Received: 10 October 2023
Revised: 17 January 2024
Accepted: 18 January 2024
DOI: 10.1111/ejh.14179
REVIEW
Systematic review of hematopoietic stem cell gene
therapy approach in thalassemia: Comparative analysis
in animal models
Indira Laksmi Maharani 1
|
1
Rakean Ahmad Kiansantang
Rizqi Najla Humaira 1
|
1
Undergraduate Medical Program, Faculty of
Medicine Universitas Indonesia, Jakarta,
Indonesia
Muhammad Hafizh Zauhari 1
| Razzan Satria Wibowo 1
Adisti Dwijayanti 2
|
|
| Imelda Rosalyn Sianipar 3,4
Abstract
Hematopoietic stem cell (HSC) gene therapy has shown potential as a therapeutic
2
Department of Medical Pharmacy, Faculty of
Medicine, Universitas Indonesia, Jakarta,
Indonesia
3
approach for thalassemia in recent years. However, a comparison of the varying gene
therapy methods of HSC gene therapy in thalassemia has never been reviewed. This
Department of Medical Physiology and
Biophysics, Faculty of Medicine, Universitas
Indonesia, Jakarta, Indonesia
study aims to evaluate the utilization of HSC gene therapy approaches in animal
4
PubMed, EBSCOHost, Science Direct, SCOPUS, and Proquest using a combination of
Stem Cell and Tissue Engineering Cluster, The
Indonesian Medical Education and Research
Institute (IMERI), Faculty of Medicine,
Universitas Indonesia, Jakarta, Indonesia
Correspondence
Indira Laksmi Maharani, Undergraduate
Medical Program, Faculty of Medicine
Universitas Indonesia, Jl. Salemba Raya
No. 6, Jakarta 10430, Indonesia.
Email: indira.laksmi01@ui.ac.id
models of thalassemia. A systematic review was conducted in five databases:
the terms hematopoietic stem cell or hematopoietic stem cell or HSC, thalassemia,
genetic therapy or gene therapy and animal model. Only journals published in English
between 2008 and 2023 were included. This literature included six studies analyzing
the use of HSC gene therapy in thalassemic mice models. The three outcomes being
assessed in this review were globin levels, hematological parameters, and red blood
cell (RBC) phenotypes. Gene therapy approaches for thalassemia using HSC showed
significant improvement in β-globin levels and RBC phenotypes. Phenotypic improvements were also observed. These outcomes indicate good efficacy in gene therapy
for thalassemia in mice models. Furthermore, more studies assessing the efficacy of
HSC gene therapy in the human model should be done in future studies.
KEYWORDS
animal model, gene therapy, hematopoietic stem cell, in vivo, thalassemia
Novelty Statement
What is the new aspect of your work?
This systematic review is the first to comprehensively compare and analyze hematopoietic stem
cell gene therapy for thalassemia in animal models.
What is the central finding of your work?
Among all types of interventions, positive outcomes were found.
What is (or could be) the specific clinical relevance of your work?
This review could be a basis for future clinical trials in humans.
848
© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
wileyonlinelibrary.com/journal/ejh
Eur J Haematol. 2024;112:848–859.
1
I N T RO DU CT I O N
|
2.2
|
Study selection
Thalassemia, a group of inherited blood disorders characterized by
Five reviewers were responsible for selecting studies to include in the
defective hemoglobin (Hb) production, remains a significant global
review. Each author made their decisions independently without
health concern, particularly in regions with a high prevalence of con-
knowing the choices of others. In case of any disagreements among
sanguineous marriages. While advancements in blood transfusion and
the reviewers, they reached a consensus through discussion. The
iron chelation therapies have improved the quality of life for individ-
information that will be extracted includes; The design of the study,
uals with thalassemia, they are far from curative.1,2 Various methods
the size of the sample, the specific mouse strain used, detailed charac-
have been studied as potential alternatives for treating thalassemia,
teristics of the mice, the type and timing of gene therapy employed,
with gene therapy being one of them. Gene therapy has emerged as a
details regarding the intervention itself, and the measured outcomes
promising solution for various diseases, including thalassemia, due to
including levels of β-globin, parameters, and phenotypic data. For each
its capacity to address the genetic roots of the condition. Different
group mean or median values should be included in the outcome data.
techniques, such as plasmid DNA, viral vectors, bacterial vectors, and
Table 1 will display the characteristics of all the studies included
even direct human gene editing, are employed in gene therapy.3
in this review. The data extraction process will be independently car-
These variations allow healthcare professionals and patients to seek
ried out by all five reviewers, with each reviewer being uninformed
the most suitable therapy for thalassemia.
about the findings of others. Additionally, three more reviewers
Hematopoietic stem cell (HSC) gene therapy, a promising therapeutic approach, has gained momentum in recent years as a potential
assessed biases using the Syrcle Risk of Bias Tool for Animal Studies
and resolved any disagreements through consensus discussions.
curative strategy for thalassemia. Many approaches have been done
to find suitable treatments using HSC gene therapy, such as transplantation of genetically modified HSC. One prime groundbreaking exam-
2.3
|
Data collection
ple is Zynteglo, HSC therapy developed for β-thalassemia treatment.4
A review for HSC gene therapy with transplantation is already avail-
The title and abstract of articles were screened by five authors. The
able with promising results, such as restoration of human erythropoie-
full text deemed to meet the inclusion criteria was retrieved by these
sis after transplantation.5 Other approaches than transplantation vary
reviewers. Disagreements were discussed. For included articles, study
in methods and vectors used. Here, we further investigate and com-
details regarding the first author, year of publication, study design,
pare other approaches in HSC gene therapy, for instance through
sample size, population, intervention, and outcome were extracted.
injection.
The key findings from each study were summarized in Table 2. Risk of
This systematic review aims to comprehensively assess and compare the outcomes of HSC gene therapy in animal models of thalasse-
bias was assessed using the Syrcle Risk of Bias Assessment. Search
flow of the literature can be seen in Figure 1.
mia. By critically analyzing the existing literature, this review seeks to
provide a comprehensive overview of the current state of research,
identify trends, and offer insights into the potential translational rele-
3
RE SU LT S
|
vance of HSC gene therapy for thalassemia patients. This comparative
analysis will contribute to our understanding of the efficacy and safety
3.1
|
Study characteristics
of this innovative approach, facilitating informed decision-making in
the pursuit of effective thalassemia treatments. Hopefully, by success-
This literature contains six studies with mice as the population of the
fully adapting suitable treatments in animal models, we can further
studies. All studies use two different types of mice, control mice and
take steps into clinical trials in humans.
thalassemia model mice. Two of the studies use female model mice,6,7
one study consists of male mice,8 one study uses both sexes,9
whereas two others have not stated the sexes.10,11 50% of control
2
METHODS
|
mice in the studies use C57BL/6 strain,8,10,11 while both of Wang
et al.6,7 studies use CD46tg strain. Bahal et al.9 use β-globin/green
2.1
|
Eligibility criteria and risk of bias
fluorescent protein fusion transgenic mice as their control mice. Using
different types of HSC sources, all of the studies also use different
Data for this systematic review were identified from PubMed, EBS-
types of gene therapy. Two out of six studies use CD34+ human
COHost, Science Direct, SCOPUS, and Proquest in September 2023.
HSCs.9,11 Characteristics of gene therapy frequencies stated two
Reference screening was also done to identify other additional rele-
studies injected the mice once every 7 days,7,10 one injected once
vant papers. Only journals published in English between 2008 and
after a week of feeding,8 whereas the other injected once every 48 h
2023 were included. The search terms used are a combination of
and 5 weeks later.6,9,11 All of these in vivo studies use injection for
hematopoietic stem cell or hematopoietic stem cell or HSC, thalasse-
their intervention by injecting the gene therapy at the mice.
mia, genetic therapy or gene therapy, and animal model. We did not
For instance, the outcomes are compared in three different outcomes:
contact the study authors.
β-globin levels, hematological parameters, and phenotype. All of these
16000609, 2024, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ejh.14179, Wiley Online Library on [31/03/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
849
MAHARANI ET AL.
2020
2019
2016
Wang
et al.
Bahal
et al.
Publication
year
In vivo correction
of anemia in
β-thalassemic
mice by γPNAmediated gene
editing with
nanoparticle
delivery
Each group consists β-globin/green
fluorescent
of 6 mice
protein (GFP)
(6 4 = 24
fusion
mice)
transgene mice
CD46tg mice
In vivo
hematopoietic
stem cell gene
therapy
ameliorates
murine
thalassemia
intermedia
Not stated
Control
CD46tg mice
Sample size
Mice strain
Not stated
Curative in vivo
hematopoietic
stem cell gene
therapy of
murine
thalassemia using
large regulatory
elements
Title
Characteristics of the included studies.
Mice containing
human b-globin
gene and
b-thalassemiaassociated
splicing mutation
at IVS2-654
Gene therapy
frequency
Male and female,
8 weeks old
CD34+ human
HSCs.
Stem cell factor
(SCF) and
gtcPNA4/donor
DNA
nanoparticles
(NPs)
Each mouse
received four
treatments given
at 48 h intervals
Hematopoietic
A 7-day
stem/progenitor
mobilization
cells (HSPC) in
approach with
vivo transduction
G-CSF 250 μg/
with HDAd-γkg i.p. (1–6 days)
globin/mgmt plus
and plerixafor
HDAd-SB followed
5 mg/kg i.p.
by in vivo
selection in
CD46+/+/
Hbbth-3 mice
Injected then in
Granulocyte-colony HDAd5/35++
vivo selection
vector containing
stimulating factor
5 weeks later
short and long
(G-CSF)β-globin locus
mobilized adult
control region
donors
(LCR)
Gene therapy type
C57BL/6 mice:
• 6–8 weeks old
• Female
• Irradiated
with 10 Gy
CD46+/+/Hbbth-3 Six- to 10-week-old Bone marrow
female CD46tg
mouse model
and CD46+/+/
expressing
Hbbth-3 females
human CD46
were used for
the in vivo
transduction/
selection studies.
Six- to tenweek-old female
C57BL/6 mice
were used as
secondary
recipients
Hbbth3 (B6.
D2-Hbbd3th/
BrkJ)
Thalassemia model
Hematopoietic
stem cell (HSC)
source
Mice
characteristics
(age, sex)
220 mg/kg of SCF
followed by a
single treatment
of 4 mg of NPs
injected
intravenously.
Then received
either blank NPs,
NPs containing
tcPNA1 and
donor DNA, or
NPs containing
gtcPNA4 and
donor DNA, with
or without SCF
IV injection of
HDAd5/35++
vectors of HSC
gene therapy
Injection of
HDAd5/35++
vector containing
short and long
β-globin LCR
with G-CSF/
AMD3100
mobilization
Intervention
MAHARANI ET AL.
16000609, 2024, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ejh.14179, Wiley Online Library on [31/03/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Wang
et al.
Author
TABLE 1
850
2010
2022
2011
Ma
et al.
Miccio
et al.
Publication
year
Control
C67BL/6 J
Sample size
Not stated
Mice strain
The GATA1-HS2
enhancer allows
persistent and
positionindependent
expression of a
β-globin
transgene
Mice were
C57BL/6 mice
transduced with
with mockeither the CMVtransduced
GFP or the
thalassemic
G-CMV-GFP
cells
vector and
transplanted in
lethally-irradiated
CD45.2
coisogenic mice
(C57BL/6; n = 9
and n = 8,
respectively)
C57BL/6 mice
control group
Reactivation of γ(n = 6), pMC.
globin expression
BESPXusing a minicircle
cytomegalovirus
DNA system to
(CMV)-SEAP (PPtreat
SEAP) group
β-thalassemia
(n = 5), and
minicircle-SEAP
(MC-SEAP) group
(n = 5).
Mobilization of
hematopoietic
stem cells in a
thalassemic
mouse model:
implications for
human gene
therapy of
thalassemia
Title
(Continued)
Male, 5 weeks old
8–12 weeks old,
sex not stated
Eight-week-old, sex
C57BL/6 mice
not stated
transplanted with
G-CMVGFP-transduced
HSCs
C57BL/6 mice
HBBth-3/HBBth-3
Thalassemia model
Mice
characteristics
(age, sex)
Human CD34+
cells were
obtained from
umbilical cord
blood of healthy
donors on
collection with
written informed
consent in
accordance with
the Declaration
of Helsinki
Human chronic
myelogenous
leukemia cell line
K562
None
Hematopoietic
stem cell (HSC)
source
Lentiviral vectors
(LV)-mediated
gene therapy
approaches
SaCas9
Recombinant
human G-CSF
Gene therapy type
Not stated
Once after 1 week
of feeding
Once a day for
7 days
Gene therapy
frequency
Intervention
Bone marrow (BM)
cells were
harvested from
C57BL/6-Ly-5.1
mice (B/6.SJLCD45a-Pep3b,
Jackson
Laboratories),
infected with
CMV-GFP and
G-CMV-GFP
vectors, and
injected into
8-week-old
C57BL/6 mice
pMC.BESPX-CMVSEAP (PP-SEAP)
or minicircleSEAP injection
Intraperitoneal
injection of
recombinant
human G-CSF
(250 μL, once a
day, 7 days)
851
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Yannaki
et al.
Author
TABLE 1
MAHARANI ET AL.
2020
2019
Author
Wang
et al.
Wang
et al.
Reticulocytes
Intervention
Hematocrit (HCT)
Intervention
13.9
9.7 ± 0.18 g/dL
± 0.63 g/dL
Control
7.1 ± 0.1
In vivo
Level of γ-globin 8.53
in comparison
± 0.29 M/
M/μL
hematopoietic
with adult
μL
stem cell gene
mouse α- and
therapy
β-globin: 10%–
ameliorates
15% of adult
murine
mouse
thalassemia
α-globin and
intermedia
β-globin; 25%
mouse β-minor
globin
Intervention
Control and
Long: higher
short: lower
Control
Control
Intervention
11.8% ± 3.7%
Before transd:
42.4%
± 1.43%
After transd:
31.13%
± 3.17%
41.7%
± 1.48%
30.7%
± 0.46%
Intervention
Normocytic
Safety precautions
Not stated
Hypochromia,
Safety of in vivo
anisopoikilocytosis
HSPC
(widely varying
transduction with
sizes and shapes),
HDAd-γ-globin/
and cell
mgmt
fragmentation
Plus HDAd-SB
followed by
O6-BG/BCNU in
vivo selection. No
overt
hematological
abnormalities
were observed.
6 weeks after the
last O6GB/BCNU
dose, all
hematological
values were within
normal ranges,
although the total
Normal bone LONG
marrow
• Normochromic,
cellular
normal shaped
distribution
• Maturing
All stages of
polychromatic
erythroid
and
differentiation
orthochromatic
are
erythroblasts
represented
Control
Peripheral
Peripheral
50%–65% Short: 40%– •
blood
blood
60%
reticulocyte:
reticulocyte:
Long: 50%–
40.9%
Short: 26.8%
65%
Long: 9.2%
•
Control
Red cell morphology
Hemoglobin (Hb)
RBC numbers
Curative in vivo HDAd-long-LCR
8–10 M/μL Short: 7–
hematopoietic • Avg 40%
9 M/μL
stem cell gene
β-globin
Long: 8–
therapy of
marking in
10 M/μL
murine
peripheral
thalassemia
RBC after 2nd
using large
cycle of in
regulatory
vivo selection
elements
• >90% in 9 out
of 10 after
3rd cycle of in
vivo selection
• 100% in
week 12
HDAd-short-LCR:
100% β-globin
marking in
RBC after 4th
cycle of in vivo
selection
Title
Outcome 1:
β-globin levels
Outcome 3: phenotype
Outcome 2: hematological parameters
Characteristics of the included studies.
Publication
Year
TABLE 2
16000609, 2024, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ejh.14179, Wiley Online Library on [31/03/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Bahal
et al.
Yannaki 2010
et al.
2016
Author
Mobilization of
not stated
hematopoietic
stem cells in a
thalassemic
mouse model:
implications
for human
gene therapy
of thalassemia
Intervention
Hematocrit (HCT)
Control
Not stated
Not stated
Not stated
Intervention
Control
Not stated
Increase in
Not stated
blank NPs
but decrease
at γtcPNA4/
donor DNA
NPs plus
SCF:
Intervention:
Day 0: 20%
Day 36: 2%
3.625 ± 1.68% 34.98%
± 14.67%
8,5 g/dL
Much highter
(γtcPNA4-Scr/
in blank NPs
donor DNA+
compared
SCF) and
with
12 g/dL
γtcPNA4/
(γtcPNA4/
donor DNA
donor DNA+
NPs plus
SCF) on Day
SCF:
140
Blank:
Day 0: 26%
Day 36: 30%
Intervention
7.21 ± 1.84 g/dL
12.01
± 2.24 g/dL
7,5 g/dL on
Day 140
Control
Control
Not stated
Not stated
Intervention
Not stated
Normocytic
Control
Not stated
The untreated group
Exhibit extreme
poikilocytosis as
well as numerous
target cells, cabot
rings,
anisochromasia
and ovalocytosis,
all changes
characteristic of
b-thalassemia
With gtcPNA4/donor
DNA and SCF
Ameliorates the
poikilocytosis and
yields a reduction
in anisocytosis,
ovalocytosis and
target cells
suggestive of
reduced α-globin
precipitation in
the RBCs
Intervention
Red cell morphology
Reticulocytes
RBC numbers
Hemoglobin (Hb)
Outcome 3: phenotype
Outcome 2: hematological parameters
3.4% of
Not stated
b-globin gene
alleles
showed the
introduced
mutation at
position
IVS2-654
Outcome 1:
β-globin levels
In vivo correction •
of anemia in
β-thalassemic
mice by
γPNAmediated
gene editing
with
nanoparticle
delivery
Title
(Continued)
Publication
Year
TABLE 2
(Continues)
• No death
• No splenic
rupture
• 65% splenic
enlargement of
G-CSF-treated
thalassemic mice
• 19,7% liver
enlargement of
G-CSF-treated
thalassemic mice
• Hemorrhagic
infarct on 12,5%
of G-CSF-treated
thalassemic mice
Not stated
WBC counts were
lower compared
with levels before
in vivo selection,
suggesting a
cytoreductive
effect of drug
treatment on
WBCs—in
particular
Safety precautions
16000609, 2024, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ejh.14179, Wiley Online Library on [31/03/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Not stated
Tables 1 and Table 2 respectively.
Not stated
Safety precautions
mia model mice. The characteristics and key findings are given in
Not stated
Intervention
Not stated
done by Yannaki et al. All of the findings on β-globin levels showed
improvement in the levels after intervention. The study by Wang
Not stated
cio et al.11 showed improvement of HbF expression in thalassemia
mice model and correction of the phenotype using low β-globin levels.
45%
Other studies showed the mutation of the β-globin gene alleles and
improvement of β-globin levels exceeds more levels than α-globin
levels.7,9,10
30%
Secondary findings of the outcome, hematological parameters,
12%–13%
Control
Not stated
Not stated
Intervention
Control
Not stated
Not stated
Intervention
β-globin levels in both measurements. Studies by Ma et al.8 and Mic-
were measured in all studies,6,7,9–11 except the study done by Ma
et al. red blood cell (RBC) numbers in three out of six studies showed
control mice had higher RBC numbers than mice with intervention.
The other three studies did not measure RBC numbers.6,7,11 Hb and
reticulocyte measurement showed two different outcomes in four
25%
Not stated
Yannaki et al. found control mice to have higher Hb levels. Hb levels
alter reticulocytes with opposite results. Hematocrit (HCT) levels are
Not stated
8–
Not stated
10 106/
μL
6 106/μL
The GATA1-HS2 The GATA1-HS2
enhancer
element
allows
improves
persistent and
correction of
positionthe murine
independent
b-thalassemia
expression of
phenotype by
a β-globin
low copy
transgene
number of a
b-globin
vector
et al. both in control and HDAd-long-LCR mice have the same results
(50%–60%),
whereas
HDAd-short-LCR
mice
have
lower
results (40%–60%). Other studies showed that control mice have
higher HCT than intervention mice.
The morphology of RBC is shown in only three studies. Three of
them stated that treated groups had normocytic and normal phenotype,
while mice with controls and untreated groups had abnormality which is
polychromatic, ovalocytosis, anisochromia, and fragmentation of the cell.
The three of them showed improvement of RBC morphology.5,6,8
4
4.1
DI SCU SSION
|
|
Globin level
The gene therapy approach to thalassemia aims to achieve high β-/γglobin expression levels. Studies included in this article reviewed the
utilization of gene therapy in reaching γ or β-globin expression levels
in thalassemia. Several studies found that γ-globin had stable expression in thalassemic mice after being injected with HDAd vectors con-
2011
taining globin genes. Studies by Wang et al.6,7 showed an increase of
Miccio
et al.
Control
Intervention
Not stated
Not stated
Control
Intervention
Not stated
HbF expression
Not stated
was
significantly
increased in
the MCSaCas9-sgRNA
group
compared with
the MCSaCas9 vector
group
Control
Title
Author
Publication
Year
lower Hb levels than mice with intervention, whereas Wang et al. and
only found in two studies which both give different results. Wang
Reactivation of
γ-globin
expression
using a
minicircle
DNA system
to treat
β-thalassemia
Reticulocytes
Hemoglobin (Hb)
RBC numbers
β-globin levels are counted in every study,6–9,11 except one study
studies. Both Wang et al. and Bahal et al. control mice models had
Outcome 1:
β-globin levels
Outcome 2: hematological parameters
(Continued)
TABLE 2
Study findings
|
et al.6 measuring HDAd-long-LCR and HDAd-short-LCR showed
Ma et al. 2022
Red cell morphology
Hematocrit (HCT)
Outcome 3: phenotype
3.2
γ-globin expression to >80% in RBCs in thalassemic mice after three
cycles of treatment with HDAd.6,7
HDAd gene transfer vector systems include features that
allow for the transduction of HSCs, mediate gene integration,
16000609, 2024, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ejh.14179, Wiley Online Library on [31/03/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
findings have a different outcome in control model mice and thalasse-
FIGURE 1
chart.
PRISMA flow
and incorporate globin genes. A study by Wang et al. 6 showed
increased the expression of human β-globin in the splenic erythroid
a significantly better result of β-globin expression in HDAd
cells of thalassemic mice. When compared with the lentiviral vector
vector containing long locus region than HDAd containing
with no GATA1-HS2 element (GLOBE), G-GLOBE showed a signifi-
short locus region. The longer locus region might prevent
cant increase in transgene expression. Fluorescence-Activated Cell
silencing and initiate transcription of the incorporated gene in
Sorting (FACS) analysis also revealed β-globin synthesis occurred in
the HDAd vector.
half of the colonies. This study showed the use of GATA1-HS2 could
Though the increasing percentage of γ-globin expression after
increase transgene expression in gene therapy using lentiviral vectors.
treatment indicates promising efficacy, it should be noted that the
However, this study did not analyze the overall globin levels in
level of γ-globin expression only achieved 10%–15% of adult
RBCs.11
mouse α- and β-globin expression.6,7 Therefore, patients with major
Current studies on gene therapy approach in thalassemia
thalassemia may require higher levels of γ-globin than current
showed that this model of therapy required the utilization
findings.
of vectors that could consistently express elevated levels of
Another study by Miccio et al. used a lentiviral vector containing
β-globin.12,13 The use of longer version of locus region and
β-globin gene. The study analyzed the ability of GATA1-HS2, an tran-
enhancer could increase the probability to express potentially cura-
scriptional activator element inserted into the lentiviral vector, to
tive amounts of globin, as shown in study by Wang et al.6 that used
increase transgene expression. The results showed that injection of
longer locus region and Miccio et al.11 that used GATA1-HS2 as
lentivirus containing β-globin gene and GATA1-HS2 (G-GLOBE)
enhancer.
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MAHARANI ET AL.
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4.2
Hematological parameters
|
significantly alter Hb values at untreated or G-CSF-treated group,
(6.46 ± 1.10 vs. 6.16 ± 1.88 g/dL), Yannaki et al.10 explain that this is
Out of the six studies relevant in this review, one did not state
probably due to the development of intense compensatory hematopoi-
the hematological parameters8 whereas the rest included the
esis from the thalassemic liver.
results.6,7,9–11 Within those five, one study only showing the relevant
graph but did not state the actual count of the parameters,11 and the
other two did not include the RBC count and Hematocrit levels.9,10
4.2.3
|
Reticulocyte percentage
In Wang et al.,6 the reticulocyte percentage resulted in significant dif-
4.2.1
|
RBC count
ferences with 40.9% vs. 26.8% vs. 9.2% for nontreated, HDAd-shortLCR-, and HDAd-long-LCR-treated Hbbth-3/+ CD46+/+ mice,
Study by Wang et al.6 is comparing between the control group, the
respectively. Indicating better results in favor of using the HDAd-
HDAd-short-LCR group, and the HDAd-long-LCR group (RBC: 8–10,
long-LCR vector.
7–9, and 8–10 M/μL; respectively). Study by Wang et al.7 are compar-
In the study by Wang et al.,7 reticulocyte staining of blood smears
ison between control and the HSPC transduction group with RBC
demonstrated an impressive reduction of reticulocyte numbers indi-
count: 8.53 ± 0.29 and 7.1 ± 0.1 M/μL; respectively.
cating the reversal of the thalassemic phenotype in treated mice
In the study by Miccio et al.11 it has been stated that the hemato-
(31.13% ± 3.17% vs. 42.4% ± 1.43%, before vs. after transduction). In
logical parameters were not significantly different in th3/+ mice
the study by Bahal et al. CBC analyses performed on blood samples
transplanted with GLOBE- and G-GLOBE-transduced HSCs. Even
taken at 36 days posttreatment from mice in each group shows
though the study did not specify the actual count of the hematological
reduced reticulocyte counts in mice treated with SCF + ɣtcPNA4/
parameters, it has shown the graph of the results. Based on the graph,
donor DNA NPs but not in the mice treated with blank NPs,
mice transplanted with GLOBE- and G-GLOBE-transduced HSCs have
(Reticulocyte percentage: 2% vs. 30%).
higher RBCs compared with untreated (control) mice. Meanwhile the
8–10
other three studies did not state the RBC count.
In Yannaki et al.10 reticulocyte frequencies between normal and
Hbbth-3/+ mice had similar properties to the Hb levels, the normal
mice didn't show significant changes in percentage when given G-CSF
or splenectomy or both. Meanwhile in thalassemic mice group, all
4.2.2
|
Hb levels
groups show high percentage of reticulocyte (normal untreated and
nonsplenectomized vs. thalassemic untreated and nonsplenectomized:
Study by Wang et al. did not specify the exact number but has stated
3.62% ± 1.68% vs. 34.98% ± 14.67%; Table 1). Within the thalassemic
that the Hb levels are indistinguishable between control and interven-
group, when given G-CSF both splenectomized and nonsplenecto-
tion groups along with WBCs, RBCs, mean corpuscular hemoglobin
mized group shows increase in percentage values (nonsplenectomy:
concentration, mean corpuscular volume, and RBC distribution width
untreated [34.98% ± 14.67%] vs. G-CSF [50.17% ± 20.26%]; splenec-
(RDW-CV). Even though the exact number wasn't specified, the Wang
tomy: untreated [35.83% ± 4.92%] vs. G-CSF [43.7% ± 17.01%])
et al.6 study also indicates that Hb levels in the HDAd-long-LCR group
indicative of intense hematopoietic activity.
In the study by Miccio et al.,11 based on the graph, mice trans-
are significantly higher than HDAd-short-LCR group.
In Wang et al. study, the resulting parameters are 13.9 ± 0.63 and
9.7 ± 0.18 g/dL, between control and intervention groups, respec-
planted with GLOBE- and G-GLOBE-transduced HSCs have lower reticulocyte percentage compared with control group (25% vs. 13%).
tively. Meanwhile, Bahal et al. results are 7.5, 8.5, and12 g/dL
(control, SCF + scrambled ɣtcPNA4-Scr/donor DNA NPs, and SCF
+ ɣtcPNA4/donor DNA NPs; respectively). Both intervention groups
4.2.4
|
Hematocrit
showed persistent correction of the anemia in the mice with elevation
of the blood Hb levels into the normal range while anemia was not
improved in any of the controls.
7
In the study by Wang et al.,6 HCT levels were higher for the HDAdlong-LCR-treated group compared with HDAd-short-LCR-treated, fur-
In Yannaki et al. Hb values between control group normal mice
ther supporting the efficacy of the vector.
and Hbbth-3/+ (thalassemic) are 12.01 ± 2.24 versus 7.21 ± 1.84 g/dL,
In the study by Wang et al.,7 HCT showed significant improve-
respectively. When both groups are treated with G-CSF, the nonsple-
ment after transduction (before vs. after: 41.3 7% ± 0.81% vs. 30.7%
nectomized normal and Hbbth-3/+ mice are not significantly affected
± 0.46%) and levels were indistinguishable with the normal mice
(Normal: 11.34 ± 1.78 vs. 12.01 ± 2.24 g/dL; Hbbth-3/+: 7.21 ± 1.84
group.
vs. 6.54 ± 1.84 g/dL). However, splenectomy in normal mice caused
anemia,
(nonsplenectomized
vs.
splenectomized:
Within the study by Miccio et al.11 HCT parameters were not sig-
12.01 ± 2.24
nificantly different in th3/+ mice transplanted with GLOBE- and
vs. 10.53 ± 1.64). Anemia then worsened in splenectomized normal
G-GLOBE-transduced HSCs, though the study did not specify the
mice after G-CSF administration (untreated vs. treated: 10.53 ± 1.64
actual count of the HCT levels, but the corresponding graph of the
vs. 9.74 ± 1.53). Meanwhile, splenectomy in thalassemic mice did not
results has shown sufficient data that supports this result.
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856
All five studies that provide the hematological parameters, even
Despite undergoing various interventions, these three studies
with different interventions, resulted in either indistinguishable levels
yielded similar results, demonstrating improvements in phenotypic
compared with the normal mice control group or closer to the normal
parameters. In two studies, conducted by Wang et al.,6,7
levels in comparison to the untreated thalassemic group.
HDAd5/35++ lentiviral vectors were employed. This well-known
stable gene transfer tool is commonly used for gene therapy, albeit
with limited insert size capacity. Wang et al.6,17 used HDAd-
4.3
long-LCR to maximize γ-globin gene expression to balance the
Phenotype
|
amount of α-globin chain and thus ameliorates thalassemia.17 ImbalThe morphology of normal RBCs is biconcave discoid shaped with pal-
ance of α-globin and non-α-globin can block differentiation and mat-
lor in their center and normocytic.14 Hence, therapies are considered
uration of erythroblasts which might be related to the differentiation
successful if the intervention group has those characteristics, which
stages seen in HDAd-long-LCR and HDAd-short-LCR.18 While using
are similar to normal ones. Therefore, the phenotype of the RBCs is
the same type of vector, Wang et al. used O6BG/BCNU as an addi-
included as one of the parameters in our studies. Here, comparison
tional therapy to sustain γ-globin+ RBCs. Unlike HDAd5/35++ and
will be made qualitatively.
O6BG/BCNU-treated mice, those who did not receive that treat-
Out of six studies, only three of them had phenotype as one of
ment were prevalent of proerythroblasts and basophilic erythro-
their parameters.6,7,9 Study by Wang, et al. compared three different
blasts which indicated blocked erythroid maturation that might be
groups consisting of control and intervention with HDAd-long-LCR
due to insufficient amount of γ-globin to balance α-globin.7,18 It is
and HDAd-short-LCR vectors. HDAd-LCR vectors are made of
stated that the HDAd5/35++
HDAd5/35++ vectors with additional elements added to maximize γ-
sufficient improvement in RBCs morphology which indicates that
globin gene expression–thus named HDAd-long-LCR. Baseline RBCs
HDAd5/35++ and O6BG/BCNU is superior in improving RBCs mor-
showed hypochromic, highly fragmented, and anisopoikilocytosis
phology.7 This is due to their unsustained amount of γ-globin+ in
RBCs. After 16 weeks of therapy, the treated mice group showed
HDAd5/35++-treated mice without O6BG/BCNU. Meanwhile, in
reversal of the thalassemic phenotype with normocytic and well-
the study by Bahal et al.,9 γtcPNA4/donor DNA and SCF-treated
shaped RBCs in it. Compared with the control group, which are
were found to be superior to empty nanoparticles, SCF-only, and
CD46tg mice, the thalassemic mice model intervened with HDAd-
γtcPNA4-scr/donor DNA plus SCF. Enhanced in vivo gene editing
long-LCR vector, which are Hbbth-3/+/CD46 mice, showed similar
with γPNA by SCF due to increased HSC mobilization and effective
results, such as normal bone marrow cellular distribution. Erythroid
delivery of γPNA by nanoparticles increased DNA repair.19 Repaired
lineages are dominated with polychromatic and orthochromatic eryth-
DNA thus ameliorated disease phenotype from poikilocytosis, aniso-
roblasts, which indicate successful maturation of the erythroid.6 This
cytosis, and target cells to improve RBCs morphology. This improve-
is in alignment with other studies that found mice proerythroblasts
ment shows that α-globin precipitation was reduced due to
undergo three mitoses that produced higher ratios for polychromatic
successful DNA repair.9 Positive results were shown in three studies
15
and orthochromatic erythroblasts.
only treated group did not have
Meanwhile, the thalassemic mice
which can be a potential treatment for future research, especially in
model intervened with HDAd-short-LCR vectors showed dominance
humans. Yet, different types of mice were used between these stud-
of erythroid lineage consisting of proerythroblasts and basophilic
ies and thus further investigations should be done on the same sam-
6
erythroblasts. Later, basophilic erythroblasts will differentiate into
ple to reach further conclusions.
polychromatic and orthochromatic erythroblasts.16 This might indicate
that the HDAd-short-LCR has a slower or blocked differentiation than
the HDAd-long-LCR. Both interventions with vectors were compara-
5
|
INTERVENTION
ble to the control group with significant differences between before
and after treatment. Study by Wang et al.7 showed similar results as
These studies include a variety of β-thalassemia treatment options.
they also found reversal of thalassemic phenotype, from hypochromia,
Both Wang et al.6 and Ma et al.8 successfully increased the levels of
anisopoikilocytosis, and cell fragmentations to near-normochromic
β-globin expression. Beta-globin LCR was inserted into an HDAd5/35
6
and well-shaped RBCs after 6 weeks of O BG/BCNU treatment in
+/+
CD46
++ vector by Wang et al.,6 which significantly increased β-globin
/Hbbth-3/+ mice using HDAd5/35++ vectors. Similar find-
expression in peripheral RBCs. A considerable rise in HbF expression
ings were found in a study conducted by Bahal et al.9 Amelioration of
was noted, according to Ma et al.8 Wang et al.'s7 analysis of the effect
thalassemic phenotype, such as microcytosis, was found in γtcPNA4/
of their intervention on γ-globin levels, in contrast, found levels at
donor DNA and SCF-treated mice with amelioration of poikilocytosis
10%–15% of adult mice α- and β-globin and 25% of mouse β-minor
and reduction of anisocytosis, ovalocytosis, and target cells 36 weeks
globin. By focusing on introducing a specific mutation in the beta-
after the last treatment. These findings were found alongside RBCs
globin gene, Bahal et al.9 were able to achieve a 3.4% mutation rate at
morphology improvement in γtcPNA4/donor DNA-treated mice,
location IVS2-654. The GATA1-HS2 element was added by Miccio
unlike other groups. Meanwhile, untreated and control groups did not
et al.11 to improve β-thalassemia repair. In comparison, Yannaki
show any anemia improvement nor amelioration of extreme poikilocy-
et al.10 offered limited outcome details concerning their use of recom-
tosis, target cells, cabot rings, anisochromasia, and ovalocytosis.
binant human G-CSF.
16000609, 2024, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ejh.14179, Wiley Online Library on [31/03/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
857
MAHARANI ET AL.
MAHARANI ET AL.
These studies illuminate a variety of interventions and out-
5.2
|
Study limitation
comes in β-thalassemia screening. Wang et al.6 and Ma et al.8
observed a higher β-globin expression level, whereas Wang et al.7
When assessing research that compares interventions in β-thalasse-
investigated the effect of γ-globin concentration. Bahal et al.9
mic animal models, it is crucial to recognize various potential pitfalls
reported on specific β-globin gene mutations, and Miccio et al.10
and limitations. First, differences in experimental design, variations in
enhanced the correction of β-thalassemia using the GATA1-HS2
animal models, administration methods, and research standards may
element. Yannaki et al.
11
provided a limited overview of the results.
Among the interventions studied, Wang et al.6 achieved
pose challenges in drawing definitive conclusions about the effectiveness of different interventions.
remarkable levels of β-globin expression using HDAd5/35++ vec-
Additionally, relying on animal models might not sufficiently cap-
tors containing β-globin LCR, showing promise in this regard. In con-
ture the complexity of human β-thalassemia, casting doubt on the
trast, Wang et al.7 in particular examined γ-globin levels, providing
translatability of findings to clinical practice. Prolonged gaps in data in
valuable insights into its modulation. Bahal et al.8 focused on the
certain studies limit our understanding of the durability and safety of
introduction of specific mutations in the β-globin gene, proposing
these interventions. Moreover, publication bias, where positive results
possible mechanisms for gene regulation. Miccio et al.11 enhanced
are more likely to be published, can undermine a consensus on the
β-thalassemia through the GATA1-HS2 element, showing promise,
success of the intervention and potentially mask a more negative
although further long-term safety studies are needed. Notably, the
outcome.
specifics of study by Yannaki et al.10 application remain unclear is
Ethical considerations of animal welfare and the translation of
still limited in the data. Ma et al.8 reported a significant increase in
their results into human trials also raise important ethical dilemmas
HbF expression, which is relevant for increased fetal Hb, but the
that must be carefully monitored Finally, the potential ability to and
long-term and safety implications require in-depth studies. The
mutually exclusive, especially when researchers have financial ties to
selection of the most appropriate intervention depends on the spe-
certain interventions or treatments, require transparency to ensure
cific aspect of β-thalassemia being studied and the intended clinical
research validity.
outcome, with a decision determining its necessity: comprehensive
While these studies provide valuable insights into interventions
comparative studies in animal models and humans, emphasis on
for β-thalassemia, variability in trial design, exposure to interpretabil-
safety studies, and comprehensive clinical trials for positive data.
ity, publication bias, ethical considerations, as well as potential conflicts of interest, hinder comparative research.
5.1
|
Safety precaution
5.3
|
Further studies and recommendation
Comparative safety precautions in research studies are critical, especially
when investigating interventions for complex medical conditions such as
Further research to enhance β-thalassemia research should focus on
β-thalassemia. In a study by Wang et al., which focused on in vivo HSC
existing highlighted gaps and challenges in comparing interventions in
gene therapy, carefully evaluated safety precautions. Their careful strat-
animal models In order to make meaningful comparisons and size
egy included in vivo HSPC transduction with HDAd-γ-globin/mgmt, in
becomes easier, researchers should establish formal procedures and
combination with HDAd-SB, followed by O6-BG/BCNU in vivo selection.
reporting guidelines, ensuring that data are collected and reported on
Notably, this procedure did not cause leukemic conditions; however, it
a regular basis. Longitudinal evaluation of interventions is necessary
was not entirely clear. Although total WBC counts decreased after treat-
to determine durability and safety data, thus increasing its clinical
ment, all blood values remained within normal ranges, indicating a con-
applicability. In addition, it is important to bridge the gap between ani-
trolled cytoreductive effect. This comprehensive safety study highlights a
mal models and human trials in order to successfully translate the
careful and systematic approach that confirmed this research.7
findings into clinical practice.
In contrast, Yannaki et al.10 investigated the mobilization of HSCs
Diversity of β-thalassemic animals should be considered as an
in a thalassemic mouse model treated with G-CSF. Their safety pre-
indicator of genetic differences of the disease, providing a broader
cautions revealed some side effects, especially splenomegaly in 65%
view of the quality of the intervention. Researchers should also
of the treated pigs, enlarged liver in 19.7%, and bleeding in 12.5% of
actively address publication bias by promoting both positive and nega-
the treated group. These findings highlight the need to be cautious
tive outcomes, and promoting transparency in research results. Ethical
about side effects and potential complications when implementing
considerations, including animal welfare and responsible translational
interventions in animal models.
practices, should play an important role in study design and implemen-
Specifically, this comparative study reveals differing safety pro-
tation. Furthermore, clear disclosure of potential conflicts of interest,
files between the two studies. Wang et al.7 demonstrated relatively
particularly financial relationships with companies involved in clinical
safe results for their intervention, whereas Yannaki et al.10 faced a
development, is important to maintain research credibility.
notable safety concern. These findings emphasize the importance of
In conclusion, fostering collaborative efforts among researchers,
safety considerations in interventions for medical conditions like
institutions, and pharmaceutical companies has the potential to expedite
β-thalassemia.7,10
the advancements in β-thalassemia research. The integration of
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858
resources and knowledge holds the key to accelerating the development
of interventions that are both safe and effective, thereby facilitating the
transition from preclinical stages to clinical use. Making recommendations in this regard could help overcome current limitations.
6
|
C O N CL U S I O N
Our findings in the studies of gene therapy for thalassemia using HSC
show a significant improvement in β-globin levels and phenotype.
Gene expression in thalassemic mice is observed. Maturation of the
erythroid also occurs, marking improvement of phenotype parameters. Hematological parameters in control mice compared with thalassemic mice have indistinguishable hematological levels. Positive
phenotypic improvements were also seen. These outcomes indicate
injection methods for HSC have a good efficacy in gene therapy for
thalassemia in mice models. Furthermore, study in the human model
should be done in future studies.
AUTHOR CONTRIBUTIONS
The idea formulation was done by ILM, MHZ, RAK, RSW, RNH, and
IRS. Data extraction was done by ILM, MHZ, RAK, RSW, RNH, IRS,
and AD. Article were written by ILM, MHZ, RAK, RSW, and RNH.
Supervision of the process was done by IRS and AD.
CONF LICT OF IN TE RE ST ST AT E MENT
Authors declare no conflict of interest.
DATA AVAI LAB ILITY S TATEMENT
Data in this research are from each article included in the review.
Availability of the data that further supports this study requires permission of the corresponding author from each article.
ORCID
Indira Laksmi Maharani
https://orcid.org/0009-0001-0446-6217
Muhammad Hafizh Zauhari
https://orcid.org/0009-0000-0681-
5348
Rakean Ahmad Kiansantang
https://orcid.org/0009-0004-1951-
7568
Razzan Satria Wibowo
Rizqi Najla Humaira
Adisti Dwijayanti
https://orcid.org/0009-0000-6376-9552
https://orcid.org/0009-0005-3474-5259
https://orcid.org/0000-0003-0612-5683
Imelda Rosalyn Sianipar
https://orcid.org/0000-0002-1438-096X
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How to cite this article: Maharani IL, Zauhari MH,
Kiansantang RA, et al. Systematic review of hematopoietic
stem cell gene therapy approach in thalassemia: Comparative
analysis in animal models. Eur J Haematol. 2024;112(6):
848‐859. doi:10.1111/ejh.14179
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MAHARANI ET AL.