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 involves the insertion of a lenti-viral (LV)/retroviral
vector that contains the whole regulatory and the β- or γ-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 γ-retroviral vectors derived from Moloney Leukemia
Viruses to introduce the functional copy of the β-globin gene.In-vitro , these vectors provided reasonable levels of β-globin
transgene expression but 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 γ-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 β or γ-globin gene and its fundamental regulatory elements are
able to correct the disease in mice with either β-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 β-globin
expression level [44].
The first clinical trial for β-thalassaemia took place in June 2007 in
France, where three patients were treated by gene therapy with the
HPV569 LV expressing the βT87TQ globin variant
[38]. In this vector, the β-globin gene was under the control of the
LCR and the β-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 βE/β0 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 106 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 β0/β0 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 β0/β0 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 β0/β0 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 β0/β0, 3 were β0/β+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 (β0/β0, β+/β+ and β0/β+) 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:
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 x106 CD34cells/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].
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 E2(PGE2). In less than 38 hours, it was shown that
PGE2 allows near-complete transduction of HSCs with LV
[78]. In vitro , the addition of PGE2 was shown to increase
VCN and/or transduction efficiency of CD34+ cells
[79]. When compared to control transduced cells, addition of PGE2
increased vector transduction of CD34+ 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].
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/m2and 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].
An innovative approach for treating β-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].
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 β-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+ HSPCs from seven patients with β-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, β-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 β-type haemoglobinopathies.
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