Correction of a mouse model of sickle cell disease: lentiviral/antisickling β-globin gene transduction of unmobilized, purified hematopoietic stem cells

Blood ◽  
2003 ◽  
Vol 102 (13) ◽  
pp. 4312-4319 ◽  
Author(s):  
Dana N. Levasseur ◽  
Thomas M. Ryan ◽  
Kevin M. Pawlik ◽  
Tim M. Townes
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Globin Gene ◽  
Bone Marrow Stem Cells ◽  
Allogeneic Bone ◽  
Cell Disease ◽  
Long Term Treatment ◽  
Gene Therapy Protocol

AbstractAlthough sickle cell anemia was the first hereditary disease to be understood at the molecular level, there is still no adequate long-term treatment. Allogeneic bone marrow transplantation is the only available cure, but this procedure is limited to a minority of patients with an available, histocompatible donor. Autologous transplantation of bone marrow stem cells that are transduced with a stably expressed, antisickling globin gene would benefit a majority of patients with sickle cell disease. Therefore, the development of a gene therapy protocol that corrects the disease in an animal model and is directly translatable to human patients is critical. A method is described in which unmobilized, highly purified bone marrow stem cells are transduced with a minimum amount of self-inactivating (SIN) lentiviral vector containing a potent antisickling β-globin gene. These cells, which were transduced in the absence of cytokine stimulation, fully reconstitute irradiated recipients and correct the hemolytic anemia and organ pathology that characterize the disease in humans. The mean increase of hemoglobin concentration was 46 g/L (4.6 g/dL) and the average lentiviral copy number was 2.2; therefore, a 21-g/L /vector copy increase (2.1-g/dL) was achieved. This transduction protocol may be directly translatable to patients with sickle cell disease who cannot tolerate current bone marrow mobilization procedures and may not safely be exposed to large viral loads. (Blood. 2003;102:4312-4319)

Effects of Sickle Cell Disease on Hematopoietic Stem Cell Function and the Bone Marrow Microenvironment.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1227-1227
Author(s):  
Elisabeth H. Javazon ◽  
Leslie S. Kean ◽  
Jennifer Perry ◽  
Jessica Butler ◽  
David R. Archer
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Cell Function ◽  
Donor Cell ◽  
Cell Disease ◽  
Hematopoietic Stem ◽  
Cell Engraftment

Abstract Gene therapy and stem cell transplantation are attractive potential therapies for sickle cell disease (SCD). Previous studies have shown that the sickle environment is highly enriched for reactive oxygen species (ROS), but have not addressed whether or not the increased ROS may alter the bone marrow (BM) microenvironment or affect stem cell function. Using the Berkeley sickle mouse model, we examined the effects of sickle cell disease on hematopoietic stem cell function and the bone marrow microenvironment. We transplanted C57BL/6 (control) BM into C57BL/6 and homozygous sickle mice. Recipients received 2 × 106 BM cells and a conditioning regimen consisting of busulfan, anti-asialo GM1, and co-stimulation blockade (anti-CD40L and CTLA4-Ig). Following transplantation, sickle mice demonstrated increased donor cell engraftment in the peripheral blood compared to normal mice (58.3% vs. 33.1%, respectively). Similarly, BMT in a fully allogeneic system also resulted in enhanced engraftment in sickle recipients. Next we analyzed whether or not engraftment defects exist within the BM stem cell population of sickle mice. In vitro colony forming assays showed a significant decrease in progenitor colony formation in sickle compared to control BM. By flow cytometry, we determined that there was a significant decrease in the KSL (c-Kit+, Sca-1+, Lineage−) progenitor population within the BM of sickle mice. Cell cycle analysis of the KSL population demonstrated that significantly fewer sickle KSL cells were in G0 phase compared to control, suggesting that there are fewer quiescent stem cells in the BM of sickle mice. To assess the potential role of ROS and glutathione depletion in sickle mice, we tested the engraftment efficiency of KSL cells from untreated and n-acetyl-cysteine (NAC) treated control, hemizygous sickle (hemi), and sickle mice in a competitive repopulation experiment. Peripheral chimerism showed an engraftment defect from both hemizygous and homozygous sickle mice such that control KSL cells engrafted > hemi > sickle at a ratio of 1 : 0.4 : 0.25. Treatment with NAC for four months prior to transplantation partially restored KSL engraftment (control : hemi : sickle; 1 : 0.97 : 0.56 ). We have demonstrated that congenic and allogeneic BMT into sickle mice result in increased donor cell engraftment in the sickle recipients. Both the decreased number of KSL cells and the decreased percentage of quiescent KSL cells in the sickle mice indicate that more stem cells in the transgenic sickle mouse model are mobilized from the BM environment. The engraftment defect of sickle KSL cells that was partially ameliorated by NAC treatment suggests that an altered redox environment in sickle mice may contribute to the engraftment deficiencies that we observed.

Selective Inhibitors of Arginine Methyl Transferase 5 (PRMT5) As a Novel Treatment for β-Thalassemia and Sickle Cell Disease.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2129-2129 ◽  
Author(s):  
Ian Street ◽  
Brendon Monahan ◽  
Hendrik Falk ◽  
Elizabeth Allan ◽  
Ylva Bergman ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Bone Marrow ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Globin Gene ◽  
Cell Disease ◽  
Globin Mrna ◽  
Cpg Dinucleotides ◽  
Radiometric Assay ◽  
Adult Bone

Abstract Abstract 2129 The developmental switch in human β-like globin gene subtype from fetal (γ) to adult (β) that begins at birth foreshadows the onset of the hemoglobinopathies, β-thalassemia and sickle cell disease (SCD). In the clinical setting it is established that β-thalassemia and SCD patients with hereditary persistence of fetal hemoglobin mutations enjoy a significant amelioration of disease severity due to the continued expression of γ-globin. This has prompted the search for therapeutic strategies to reverse γ-globin gene silencing. Central to the mechanism of γ-gene silencing is DNA methylation, which marks critical CpG dinucleotides flanking the γ-gene transcriptional initiation site in adult bone marrow erythroid cells. These marks are established by recruitment of DNMT3A, a DNA methyltransferase, to the γ-globin promoter by protein arginine methyltransferase 5 (PRMT5)[Zhao Q et al. Nat Struct Mol Biol. 2009;16(3):304–311]. PRMT5 catalyses the symmetric dimethylation of arginine 3 of Histone 4 (H4R3me2), which serves as a template for direct binding of DNMT3A and the subsequent DNA methylation of the γ-gene promoter. Loss of PRMT5 or its enzymatic activity is sufficient to induce demethylation of the CpG dinucleotides and reactivation of γ-globin gene expression [Rank, G., et al. Blood, 116(9), 1585–92]. Based on these observations we hypothesize that small molecule inhibitors of PRMT5 activity could provide a beneficial treatment for β-thalassemia and SCD. To identify small molecule inhibitors of PRMT5 a high throughput screen (HTS) was performed. Both radiometric and non-radiometric assay formats were developed to support the screening campaign. The radiometric assay format measures the ability of PRMT5 purified from K562 cells to catalyse the labelling of a short peptide based on the N-terminal sequence of Histone H4 by 3H-Methyl-S-Adenosyl-L-methionine (SAM). In contrast, the non-radiometric assay format employs recombinant PRMT5/MEP50 and measures the production of S-adenosyl-L-homocysteine (SAH), which is generated by PRMT5-catalysed methylation of H4 peptide. SAH is measured with Transcreener EPIGEN” and the assay is formatted in 1536-well microtitre plates in a total assay volume of 4 μL. Using these assays, a chemical library of 350,000 lead-like molecules and known pharmacologically active agents was screened to identify inhibitors of PRMT5 methyltransferase activity. A number of compounds with low micromolar or submicromolar inhibitory activity were identified by the HTS campaign, and six were selected for re-synthesis. The inhibitory activity of five of the six compounds was confirmed. To provide an initial appraisal of inhibitor selectivity the five active compounds were subsequently tested against a panel of enzymes consisting of 23 protein and DNA methyltransferases and 12 kinases. These compounds were found to be remarkably selective PRMT5 inhibitors, inhibition of MLL4 being the only significant off-target activity noted for one of the scaffolds. We have established a critical path for selection and progression of new chemical analogues which entails testing the compounds for: i) inhibition of PRMT5, other protein methyl transferases and kinases; ii) the ability to induce expression of γ-globin mRNA in the K562 erythroleukemic cell line; iii) the ability to induce expression of γ-globin mRNA in adult bone marrow erythroid cells; and iv) the induction of γ-globin in vivo in β-YAC mice, a transgenic model which carries the 250-kb human globin locus. In parallel, the physicochemical, metabolism, and pharmacokinetic properties of the most promising compounds are also determined. Medicinal chemistry efforts have now produced molecules with > 100-fold increased inhibitory potency against PRMT5 compared to the original hits, and preliminary results indicate that the more potent compounds have the ability to induce γ-globin mRNA in our cell based models. These early results illustrate the potential of PRMT5 inhibitors as a novel approach for the treatment of β-thalassemia and sickle cell disease. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Graves’ Disease After Hematopoietic Allogeneic Bone Marrow Transplant in a Patient With Sickle Cell Disease

2021 ◽  
Vol 5 (Supplement_1) ◽  
pp. A930-A931
Author(s):  
Majid Alameri
Keyword(s):  
Bone Marrow ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Marrow Transplant ◽  
Graves Disease ◽  
Allogeneic Bone ◽  
Cell Disease ◽  
Free T4 ◽  
Post Transplant

Abstract Endocrinopathies are among the most recognized late complications post hematopoietic stem cell transplant (HSCT). Dysfunctions of hormonal axes including the hypothalamus, pituitary, gonads, thyroid and adrenals reported. Moreover, thyroid dysfunctions including thyroiditis, hypothyroidism and hyperthyroidism has been reported to develop 8-32 months after HSCT. We report a 27-year-old male with sickle cell disease diagnosed at age of 5. He had multiple painful vasoocclusive sickle crises treated with blood transfusions, folic acid and rituximab. At age of 21, he presented with sudden right sided weakness and slurred speech. Further investigations, including magnetic resonance imaging of brain revealed occlusion of the left middle cerebral artery resulting in ischemic infarction. Subsequently, he had multiple red blood cell exchange transfusions on regular basis. He remained with residual weakness and slurred speech after rehabilitation. Bone marrow transplant was recommended as a curative treatment for his sickle cell disease by haematology team. A year later, he underwent a geno-identical allogeneic bone marrow transplantation harvested from his brother. He remained well for 22 months post-transplant without any evidence of graft versus host disease. 23 months post-transplant, he presented with loose motions, 2 kg wight loss and fine tremors. He was referred to endocrine department for further workup. Physical examination revealed a small smooth goitre. He had discrete exophthalmos of his left eye without any signs of active inflammation. Thyroid function tests confirmed diagnosis of Graves’ disease with TSH<0.01 milli IU/L, Free T4=23.9 pmol/L, and TSH receptor antibodies of 3.79 IU/ml. Ophthalmological consultation suggested 6 months of selenium supplementation (200 mcg/day) with regular follow up. There has been no family history of autoimmune diseases or thyroid disorders. He started carbimazole (CMZ) 30 mg daily. His symptoms improved within 8 weeks, with normalization of Free T4 and Free T3 (TSH remained suppressed). 18 months later, he remained asymptomatic on carbimazole. He had recurrence of hyperthyroidism symptoms after 4 weeks trail of stopping carbimazole with elevation of Free T4 and Free T3. Carbimazole was restarted and he has been offered other treatment modalities of Graves’ disease. He elected to undergo total thyroidectomy. His sickle cell and blood counts remained stable during follow up period. Conclusion: Transplanted patients carries a life-long risk for developing endocrinopathies post initial transplant therapy. Acknowledging the wide spectrum of post-transplant endocrinopathies, an individualized case based periodic screening can be helpful to improve health outcomes of such patients. Because of the usual late presentation of such endocrine complications, transplanted patients might need life-long endocrine follow-up.

Preclinical Studies for Sickle Cell Disease Gene Therapy Using Bone Marrow CD34+ Cells Modified with a βAS3-Globin Lentiviral Vector

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3119-3119
Author(s):  
Fabrizia Urbinati ◽  
Zulema Romero Garcia ◽  
Sabine Geiger ◽  
Rafael Ruiz de Assin ◽  
Gabriela Kuftinec ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Blood Cells ◽  
Globin Gene ◽  
Cell Disease ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Abstract 3119 BACKGROUND: Sickle cell disease (SCD) affects approximately 80, 000 Americans, and causes significant neurologic, pulmonary, and renal injury, as well as severe acute and chronic pain that adversely impacts quality of life. Because SCD results from abnormalities in red blood cells, which in turn are produced from adult hematopoietic stem cells, hematopoietic stem cell transplant (HSCT) from a healthy (allogeneic) donor can benefit patients with SCD, by providing a source for life-long production of normal red blood cells. However, allogeneic HSCT is limited by the availability of well-matched donors and by immunological complications of graft rejection and graft-versus-host disease. Thus, despite major improvements in clinical care, SCD continues to cause significant morbidity and early mortality. HYPOTHESIS: We hypothesize that autologous stem cell gene therapy for SCD has the potential to treat this illness without the need for immune suppression of current allogeneic HSCT approaches. Previous studies have demonstrated that addition of a β-globin gene, modified to have the anti-sickling properties of fetal (γ-) globin (βAS3), to bone marrow (BM) stem cells in murine models of SCD normalizes RBC physiology and prevents the manifestations of sickle cell disease (Levassuer Blood 102 :4312–9, 2003). The present work seeks to provide pre-clinical evidence of efficacy for SCD gene therapy using human BM CD34+ cells modified with the bAS3 lentiviral (LV) vector. RESULTS: The βAS3 globin expression cassette was inserted into the pCCL LV vector backbone to confer tat-independence for packaging. The FB (FII/BEAD-A) composite enhancer-blocking insulator was inserted into the 3' LTR (Ramezani, Stem Cells 26 :32–766, 2008). Assessments were performed transducing human BM CD34+ cells from healthy or SCD donors with βAS3 LV vectors. Efficient (1–3 vector copies/cell) and stable gene transmission were determined by qPCR and Southern Blot. CFU assays demonstrated that βAS3 gene modified SCD CD34+ cells are fully capable of maintaining their hematopoietic potential. To demonstrate the effectiveness of the erythroid-specific bAS3 gene in the context of human HSPC (Hematopoietic Stem and Progenitor Cells), we optimized an in vitro model of erythroid differentiation of huBM CD34+ cells. We successfully obtained an expansion up to 700 fold with >80% fully mature enucleated RBC derived from CD34+ cells obtained from healthy or SCD BM donors. We then assessed the expression of the βAS3 globin gene by isoelectric focusing: an average of 18% HbAS3 over the total globin present (HbS, HbA2) per Vector Copy Number (VCN) was detected in RBC derived from SCD BM CD34+. A qRT-PCR assay able to discriminate HbAS3 vs. HbA RNA, was also established, confirming the quantitative expression results obtained by isoelectric focusing. Finally, we show morphologic correction of in vitro differentiated RBC obtained from SCD BM CD34+ cells after βAS3 LV transduction; upon induction of deoxygenation, cells derived from SCD patients showed the typical sickle shape whereas significantly reduced numbers were detected in βAS3 gene modified cells. Studies to investigate risks of insertional oncogenesis from gene modification of CD34+ cells by βAS3 LV vectors are ongoing as are in vivo studies to demonstrate the efficacy of βAS3 LV vector in the NSG mouse model. CONCLUSIONS: This work provides initial evidence for the efficacy of the modification of human SCD BM CD34+ cells with βAS3 LV vector for gene therapy of sickle cell disease. This work was supported by the California Institute for Regenerative Medicine Disease Team Award (DR1-01452). Disclosures: No relevant conflicts of interest to declare.

Altered Hematopoiesis in Murine Sickle Cell Disease

Blood ◽  
1999 ◽  
Vol 94 (4) ◽  
pp. 1451-1459 ◽  
Author(s):  
Marie-José Blouin ◽  
Monique E. De Paepe ◽  
Marie Trudel
Keyword(s):  
Bone Marrow ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Peripheral Blood ◽  
Fold Increase ◽  
Compensatory Mechanism ◽  
Allogeneic Bone ◽  
Cell Disease ◽  
Multipotent Cells ◽  
Colony Forming Units

Abstract We investigated the mechanisms of sickle cell disease (SCD) hematopoietic/erythropoietic defects using bone marrow, spleen, and/or peripheral blood from the transgenic SAD mouse model, which closely reproduces the biochemical and physiological disorders observed in human SCD. First, the erythropoietic lineage late precursors (polychromatophilic normoblasts to the intramedullary reticulocytes) of SAD mouse bone marrow were significantly altered morphologically. These anomalies resulted from high levels of hemoglobin polymers and were associated with increased cell fragmentation occurring during medullary endothelial migration of reticulocytes. Secondly, analysis of bone marrow erythropoiesis in earlier stages showed a marked depletion in SAD erythroid burst-forming units (BFU-E; of ∼42%) and erythroid colony-forming units (CFU-E; of ∼23%) progenitors, despite a significant increase in their proliferation, suggesting a compensatory mechanism. In contrast to the bone marrow progenitor depletion, we observed (1) a high mobilization/relocation of BFU-E early progenitors (∼4-fold increase) in peripheral blood of SAD mice as well as of colony-forming units–granulocyte-macrophage (CFU-GM) and (2) a 7-fold increase of SAD CFU-E in the spleen. Third, and most importantly, SAD bone marrow multipotent cells (spleen colony-forming units [CFU-S], granulocyte-erythroid-macrophage-megakaryocyte colony-forming units [CFU-GEMM], and Sca+Lin−) were highly mobilized to the peripheral blood (∼4-fold increase), suggesting that peripheral multipotent cells could serve as proliferative and autologous vehicles for gene therapy. Therefore, we conclude the following. (1) The abnormal differentiation and morphology of late nucleated erythroid precursors result in an ineffective sickle erythropoiesis and likely contribute to the pathophysiology of sickle cell disorders; this suggests that transfer of normal or modified SCD bone marrow cells may have a selective advantage in vivo. (2) A hematopoietic compensatory mechanism exists in SAD/SCD pathology and consists of mobilization of multipotent cells from the bone marrow to the peripheral blood and their subsequent uptake into the spleen, an extramedullary hematopoietic site for immediate differentiation. Altogether, these results corroborate the strong potential effectiveness of both autologous and allogeneic bone marrow transplantation for SCD hematopoietic therapy.

scholarly journals Correction of sickle cell disease by homologous recombination in embryonic stem cells

Blood ◽  
2006 ◽  
Vol 108 (4) ◽  
pp. 1183-1188 ◽  
Author(s):  
Li-Chen Wu ◽  
Chiao-Wang Sun ◽  
Thomas M. Ryan ◽  
Kevin M. Pawlik ◽  
Jinxiang Ren ◽  
...  
Keyword(s):  
Stem Cells ◽  
Sickle Cell Disease ◽  
Homologous Recombination ◽  
Sickle Cell ◽  
Insertional Mutagenesis ◽  
Es Cells ◽  
Globin Gene ◽  
Embryonic Stem ◽  
Cell Disease ◽  
Human Es Cells

Abstract Previous studies have demonstrated that sickle cell disease (SCD) can be corrected in mouse models by transduction of hematopoietic stem cells with lentiviral vectors containing antisickling globin genes followed by transplantation of these cells into syngeneic recipients. Although self-inactivating (SIN) lentiviral vectors with or without insulator elements should provide a safe and effective treatment in humans, some concerns about insertional mutagenesis persist. An ideal correction would involve replacement of the sickle globin gene (βS) with a normal copy of the gene (βA). We recently derived embryonic stem (ES) cells from a novel knock-in mouse model of SCD and tested a protocol for correcting the sickle mutation by homologous recombination. In this paper, we demonstrate the replacement of the human βS-globin gene with a human βA-globin gene and the derivation of mice from these cells. The animals produce high levels of normal human hemoglobin (HbA) and the pathology associated with SCD is corrected. Hematologic values are restored to normal levels and organ pathology is ameliorated. These experiments provide a foundation for similar studies in human ES cells derived from sickle cell patients. Although efficient methods for production of human ES cells by somatic nuclear transfer must be developed, the data in this paper demonstrate that sickle cell disease can be corrected without the risk of insertional mutagenesis.

Altered Hematopoiesis in Murine Sickle Cell Disease

Blood ◽  
1999 ◽  
Vol 94 (4) ◽  
pp. 1451-1459
Author(s):  
Marie-José Blouin ◽  
Monique E. De Paepe ◽  
Marie Trudel
Keyword(s):  
Bone Marrow ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Peripheral Blood ◽  
Fold Increase ◽  
Compensatory Mechanism ◽  
Allogeneic Bone ◽  
Cell Disease ◽  
Multipotent Cells ◽  
Colony Forming Units

We investigated the mechanisms of sickle cell disease (SCD) hematopoietic/erythropoietic defects using bone marrow, spleen, and/or peripheral blood from the transgenic SAD mouse model, which closely reproduces the biochemical and physiological disorders observed in human SCD. First, the erythropoietic lineage late precursors (polychromatophilic normoblasts to the intramedullary reticulocytes) of SAD mouse bone marrow were significantly altered morphologically. These anomalies resulted from high levels of hemoglobin polymers and were associated with increased cell fragmentation occurring during medullary endothelial migration of reticulocytes. Secondly, analysis of bone marrow erythropoiesis in earlier stages showed a marked depletion in SAD erythroid burst-forming units (BFU-E; of ∼42%) and erythroid colony-forming units (CFU-E; of ∼23%) progenitors, despite a significant increase in their proliferation, suggesting a compensatory mechanism. In contrast to the bone marrow progenitor depletion, we observed (1) a high mobilization/relocation of BFU-E early progenitors (∼4-fold increase) in peripheral blood of SAD mice as well as of colony-forming units–granulocyte-macrophage (CFU-GM) and (2) a 7-fold increase of SAD CFU-E in the spleen. Third, and most importantly, SAD bone marrow multipotent cells (spleen colony-forming units [CFU-S], granulocyte-erythroid-macrophage-megakaryocyte colony-forming units [CFU-GEMM], and Sca+Lin−) were highly mobilized to the peripheral blood (∼4-fold increase), suggesting that peripheral multipotent cells could serve as proliferative and autologous vehicles for gene therapy. Therefore, we conclude the following. (1) The abnormal differentiation and morphology of late nucleated erythroid precursors result in an ineffective sickle erythropoiesis and likely contribute to the pathophysiology of sickle cell disorders; this suggests that transfer of normal or modified SCD bone marrow cells may have a selective advantage in vivo. (2) A hematopoietic compensatory mechanism exists in SAD/SCD pathology and consists of mobilization of multipotent cells from the bone marrow to the peripheral blood and their subsequent uptake into the spleen, an extramedullary hematopoietic site for immediate differentiation. Altogether, these results corroborate the strong potential effectiveness of both autologous and allogeneic bone marrow transplantation for SCD hematopoietic therapy.

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