β-Thalassaemia is caused by over 300 autosomal recessively inherited mutations in and around the β-globin gene in chromosome 11 (Taher et al., 2021). Homozygous or compound heterozygous inheritance of these mutations leads to β-thalassaemia major, a transfusion-dependent form of thalassaemia (Mettananda and Higgs, 2018). Compound heterozygous inheritance of one β-thalassaemia mutation with the haemoglobin E mutation results in haemoglobin E β-thalassaemia, another common variant of β-thalassaemia (Fucharoen and Weatherall, 2012).

Adult haemoglobin is a tetramer of two α- and two β-globin chains. In normal human erythroid cells, a tight balance between the α-globin and β-like globin (β-globin and γ-globin) chains is maintained (Weatherall, 2010). Disequilibrium of the globin chains due to reduced or absent synthesis of β-globin chains in erythroid cells is the primary pathophysiology of β-thalassaemia (Schrier, 2002). Consequently, free α-globin chains that do not have adequate β-like globin chains to pair with, form insoluble α-hemichromes that trigger a cascade of events through the generation of reactive oxygen species leading to premature destruction of erythroid cells (Figure 1); (Schrier et al., 2003). Thus, excess free α-globin chains are the main mediators of ineffective erythropoiesis and haemolysis in β-thalassaemia (Allen et al., 2021).

Several clinical genetic studies done among patients with β-thalassaemia major and haemoglobin E β-thalassaemia have conclusively identified coinheritance of α-thalassaemia as a favourable genetic modifier that ameliorates the disease phenotype of β-thalassaemia (Mettananda et al., 2015). When coinherited, deletion of one or two (out of four) α-globin genes reduces the free α-globin pool and minimises the haemolysis and ineffective erythropoiesis (Premawardhena et al., 2005). Thus, a patient who would otherwise develop transfusion-dependent thalassaemia will transform into a less severe phenotype of non-transfusion dependent thalassaemia. Specifically, deletion of two α-globin genes (--/αα or -α/-α) reduces the severity in most patients with β-thalassaemia, whereas deletion of a single α-globin gene (-α/αα) ameliorates the disease phenotype in all patients except those with the most severe β⁰/β⁰-thalassaemia phenotype (Ho et al., 1998; Galanello et al., 2009; Mettananda et al., 2015). Also, the coinheritance of α-thalassaemia improves the RBC indices and haemoglobin levels among individuals with heterozygous β-thalassaemia trait (Mettananda et al., 2018a). In summary, a 25–50% reduction in α-globin chain output in RBC could significantly lessen the severity of β-thalassaemia (Mettananda et al., 2015).

The human α-globin genes are in the short arm of chromosome 16 (16p13.3), approximately 150 kb from the telomere (Hughes et al., 2005). Two α-globin genes, HBA1 and HBA2, are in each chromosome; therefore, each human cell have four copies of α-globin genes. The expression of α-globin genes is regulated by a complex interplay between gene promoters, enhancers, boundary elements, transcription factors and epigenetic modifications of the associated chromatin (Mettananda et al., 2016; Oudelaar and Higgs, 2021). It is believed that during transcription, enhancers and promoters are brought into close physical proximity to each other by chromatin looping (Mettananda et al., 2016).

Several in vitro, transgenic mice and translational research studies have identified four distant enhancers designated as multispecies conserved sequence (MCS) R1 to R4 located 10–48 kb upstream to the gene locus, critical for the expression of α-globin genes (Figure 2). Of these enhancers, the MCS-R2 located 40 kb upstream of the α-globin gene has been shown as the most critical enhancer influencing the expression of α-globin genes. In transgenic humanised mice, deletions involving the MCS-R2 region led to a marked reduction of the α-globin expression to very low levels (Wallace et al., 2007; Farashi and Harteveld, 2018). A rare naturally occurring homozygous deletion of the MCS-R2 region resulted in severe α-thalassaemia despite having all four α-globin genes intact (Coelho et al., 2010).

Epigenetic modifications play a pivotal role in regulating the α-globin gene expression (Mettananda, 2018b). The human α-globin gene cluster is in a gene dense, early replicating, open chromatin region of the genome, therefore, requires active silencing through epigenetic mechanisms (Figure 3); (De Gobbi et al., 2007). The α-globin genes in non-erythroid cells are silenced through binding of the Polycomb repressive complex 2 (PRC2), which is associated with histone methyltransferase that increases the repressive chromatin signature Histone 3 Lysine 27 trimethylation (H3K27me3) (Garrick et al., 2008). In erythroid cells, when α-globin gene is activated, PRC2 is displaced, and the H3K27me3 chromatin signatures are removed by histone lysine demethylase (KDM) 6B (Vernimmen et al., 2011).

Altering these epigenetic signatures using epigenetic inhibitor drugs to silence the α-globin has been attempted. Inhibition of histone lysine demethylation by a novel molecule IOX1 has shown to downregulate the expression of α-globin in vitro in primary human erythroid cells (Mettananda et al., 2017a). IOX1 is a pan-histone demethylase inhibitor that inhibits a number of KDM enzymes at various concentrations. Thus, inhibition of KDM6B is believed to be the mechanism of action of IOX1 in human erythroid cells.

Similarly, histone deacetylase (HDAC) inhibitor vorinostat has been shown to silence α-globin whilst upregulating the expression of γ-globin in human erythroid cells (Mettananda et al., 2019c). Vorinostat is a hydroxamic acid-group pan-HDAC inhibitor that causes significant upregulation of γ-globin similar to other HDAC inhibitors (Okam et al., 2015). Although the exact mechanism of downregulation of α-globin by vorinostat is uncertain, it may act through the KDM6B. In summary, both IOX1 and vorinostat have shown considerable promise in in vitro pre-clinical studies in human erythroid cells. However, their clinical usefulness is yet to be tested in clinical trials.

β-Thalassaemia has several salient features for it to become a promising candidate disease for the development of genome editing therapies (Doudna, 2020). Firstly, the genetic basis and the molecular pathology of β-thalassaemia is well understood (Taher et al., 2021). Secondly, the genome edits are only needed to be performed in HSC, which can easily be mobilised from patients for ex vivo manipulations. Thirdly, the technologies for mobilisation and subsequent transplantation of HSCs are highly advanced at present due to the long-standing clinical use of HSCT for multiple indications (Ferrari et al., 2021). Therefore, β-thalassaemia has been at the forefront to bring genome editing therapies to the bedside. Any genome editing treatment aiming to cure β-thalassaemia shall involve mobilisation and collection of HSCs, introducing the desired genome edits ex vivo and transplanting back to the patients after myeloablative conditioning (Mettananda, 2020).

Genome editing to upregulate γ-globin expression with a view of increasing HbF synthesis has been successful in clinical trials (Demirci et al., 2020a). The scientific rationale behind this is to increase the production of γ-globin chains, which would then mop-up the excess of α-globin chains to reduce free α-globin chains, thus improving the α/β-like globin disequilibrium in RBCs (Bauer et al., 2012). XmnI polymorphism (rs7482144) located within the promoter of γ-globin gene, HBS1L-MYB intergenic region on chromosome 6q23 and BCL11A gene located on chromosome 2p16 are the genomic targets to upregulate γ-globin (Menzel et al., 2007; Thein et al., 2007; Sankaran et al., 2008; Demirci et al., 2020b; Zeng et al., 2020).

Therapeutic disruption of the expression of B-cell lymphoma/leukaemia 11A protein (BCL11A), which is a critical transcription factor involved in postnatal silencing of γ-globin, has been the target of recent clinical trials (Psatha et al., 2018; Canver et al., 2015). One of these clinical trials (ClinicalTrials.gov Identifier NCT03432364) utilised zinc finger nucleases (ZFN) as the genome editing tool. In this trial, CD34⁺ HSCs were transfected with mRNA encoding ZFNs, which has binding sites for the GATA-binding region of the enhancer of BCL11A. Preliminary results of this trial reports elevation of HbF in two patients after receiving infusions of autologous genome edited CD34⁺ HSC product ST-400. However, at least one patient failed to remain transfusion independent (Smith et al., 2019).

In contrast, another clinical trial (ClinicalTrials.gov Identifier NCT03655678) used CRISPR/Cas9 gene editing to disrupt an erythroid-specific enhancer region of BCL11A. In this trial, a female patient with transfusion-dependent β-thalassaemia who received the autologous genome edited CD34⁺ HSC product CTX001 showed sustained elevations of HbF and remained transfusion independent (last reviewed after 18 months post-transplant) (Frangoul et al., 2021). In addition to these clinical trials, the disruption of a γ-globin gene promoter motif bound by BCL11A by CRISPR/Cas9 has induced HbF to potentially therapeutic levels in in vitro studies (Métais et al., 2019).

Furthermore, CRISPR based genome editing has been used to correct some β-thalassaemia mutations in vitro. Allelic disruption of aberrant splice sites of IVS1-110G > A and IVS2-654C > T mutations and repair of CD39 (CAG > TAG) mutation using a single-strand donor DNA template have shown promising results in pre-clinical studies (Xu et al., 2019; Cosenza et al., 2021).

Genome editing of α-globin gene to improve the α/β globin imbalance is a promising alternative strategy to treat β-thalassaemia. This strategy aims to decrease the α-globin output by 25–50% to reproduce the beneficial natural effect of α-thalassaemia trait on transfusion-dependent β-thalassaemia. CRISPR/Cas9 genome editing to down-regulate α-globin by deleting one of the two α-globin genes (HBA2) to recreate an α-thalassemia trait was reported in a recent in vitro study (Pavani et al., 2021). In this study, the investigators designed a single guide RNA (sgRNA) to target the 5′UTR of HBA1 and HBA2 genes, thus removing the HBA2 gene (Figure 2). Erythroid cells generated from a β-thalassaemia HSC line (HUDEP-2 β⁰) transfected with ribonucleoprotein containing this sgRNA resulted in a dramatic reduction of α-globin messenger RNA and a significant decrease in α-globin precipitates in erythroid cells. Transfection of HSC of β⁺-thalassaemia with these ribonucleoproteins and differentiation of edited HSC into erythroid cells resulted in correction of the pathologically high α/β-globin ratios.

Another approach to reduce α-globin chain synthesis in human erythroid cells is to genome edit the MCS-R2 critical α-globin enhancer. As postulated by natural mutations involving the MCS-R2 enhancer, a single allelic genome editing of the MCS-R2 decreases the α-globin synthesis by up to 50%, whereas homozygous biallelic mutation would reduce the α-globin synthesis to a level seen in patients with haemoglobin H disease. A recent in vitro study reported successful replication of natural mutations by creating a targeted deletion of the MCS-R2 enhancer using CRISPR/Cas9 genome editing in primary human erythroid cells [(Mettananda et al., 2017b), (Mettananda et al., 2018b)]. In this study, the erythroid cells differentiated from genome edited CD34⁺ HSCs resulted in over 60% knockdown of α-globin chain synthesis following the monoallelic deletion of the MCS-R2 enhancer. Biallelic deletion of the enhancer resulted in a pronounced (∼90%) knockdown of α-globin chain synthesis. The deletion of the MCS-R2 enhancer in erythroid cells of patients with β-thalassaemia could restore the α/β-globin chain imbalance to normal levels. Furthermore, multi-lineage engraftment of genome edited HSC was demonstrated in secondary serial xenotransplantation assays to confirm editing of long-term repopulating HSCs.

Several genome editing approaches include insertion of β-globin transgene, upregulation of γ-globin and downregulation of α-globin are beneficial to restore the globin balance in β-thalassaemia. Therefore, these approaches could be used in combination to improve the disease severity of patients. In fact, a recent in vitro study used CRISPR/Cas9 genome editing to delete one of the two α-globin genes and to integrate a β-globin transgene (β^AS3) under the control of the endogenous HBA promoter (Pavani et al., 2021). Co-transfection of HSC with this β-globin transgene and ribonucleoprotein containing sgRNA designed to delete the HBA2 gene resulted in an improvement of α/β globin chain to near-normal levels in erythroid cells of patients with β⁺-thalassaemia and β⁰-thalassaemia. Another recent study demonstrated successful use of the combined Cas9/AAV6 genome editing method to replace the endogenous HBA1 gene with a full-length HBB transgene (Cromer et al., 2021). Although the genome editing frequency was below the desired levels, erythroid cells differentiated from edited β-thalassaemia patient HSCs demonstrated normalisation of α/β-globin mRNA and protein ratios and restoration of functional adult haemoglobin tetramers.

Similarly, various combinations of gene therapy, genome editing and other genetic technologies could synergistically treat β-thalassaemia. A recent study demonstrated the use of a modified vector comprised of the standard β^A−T87Q-globin transgene and an intron-embedded miR-30-based short hairpin RNA designed to target the α2-globin mRNA expression (Nualkaew et al., 2021). Use of this vector showed 1.7-fold greater potency to improve α/β-globin ratios resulting in a greater therapeutic efficacy for β-thalassaemia. Also, genome editing approaches that target downregulation of α-globin can be combined with genome editing approaches of HbF induction to have additive effects. These methods can mitigate the lack of efficacy of individual systems to achieve a precise balance of α to β-like globin chain ratio in RBCs.

Although genetic and epigenetic therapies have shown considerable promise, several challenges still limit their ability to develop into standard clinical care (Dong et al., 2013). All genome editing approaches would require the process of HSC transplantation (High and Roncarolo, 2019). Although the risk of graft vs. host disease and long-term immunosuppression is eliminated, patients undergoing genome editing still need to undergo myeloablative conditioning before the autologous HSC transplantation. Also, these therapies are technologically demanding thus, are associated with very high costs (Ferrari et al., 2021). Therefore, they would not be available to patients in developing countries who need these treatments most.

Genotoxicity due to undesired DNA modifications is a significant challenge of genome editing. Although most of these changes occur as non-specific off-target effects, a recent study has shown that chromothripsis, a catastrophic genomic rearrangement of chromosomes in a haphazard order, could occur due to on-target effects of genome editing (Leibowitz et al., 2021). Other reported genotoxicity effects of CRISPR/Cas9 genome editing include large-scale DNA deletions or inversions, activation of proto-oncogenes and induction of large chromosomal truncations (Haapaniemi et al., 2018; Kosicki et al., 2018; Cullot et al., 2019). Although newer systems that include high-fidelity Cas9 nucleases and base or prime editing approaches minimises the risk of off-target effects, none of the genome editing tools can be considered hazard free at present (Komor et al., 2016; Anzalone et al., 2019; Magrin et al., 2019).

Another limitation inherent to the current approach is difficulty in titrating the α-globin knockdown to the desirable level. Studies have shown that a 25–50% reduction in α-globin output is the most desirable for optimal α/β globin balance and to ameliorate β-thalassaemia (Mettananda et al., 2015). Too much knockdown will lead to lack of α-globin production and subsequent worsening of anaemia (Mettananda, 2017). Therefore, all genome editing and epigenetic therapies on development towards curing β-thalassaemia by silencing α-globin has a very narrow therapeutic window.