b-thalassemia is a hereditary disorder caused by defective production of b-globin chains of hemoglobin (Hb) that leads to an increased a/b globins ratio with subsequent free a-globins. Alpha globin ...excess causes oxidative stress, RBCs membrane damage, premature death of late-stage erythroid precursors, resulting in ineffective erythropoiesis.
The transforming growth factor b (TGF-b) superfamily signaling acts on biological processes, such as cell quiescence, apoptosis, proliferation, differentiation, and migration, and also plays an important role in the regulation of hematopoiesis. This pathway can lose its physiologic regulation in pathologic conditions, leading to anemia and ineffective erythropoiesis. Activin receptor ligand trap molecules such as Sotatercept and Luspatercept downregulate the TGF-b pathway by inhibiting the Smad2/3 cascade, thus alleviating anemia in patients with b-thalassemia and myelodysplastic syndromes.
In this review, we describe in extenso the TGF-b pathway, as well as the molecular and biological basis of activin receptors ligand traps, focusing on their role in various b-thalassemia experimental models. The most recent results from clinical trials on sotatercept and luspatercept will also be reviewed.
Juvenile hemochromatosis (JH) is a rare autosomal recessive disorder of iron metabolism, genetically heterogeneous. In JH, symptomatic organ involvement occurs as early as the second decade of life. ...Heart failure and/or arrhythmias are the most frequent causes of death. Phlebotomy is the safest, most effective, and most economic therapeutic approach in hemochromatosis patients but is not indicated during the treatment of severe congestive heart failure with unstable hemodynamic status. The treatment of iron overload in these prohibitive clinical situations has to be carried out using iron chelators. We report a case of heart failure in the setting of unrecognized juvenile hemochromatosis successfully treated by the simultaneous administration of deferoxamine and deferiprone. To our knowledge, this is the first patient affected by JH treated with combined chelation regimen.
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.
Gaucher disease type 1 is the most common inherited lysosomal storage disorder caused by the deficiency of lysosomal β-glucocerebrosidase (GBA, acid-β-glucosidase), required for the degradation of ...glycosphingolipids. The deficiency of the enzyme results in the widespread accumulation of glucosylceramide in macrophages, leading to anemia, thrombocytopenia and coagulopathy, visceral (hepatosplenomegaly, lungs) and skeletal manifestations (deformities, fractures, avascular osteonecrosis). However, GD manifestations are caused not only by the burden of glucosylceramide storage, but also by macrophage activation. It seems that GD reflects the downstream consequences of inappropriate macrophage activation, with the release of pro-inflammatory cytokines and other responses to storage material.
The aim of this study is to assess in vitro phenotypic characterization, functional properties and gene expression anaysis of GD1 macrophages in order to understand their possible role in inflammation and in impairment of iron metabolism occurring in GD patients.
Monocytes were isolated from buffy coats of GD patients (n=3) and controls (n=3) by applying an in vitro model protocol. Monocytes were expanded in ImmunoCult™ medium and 1% ZellShield® antibiotic cocktail. Differentiation was induced in ImmunoCult™ medium, 50 ng/mL M-CSF and 50 ng/mL GM-CSF. To mimic the in vivo condition, macrophage population was loaded with erythrocytes ghosts isolated from GD patients. Cell morphology was analyzed on cytocentrifuged preparations stained with May-Grünwald Giemsa (MGG). Surface marker expression (CD11, CD33, CD68, CD64) were examined by flow cytometry to evaluate macrophages differentiation and phenotype. Gene expression analysis of iron metabolism-related genes was evaluated through Real-Time Quantitative PCR. Biochemical indices (NTBI, GDF15, sTfR and chitotriosidase) were analyzed in supernatant through ELISA assay.
Flow cytometry analysis (Fig.1) revealed that without erythrocytes ghosts (preM∅), the proportion of CD11+/CD68+ macrophage population was similar between GD patients and control. However, in GD macrophages, when loaded with Gaucher erythrocytes ghosts (M∅+ghosts), the proportion of CD11++/CD68++ cells increased (36.4%), as a reflection of a more pro-inflammatory phenotype. Treated controls showed no differences. To further characterize these different macrophages subpopulations in GD patients, we used an additional parameter, CD64, because CD64/CD68 markers are specific for M1/M2 polarization. GD M∅+ghosts showed an higher percentage of CD64++/CD68++ population (78%) in comparison of GD pre-M∅ (45%) and controls (36%), confirming a M1 proinflammatory phenotype of GD macrophages loaded with erythrocytes ghosts. Under microscope evaluation, GD M∅+ghosts presented high number of spindle-shaped fibroblastoid, typically M1 phenotype cells, rather than large flat-round cells (M2 cells). Moreover, morphological staining of these cells confirmed the typical features of GD cells with basophilic cytoplasm with characteristic crinkles. Controls showed no differences in macrophage features when loaded with erythrocytes ghosts. To confirm the pro-inflammatory potential of GD M∅+ghosts, high levels of pro-inflammatory mediators TNF-α, IL-1β and (s)TfR) were found in supernatant of GD M∅+ghosts (72,9±8,5, 24,7±2,7 and 3,1±4,4, respectively) compared to pre-M∅ and controls. High GDF15 expression in GD M∅+ghosts (14,77±5,87) respect to preM∅ (1,80±1,1) and controls (1,67±0,8) was observed. By gene expression analysis we observed in GD M∅+ghosts an higher HAMP expression (28,13±5,63) compared to preM∅ (2,49±1,5) and controls (0,71±0,02), and a lower SLC40A1 expression (0,01±0,00) compared to GD preM∅ (0,16±0,05) and controls (0,79±0,25). No significant differences in TFRC and GDF11 expression between GD preM∅ and M∅+ghosts in GD patients were observed.
These preliminary data suggest that GD macrophages, when stimulated, display a proinflammatory potential. These activated M1 macrophages could contribute to an inflammatory-producing environment, triggering hepcidin and ferroportin expression by an autocrine/paracrine mechanism and leading to dysregulation of iron distribution.
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Cappellini:CRISPR Therapeutics: Membership on an entity's Board of Directors or advisory committees; Vifor Pharmaceutical: 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; Genzyme/Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees. Motta:Sanofi-Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees.
β-thalassemia is a hereditary disorder caused by defective production of β-globin chains of hemoglobin (Hb) that leads to an increased α/β globins ratio with subsequent free α-globins. Alpha globin ...excess causes oxidative stress, red blood cells membrane damage, premature death of late-stage erythroid precursors, resulting in ineffective erythropoiesis.
The transforming growth factor β (TGF-β) superfamily signaling acts on biological processes, such as cell quiescence, apoptosis, proliferation, differentiation, and migration, and plays an essential role in regulating the hematopoiesis. This pathway can lose its physiologic regulation in pathologic conditions, leading to anemia and ineffective erythropoiesis. Activin receptor-ligand trap molecules such as Sotatercept and Luspatercept downregulate the TGF-β pathway, thus inhibiting the Smad2/3 cascade and alleviating anemia in patients with β-thalassemia and myelodysplastic syndromes.
In this review, we describe
in extenso
the TGF-β pathway, as well as the molecular and biological basis of activin receptors ligand traps, focusing on their role in various β-thalassemia experimental models. The most recent results from clinical trials on sotatercept and luspatercept will also be reviewed.
In β-thalassemia, accumulated free α-globin forms intracellular precipitates that impair erythroid cell maturation and viability. Protein quality control systems mitigate β-thalassemia ...pathophysiology by degrading toxic free α-globin, although the associated mechanisms are poorly understood. We show that loss of the autophagy-activating Unc-51-like kinase 1 (
) gene in β-thalassemic mice reduces autophagic clearance of α-globin in red blood cell precursors and exacerbates disease phenotypes, whereas inactivation of the canonical autophagy-related 5 (
) gene has relatively minor effects. Systemic treatment with the mTORC1 inhibitor rapamycin reduces α-globin precipitates and lessens pathologies in β-thalassemic mice via an ULK1-dependent pathway. Similarly, rapamycin reduces free α-globin accumulation in erythroblasts derived from CD34
cells of β-thalassemic individuals. Our findings define a drug-regulatable pathway for ameliorating β-thalassemia.
Introduction. Gaucher disease type 1 (GD, OMIM #230800) is the most common inherited lysosomal storage disorder resulting from lysosomal glucocerebrosidase (GBA, acid-β-glucosidase) deficiency and ...subsequent accumulation of glucosylceramide in macrophages. This leads to anemia, thrombocytopenia and coagulopathy, visceral (hepatosplenomegaly, lungs) and skeletal manifestations (deformities, fractures, avascular osteonecrosis). Anemia is attributed to bone marrow infiltration by Gaucher cells, to inflammation and to the presence of splenomegaly; however the underlying pathophysiological mechanism hasn't been fully elucidated.
Aim. The aim of this study is to evaluate the in vitro erythroid status at baseline (T0) and the response to Enzyme Replacement Therapy (ERT) after 6 months (T1) and 1 year (T2). We evaluated the expansion and differentiation in erythroid cultures of CD34+ cells obtained from peripheral blood of GD type 1 patients at diagnosis before starting ERT (T0), at 6 months (T1) and at 1 year (T2) of ERT.
Methods. CD34+ cells were obtained by immunomagnetic separation from peripheral blood mononuclear cells of GD patients (n=3) and controls (n=5). Isolated CD34+ were evaluated by in vitro clonogenic capacity assays using the methylcellulose semisolid culture medium after 14-16 days of culture. Hematopoietic colonies (burst-forming unit-erythroid (BFU-E) and colony-forming unit-macrophage (CFU-M)) were scored with an inverted microscope. CD34+ cells were expanded in StemSpan medium, 1% StemSpan CC100 cytokine cocktail, 2 U/ml Erythropoietin (EPO) and 1 μM dexamethasone. Differentiation was induced in StemSpan medium with 10 ng/ml SCF and 10 U/ml EPO. Proliferation and surface marker expression (CD34, CD45, Glycophorine A, CD71) were examined by flow cytometry. Cell morphology was analyzed on cytocentrifuged smears stained with May-Grunwald-Giemsa.
Results. At T0 we observed a higher number of BFU-E in GD samples (126±23 BFU-E/105 cells) compared to controls (49±12 BFU-E/105 cells) (Tab.1). Similarly, higher macrophage CFU-M were observed in GD samples (29±12 CFU-M/105 cells) compared to controls (10±1 CFU-M/105 cells) (Tab.1). The BFU-E and CFU-M morphology showed no difference between GD patients and controls. The increased proliferation of erythroid precursors of GD patients was associated to impaired ability to differentiate in liquid cultures after 14 days (45,9% proerythroblasts and only 4% polichromatophilic erythroblasts) compared to controls (27% and 19%, respectively) (Tab.1). Flow cytometry confirmed an impaired differentiation of GD erythroblasts compared to controls at day 14 of the erythroid liquid cultures, as shown by high percentage of CD71high/GlyAlow early erythroid precursor cells and low percentage of CD71high/GlyAhigh mature erythroblasts (GD: 73,9 and 4,9%, respectively; controls: 67,2 and 22.4%, respectively) (Fig.1). In samples from GD patients, after 1-year treatment with ERT (T2), we observed a reduction in the number of BFU-E compared to T0, reaching values similar to controls (GD T0: 126±23 BFU-E/105 cells, T2: 41±11 BFU-E/105 cells; controls: 49±12 BFU-E/105 cells) and an increased number of mature erythroid cells generated in GD liquid cultures (Tab. 1). Also CFU-M were reduced at T2 compared to T0 (GD T0: 29±12 CFU-M/105 cells, T2: 8±5 CFU-M/105 cells; controls: 10±1 CFU-M/105 cells) (Tab.1). An increased percentage of more differentiated erythroid cells at T2 compared to T0 was confirmed by flow cytometry in cultures from GD patients, as shown by low percentage of the CD71high/GlyAlow early erythroid precursor cells and high percentage of CD71high/GlyAhigh mature erythroblasts on day 14 (GD: 44,7 and 45,6%, respectively; controls: 67,2 and 22.4%, respectively) (Fig.1).
Conclusions. These preliminary data suggest that anemia in GD might partly be related to a delay in erythroid differentiation. ERT treatment seems to correct the dyserythropoiesis by improving erythroid differentiation. Since glucosylceramide accumulates in macrophages, that are key elements in the erythroid niche, impaired erythroid differentiation might be related to the pathological micro-environment, rather than intrinsic hematopoietic stem cell defect. More extensive studies are needed to better understand the pathophysiology of anemia and pancytopenia of this rare disease.This study was partially supported by Sanofi Genzyme.
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Cappellini:Celgene: Honoraria; Novartis: Speakers Bureau; Vifor: Honoraria; Sanofi-Genzyme: Honoraria, Research Funding, Speakers Bureau.
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.
Summary
Transmembrane Protease, Serine 6 (TMPRSS6) has an important role in iron homeostasis and its mutations, performed in TMPRSS6‐deficient mice, have been recently associated with iron‐refractory ...iron deficiency anaemia (IRIDA). Several variants of TMPRSS6 have been already identified; however the role of polymorphisms and TMPRSS6 haplotypes, causing iron deficiency anaemia, have not yet been investigated. This study sequenced the TMPRSS6 gene in 16 subjects with IRIDA phenotype and identified 27 DNA polymorphisms. Eight single nucleotide polymorphisms and four haplotypes were significantly associated with iron‐refractory anaemia (P < 0·001). Our preliminary results suggest a possible association between specific haplotypes of TMPRSS6 and IRIDA.
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