Introduction

Despite major advances in the treatment of thalassaemia, resulting in prolongation of the life expectancy of affected patients, a definitive cure available to all patients remained elusive until very recently. For years, the only curative treatment has been allogeneic bone marrow transplantation, limited however by its availability only to young patients with a well-matched donor (Lucarelli, Andreani and Angelucci, 2002) and the requirement for long-term immunosuppression to prevent or treat transplant-related immunological complications such as graft-vs-host-disease (GvHD) and rejection, carrying a non-negligible risk of mortality (Galanello and Origa, 2010).

Given the widespread elucidation of the genetic basis of haemoglobinopathies (Stamatoyannopoulos et al., 2001; Weatherall and Clegg, 2001), gene therapy approaches have long been envisioned by pioneers such as late Dr George Stamatoyannopoulos (Higgs, Engel and Stamatoyannopoulos, 2012). Gene therapy aspires to provide cure for thalassaemia through the manipulation of the genome of haematopoietic stem cells, thus compensating for the inadequate or faulty function of mutated genes. This can be achieved either by gene addition via a semi-random insertion of a healthy copy of the therapeutic gene into the cells using viral vectors (Dong and Rivella, 2017) or gene editing via a precisely directed mutation that repairs the gene in situ or induces a disease-modifying effect (i.e. reactivation of Hb F synthesis) using site-specific nucleases (Porteus, 2017; Antoniani et al., 2018; Boulad et al., 2018).

Ex vivo gene therapy for thalassaemia, either by gene addition or gene editing, is a haematopoietic stem cell (HSC) transplantation-based procedure. The autologous (patient-derived) HSCs are mobilised with granulocyte colony-stimulating factor (G-CSF) plus plerixafor, harvested by cytapheresis, CD34+cell-enriched by immunomagnetic separation and genetically modified ex vivo (Yannaki et al., 2012; Psatha et al., 2014; Karponi et al., 2015). The patient undergoes myeloablative conditioning followed by infusion of the gene-modified cells and remains hospitalised until the haematopoietic recovery (Finotti et al., 2015; Malik, 2016; Mansilla-Soto et al., 2016; Ferrari, Cavazzana and Mavilio, 2017; Biffi, 2018; Cavazzana and Mavilio, 2018) (Figure 1, left panel).

Figure 1

Ex vivo and in vivo gene therapy for haemoglobinopathies: the concept. G-CSF, granulocyte colony-stimulating factor; HSCs, haematopoietic stem cells; iv, intravenous; SIN-LV, self-inactivating lentiviral vectors.

Recently, in vivo approaches for gene therapy of thalassaemia, either by gene addition or gene editing, have also been preclinically pursued, aiming to overcome the current limitations of ex vivo gene therapy including the need for myeloablative conditioning, the need for transplantation expertise and high costs (Li et al., 2018; Wang et al., 2019) (Figure 1, right panel).

Gene therapy by gene addition

Gene therapy by gene addition, in other words, the transfer of a healthy copy of the β (or γ) globin gene along with its important regulatory genomic elements into target-cells, using modified viral constructs as delivery vehicles (Higgs, Engel and Stamatoyannopoulos, 2012; Mansilla-Soto et al., 2016; Cavazzana, Antoniani and Miccio, 2017), has been the most developed gene therapy approach up-to-now. After many years of preclinical evaluation, self-inactivating lentiviral vectors (SIN-LVs) have proven to be the safest and most effective means of delivery (Zufferey et al., 1997; Naldini, 2011; Naldini, Trono and Verma, 2016). In contrast to γ-retroviral vectors favouring integration in transcription start sites (TSS) and having been associated with insertional mutagenesis in gene therapy trials of X-linked severe combined immunodeficiency (SCID) (Hacein-Bey-Abina et al., 2010; Zhang, Thrasher and Gaspar, 2013) and Wiskott–Aldrich syndrome (Boztug et al., 2010; Braun et al., 2014), SIN-LVs, which preferentially integrate into actively transcribed genes rather than TSS, have not been associated with genotoxicity so far (Montini et al., 2006, 2009; Cesana et al., 2014). Extensive integration site analysis from preclinical and clinical studies involving SIN-LVs and in various disease settings, has demonstrated polyclonal haematopoietic reconstitution patterns (Aiuti et al., 2013; Biffi et al., 2013; Thompson et al., 2018; Marktel et al., 2019). Short genomic elements termed chromatin insulators have been used (Chung, Whiteley and Felsenfeld, 1993; Cavazzana-Calvo et al., 2010) or are under evaluation (Emery et al., 2000; Liu et al., 2015), concerning their ability to act as additional safety features, shielding the transgene from the influence of its surrounding genomic environment (Rivella et al., 2000).

Gene therapy by Gene editing

In gene editing approaches, site-specific nucleases − (zinc-finger nucleases (ZFN) (Carroll, 2011), transcription activator-like effector nucleases (TALEN) (Ma et al., 2013) and clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9) complex (Dever et al., 2016) are engineered to precisely identify specific genomic sequences where they create a double-strand break (DSB), which can subsequently be repaired by endogenous cellular repair mechanisms: i) in non-homologous end joining (NHEJ), a direct ligation of the two ends takes place, in an error-prone process that causes insertions or deletions (indels) leading to frameshift mutations and rendering the locus non-functional (i.e. gene knockout); ii) in homology directed repair (HDR), a donor DNA template is used and high fidelity, albeit substantially less efficient, repair of the DSB takes place, due to the resistance of haematopoietic stem cells to HDR; iii) another approach is the targeted integration of a therapeutic gene in a predetermined genomic ’safe harbour’ that supports long-term expression without interfering with the transcriptional activity of endogenous loci (Osborn et al., 2016) (Figure 2).

Figure 2

Therapeutic approaches involving genetic modification of HSCs in patients with haemoglobinopathies. a) LV addition of a therapeutic β- or γ-globin gene into HSCs b) Mutation correction by a nuclease-template genome editing method, c) Targeted (more...)

Genome editing eliminates the need for semi-randomly integrating viral vectors and, reasonably, the risk of insertional mutagenesis; however, the main safety concern with this approach is the unintended ‘off target’ mutagenesis that could generate additional mutations in undesired genomic loci with potential unexpected consequences.

Gene editing strategies for β thalassaemia have primarily focused on inducing reactivation of the γ globin gene (Wienert et al., 2018), aiming to correct the imbalance of the α-like/β-like globin chain ratio, the main pathophysiological cause of the disease. This can be achieved by inhibiting factors that repress the expression of the γ globin gene (i.e. BCL11A), thus mimicking hereditary persistence of fetal haemoglobin (HPFH) in which high levels of haemoglobin (Hb) F are maintained throughout adult life and compensate for the low levels of β globin expression in patients carrying thalassaemic mutations. Disrupting the binding site or the enhancer of BCL11 causes upregulation of HbF to therapeutic levels (Psatha et al., 2018). Both CRISPR/Cas9 and ZFNs are being used in ongoing gene editing clinical trials for β thalassaemia and sickle cell disease (SCD). Several other interventions at the molecular level that may provide a therapeutic result (e.g. RNA interference and forced chromatin looping) (Yannaki, Emery and Stamatoyannopoulos, 2010) are also being currently tested (Figure 2).

Gene therapy clinical trials

B. Gene editing trials

CRISPR Therapeutics uses the CRISPR/Cas9 technology for HbF reactivation in two phase I/II clinical trials for β thalassaemia (CTX001-111) and SCD (CTX001-121), recruiting 45 patients each, aged 18-35 years. A putative γ-δ intergenic HbF silencer, binding site of BCL11A, is targeted and disrupted by CRISPR/Cas9 (Antoniani et al., 2018). One β thalassaemia patient has been treated and reported transfusion-free for over 4 months post-treatment. Sangamo in another clinical trial (Thales), exploits ZFNs to disrupt the BCL11A erythroid enhancer. The study anticipates the enrolment of six patients with β thalassaemia (Holmes et al., 2018), one of whom has been reported transfusion-free for 5 weeks post-treatment with a significant increase in HbF (31%) and stable total haemoglobin levels (~90 g/l).

The long-term safety of gene therapy

Patients with hemoglobinopathies undergoing gene therapy by gene addition or gene editing need to be prospectively evaluated for at least 15 years, so as both the sustainability of clinical benefit and the long-term safety be addressed. Such patients maybe at an increased risk of developing malignancies for several reasons; first, host factors generated by the disease background or/and treatment, as in SCD, the chronic hypoxia and inflammation, clonal hematopoiesis of indeterminate potential (CHIP)-related mutations (Ghannam et al., 2020) and chronic exposure to hydroxyurea or as in thalassemia solid organ hemosiderosis, may create a permissive environment for the development of malignancies. Second, the transplant-procedure itself, either the conditioning regimen (myeloablative or non-myeloablative) or/and a low engraftment of gene-corrected HSCs allowing for clonal proliferation of the endogenous cells, constitute additional risk factors for oncogenesis. Finally, gene therapy by gene addition is associated with a low, but existing risk of insertional mutagenesis while gene therapy by gene editing has the potential to cause double strand breaks at other than the desired locations (“off-target” effects).

Up to date, in the BBB trials with the longest follow up, there have been 3 SCD patients who have developed myeloid malignancies following gene therapy by gene addition. The malignancies developed in the HGB-206 trial, at 3 and 5.5 years (MDS progressing to AML and AML, respectively) or 6 months (MDS) post gene therapy. In the first case of MDS that subsequently progressed to acute leukemia, the investigation demonstrated that the event was not gene therapy-related due to absence of vector integration in the CD34+ blast cells (Hsieh et al., 2020). In the recent case of AML, investigation has demonstrated that the vector integrated within the vesicular-associated membrane protein 4 (VAMP4) gene which has no known role in the development of malignancies whereas the patient presented several well-known genetic mutations and chromosomal abnormalities commonly observed in AML. In the early onset post-gene therapy MDS event, the diagnosis was based on trisomy 8, an MDS-related cytogenetic abnormality, but no blast cells were seen in the bone marrow and the patient is followed for the true impact of this abnormality.

Data so far suggest that in SCD patients who developed hematologic malignancies, it is unlikely the integration site of BB305 lentiviral vector to have played a role and more likely malignancies have been triggered by host factors and been exacerbated by the busulfan conditioning probably in combination with the chronic use of Hydroxyurea. There is accumulating evidence that sickle patients may be more prone to the development of myeloid malignancies (3.6 fold increase over the general population, Brunson et al., 2017), although thalassemia also, especially if transfusion-dependent, has also been associated with an increased risk of malignancies, especially hematologic ones (1.47 fold increase over the general population, Hodroj MH, Blood Reviews 2019)

Gene editing clinical studies are still in their beginning and longer follow-up in this setting also, will address the safety and the sustainability of the clinical effect.

Regulatory approval and marketing authorisation

Based on the successful clinical data from 32 adults and adolescents treated in Bluebird bio’s clinical trials, the European Commission granted conditional approval, in June 2019, for the Bluebird bio gene therapy product (autologous CD34+ cells encoding the β A-T87Q-globin gene) under the commercial name Zynteglo, to treat transfusion-dependent β+ thalassaemia in patients 12 years and older, who are eligible for stem cell transplantation but do not have a matched related donor.

The recent marketing authorization of Zynteglo and also other gene therapy products in different disease settings, with burdensome price tags has economically challenged the health system payers − even in higher income countries − who are tasked to pay a large price once, instead of paying smaller amounts repeatedly for a life-time. To encourage patient access to such highly expensive genetic treatments, new models of payment have been proposed by the stakeholders. Bluebird bio, on an outcome-based model of payment, suggested for Zynteglo five annual dose payments for patients who become transfusion-free or, as an alternative, reimbursement of only the first dose.

It remains however to be shown in the long run and on the basis of the percentage of patients becoming transfusion independent, developing a thalassaemia intermedia phenotype or failing to respond, whether indeed, such an one-time expense will be cost-effective overall, compared to the cumulative costs of life-long conventional treatment of the disease and its complications.

The current challenge of applying gene therapy

After almost 4 decades of strenuous research on haemoglobinopathies, the long-awaited gene therapy is entering the realm of clinical practice. With one gene therapy product already officially approved for β thalassaemia in Europe and more under development with promising and encouraging results in clinical trials so far, the future looks brighter than ever (Psatha, Papayanni and Yannaki, 2018). However, new challenges emerge for the thalassaemia community globally, including the costs of application of these expensive therapies on a massive scale, selection of patients clinically capable of undergoing such procedures, global access to the treatment and need for transplantation expertise to administer these sophisticated cellular treatments.

Moreover, the striking imbalance between developed and developing countries, both in terms of patient demographics (i.e. large populations of young patients in low-income countries and of adult, aged patients in high-income countries) and financial potential of health systems, makes the situation an even more complicated conundrum. Close collaboration among all stakeholders involved (healthcare professionals, patients, the pharmaceutical industry, policy makers and healthcare providers) is mandatory.

Box

Summary and Recommendations.

Table 1

Gene therapy and gene editing clinical trials for β-thalassemia. TDT: transfusion dependent thalassemia; PK pharmacokinetics; hHSPCs: Human Hematopoietic Stem and Progenitor Cells; ZFN: zinc finger nuclease ^§as of March 3, 2021 from ClinicalTrials.gov. (more...)

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