Novel therapies for β-thalassaemia
Compounds that modulate foetal haemoglobin
After 2 years of age, the main type of haemoglobin present is adult Hb
(HbA) together with HbA2 (<3.3%) and small amounts of foetal
haemoglobin (HbF, <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 γ-globin decreases the α/β-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 (HBS1L-MYB ) on chromosome 6
[23], [24], the B-cell CLL/lymphoma11A (BCL11A ) locus on
chromosome 2 [26] and the Kruppel-like factor (KLF1) on chromosome
19 [27]. The role of 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 HBB locus as a preferential activator of theHBB gene and secondly it acts indirectly by activating the
expression of BCL11A which, in turns, represses the gamma globin
genes [27], [28]. The 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 γ-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 β-thalassaemia is the S-phase cell-cycle inhibitor hydroxyurea
(HU). An increase of 2- to 9- fold change in γ-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/β-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
β-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 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 β-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 β-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 β-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
β-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 β-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)