Figure 1: Schematic representation of gene therapy in beta-thalassemia. Mutations in the beta globin gene give rise to beta-thalassaemia. Conventional therapies include transfusion followed by iron chelation therapy. Autologous transplantation of HSCs genetically modified by gene addition or gene editing is another therapy for patients affected by beta-thalassaemia.
Gene-addition
Gene addition involves the insertion of a lenti-viral (LV)/retroviral vector that contains the whole regulatory and the β- or γ-producing genets into autologous human stem cells in-vitro and then infusing these modified stem cells back into the patient. Early gene therapy studies used recombinant γ-retroviral vectors derived from Moloney Leukemia Viruses to introduce the functional copy of the β-globin gene.In-vitro , these vectors provided reasonable levels of β-globin transgene expression but in vivo , it resulted in a limited variable expression [47], [48], [49]. These types of vectors have a number of defects: (i) instability, (ii) limited cargo capacity and (iii) an inability to transduce non-dividing cells [38]. The use of γ-retroviral vectors was abandoned and in the mid-1990s, lentiviral vectors were derived from human immunodeficiency virus type 1 (HIV1). These types of lentiviral vectors were able to transfer much longer sequences and also were able to transduce cells arrested at the G1-S boundary of the cell cycle [50], [51].
Several studies have shown that self-inactivating lentiviral carrying the human β or γ-globin gene and its fundamental regulatory elements are able to correct the disease in mice with either β-thalassaemia intermedia or major [52], [53], [54]. Several investigators continued to work hard to improve LV in terms of higher transgene expression, acting mostly on regulatory transcriptional components and the addition of insulators to prevent negative chromatin position effects. These included larger HS2 and HS4 elements of the Locus Control Region (LCR) and incorporation of insulator elements and inclusion of HS1 or GATA-1 elements. In most cases, although increased transgenic expression was achieved this happened at the cost of a reduced transduction efficiency and vector copy number/cell [55]. The final approved LVs for clinical trials were the result of an acceptable compromise between good manufacturing yield, title and β-globin expression level [44].
The first clinical trial for β-thalassaemia took place in June 2007 in France, where three patients were treated by gene therapy with the HPV569 LV expressing the βT87TQ globin variant [38]. In this vector, the β-globin gene was under the control of the LCR and the β-globin promoter and was surrounded by tandem copies of the cHS4 core element [56]. The first patient to be treated was an 18-year-old βE0 who had been transfusion dependent and on parenteral iron chelation since early childhood. Bone marrow cells were processed for CD34+ selection and transduced with the HPV569 LC. After busulfan conditioning the patient received an infusion of 3.9 x 106 cells/kg. After one year, the patient became transfusion independent and his haemoglobin was stable at about 8-9 g/dl after 7 years from gene therapy [57], [58], [59]. Integration site analysis revealed expansion of a single clone in which the provirus was inserted at the high mobility group AT-hook 2. This benign clonal dominance persisted for 9 years after which it started to decline. In 2011, another patient underwent transplantation but the vector copy number was lower and this patient is still transfusion dependent [59].
To achieve higher transduction efficacy the original LV was further improved. This was done by the removal of the cHS4 from HPV569 LV and this yielded
The HGB-204 clinical trial started in 2012 and completed in February 2018. Data from up to 5 years of follow-up from the completed Phase 1/2 as of December, 2019 (ASH, 2019) it was shown that 8 out of 10 treated patients who did not have a β0/β0 genotype achieved and continued to maintain transfusion independency for up to 51.3 months with a median Hb of 10.3 g/dL. In the other two patients, who did not achieve transfusion independency, the transfusion volumes were reduced by 79% and 52%. Three out of 8 patients that were β0/β0 achieved and continued to maintain transfusion independence up to 30.4 months with a median Hb of 9.9g/dL [61].
The clinical trial HGB-207 started in July 2016 and it is estimated to be completed in February 2022. Recruitment is completed, and 23 beta-thalassaemia transfusion dependent who are not β0/β0 genotype between the age of 4 and 34 were transfected with BB305 Lenti-globin after myeloablative conditioning with busulfan. As of June 2020 (EHA, 2020) 19 out of the 23 patients were evaluable for transfusion independence while the other 4 do not have sufficient follow up. Seventeen out of the 19 evaluable patients, achieved transfusion independence with median Hb levels of 11.9g/dL. Improved iron levels were also observed while over half of the patients stopped chelating therapy [62].
The other clinical trial using the BB305 Lenti-globin is HGB-212 which started in June 2017 and it is estimated to be completed by June 2022. As of June 2020 (EHA, 2020) 15 patients with different beta-genotypes were treated and had a median follow up of 14.4 months. Nine out of the 15 were β0/β0, 3 were β0/β+IVS1-110 and 3 IVS1-110 homozygous. Six of eight evaluable patients achieved transfusion independence with median Hb of 11.5 g/dL. 11 out of 13 patients with at least 7 months of follow up did not receive a transfusion [62].
In 2012, a trial opened at MSKCC in New York (NCT01639690) where four beta-thalassaemia transfusion dependent patients were treated with gene therapy using the TNS9.3.55 LV. This protocol differs from the other clinical trials with the BB305 LV because in this trial a reduced intensity conditioning was used rather than a fully myeloablative regimen. This led to insufficient gene marking with minimal clinical benefit [63], [64]. In 2015, the clinical trial NCT02453477 (TIGET BTHAL) started in Italy and it was completed in August 2019. Nine patients with different genotypes (β0/β0, β+/β+ and β0/β+) 3 adults followed by 6 minors were treated. This clinical trial was based on the autologous transplantation of G-CSF and plerixafor mobilized HSCs engineered by the GLOBE lentiviral vector. Following myeloablative conditioning by treosulfan and thiotepa the transduced cells were infused by intraosseous injection. As for April 2019, the three adult patients had reduction of transfusion requirement while 4 paediatric patients are transfusion independent and two are still receiving blood transfusions [65].
The main goal of gene therapy in beta-thalassaemia is to achieve stable introduction of functional globin genes in the patient’s own HSCs in order to abolish transfusions by correcting ineffective erythropoiesis and haemolytic anaemia. This can be achieved by taking into consideration a number of critical issues such:
Source of haematopoietic stem/progenitor cells
Nowadays, the preferred source for many autologous and allogenic transplantation approaches is peripheral blood mobilized HSCs. When compared to conventional bone marrow harvest, this minimally invasive procedure provides several-fold higher numbers of HSCs [66], [67]. In adult patients, HSC procurement is critical since the minimal target dose of 2-3 x106 CD34cells/kg poses a challenge for steady-state bone-marrow. Also, favourable gene therapy results are correlated with the dose of transfused cells infused and engrafted [44]. The growth factor granulocyte-colony stimulating factor (G-CSF), was the standard agent used to mobilize HSCs for transplantation. This stimulating factor, results in rapid mobilization within hours following administration [68]. Although it is known to be well-tolerated it raised concerns for its safety in thalassaemia due to the rare events of splenic rupture or thrombosis during mobilization in normal donors and in patients with haematological malignancies [69], [70], [71].
Plerixafor (AMD3100, MozbilTM) a bicyclam molecule that mobilizes HSCs by selectively and reversibly antagonizing the binding of stromal cell derived factor-1 to chemokine CXC receptor-4 (CXCR4) is considered as an alternative mobilization agent [72]. In conditions resulting in poor mobilization, this agent is approved by FDA and European Medical Agency (EMA) to be used together with G-CSF [73]. A number of mobilization trials took place to define the optimal mobilization approach for adult patients with beta-thalassaemia before implementation of gene therapy. From these trials it was established that the optimal mobilization approach for these patients is either Plerixafor alone or in combination with G-CSF [74], [75], [76], [77].
Dose of transduced HSCs and transduction methods
Two important factors that might affect gene therapy outcome are the transduced cell dose as well as the stem cell source. Determining the dose of the optimal genetically modified cells to transduce still remains unclear. Based on the results available until now from clinical trials we still do not have a direct correlation between transduced cell dose and clinical outcome. In clinical trials, beta-thalassaemia patients have been treated with different cell doses and the vector/copy number in drug products is also variable. Other different variables such as severity of genotype/phenotype, comorbidities, secondary modifiers of the pathology, proportion of engrafting genetically modified long-term analysis may also influence the clinical trial. From clinical trials, preliminary indications show that patients that receive the highest dose of transduce with the highest vector/copy number should have the better outcome [44].
A near-complete transduction of the purified HSCs with LV can be achieved by adding Prostaglandin E2(PGE2). In less than 38 hours, it was shown that PGE2 allows near-complete transduction of HSCs with LV [78]. In vitro , the addition of PGE2 was shown to increase VCN and/or transduction efficiency of CD34+ cells [79]. When compared to control transduced cells, addition of PGE2 increased vector transduction of CD34+ cells approximately to 2-fold [80]. Addition of rapamycin which is an inhibitor of the mammalian mTOR pathway also resulted in significantly enhanced transduction without alterations in lentiviral integration profile [81],[82].
Bone-marrow conditioning and HSC administration
Before administration of transduced HSCs, conditioning of the bone marrow must take place. Since beta-thalassaemia is a non-malignant disease, it requires complete elimination of endogenous HSCs to confer therapy. Initially, it was discussed whether a partial or full myeloablation is needed. Non-myeloablation treatment is preferred to conventional fully myeloablation due to the high risk of non-haematological toxicity but it can lead to mixed chimerism. Also, despite the encouraging engraftment rates in mouse-models achieved under non-myeloablative settings with various vectors, this preparative regimen could not be used in vivo [83], [84], [85]. Most of the clinical trials for gene-therapy in beta-thalassaemia nowadays, are using reduced intensity conditioning with Busulfan. The MSKCC trial used Busulfan at 8mg/kg while the other clinical trials with the either HPV569 LV or BB305 LV used Busulfan 12.8mg/kg. The clinical trial of TIGET-BTHAL conditioning was done by Treosulfan 42g/m2and Thiotepa 8mg/kg.
Following myeloablative treatment, administration of the HSCs is usually given by intravenous injection. The systematic delivery via peripheral circulation is an easy procedure for the dissemination of transplanted cells that home and engraft in the bone marrow niche. On the other hand, one of the drawbacks of this administration, is the loss of a significant proportion of injected cells. These cells are lost due to the major filter organs trapping such as the lungs and spleen. The TIGET BTAL clinical trial used an alternative route of administration to overcome cell loss. In this clinical trial the transduced cells were delivered by intraosseous injection bilaterally in the iliac crests [44].
Gene editing
An innovative approach for treating β-thalassaemia is gene editing. In the last decades, several nucleases such as zinc-finger nucleases (ZFNs), transcription-activator-like effector nucleases (TALENs) were developed for genetic engineering with CRISRP/CAS9 system being the most novel approach. The CRISPR/Cas9 system uses a single or multiple short guide RNAs (gRNA) with 20bp complementary to DNA sequence target. The nucleases will than create site specific double strand breaks which can be repaired either by non-homologous end joining (NHEJ) or by homologous directed repair (HDR) [86], [87].
In-vitro , in the context of β-haemoglobinopathies a number of gene editing strategies have been successful. These include correction of the β-globin gene mutation [88], [89], [90] or induction of endogenous foetal haemoglobin [91]. A recent study used the CRISPR/Cas9 system to mutate a 13-nucleotide sequence in the promoters of the gamma genes. This was done via microhomoglogy-mediated end joining leading to HbF increase to potentially therapeutic levels [92]. Although many labs have been successfully performing gene editing of immortalized cells, gene editing of primary cells remains a challenge due to toxicity caused by gene-editing reagents, off-target mutations and low efficiency of transfection [93].
In the past 5 years, gene correction of patient-specific induced pluripotent stem cells (iPSCs) by CRISPR/CAS9 and cell transplantation has appeared as a promising therapeutic solution for many diseases including β-thalassemia [94], [95]. In 2019, the group of Bauer at the Dana-Farber/Boston Children’s Cancer and Blood Disorders Centre at the University of Massachuseets Medical School edited CD34+ HSPCs from seven patients with β-thalassaemia of varying genotypes using CRISPR-CAS9 gene editing. After overcoming technical challenges associated with editing of blood stem cells they have demonstrated that CAS9:single guide RNA ribonucleoprotein (RNP)-mediated cleavage within a GATA1 binding site at the +58 BCL11A erythroid enhancer resulted in a highly penetrant disruption of this motif. The reduced BCL11A expression gave rise to an increase in gamma-globin levels between 35.3 and 75.1% and an increase in gamma protein levels between 27.5 and 46.9% [96]. In the context of beta-thalassaemia two clinical trials one utilizing a CRISPR/CAS9 system and one implementing ZFN are currently in the recruitment phase. The clinical trial NCT03655678 is a phase 1/2 study of the safety and efficacy of a single dose of autologous CRISPR-Cas9 Modified CD34+ HPSPc in 45 subjects with Transfusion-Dependent beta thalassaemia. The other clinical trial, NCT03432364, will assess ST-400 in 6 subjects with transfusion-dependent beta-thalassaemia. ST-100 will be composed of the patient’s own blood stem cells which are genetically modified in the lab using Sangamo’s zinc finger nuclease technology to disrupt a precise and specific sequence of the enhancer of the BCL11A.
As can be seen from this review, β-thalassaemia has offered a very robust model on which novel therapies and treatments can be explored with a great impact that holds promise on a clinical level. In conclusion, whether an approach to re-induce high levels of HbF in vivo, or a gene editing approach that amount to gene replacement or correction it is now envisaged that such activities are all fruitful approaches to augment HbF levels in individuals with β-type haemoglobinopathies.
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