek}β \selectlanguage{english}globin gene alleles are affected, no \selectlanguage{greek}β
\selectlanguage{english}globin is produced and these patients are referred to as \selectlanguage{greek}β-\selectlanguage{english}thalassaemia
homozygotes or major. \selectlanguage{greek}β-\selectlanguage{english}thalassaemia major presents itself within the
first 2 years of age with severe anaemia, poor growth and skeletal
abnormalities if left untreated {[}4{]}

Due to the imbalance in globin chain synthesis, an excess of freed
\selectlanguage{greek}α-\selectlanguage{english}globin chains accumulates within erythroid cells. Aggregation,
denaturation and degradation of these excess \selectlanguage{greek}α-\selectlanguage{english}chains leads to the
formation of methaemoglobin and insoluble hemichromes resulting in free
iron which catalyses the formation of reactive oxygen species. The
reactive oxygen species damages the cell membrane leading to ineffective
erythropoiesis in the bone marrow and haemolysis of red cells within
circulation. This triggers loss of red blood cells in the spleen due to
the binding of immunoglobulins and complement components to defective
red cell membranes. The resulting severe anaemia leads to reduced tissue
oxygenation, increased erythropoietin and bone marrow expansion. This
leads to skeletal deformities and osteopenia. Substances released from
degenerating red cells increase iron absorption, which contributes to
iron overload which can cause liver and heart disease {[}5{]},{[}6{]}.

\selectlanguage{greek}\textbf{Management of β-thalassaemia}\selectlanguage{english}

Without treatment, \selectlanguage{greek}β-\selectlanguage{english}thalassaemia major is lethal within the first
decade of life due to the complex pathophysiology which leads to wide
clinical manifestations. Current management strategies for these
patients comprise blood transfusion, iron chelation therapy and, for a
subset of patients; allogenic bone marrow transplantation also known as
haemopoietic stem cell transplantation (HSCT). In recent years, a better
understanding of the pathogenesis and clinical effects of the disease
has enthused research into a number of promising novel therapies such as
therapies that modulate foetal haemoglobin (HbF) induction, gene therapy
and gene editing {[}5{]}, {[}7{]}, {[}8{]}. For this review we will talk
about the current management strategies and focus on novel therapies.

Blood Transfusions

Patients with \selectlanguage{greek}β-\selectlanguage{english}thalassaemia major require lifelong, regular blood
transfusions administered every 2-5 weeks to maintain haemoglobin levels
of at least 9 -- 10.5 g/dL. Transfusion is usually started before the
age of 2 years and it provides normal red blood cells and suppresses
ineffective erythropoiesis, also it enables normal growth and physical
activities and helps to reduce hepatosplenomegaly and bone deformities
{[}9{]}. It is estimated that approximately 100,000 patients currently
receive regular transfusions for beta-thalassemia worldwide and in some
countries transfusion therapies can be a huge burden. Although processes
for screening, preparation and administration of blood have improved,
blood transfusions expose patients to a number of risks such as
blood-borne infection, alloimmunization and iron overload {[}7{]},
{[}9{]}.

Iron Chelation

In \selectlanguage{greek}β-\selectlanguage{english}thalassaemia patients, iron stores are increased far beyond normal
physiological levels primarily due to increased iron absorptions and
secondary due to transfusions. Unless effective iron chelation therapy
is provided, these patients can suffer from iron overload that affects
the heart, liver and endocrine tissues. Three iron chelators are
currently approved by the regulatory authorities for the treatment of
iron overload in beta-thalassemia {[}7{]}. Deferoxamine (DFO), the first
commercially iron chelator is a hexadentate iron chelator that binds
iron in 1:1 complex. It is administered subcutaneously or intravenously
at a dose of 20-50 mg/kg/day. It has a short plasma half-life of 20-30
minutes and therefore it should be administered over a span of 8-10
hours a day on 5-7 days a week {[}10{]}. Although the benefits of
deferoxamine are well documented, the demanding regime leads to poor
compliance.

The introduction of two orally active iron chelators; Deferiprone and
Deferasirox were of a great advance in the management of patients with
beta thalassaemia. Deferiprone (DFP), the first oral iron chelator was
approved in 2011, it is a bidentate iron chelator that forms 3:1
complex. It is usually given at a dose of 75-100mg/kg/day three times
daily. DFP may cause gastrointestinal disturbances, increased
liver-enzymes, agranulocytosis and neutropenia and therefore patients on
DFP should be closely monitored Deferasirox (DFX), the other oral iron
chelator is a tridentate that forms 2:1complex. It is given at a dose of
20-40 mg/kg once daily and the most common side effects are
gastrointestinal disturbances, rash and mild increases in serum
creatinine. Sometimes a combination of both iron chelators is used
{[}7{]},{[}11{]}. Although iron-chelation therapy is available in most
countries, death due to iron overload remains an issue in
beta-thalassaemia patients.

Haemopoietic Stem Cell Transplantation (HSCT)

Until now the only available curative therapeutic approach for patients
with beta-thalassaemia is HSCT. The first HSCT in patients with beta
thalassaemia major took place in the 1980s and early 1990s at the
transplant centre in Pesaro, Italy. Initially, transplant experiences
were limited to good risk young thalassaemia patients with limited
morbidities and from matched sibling donors (MSD)
{[}12{]},{[}13{]},{[}14{]}. With today advances in medicine, HSCT is not
only limited to patients with matched sibling donors but also from
unrelated donors and cord blood transplantation {[}15{]}. Instead of
bone marrow, it was proposed to use peripheral blood stem cell
transplantation from HLA-matched siblings but studies showed that this
increased risk of cGVHD {[}16{]}, {[}17{]}. In 2003, the possibility of
using HLA-identical sibling cord blood for HSCT in thalassaemia major
was reported that this type of allograft was associated with decreased
risk of both acute GvHD and chronic GvHD {[}18{]}.

The best clinical outcomes of HSCT among patients with beta thalassaemia
are reported in those aged under 14 years at transplantation, in fact in
the last decade, almost all transplant centres try to perform HSCT in
the first years of life before iron-related complications develop.
Experience of HSCT in adult patients is still very limited and only few
centres perform HSCT in patients over the age of 18 years. In-fact it is
recommended that HSCT in adults is done only in patients who have been
well-chelated since infancy. About 25-30\% of patients with thalassaemia
major have an available MSD but this is not always the case. The
experience of HSCT from HLA-disparate relatives is still very limited
and the results obtained are inferior than those obtained with
HLA-identical siblings as donors. A small study from related donors that
were not MSD had a thalassemia major free survival of 94\%. This was
obtained by using a pre-conditioning phase with hydroxyurea, azathiprone
and fludarabine while the conditioning regime included busulfan,
thiotepa, cyclophosphamide and rabbit ATG {[}19{]}, {[}20{]}. In case
that unrelated donors are used for HSCT, the donor must be selected
using high-resolution molecular typing for both HLA class I and II
molecules and a stringent criterion of compatibility with the recipient
{[}20{]}.

Although HSCT is the only available curative approach for thalassemia,
nevertheless it has been limited by the high cost and significant
drawbacks associated with its implementation which include limited
availability of MHC-matched donors, the need for long-term
immunosuppression and increased risk of immunological complications.
Understanding the pathophysiology and clinical effects of
beta-thalassaemia has stimulated research into a number of promising
therapeutic approaches to tackle this disease.

\selectlanguage{greek}\textbf{Novel therapies for β-thalassaemia}\selectlanguage{english}

Compounds that modulate foetal haemoglobin

After 2 years of age, the main type of haemoglobin present is adult Hb
(HbA) together with HbA2 (\textless{}3.3\%) and small amounts of foetal
haemoglobin (HbF, \textless{}1). In some individuals, the HbF levels
exceed this threshold and genome wide association studies (GWAS) have
shown that variant HbF levels are highly inheritable {[}21{]},{[}22{]}.
It is well known that beta-thalassaemia patients with inherited
persistent high levels of HbF production have a milder clinical
progression than other patients with this disease, and many of these
patients do not require transfusions. This is because increased
production of \selectlanguage{greek}γ-\selectlanguage{english}globin decreases the \selectlanguage{greek}α/β-\selectlanguage{english}chain imbalance. Reactivation
of HbF is one of the novel therapeutic approaches for beta-thalassaemia.
Various studies have been carried out to identify quantitative trait
loci (QTL) that increases HbF levels to help in the development of
clinical HbF inducers.

A number of genomic loci have been identified which include the beta
globin (HBB), the HBS1-like translational GTPase-v-myb avain
myeloblastosis viral oncogene homolog (\emph{HBS1L-MYB} ) on chromosome
6 {[}23{]}, {[}24{]}, the B-cell CLL/lymphoma11A (\emph{BCL11A} ) locus
on chromosome 2 {[}26{]} and the Kruppel-like factor (KLF1) on
chromosome 19 {[}27{]}. The role of \emph{KLF1} in globin gene switching
was shown in a large Maltese family with hereditary persistence of
foetal haemoglobin (HPFH). Ten out of twenty-seven family members had
HbF levels between 3 and 19\% which was due to a truncation mutation in
the KLF1 gene. Functional work showed that KLF1 have a dual role in the
regulation of foetal-to-adult globin gene switching. First, it acts
directly on the \emph{HBB} locus as a preferential activator of
the\emph{HBB} gene and secondly it acts indirectly by activating the
expression of \emph{BCL11A} which, in turns, represses the gamma globin
genes {[}27{]}, {[}28{]}. The \emph{MYB} gene is a key regulatory of the
balance between proliferation and differentiation during erythropoiesis.
MYB inhibits HbF expression through regulating KLF1 {[}29{]}.

These regulatory transcription factors involved in \selectlanguage{greek}γ-\selectlanguage{english}globin regulation
are potential targets for HbF increase. Although a lot of research is
focusing in this area, it remains difficult to modulate the function of
factors other than enzymes or signal-dependent nuclear factors by
disrupting DNA/protein interactions or protein/protein interaction.
Furthermore, any interference with erythroid transcription factors may
disrupt erythropoiesis, and therefore efforts are being made to design
endonucleases capable of precisely disrupt the genomic sequences
involved in the expression of gamma globin repressors {[}30{]}.

One drug that has proved to be clinically effective in some patients
with \selectlanguage{greek}β-\selectlanguage{english}thalassaemia is the S-phase cell-cycle inhibitor hydroxyurea
(HU). An increase of 2- to 9- fold change in \selectlanguage{greek}γ-\selectlanguage{english}mRNA expression was noted
in some thalassaemia patients, but the increases in HbF did not always
correlate with an increase in total haemoglobin. {[}31{]}, {[}32{]}. HU
treatment of 10-20mg/kg per day has been reported to lead to 40-70\%
decrease in transfusion needs in thalassaemia major patients. This was
mostly noted in individuals with the HbE/\selectlanguage{greek}β-\selectlanguage{english}thalassaemia genotypes
{[}33{]}. A number of side effects have been reported which include
cytopenia, hyperpigmentation, opportunistic infections, marked
hypomagnesemia and in about 80\% of men azoospermia. Although it is
believed that HU is teratogenic, there is little or no risk of leukaemia
{[}34{]}. Given that reported efficacy was seen in only a subset of
patients, and its potential side effects, it is important to identify
likely responders and non-responders before initiating treatment. Some
studies noted that the increase in HbF levels following HU treatment in
\selectlanguage{greek}β-\selectlanguage{english}thalassaemia major patients was correlated with the XmnI polymorphism
while in a study of beta-thalassaemia patients of Iran origin, HU
response was correlated with \emph{BCL11A} SNPs {[}35{]}, {[}36{]},
{[}37{]}. Since only a few studies have investigated the correlation
between polymorphism and response to HU treatment and conflicting
results have been obtained, the criteria for treatment with HU should
not be based on these genetic characteristics {[}38{]}.

Thalidomide, a synthetic glutamic acid derivative, was introduced in the
late fifties and was later abandoned because of its teratogenic effect.
The first report on its use in thalassaemia patients appeared in 2008
where a 21-year old transfusion dependent woman with beta-thalassaemia
major was treated with 100mg/day thalidomide. After 3 months her
haemoglobin levels went up from 2.9g/dL to 7g/dL and her HbF went up
from 62.3\% to almost 100\%. In the paper they reported that she was
given thalidomide uninterrupted and was never transfused again {[}39{]}.
In 2017, a centre in India did a retrospective study on 104 thalassaemic
patients who received thalidomide between January 2006 and April 2016 to
see the effect of thalidomide on ferritin levels. It was found that the
ferritin levels reduced to 51\% in all patients {[}40{]}. In another
prospective study between October 2017 and April 2018, 70 known cases of
transfusion dependent \selectlanguage{greek}β-\selectlanguage{english}thalassaemia major were given thalidomide at a
dose of 2mg/kg to 10mg/kg for 6 months. It was found that thalidomide
increased amelotin levels while it reduced ferritin levels in these
patients {[}41{]}. In another study, a cohort of 37 patients with
symptomatic \selectlanguage{greek}β-\selectlanguage{english}thalassaemia were put on low dose Thalidomide (2-10mg/kg)
and followed for a minimum of 8 months. In non-transfusion dependent
patients, a significant increase in haemoglobin was noted while in
transfusion dependent patients, there was a significant drop in yearly
transfusions {[}42{]}. The risk-benefit of thalidomide still needs to be
established and therefore this drug should be administered in the
context of clinical studies {[}43{]}.

Sirolimus is another potential drug for inducing HbF production in
thalassaemia patients. Sirolimus is a macrolide compound that acts by
selectively blocking the transcriptional activation of cytokines thus
inhibiting cytokine production. It is only bioactive when bound to
immunophilins and they are potent immunosuppressants and possess both
antifungal and antineoplastic properties. Right now, there are two
ongoing clinical trials to test Sirolimus in beta-thalassaemia patients.
The first trial, NCT03877809 started in June 2019 were 20 beta
thalassaemia major patients between the age of 18 and 65 who are on
regular transfusion since at least 6 years were recruited. They were
administered 0.5mg of Sirolimus daily. After 360 days the HbF will be
measured and compared to the HbF before treatment. The second trial,
NCT04247750 started in January 2020 and will recruit 15 beta
thalassaemia major patients between the age of 18 and 65 and these
patients will be administered also 0.5mg of Sirolimus daily.

A number of other non-selective compounds such as cytotoxic compounds,
short-fatty acid derivatives and hypomethylating agents are being
investigated in-vitro to see their effect on foetal haemoglobin.

Gene therapy

Gene therapy for \selectlanguage{greek}β-\selectlanguage{english}thalassaemia is currently based on transplantation of
autologous haemopoietic stem cells (HSCs) genetically modified with
integrating viral vectors expressing the transgene of interest
{[}44{]}.The concept of gene therapy for beta-thalassemia emerged a long
time ago, in 1978 at the University of California at Los Angeles was
included in a plan for the viable treatment for haemoglobinopathies
{[}45{]}. A number of major technical issues were encountered and early
attempts in 1980 to treat beta-thalassaemia patients by inserting the
\selectlanguage{greek}β-\selectlanguage{english}globin gene into the bone marrow cells were completely unsuccessful
and received a lot of criticism {[}46{]}. At that time, the regulatory
globin sequences required for high levels of production and efficient
methods for gene introduction were not available. After 25 years, these
goals were achieved and made possible by the characterization, size
reduction, isolation and amalgamation of the \selectlanguage{greek}β-\selectlanguage{english}globin locus regulatory
elements and the advent of lentiviral vectors which can transfer complex
sequences into hematopoietic stem cells {[}38{]}. Currently, the
gene-therapy approaches can be divided into two broad groups -- (i) gene
addition and (b) gene-editing approaches (figure 1)\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

Figure 1: Schematic representation of gene therapy in beta-thalassemia.
Mutations in the beta globin gene give rise to beta-thalassaemia.
Conventional therapies include transfusion followed by iron chelation
therapy. Autologous transplantation of HSCs genetically modified by gene
addition or gene editing is another therapy for patients affected by
beta-thalassaemia.

Gene-addition

Gene addition involves the insertion of a lenti-viral (LV)/retroviral
vector that contains the whole regulatory and the \selectlanguage{greek}β- \selectlanguage{english}or \selectlanguage{greek}γ-\selectlanguage{english}producing
genets into autologous human stem cells in-vitro and then infusing these
modified stem cells back into the patient. Early gene therapy studies
used recombinant \selectlanguage{greek}γ-\selectlanguage{english}retroviral vectors derived from Moloney Leukemia
Viruses to introduce the functional copy of the \selectlanguage{greek}β-\selectlanguage{english}globin
gene.\emph{In-vitro} , these vectors provided reasonable levels of
\selectlanguage{greek}β-\selectlanguage{english}globin transgene expression but \emph{in vivo} , it resulted in a
limited variable expression {[}47{]}, {[}48{]}, {[}49{]}. These types of
vectors have a number of defects: (i) instability, (ii) limited cargo
capacity and (iii) an inability to transduce non-dividing cells
{[}38{]}. The use of \selectlanguage{greek}γ-\selectlanguage{english}retroviral vectors was abandoned and in the
mid-1990s, lentiviral vectors were derived from human immunodeficiency
virus type 1 (HIV1). These types of lentiviral vectors were able to
transfer much longer sequences and also were able to transduce cells
arrested at the G1-S boundary of the cell cycle {[}50{]}, {[}51{]}.

Several studies have shown that self-inactivating lentiviral carrying
the human \selectlanguage{greek}β \selectlanguage{english}or \selectlanguage{greek}γ-\selectlanguage{english}globin gene and its fundamental regulatory elements are
able to correct the disease in mice with either \selectlanguage{greek}β-\selectlanguage{english}thalassaemia
intermedia or major {[}52{]}, {[}53{]}, {[}54{]}. Several investigators
continued to work hard to improve LV in terms of higher transgene
expression, acting mostly on regulatory transcriptional components and
the addition of insulators to prevent negative chromatin position
effects. These included larger HS2 and HS4 elements of the Locus Control
Region (LCR) and incorporation of insulator elements and inclusion of
HS1 or GATA-1 elements. In most cases, although increased transgenic
expression was achieved this happened at the cost of a reduced
transduction efficiency and vector copy number/cell {[}55{]}. The final
approved LVs for clinical trials were the result of an acceptable
compromise between good manufacturing yield, title and \selectlanguage{greek}β-\selectlanguage{english}globin
expression level {[}44{]}.

The first clinical trial for \selectlanguage{greek}β-\selectlanguage{english}thalassaemia took place in June 2007 in
France, where three patients were treated by gene therapy with the
HPV569 LV expressing the \selectlanguage{greek}β\selectlanguage{english}\textsuperscript{T87TQ} globin variant
{[}38{]}. In this vector, the \selectlanguage{greek}β-\selectlanguage{english}globin gene was under the control of the
LCR and the \selectlanguage{greek}β-\selectlanguage{english}globin promoter and was surrounded by tandem copies of the
cHS4 core element {[}56{]}. The first patient to be treated was an
18-year-old \selectlanguage{greek}β\selectlanguage{english}\textsuperscript{E}/\selectlanguage{greek}β\selectlanguage{english}\textsuperscript{0} who had been
transfusion dependent and on parenteral iron chelation since early
childhood. Bone marrow cells were processed for CD34+ selection and
transduced with the HPV569 LC. After busulfan conditioning the patient
received an infusion of 3.9 x 10\textsuperscript{6} cells/kg. After one
year, the patient became transfusion independent and his haemoglobin was
stable at about 8-9 g/dl after 7 years from gene therapy {[}57{]},
{[}58{]}, {[}59{]}. Integration site analysis revealed expansion of a
single clone in which the provirus was inserted at the high mobility
group AT-hook 2. This benign clonal dominance persisted for 9 years
after which it started to decline. In 2011, another patient underwent
transplantation but the vector copy number was lower and this patient is
still transfusion dependent {[}59{]}.

To achieve higher transduction efficacy the original LV was further
improved. This was done by the removal of the cHS4 from HPV569 LV and
this yielded

The HGB-204 clinical trial started in 2012 and completed in February
2018. Data from up to 5 years of follow-up from the completed Phase 1/2
as of December, 2019 (ASH, 2019) it was shown that 8 out of 10 treated
patients who did not have a \selectlanguage{greek}β\selectlanguage{english}0/\selectlanguage{greek}β\selectlanguage{english}0 genotype achieved and continued to
maintain transfusion independency for up to 51.3 months with a median Hb
of 10.3 g/dL. In the other two patients, who did not achieve transfusion
independency, the transfusion volumes were reduced by 79\% and 52\%.
Three out of 8 patients that were \selectlanguage{greek}β\selectlanguage{english}0/\selectlanguage{greek}β\selectlanguage{english}0 achieved and continued to
maintain transfusion independence up to 30.4 months with a median Hb of
9.9g/dL {[}61{]}.

The clinical trial HGB-207 started in July 2016 and it is estimated to
be completed in February 2022. Recruitment is completed, and 23
beta-thalassaemia transfusion dependent who are not \selectlanguage{greek}β\selectlanguage{english}0/\selectlanguage{greek}β\selectlanguage{english}0 genotype
between the age of 4 and 34 were transfected with BB305 Lenti-globin
after myeloablative conditioning with busulfan. As of June 2020 (EHA,
2020) 19 out of the 23 patients were evaluable for transfusion
independence while the other 4 do not have sufficient follow up.
Seventeen out of the 19 evaluable patients, achieved transfusion
independence with median Hb levels of 11.9g/dL. Improved iron levels
were also observed while over half of the patients stopped chelating
therapy {[}62{]}.

The other clinical trial using the BB305 Lenti-globin is HGB-212 which
started in June 2017 and it is estimated to be completed by June 2022.
As of June 2020 (EHA, 2020) 15 patients with different beta-genotypes
were treated and had a median follow up of 14.4 months. Nine out of the
15 were \selectlanguage{greek}β\selectlanguage{english}0/\selectlanguage{greek}β\selectlanguage{english}0, 3 were \selectlanguage{greek}β\selectlanguage{english}0/\selectlanguage{greek}β\selectlanguage{english}+IVS1-110 and 3 IVS1-110 homozygous. Six of
eight evaluable patients achieved transfusion independence with median
Hb of 11.5 g/dL. 11 out of 13 patients with at least 7 months of follow
up did not receive a transfusion {[}62{]}.

In 2012, a trial opened at MSKCC in New York (NCT01639690) where four
beta-thalassaemia transfusion dependent patients were treated with gene
therapy using the TNS9.3.55 LV. This protocol differs from the other
clinical trials with the BB305 LV because in this trial a reduced
intensity conditioning was used rather than a fully myeloablative
regimen. This led to insufficient gene marking with minimal clinical
benefit {[}63{]}, {[}64{]}. In 2015, the clinical trial NCT02453477
(TIGET BTHAL) started in Italy and it was completed in August 2019. Nine
patients with different genotypes (\selectlanguage{greek}β\selectlanguage{english}0/\selectlanguage{greek}β\selectlanguage{english}0, \selectlanguage{greek}β\selectlanguage{english}+/\selectlanguage{greek}β\selectlanguage{english}+ and \selectlanguage{greek}β\selectlanguage{english}0/\selectlanguage{greek}β\selectlanguage{english}+) 3 adults
followed by 6 minors were treated. This clinical trial was based on the
autologous transplantation of G-CSF and plerixafor mobilized HSCs
engineered by the GLOBE lentiviral vector. Following myeloablative
conditioning by treosulfan and thiotepa the transduced cells were
infused by intraosseous injection. As for April 2019, the three adult
patients had reduction of transfusion requirement while 4 paediatric
patients are transfusion independent and two are still receiving blood
transfusions {[}65{]}.

The main goal of gene therapy in beta-thalassaemia is to achieve stable
introduction of functional globin genes in the patient's own HSCs in
order to abolish transfusions by correcting ineffective erythropoiesis
and haemolytic anaemia. This can be achieved by taking into
consideration a number of critical issues such:

Source of haematopoietic stem/progenitor cells

Nowadays, the preferred source for many autologous and allogenic
transplantation approaches is peripheral blood mobilized HSCs. When
compared to conventional bone marrow harvest, this minimally invasive
procedure provides several-fold higher numbers of HSCs {[}66{]},
{[}67{]}. In adult patients, HSC procurement is critical since the
minimal target dose of 2-3 x10\textsuperscript{6}
CD\textsuperscript{34}cells/kg poses a challenge for steady-state
bone-marrow. Also, favourable gene therapy results are correlated with
the dose of transfused cells infused and engrafted {[}44{]}. The growth
factor granulocyte-colony stimulating factor (G-CSF), was the standard
agent used to mobilize HSCs for transplantation. This stimulating
factor, results in rapid mobilization within hours following
administration {[}68{]}. Although it is known to be well-tolerated it
raised concerns for its safety in thalassaemia due to the rare events of
splenic rupture or thrombosis during mobilization in normal donors and
in patients with haematological malignancies {[}69{]}, {[}70{]},
{[}71{]}.

Plerixafor (AMD3100, Mozbil\textsuperscript{TM}) a bicyclam molecule
that mobilizes HSCs by selectively and reversibly antagonizing the
binding of stromal cell derived factor-1 to chemokine CXC receptor-4
(CXCR4) is considered as an alternative mobilization agent {[}72{]}. In
conditions resulting in poor mobilization, this agent is approved by FDA
and European Medical Agency (EMA) to be used together with G-CSF
{[}73{]}. A number of mobilization trials took place to define the
optimal mobilization approach for adult patients with beta-thalassaemia
before implementation of gene therapy. From these trials it was
established that the optimal mobilization approach for these patients is
either Plerixafor alone or in combination with G-CSF {[}74{]}, {[}75{]},
{[}76{]}, {[}77{]}.

Dose of transduced HSCs and transduction methods

Two important factors that might affect gene therapy outcome are the
transduced cell dose as well as the stem cell source. Determining the
dose of the optimal genetically modified cells to transduce still
remains unclear. Based on the results available until now from clinical
trials we still do not have a direct correlation between transduced cell
dose and clinical outcome. In clinical trials, beta-thalassaemia
patients have been treated with different cell doses and the vector/copy
number in drug products is also variable. Other different variables such
as severity of genotype/phenotype, comorbidities, secondary modifiers of
the pathology, proportion of engrafting genetically modified long-term
analysis may also influence the clinical trial. From clinical trials,
preliminary indications show that patients that receive the highest dose
of transduce with the highest vector/copy number should have the better
outcome {[}44{]}.

A near-complete transduction of the purified HSCs with LV can be
achieved by adding Prostaglandin
E\textsubscript{2}(PGE\textsubscript{2}). In less than 38 hours, it was
shown that PGE\textsubscript{2} allows near-complete transduction of
HSCs with LV {[}78{]}. \emph{In vitro} , the addition of PGE2 was shown
to increase VCN and/or transduction efficiency of
CD34\textsuperscript{+} cells {[}79{]}. When compared to control
transduced cells, addition of PGE2 increased vector transduction of
CD34\textsuperscript{+} cells approximately to 2-fold {[}80{]}. Addition
of rapamycin which is an inhibitor of the mammalian mTOR pathway also
resulted in significantly enhanced transduction without alterations in
lentiviral integration profile {[}81{]},{[}82{]}.

Bone-marrow conditioning and HSC administration

Before administration of transduced HSCs, conditioning of the bone
marrow must take place. Since beta-thalassaemia is a non-malignant
disease, it requires complete elimination of endogenous HSCs to confer
therapy. Initially, it was discussed whether a partial or full
myeloablation is needed. Non-myeloablation treatment is preferred to
conventional fully myeloablation due to the high risk of
non-haematological toxicity but it can lead to mixed chimerism. Also,
despite the encouraging engraftment rates in mouse-models achieved under
non-myeloablative settings with various vectors, this preparative
regimen could not be used in vivo {[}83{]}, {[}84{]}, {[}85{]}. Most of
the clinical trials for gene-therapy in beta-thalassaemia nowadays, are
using reduced intensity conditioning with Busulfan. The MSKCC trial used
Busulfan at 8mg/kg while the other clinical trials with the either
HPV569 LV or BB305 LV used Busulfan 12.8mg/kg. The clinical trial of
TIGET-BTHAL conditioning was done by Treosulfan
42g/m\textsuperscript{2}and Thiotepa 8mg/kg.

Following myeloablative treatment, administration of the HSCs is usually
given by intravenous injection. The systematic delivery via peripheral
circulation is an easy procedure for the dissemination of transplanted
cells that home and engraft in the bone marrow niche. On the other hand,
one of the drawbacks of this administration, is the loss of a
significant proportion of injected cells. These cells are lost due to
the major filter organs trapping such as the lungs and spleen. The TIGET
BTAL clinical trial used an alternative route of administration to
overcome cell loss. In this clinical trial the transduced cells were
delivered by intraosseous injection bilaterally in the iliac crests
{[}44{]}.

Gene editing

An innovative approach for treating \selectlanguage{greek}β-\selectlanguage{english}thalassaemia is gene editing. In
the last decades, several nucleases such as zinc-finger nucleases
(ZFNs), transcription-activator-like effector nucleases (TALENs) were
developed for genetic engineering with CRISRP/CAS9 system being the most
novel approach. The CRISPR/Cas9 system uses a single or multiple short
guide RNAs (gRNA) with 20bp complementary to DNA sequence target. The
nucleases will than create site specific double strand breaks which can
be repaired either by non-homologous end joining (NHEJ) or by homologous
directed repair (HDR) {[}86{]}, {[}87{]}.

\emph{In-vitro} , in the context of \selectlanguage{greek}β-\selectlanguage{english}haemoglobinopathies a number of
gene editing strategies have been successful. These include correction
of the \selectlanguage{greek}β-\selectlanguage{english}globin gene mutation {[}88{]}, {[}89{]}, {[}90{]} or induction
of endogenous foetal haemoglobin {[}91{]}. A recent study used the
CRISPR/Cas9 system to mutate a 13-nucleotide sequence in the promoters
of the gamma genes. This was done via microhomoglogy-mediated end
joining leading to HbF increase to potentially therapeutic levels
{[}92{]}. Although many labs have been successfully performing gene
editing of immortalized cells, gene editing of primary cells remains a
challenge due to toxicity caused by gene-editing reagents, off-target
mutations and low efficiency of transfection {[}93{]}.

In the past 5 years, gene correction of patient-specific induced
pluripotent stem cells (iPSCs) by CRISPR/CAS9 and cell transplantation
has appeared as a promising therapeutic solution for many diseases
including \selectlanguage{greek}β-\selectlanguage{english}thalassemia {[}94{]}, {[}95{]}. In 2019, the group of Bauer
at the Dana-Farber/Boston Children's Cancer and Blood Disorders Centre
at the University of Massachuseets Medical School edited
CD34\textsuperscript{+} HSPCs from seven patients with \selectlanguage{greek}β-\selectlanguage{english}thalassaemia of
varying genotypes using CRISPR-CAS9 gene editing. After overcoming
technical challenges associated with editing of blood stem cells they
have demonstrated that CAS9:single guide RNA ribonucleoprotein
(RNP)-mediated cleavage within a GATA1 binding site at the +58 BCL11A
erythroid enhancer resulted in a highly penetrant disruption of this
motif. The reduced BCL11A expression gave rise to an increase in
gamma-globin levels between 35.3 and 75.1\% and an increase in gamma
protein levels between 27.5 and 46.9\% {[}96{]}. In the context of
beta-thalassaemia two clinical trials one utilizing a CRISPR/CAS9 system
and one implementing ZFN are currently in the recruitment phase. The
clinical trial NCT03655678 is a phase 1/2 study of the safety and
efficacy of a single dose of autologous CRISPR-Cas9 Modified CD34+ HPSPc
in 45 subjects with Transfusion-Dependent beta thalassaemia. The other
clinical trial, NCT03432364, will assess ST-400 in 6 subjects with
transfusion-dependent beta-thalassaemia. ST-100 will be composed of the
patient's own blood stem cells which are genetically modified in the lab
using Sangamo's zinc finger nuclease technology to disrupt a precise and
specific sequence of the enhancer of the BCL11A.

As can be seen from this review, \selectlanguage{greek}β-\selectlanguage{english}thalassaemia has offered a very
robust model on which novel therapies and treatments can be explored
with a great impact that holds promise on a clinical level. In
conclusion, whether an approach to re-induce high levels of HbF in vivo,
or a gene editing approach that amount to gene replacement or correction
it is now envisaged that such activities are all fruitful approaches to
augment HbF levels in individuals with \selectlanguage{greek}β-\selectlanguage{english}type haemoglobinopathies.

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