Beta-thalassemia and sickle cell anemia are two of the most common diseases related to the hemoglobin protein. In these diseases, the beta-globin gene is mutated, causing severe anemia and ...ineffective erythropoiesis. Patients can additionally present with a number of life-threatening co-morbidities, such as stroke or spontaneous fractures. Current treatment involves transfusion and iron chelation; allogeneic bone marrow transplant is the only curative option, but is limited by the availability of matching donors and graft-versus-host disease. As these two diseases are monogenic diseases, they make an attractive setting for gene therapy. Gene therapy aims to correct the mutated beta-globin gene or add back a functional copy of beta- or gamma-globin. Initial gene therapy work was done with oncoretroviral vectors, but has since shifted to lentiviral vectors. Currently, there are a few clinical trials underway to test the curative potential of some of these lentiviral vectors. This review will highlight the work done thus far, and present the challenges still facing gene therapy, such as genome toxicity concerns and achieving sufficient transgene expression to cure those with the most severe forms of thalassemia.
β-thalassemia is a disorder caused by altered hemoglobin protein synthesis and affects individuals worldwide. Severe forms of the disease, left untreated, can result in death before the age of 3 ...years (1). The standard of care consists of chronic and costly palliative treatment by blood transfusion combined with iron chelation. This dual approach suppresses anemia and reduces iron-related toxicities in patients. Allogeneic bone marrow transplant is an option, but limited by the availability of a highly compatible HSC donor. While gene therapy is been explored in several trials, its use is highly limited to developed regions with centers of excellence and well-established healthcare systems (2). Hence, there remains a tremendous unmet medical need to develop alternative treatment strategies for β-thalassemia (3). Occurrence of aberrant splicing is one of the processes that affects β-globin synthesis in β-thalassemia. The (C>G) IVS-2-745 is a splicing mutation within intron 2 of the β-globin gene. It leads to an aberrantly spliced mRNA that incorporates an intron fragment. This results in an in-frame premature termination codon that inhibits β-globin production. Here, we propose the use of uniform 2'-O-methoxyethyl (2'-MOE) splice switching oligos (SSOs) to reverse this aberrant splicing in the pre-mRNA. With these lead SSOs we show aberrant to wild type splice switching. This switching leads to an increase of adult hemoglobin (HbA) up to 80% in erythroid cells from patients with the IVS-2-745 mutation. Furthermore, we demonstrate a restoration of the balance between β-like- and α-globin chains, and up to an 87% reduction in toxic α-heme aggregates. While examining the potential benefit of 2'-MOE-SSOs in a mixed sickle-thalassemic phenotypic setting, we found reduced HbS synthesis and sickle cell formation due to HbA induction. In summary, 2'-MOE-SSOs are a promising therapy for forms of β-thalassemia caused by mutations leading to aberrant splicing.
Hemoglobinopathies are genetic inherited conditions that originate from the lack or malfunction of the hemoglobin (Hb) protein. Sickle cell disease (SCD) and thalassemia are the most common forms of ...these conditions. The severe anemia combined with complications that arise in the most affected patients raises the necessity for a cure to restore hemoglobin function. The current routine therapies for these conditions, namely transfusion and iron chelation, have significantly improved the quality of life in patients over the years, but still fail to address the underlying cause of the diseases. A curative option, allogeneic bone marrow transplantation is available, but limited by the availability of suitable donors and graft-vs-host disease. Gene therapy offers an alternative approach to cure patients with hemoglobinopathies and aims at the direct recovery of the hemoglobin function via globin gene transfer. In the last 2 decades, gene transfer tools based on lentiviral vector development have been significantly improved and proven curative in several animal models for SCD and thalassemia. As a result, clinical trials are in progress and 1 patient has been successfully treated with this approach. However, there are still frontiers to explore that might improve this approach: the stoichiometry between the transgenic hemoglobin and endogenous hemoglobin with respect to the different globin genetic mutations; donor cell sourcing, such as the use of induced pluripotent stem cells (iPSCs); and the use of safer gene insertion methods to prevent oncogenesis. With this review we will provide insights about (1) the different lentiviral gene therapy approaches in mouse models and human cells; (2) current and planned clinical trials; (3) hurdles to overcome for clinical trials, such as myeloablation toxicity, insertional oncogenesis, and high vector expression; and (4) future perspectives for gene therapy, including safe harbors and iPSCs technology.
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Introduction: Sickle cell disease (SCD) is a genetic red blood cell (RBC) disorder that causes chronic hemolytic anemia, progressive organ damage, and life-threatening acute complications such as ...painful vaso-occlusive crises. Allogeneic hematopoietic stem cell transplant (allo-HSCT) with myeloablative conditioning remains the only curative therapy for SCD but has several limitations including low donor availability and conditioning-related toxicity. Genetic modification of autologous hematopoietic system cells (HSCs) with reduced-intensity conditioning (RIC) using a high-potency drug product may address these limitations.
ARU-1801 is a gene therapy that uses a modified γ-globin lentiviral vector to produce HbF G16D within autologous CD34+ HSCs. Preclinical studies in SCD mice have shown the G16D mutation enables γ-globin G16D to bind α-globin with higher affinity; lentiviral transfer of γ-globin G16D resulted in 1.5-2x more HbF per vector copy number (VCN) compared to analogous wild-type γ-globin vector. Early studies also suggested HbF G16D may be more potent for anti-sickling than HbF, lowering reticulocyte counts in SCD mice to a greater extent at similar protein levels. We hypothesize ARU-1801 with RIC could lessen toxicities and resource utilization relative to myeloablative approaches, allowing expanded access to gene therapy for a broader group of SCD patients. Updated data from patients in the ongoing Phase 1/2 study (NCT02186418) including laboratory and clinical markers of efficacy are presented here.
Methods: Adults (18-45 years old) with severe SCD (defined by recurrent vaso-occlusive events VOE and acute chest syndrome) were screened for eligibility. Prior to ARU-1801 drug product (DP) infusion, all patients received a single IV dose of RIC melphalan (140 mg/m 2). Endpoints included measures of safety, engraftment, VCN, hemoglobin sub-fractions, and SCD-related outcomes. Patients were weaned off transfusions 3-6 months after DP infusion. Levels of anti-sickling globins (including HbF G16D) are presented as proportions of non-transfused total hemoglobin.
Results: As of 28 July 2021, four patients (mean, 26 19-35 years old) have been treated with ARU-1801 gene therapy for SCD with three patients followed for ≥12 months post-transplant. Transient neutropenia and thrombocytopenia were the predominant adverse events, lasting a median seven days each. There have been no other serious adverse events related to chemotherapy or ARU-1801 to date.
At 36 months post-transplant, Patient 1 has shown stable HbF expression (27%) and 64% F-cells. Patient 2 has maintained 14% HbF and 37% F-cells at 36 months despite lower engraftment of ARU-1801 due to renal hyperfiltration (eGFR = 200 mL/min/1.73 m 2) at time of conditioning, which resulted in lower melphalan exposure. Both patients saw marked improvements in SCD manifestations, including 93% and 85% fewer annualized VOEs, respectively, in the two years after receiving ARU-1801 gene therapy compared to two years prior.
Patient 3 received ARU-1801 manufactured with several process modifications (including improvements of HSC collection timing and lentiviral production) and has maintained 36% HbF at month 15 with pancellular distribution (96% F-cells). To date, Patient 3 has had no VOEs since ARU-1801 administration, representing 100% reduction from baseline.
Conclusion: Amelioration of SCD phenotype and engraftment of ARU-1801 gene-modified HSCs is possible with a single RIC dose of melphalan, as demonstrated in three patients. The first patient shows 27% HbF expression at three years, and 93% reduction in VOEs. The second patient had lower HSC engraftment due to below-target melphalan exposure (likely caused by renal hyperfiltration), with 14% HbF and 5% HbA2 at three years. Nonetheless, an 85% reduction in VOEs in Patient 2 demonstrates significant clinical benefit. Dose-adjusted melphalan has the potential to improve engraftment in SCD patients with renal hyperfiltration. Following manufacturing process improvements, the third patient has shown the highest HbF (36%) at one year, the highest F-cells (96%), and no VOEs since receiving ARU-1801. ARU-1801, with RIC melphalan conditioning, is a promising alternative to myeloablative transplants for achieving durable responses with a favorable safety profile in patients with severe SCD. Longer follow-up and additional patients will be presented.
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Asnani: Avicanna Ltd.: Research Funding; Aruvant Sciences: Research Funding. Lutzko: Aruvant Sciences: Patents & Royalties: preclinical vector development. Quinn: Forma Therapeutics: Consultancy; Emmaus Medical: Research Funding; Novo Nordisk: Consultancy; Aruvant: Research Funding. Lo: Aruvant Sciences: Current Employment. Little: Aruvant Sciences: Current Employment. Dong: Aruvant Sciences: Current Employment. Malik: Aruvant Sciences: Consultancy; Forma Therapeutics: Consultancy; Aruvant Sciences: Patents & Royalties; CSL Behring: Patents & Royalties.
Plerixafor was used for stem cell mobilization. Melphalan was used as chemotherapy conditioning prior to autologous transplant with ARU-1801
Ongoing clinical trials for treatment of beta-globinopathies by gene therapy involve the transfer of the beta-globin gene, which requires integration of three to four copies per genome in most target ...cells. This high proviral load may increase genome toxicity, potentially limiting the safety of this therapy and relegating its use to total body myeloablation. We hypothesized that introducing an additional hypersensitive site from the locus control region, the complete sequence of the second intron of the beta-globin gene, and the ankyrin insulator may enhance beta-globin expression. We identified a construct, ALS20, that synthesized significantly higher adult hemoglobin levels than those of other constructs currently used in clinical trials. These findings were confirmed in erythroblastic cell lines and in primary cells isolated from sickle cell disease patients. Bone marrow transplantation studies in beta-thalassemia mice revealed that ALS20 was curative at less than one copy per genome. Injection of human CD34+ cells transduced with ALS20 led to safe, long-term, and high polyclonal engraftment in xenograft experiments. Successful treatment of beta-globinopathies with ALS20 could potentially be achieved at less than two copies per genome, minimizing the risk of cytotoxic events and lowering the intensity of myeloablation.
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Increase of beta-globin expression by gene addition is critical to successfully curing patients with beta-thalassemia and sickle cell disease. Here, Breda and colleagues report genomic features that maximize transgene expression at low genome integration rates, preserving efficacy and safety and potentially reducing the burden of autologous bone marrow transplantation.
β-thalassemia is a disorder caused by altered hemoglobin protein synthesis which affects individuals worldwide. Severe forms of the disease, left untreated, can result in death before the age of 3 ...years.
1
The standard of care consists of chronic and costly palliative treatment by blood transfusion combined with iron chelation. This dual approach suppresses anemia and reduces iron-related toxicities in patients. Allogeneic bone marrow transplant is an option, but limited by the availability of a highly compatible hematopoietic stem cell donor. While gene therapy is being explored in several trials, its use is highly limited to developed regions with centers of excellence and well-established healthcare systems.
2
Hence, there remains a tremendous unmet medical need to develop alternative treatment strategies for b-thalassemia.
3
Occurrence of aberrant splicing is one of the processes that affects b-globin synthesis in b-thalassemia. The (C>G) IVS2-745 is a splicing mutation within intron 2 of the b-globin (
HBB
) gene. It leads to an aberrantly spliced mRNA that incorporates an intron fragment. This results in an in-frame premature termination codon that inhibits b-globin production. Here, we propose the use of uniform 2'-O-methoxyethyl (2'-MOE) splice switching oligos (SSO) to reverse this aberrant splicing in the pre-mRNA. With these SSO we show aberrant to wild-type splice switching. This switching leads to an increase of adult hemoglobin up to 80% in erythroid cells from patients with the IVS2-745
HBB
mutation. Furthermore, we demonstrate a restoration of the balance between b-like- and α-globin chains, and up to an 87% reduction in toxic heme aggregates. While examining the potential benefit of 2'-MOE-SSO in a mixed sickle-thalassemic phenotypic setting, we found reduced sickle hemoglobin synthesis and sickle cell formation due to HbA induction. In summary, 2'-MOE-SSO are a promising therapy for forms of b-thalassemia caused by mutations leading to aberrant splicing.
The β thalassemia trait is associated with over 300 mutations in the β-globin gene that lead to reduced (β+ allele) or absent (β0 allele) synthesis of the β globin chain. A subset of these mutations ...affect the canonic splicing of the β globin mRNA. Such mutations activate aberrant splice sites, which lead to an altered splicing pathway and consequently affects protein synthesis.
The (C>G) IVS-2-745 mutation is common in South Eastern Europe, Cyprus, Lebanon, India, Malaysia, and Indonesia. This mutation, located within intron 2 of the β-globin gene, creates an aberrant 5‘ splice site at nucleotide 745 of intron 2 and activates a cryptic 3‘ splice site within the same intron. Portions of the intronic sequence are incorrectly retained in the spliced mutant mRNA. The mutation results in a premature stop codon that prevents proper mRNA translation and causes a β‐globin deficiency, resulting in β‐thalassemia. The IVS-2-745 allele has the functional splice sites preserved, but produces a significantly reduced level of correctly spliced β-globin mRNA and results in only marginal synthesis of HbA. Therefore, the IVS-2-745 mutation in homozygosity leads to severe transfusion-dependent thalassemia major. Taking advantage of conserved canonical splice sites in defective β‐globin genes, such as IVS-2-745, recently developed approaches show that by targeting the aberrant splice sites it is possible to circumvent the aberrant splice site and restore the normal β-globin splicing pattern.
We sought to use uniform 2'-O-methoxyethyl (2'-MOE) splice switching oligos (SSOs) to reverse aberrant splicing in the pre-mRNA for the IVS-2-745 mutation. Using these SSOs, we show effective aberrant-to-wild-type splice switching. This leads to an increase in adult hemoglobin (HbA) by up to 80% in erythroid cells from patients with the IVS-2-745 mutation. Furthermore, we demonstrate a restoration of the balance between β-like- and α-globin chains, and up to an 87% reduction in α-heme aggregates. While examining the potential benefit of 2'-MOE-SSOs in a sickle/IVS-2-745-thalassemic genotype setting, we found that use of these oligos restored production of HbA and reduced HbS synthesis, which ultimately lessened cell sickling under hypoxic conditions. We confirmed increased WT β-globin expression in specimens treated with 2'-MOE-SSOs with semi- and quantitative methods (RT and Q-PCR), and further supported this evidence using a direct quantification method (ddPCR). Compared to treated specimens heterozygous for IVS-2-745 , homozygous specimens showed elevated WT HbA, reflecting the additive effect of targeting the aberrant splicing of both alleles as opposed to a single IVS-2-745 allele. In fact, while 2'-MOE-SSOs significantly reduced aberrant splicing, leading to a consequent 60% increase in HbA levels in specimens from patients with a β0/IVS-2-745 genotype, the same oligos produced a more robust effect in specimens with a homozygous IVS-2-745 genotype, resulting in an 80% increase in HbA levels. This level of increase could potentially be curative for patients with this particular genotype.
Moreover, we compared the effect of 2‘-MOE-SSOs treatment to a lentiviral vector carrying a WT β-globin gene. In this comparative assay, β0/IVS-2-745 cells treated with 2‘-MOE-SSOs or the lentivector (with 1.13 copies integrated per genome) lead to a similar increase in HbA (50%). This suggests that the oligo-based technology is a competitive approach and a viable alternative to gene addition therapy to overcome anemia in IVS-2-745 β-thalassemia.
In summary, 2'-MOE-SSOs are promising therapeutic tools for certain forms of β-thalassemia caused by aberrant splicing. Their ability to correct the underlying splicing defect offers a pharmacological treatment that is direct, specific, and accessible. In comparison, gene therapy approaches utilizing gene addition or editing are primarily available in advanced medical care environment resulting in an unfulfilled demand in regions where such conditions are not readily available. The restoration of target gene activity reported here suggests that this treatment strategy could be applicable to other forms of thalassemia resulting from mutations affecting splicing. This could have, with an effective method of delivery, potential clinical utility in helping patients reduce their transfusion dependence or even achieving transfusion independence.
Dong:Aruvant Sciences INC: Employment. Motta:Sanofi-Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees. Guo:Ionis Pharmaceutical, INC: Employment, Other: shareholders. Peralta:Ionis Pharmaceutical, Inc: Employment. Freier:Ionis Pharmaceuticals: Employment. Watt:Ionis Pharmaceuticals: Employment. Manwani:Novartis: Consultancy; Pfizer: Consultancy; GBT: Consultancy, Research Funding. Cappellini:Genzyme/Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Honoraria; Vifor Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; CRISPR Therapeutics: Membership on an entity's Board of Directors or advisory committees. Abdulmalik:The Children's Hospital of Philadelphia: Patents & Royalties: Provisional Patent. Rivella:Meira GTx, Ionis Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Disc medicine, Protagonist, LIPC, Meira GTx: Consultancy.
Beta-thalassemia is one of the most common diseases related to the hemoglobin protein. In this disease, the beta-globin gene is mutated, causing severe anemia and ineffective erythropoiesis. Patients ...can additionally present with a number of life-threatening co-morbidities, such as anemia and iron overload. Current treatment involves transfusion and iron chelation; allogeneic bone marrow transplant is the only curative option, but is limited by the availability of matching donors and graft-versus-host disease. As this diseases is a monogenic disease, it makes an attractive setting for genetic therapy at the RNA and DNA level. At the DNA level, gene therapy aims to correct the mutated beta-globin or add back a functional copy of beta-globin. Initial gene therapy work was done with oncoretroviral vectors, but has since shifted to lentiviral vectors. Currently, there are a few clinical trials and preclinical tests underway to test the curative potential of some of these lentiviral vectors, including our new gene therapy vector described here with the ankyrin insulator. IVS2-745 is a splicing mutation that occurs in intron 2 of the beta-globin gene. It creates an aberrantly spliced form that incorporates an extra exon and leads to a premature stop codon. Here we report novel uniform 2’-O-methoxyethyl (2’-MOE) splice switching oligos (SSOs) to reverse this aberrant splicing. Lead 2’-MOE SSOs were generated to redirect splicing in the IVS2-745 pre-mRNA. With these lead SSOs we have demonstrated aberrant to wild type splice switching. This switching led to an increase from 3-6% to up to 80% HbA in erythroid cells from thalassemic patients. Furthermore, we have demonstrated a restoration of the balance between beta-like chains and alpha chains, and up to an 87% reduction in toxic alpha-heme aggregates. While next examining the potential benefit of 2’MOE SSOs to patients with a sickle-thalassemic genotype, we observed reduced sickling as a result of 2’MOE SSO induced HbA. In summary, 2’MOE-SSOs are a promising therapy for splicing forms of beta-thalassemia. Their ability to functionally modulate the thalassemia phenotype by correcting the underlying splicing cause offers a pharmacological treatment that is both direct and specific.
Hemoglobinopathies are the most common inherited blood disorders. World Health Organization statistics show that in the Mediterranean, Eastern European, and Middle Eastern regions, frequencies range ...from 0.1 to 4.9/1000 of live births. The mutation known as IVS2-745 is relatively common in the regions of Spain, Jordan, Romania, and Serbia (Ithanet Database, http://www.ithanet.eu/db/ithamaps), reaching as high as 15-20% of beta-thalassemia mutations in these regions. The IVS2-745 is a splicing mutation that occurs in intron 2 of the beta-globin gene and results in an aberrantly spliced mRNA that incorporates an extra exon and premature stop codon. Here we report novel uniform 2'-O-methoxyethyl (2'-MOE) splice switching oligos (SSOs) that reverse the aberrant splicing and restore up to 80% adult hemoglobin (HbA) production in vitro.
Uniform 2'-MOE SSOs do not mediate RNase H degradation when they bind their targets; therefore, they can be used to redirect the splicing machinery and restore WT splicing. After generating mouse erythroleukemia cells that carry the human IVS2-745 mutated beta-globin gene, lead 2'-MOE SSOs targeting the 745 mRNA were raised against these cell lines. With these lead SSOs we have demonstrated aberrant 745 to WT splice switching in 5 patient samples. CD34+ cells were isolated from the blood of four 745/β0 compound heterozygotes and one 745/ 745 homozygote (Breda et al, PloS One 2012). After CD34+ expansion, cells were differentiated to the red cell lineage and treated via syringe loading or lipofectamine transfection with 2'-MOE SSOs. Up to 80% HbA protein production was restored with 2'MOE-SSO treatment in the 745 homozygote patient sample (Figure 1), and up to 60% HbA in multiple 745 compound heterozygote specimens. Compared to 3-6% HbA in scramble treated controls, this represents up to a 20-fold increase in HbA with treatment.
In addition to HbA production, we have shown improvement in other parameters characteristic in beta-thalassemia, such as the imbalance of alpha and beta chains and the accumulation of toxic alpha-only homotetramers. 2'MOE SSOs are able to reinstate balance of beta- to alpha-like chains, which resulted in a near elimination of toxic alpha-only homotetramers in the homozygote cell lysate as detected by HPLC (Figure 1). We further proved the benefit of 2'MOE SSOs in a 745/Sickle model system, where in vitro sickling was significantly reduced as a result of increased levels of HbA. To create this model system, we transduced CD34+ cells from a homozygous sickle patient specimen with a lentivirus expressing human IVS2-745 beta-globin. With vector copy numbers ~2, this system replicates what a single allele would do, as the 2 endogenous sickle alleles are equally matched. Upon differentiation and exposure to hypoxia, in vitro sickling was reduced by 50% in 2'MOE-SSO treated samples as compared to scramble controls (Figure 2).
In summary, 2'MOE-SSOs are a promising therapy for certain splicing forms of beta-thalassemia. Their ability to correct the underlying splicing cause offers a pharmacological treatment that is both direct and specific. As such, this therapy could help patients reduce their transfusion dependence or even reach transfusion independence.
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Guo:Ionis Pharmaceuticals: Employment, Equity Ownership. Peralta:Ionis Pharmacueticals: Employment. Cappellini:Celgene: Membership on an entity's Board of Directors or advisory committees; Genzyme-Sanofi: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.
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