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)