The development of hematopoietic stem cell (HSCs) gene therapy for DNA repair disorders, such as Fanconi anemia and Bloom syndrome, is challenging because of the induction of HSCs apoptosis by ...cytokine stimulation. Although the Baboon envelope pseudotyped lentiviral vector (BaEV-Rless-LV) has been reported as a non-stimulatory gene transfer tool, the virus titer of BaEV-Rless-LV is too low for use in clinical applications. Transfected 293 T cells with helper plasmids, including the BaEV-Rless plasmid, showed morphological changes, such as syncytium formation and detachment. To establish a novel protocol for producing a high titer of BaEV-Rless-LV, we optimized three aspects of a basic virus production protocol by focusing on modifying culture conditions and the use of reagents: the virus titer increased 3-fold when the amount of BaEV-Rless plasmid was increased 1.2-fold; the highest titer was obtained when the viral supernatant was harvested at 48-h post-transfection, despite complete syncytium formation and detachment of the 293 T cells; and the use of poly-L-lysine-coated culture plates to enhance the adhesion and proliferation of 293 T cells and prevent detachment doubled the titer. Collectively, our novel protocol resulted in a 10-fold titer increase compared to the basic protocol and may be useful in clinical applications for treating DNA repair disorders.
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•Hematopoietic stem cell gene therapy for DNA repair disorders is challenging.•BaEV-Rless pseudotyped lentiviral vector could solve the issue by cytokine depletion.•Although the titer is too low, novel virus production protocol is needed.•We modified protocol focusing on syncytium and detachment of transfected 293 T cells.•Our novel protocol resulted in a 10-fold titer increase compared to basic protocol.
Inherited hemoglobin disorders, including beta-thalassemia (BT) and sickle-cell disease (SCD) are the most common monogenic diseases worldwide, with a global carrier frequency of over 5%. With ...migration they are becoming more common worldwide, making their management and care an increasing concern for health care systems.
BT is characterized by an imbalance in the α/β-globin chain ratio, ineffective erythropoiesis, chronic hemolytic anemia, and compensatory haemopoietic expansion. Globally, there are over 25,000 births each year with transfusion-dependent thalassemia (TDT). The current available treatment for TDT is lifelong transfusions and iron chelation therapy or allogenic bone marrow as curative option. SCD affects 300 million people worldwide and severely impacts the quality of life of patients, who experience unpredictable, recurrent acute and chronic severe pain, stroke, infections, pulmonary disease, kidney disease, retinopathy, and other complications. While survival has been dramatically extended, quality of life is markedly reduced by disease- and treatment-associated morbidity.
The development of safe, tissue specific and efficient vectors, and efficient gene editing technologies have led to the development of several gene therapy trials for BT and SCD. Yet, the complexity of the approach presents its hurdles. Fundamental factors at play include the requirement for myeloablation on a patient with a benign disease, the age of the patient and consequent bone marrow microenvironment. A successful path from proof-of-concept studies to commercialization must render gene therapy a sustainable and accessible approach for a large number of patients. Furthermore, the cost of these therapies is a considerable challenge for the health care system. While new promising therapeutic options are emerging and many others are on the pipeline5, gene therapy can potentially cure patients. We herein provide an overview of the most recent potentially curative therapies for hemoglobinopathies and a summary of the challenges that these approaches entail.
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.
Fe-S clusters are essential cofactors for mitochondria functions, and mitochondria are required for Fe-S cluster synthesis. Additionally, mitochondria biogenesis demands cellular iron uptake, which ...is negatively regulated by Fe-S clusters. Fe-S clusters are synthesized in the mitochondria and cytosol by two different machineries. However, cytosolic Fe-S cluster synthesis necessitates the mitochondrial Fe-S cluster assembly machinery.
PGC-1α is a transcriptional coactivator and a master regulator of mitochondria biogenesis. We confirmed that overexpression of PGC-1α in adipocytes and hepatocytes stimulated mitochondria biogenesis, as measured by Mitotrack Green and Deep Red staining, which label total and alive mitochondria, respectively. We further measured Fe-S cluster synthesis by monitoring the gene expression of Fe-S cluster assembly machinery. By using RT-qPCR and Western Blot analyses, we confirmed that PGC-1α expression increases expression of ABCB7, ISCA1, ISCA2, ISD11, Nfu1 and ISCU, components of the Fe-S assembly machinery, suggesting a coordination between mitochondria biogenesis and Fe-S cluster synthesis.
Iron Regulatory Proteins (IRP1 and IRP2) control iron metabolism by binding to specific non-coding sequences within an mRNA, known as iron-responsive elements (IRE). In the absence of Fe-S clusters, IRP1 acts as an aconitase (aka ACO1), while IRP2 is degraded by ubiquitination. Aconitases, represented by the cytosolic form ACO1 and mitochondrial form ACO2, catalyze the isomerization of citrate to isocitrate and require Fe-S clusters to be enzymatically active. PGC-1α overexpression enhanced aconitase activity but not their protein levels, corroborating the notion that Fe-S cluster synthesis was increased.
To explore whether this coordination solely depends on PGC-1α, we evaluated the Fe-S cluster synthesis status during brown adipocyte maturation, which is characterized by enhanced mitochondria biogenesis and has been suggested to be PGC-1α-independent. We found that the synthesis of Fe-S cluster assembly machinery increased during maturation in both wild-type and PGC-1α-knockout brown adipocytes, indicating that Fe-S cluster synthesis coordinates with mitochondria biogenesis even in the absence of PGC-1α.
To explore the impact of Fe-S cluster synthesis on iron acquisition under enhanced mitochondria biogenesis, we evaluated the expression of the iron importer transferrin receptor 1 (TfR1). TfR1 mRNA contains IREs in the 3' untranslated region (UTR). These 3'UTR IREs can be bound by IRPs and responsible for the subsequent stabilization of TfR1 mRNA. Therefore, if IRP1 associates with Fe-S cluster and converted into ACO1, it is expected that both TfR1 mRNA and protein levels would decrease. In contrast, we found that stimulated Fe-S cluster synthesis increased levels of the TfR1 protein, despite reduced IRP1 activity and destabilized TfR1 mRNA. This suggests that Fe-S cluster synthesis coordinates with mitochondria biogenesis but does not block iron uptake.
Moreover, we extended our work to erythropoiesis by using murine erythroleukemia (MEL) cells. Stimulated mitochondria biogenesis enhanced expression of the Fe-S cluster assembly machinery and Fe-S cluster synthesis in these cells. TfR1 protein levels were increased despite elevated Fe-S cluster synthesis and reduced IRP activity. We also found increases in heme levels and the expression of aminolevulinic acid synthase 2 (ALAS2), the rate-limiting enzyme for erythroid heme synthesis. Of note, the ALAS2 mRNA contains IRE at the 5'UTR; binding of IRPs to the IRE inhibits translation while high Fe-S cluster levels lead to release. Moreover, as α- and β-globins chain expression is stimulated by increased heme availability, we also observed that mitochondria biogenesis was associated with increased synthesis of these two proteins and hemoglobinization. These data suggests that erythroid heme synthesis, hemoglobin expression and hemoglobinization coordinates with mitochondria biogenesis via Fe-S cluster synthesis.
In conclusion, our data show that Fe-S cluster synthesis coordinates with mitochondria biogenesis but does not block cellular iron uptake, thus suggesting a potential unidentified iron regulator to ensure adequate iron for mitochondria biogenesis. Moreover, our work suggests a mechanism underlying the essential role of mitochondria biogenesis in erythropoiesis.
Rivella:Disc Medicine: Consultancy; MeiraGTx: Other: SAB; Ionis Pharmaceuticals, Inc: Consultancy; Protagonist: Consultancy.
Inherited monogenic disorders such as beta-hemoglobinopathies (BH) are fitting candidates for treatment via gene therapy by gene transfer or gene editing. The reported safety and efficacy of ...lentiviral vectors in preclinical studies have led to the development of several clinical trials for the addition of a functional beta-globin gene. Across trials, dozens of transfusion-dependent patients with sickle cell disease (SCD) and transfusion-dependent beta-thalassemia (TDT) have been treated via gene therapy and have achieved reduced transfusion requirements. While overall results are encouraging, the outcomes appear to be strongly influenced by the level of lentiviral integration in transduced cells after engraftment, as well as the underlying genotype resulting in thalassemia. In addition, the method of procurement of hematopoietic stem cells can affect their quality and thus the outcome of gene therapy both in SCD and TDT. This suggests that new studies aimed at maximizing the number of corrected cells with long-term self-renewal potential are crucial to ensure successful treatment for every patient. Recent advancements in gene transfer and bone marrow transplantation have improved the success of this approach, and the results obtained by using these strategies demonstrated significant improvement of gene transfer outcome in patients. The advent of new gene-editing technologies has suggested additional therapeutic options. These are primarily focused on correcting the defective beta-globin gene or editing the expression of genes or genomic segments that regulate fetal hemoglobin synthesis. In this review, we aim to establish the potential benefits of gene therapy for BH, to summarize the status of the ongoing trials, and to discuss the possible improvement or direction for future treatments.
Iron-sulfur (Fe-S) clusters are required for mitochondrial function. Fe-S cluster synthesis occurs in the mitochondria and iron uptake is required for mitochondrial biogenesis. However, Fe-S clusters ...inhibit the expression of the iron importer transferrin receptor 1 (TfR1), whereas lack of the Fe-S cluster stimulates TfR1 expression. Yet, it is unclear whether Fe-S cluster synthesis increases with mitochondria biogenesis and, in turn, whether this negatively modulates TfR1 expression. We manipulated peroxisome proliferator-activated receptor-gamma coactivator-1α expression to control mitochondrial biogenesis in a variety of cell types, including erythroid cells. We demonstrated that Fe-S cluster synthesis increases with mitochondria biogenesis but does not interfere with increasing TfR1 expression. In fact, TfR1 expression is stimulated through alternative means to meet iron requirement for mitochondria biogenesis. Furthermore, under enhanced mitochondria biogenesis, increased Fe-S cluster synthesis inhibits the function of iron-regulating protein (IRP)1 and hence stimulates the expression of 5'-aminolevulinate synthase 2 (ALAS2), a target of IRP1 and rate-limiting enzyme in erythroid heme biogenesis. Increased ALAS2 expression leads to enhanced heme production, hemoglobinization, and erythropoiesis. Therefore, our study also provides a mechanism to link mitochondrial biogenesis with erythropoiesis and has a potential therapeutic value in the treatment of blood disorders.
Alpha thalassemia (α-thal) is caused by insufficient production of the α-globin protein because of either deletional or non-deletional inactivation of endogenous α-globin genes. Clinical presentation ...of deletional α-thal varies from an asymptomatic condition (one inactivated α-globin gene) to a complete knockout (Hb Bart's Hydrops Fetalis). In patients with severe α-thal, a blood transfusion independent state is achievable through allogeneic bone marrow transplantation.
The aims of this study are to develop a novel adult mouse model of α-thal and a gene therapy approach for this disease.
We generated adult animals that do not produce α-globin chains (α-KO) through transplantation of homozygous B6.129S7-Hbatm1Paz/J fetal liver cells (FLC; isolated at E14.5) into WT recipient mice. These animals demonstrate a worsening phenotype, paradoxically showing elevated hematocrit, high reticulocyte count and a high number of red blood cells (RBC) which expressed only β-globin chains (HbH). RBC show aberrant morphology and aggregation of α- globin tetramers on RBC membranes. Due to severe inability of these RBC to deliver oxygen, the mice eventually succumb to anemia, showing splenomegaly and other organ pathologies, including vaso-occlusive events. These animals show iron deposition in the liver and kidney, in agreement with very low levels of hepcidin expression in the liver, and elevated erythropoietin (EPO) in the kidney.
Interestingly, α-KO embryos show lower numbers of FLC compared to WT embryos, lower frequency of engraftable hematopoietic stem cells (HSC; Lin-Sca-1+c-kit+CD48-), decreased clonogenic potential (fewer class 4 CFUs) and elevated erythroferrone. Lethally irradiated mice transplanted with FLC-KO require 5-6x as many cells as those transplanted with FLC-WT for recovery, further suggesting some level of engraftment impairment. Our current hypothesis is that excessive hypoxia in the embryos impairs HSC function and stem cell fitness. Additional assays are in progress to assess the nature of this impairment.
To generate a gene therapy tool to rescue these animals and eventually cure severe human α-thal patients, we screened multiple lentiviral vectors to identify the variant capable of producing the highest human α-globin protein per copy. The selection was conducted in murine erythroleukemia cells and human umbilical cord derived erythroid progenitor (HUDEP) cells, modified by knocking out all the human α-globin genes. We identified ALS20α, a vector where α-globin is under control of the β-globin promoter and its locus control region, as the most efficient vector. One copy of ALS20α produces exogenous α-globin at a level comparable to that produced by one endogenous α-globin gene. These results suggest that a relatively low VCN could result in dramatic therapeutic benefits. Transplantation of ALS20α transduced murine BM-KO results in correction of the disease phenotype in a dose-dependent manner. At VCN<1 we observe a delay in death proportional to the VCN value, while at VCN>1 we observe phenotypic normalization, including Hb, hepcidin and EPO levels.
We tested ALS20α in CD34 cells isolated from four patients with both deletional and non- deletional HbH disease. We measured the change of β/α-globin mRNA ratio (β/αR) and protein level by HPLC in erythroblasts derived from these cultures. For the specimen with mutational HbH, the initial β/αR matches that of healthy controls, as the mutations do not eliminate the ability for the gene to produce aberrant mRNA transcripts, and decreased with increasing VCN. Erythroblasts with deletional HbH have a β/αR approximately 3x higher than normal cells, decreasing in a dose dependent manner with increasing VCN. HPLC detection of HbH (β4), a hallmark of HbH disease, is observed in hemolysis products from all non-transduced α−thal erythroblasts. A ~50% reduction of HbH is detected in the very same specimens upon integration of ALS20α (VCN between 1 and 2).
In conclusion, we generated an adult mouse model of lethal α-thal and, in preliminary experiments, we rescue it with ALS20α. Furthermore, ALS20α successfully improves α-globin levels in patient cells. Further experiments are in progress to establish the consistency of our vector's expression in vivo, as well as to demonstrate its ability to transduce bona fide long-term HSCs.
Kattamis: Agios Pharmaceuticals: Consultancy; IONIS: Consultancy; VIFOR: Consultancy; CRISPR/Vertex: Consultancy, Honoraria; BMS/Celgene: Consultancy, Honoraria, Research Funding; Chiesi: Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Amgen: Consultancy. Rivella: Celgene Corporation: Consultancy; Keros Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Disc Medicine: Consultancy, Membership on an entity's Board of Directors or advisory committees; MeiraGTx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Forma Theraputics: Consultancy; Incyte: Consultancy; Ionis Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees.
-Thalassemia (AT) is one of the most commonly occurring inherited hematological diseases. However, few treatments are available, and allogeneic bone marrow transplantation (BMT) is the only ...available therapeutic option for patients with severe AT. Research into AT has remained limited due to a lack of adult mouse models, with severe AT typically resulting in in utero lethality. By using a lipid nanoparticle (LNP) targeting the receptor CD117 and delivering a Cre mRNA (mRNACreLNPCD117), we were able to delete floxed -globin genes at high efficiency in hematopoietic stem cells (HSC) ex vivo. These cells were then engrafted in the absence or presence of a novel α-globin expressing lentiviral vector (ALS20I). Myeloablated mice transplanted with mRNACreLNPCD117-treated HSC showed a complete knockout of -globin genes. They demonstrated a phenotype characterized by the synthesis of hemoglobin H (-tetramers, or HbH), aberrant erythropoiesis, and abnormal organ morphology, culminating in lethality approximately eight weeks following engraftment. Mice receiving mRNACreLNPCD117-treated HSC with at least one copy of ALS20I survived long-term with normalization of erythropoiesis, decreased the production of HbH, and ameliorated the abnormal organ morphology. Furthermore, we tested ALS20I in erythroid progenitors derived from -globin-KO CD34+ and cells isolated from patients with both deletional and non-deletional HbH disease, demonstrating improvement in -globin/-globin mRNA ratio and reduction in the formation of HbH by HPLC. Our results demonstrate the broad applicability of LNP for disease modeling, characterization of a novel severe mouse model of AT, and the efficacy of ALS20I for treating AT.
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