scholarly journals CRISPR-mediated gene modification of hematopoietic stem cells with beta-thalassemia IVS-1-110 mutation

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Hala Gabr ◽  
Mona Kamal El Ghamrawy ◽  
Abdulrahman H. Almaeen ◽  
Ahmed Samir Abdelhafiz ◽  
Aya Osama Saad Hassan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Genetic Disorders ◽  
Gene Mutations ◽  
Erythroid Differentiation ◽  
Beta Thalassemia ◽  
Gene Modification ◽  
Hematopoietic Stem ◽  
Knock Out ◽  
Guide Rna

Abstract Background β-Thalassemias represent a group of genetic disorders caused by human hemoglobin beta (HBB) gene mutations. The radical curative approach is to correct the mutations causing the disease. CRISPR-CAS9 is a novel gene-editing technology that can be used auspiciously for the treatment of these disorders. The study aimed to investigate the utility of CRISPR-CAS9 for gene modification of hematopoietic stem cells in β-thalassemia with IVS-1-110 mutation. Methods and results We successfully isolated CD34+ cells from peripheral blood of β-thalassemia patients with IVS-1-110 mutation. The cells were transfected with Cas9 endonuclease together with guide RNA to create double-strand breaks and knock out the mutation. The mutation-corrected CD34+ cells were subjected to erythroid differentiation by culturing in complete media containing erythropoietin. Conclusion CRISPR/Cas-9 is an effective tool for gene therapy that will broaden the spectrum of therapy and potentially improve the outcomes of β-thalassemia.

scholarly journals A Universal Approach to Target Various HBB Gene Mutations in Hematopoietic Stem/Progenitor Cells for Beta-Thalassemia Gene Therapy by CRISPR/Cas9 and the rAAV6 Vector

2020 ◽  
Author(s):  
Li-Na He ◽  
Yi Yang ◽  
Yi Cheng ◽  
Han Wu ◽  
Shou-Heng Lin ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Gene Mutations ◽  
Erythroid Differentiation ◽  
Beta Thalassemia ◽  
Hematopoietic Stem ◽  
Guide Rna ◽  
Universal Approach ◽  
Optimizing Design

Abstract Background: Engineered nuclease-mediated gene targeting through homology-directed repair (HDR) in autologous hematopoietic stem and progenitor cells (HSPCs) has the potential to cure β-thalassemia (β-thal). Although previous studies have precisely corrected site-specific HBB mutations by HDR in vitro and in vivo, targeting the various HBB mutations in β-thal is still challenging. Here, we devised a universal strategy to achieve repaired most types of HBB mutations through the CRISPR/Cas9 and the rAAV6 donor.Methods: Using cord blood-derived HSPCs from health donors, we tested the strategy to achieved highly efficient targeted integration by optimizing design and delivery parameters of a ribonucleoprotein (RNP) complex comprising Cas9 protein and modified single guide RNA, together with a rAAV6 donor. We assessed the edited HSPCs function in vitro by methylcellulose colonies assay, CFU assay, differentiation experiment and Wright-Giemsa staining. Edited HSPCs transplanted into NSI mice to assess the long-term reconstitution in vivo. Whole-genome sequencing was used to analysis the off-target mutagenesis of edited HSPCs.Results: Edited HSPCs exhibited normal multilineage formation and erythroid differentiation abilities without off-target mutagenesis and retained the ability to engraft. Moreover, we used the strategy to efficiently correct the β-CD41/42 mutation of patient-derived HSPCs, erythrocytes differentiation from which expressed more HBB mRNA than uncorrected cells.Conclusion: This strategy demonstrated a universal approach to correct most types of HBB gene mutations in β-thal.

scholarly journals Beta thalassemia and role of herbals and hematopoietic stem cells in its remedy

2018 ◽  
Vol 6 (5) ◽  
Author(s):  
Pooja Rai ◽  
Kamal Uddin Zaidi ◽  
Vijay Thawani
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
High Efficiency ◽  
Genetic Disorders ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Beta Thalassemia ◽  
The Novel ◽  
Hematopoietic Stem ◽  
Drug Treatments

Genetic disorders caused by mutations in the β-globin gene are widely known as the human β-hemoglobinopathies, in which there is β-thalassemia. In recent years, effort has been made to identify the natural inducers and drug treatments which can increase the synthesis of fetal hemoglobin and promote the expression of fetal γ-globin gene. This review aims to reveal the novel screening platforms for identifying potential herbal inducers with high efficiency and accuracy and to describe the hematopoietic stem cells remedies to provide perspectives in fetal hemoglobin reactivation for treating β-thalassemia.

scholarly journals High-level correction of the sickle mutation amplified in vivo during erythroid differentiation

2018 ◽  
Author(s):  
Wendy Magis ◽  
Mark A. DeWitt ◽  
Stacia K. Wyman ◽  
Jonathan T. Vu ◽  
Seok-Jin Heo ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Genetic Disorders ◽  
Erythroid Differentiation ◽  
Hematopoietic Stem ◽  
Curative Therapy ◽  
Human Hscs ◽  
High Level

ABSTRACTSickle Cell Disease (SCD), one of the world’s most common genetic disorders, causes anemia and progressive multiorgan damage that typically shortens lifespan by decades; currently there is no broadly applicable curative therapy. Here we show that Cas9 RNP-mediated gene editing with an ssDNA oligonucleotide donor yields markerless correction of the sickle mutation in more than 30% of long-term engrafting human hematopoietic stem cells (HSCs), using a selection-free protocol that is directly applicable to a clinical setting. We further find that in vivo erythroid differentiation markedly enriches for corrected ß-globin alleles. Adoption of a high-fidelity Cas9 variant demonstrates that this approach can yield efficient editing with almost no off-target events. These findings indicate that the sickle mutation can be corrected in human HSCs at curative levels with a streamlined protocol that is ready to be translated into a therapy.ONE SENTENCE SUMMARYCas9-mediated correction of the sickle mutation in human hematopoietic stem cells can be accomplished at curative levels.

scholarly journals A Universal Approach to Target Various HBB Gene Mutations in Hematopoietic Stem/Progenitor Cells for Beta-Thalassemia Gene Therapy by CRISPR/Cas9 and the rAAV6 Vector

2020 ◽  
Author(s):  
Li-Na He ◽  
Yi Yang ◽  
Yi Cheng ◽  
Han Wu ◽  
Shou-Heng Lin ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Gene Mutations ◽  
Erythroid Differentiation ◽  
Beta Thalassemia ◽  
Hematopoietic Stem ◽  
Guide Rna ◽  
Universal Approach ◽  
Optimizing Design

Abstract Background Engineered nuclease-mediated gene targeting through homology-directed repair (HDR) in autologous hematopoietic stem and progenitor cells (HSPCs) has the potential to cure β-thalassemia (β-thal). Although previous studies have precisely corrected site-specific HBB mutations by HDR in vitro and in vivo, targeting the various HBB mutations in β-thal is still challenging. Here, we devised a universal strategy to achieve repaired most types of HBB mutations through the CRISPR/Cas9 and the rAAV6 donor. Methods Using cord blood-derived HSPCs from health donors, we tested the strategy to achieved highly efficient targeted integration by optimizing design and delivery parameters of a ribonucleoprotein (RNP) complex comprising Cas9 protein and modified single guide RNA, together with a rAAV6 donor. We assessed the edited HSPCs function in vitro by methylcellulose colonies assay, CFU assay, differentiation experiment and Wright-Giemsa staining. Edited HSPCs transplanted into NSI mice to assess the long-term reconstitution in vivo. Whole-genome sequencing was used to analysis the off-target mutagenesis of edited HSPCs. Results Edited HSPCs exhibited normal multilineage formation and erythroid differentiation abilities without off-target mutagenesis and retained the ability to engraft. Moreover, we used the strategy to efficiently correct the β-CD41/42 mutation of patient-derived HSPCs, erythrocytes differentiation from which expressed more HBB mRNA than uncorrected cells. Conclusion This strategy demonstrated a universal approach to correct most types of HBB gene mutations in β-thal.

scholarly journals Hematopoietic Stem Cells Are Endowed with Erythroid Signature in Beta-Thalassemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 31-31
Author(s):  
Maria Rosa Lidonnici ◽  
Giulia Chianella ◽  
Francesca Tiboni ◽  
Matteo Barcella ◽  
Ivan Merelli ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Gene Set Enrichment Analysis ◽  
Lineage Commitment ◽  
Erythroid Cell ◽  
Beta Thalassemia ◽  
Hematopoietic Stem ◽  
Multipotent Progenitors

Background Beta-thalassemia (Bthal) is a genetic disorder due to mutations in the ß-globin gene, leading to a reduced or absent production of HbA, which interferes with erythroid cell maturation and limits normal red cell production. Patients are affected by severe anemia, hepatosplenomegaly, and skeletal abnormalities due to rapid expansion of the erythroid compartment in bone marrow (BM) caused by ineffective erythropoiesis. In a classical view of hematopoiesis, the blood cell lineages arise via a hierarchical scheme starting with multipotent stem cells that become increasingly restricted in their differentiation potential through oligopotent and then unipotent progenitors. In human, novel purification strategies based on differential expression of CD49f and CD90 enrich for long-term (49f+) and short-term (49f−) repopulating hematopoietic stem cells (HSCs), with distinct cell cycle properties, but similar myeloid (My) and lymphoid (Ly) potential. In this view, it has been proposed that erythroid (Ery) and megakaryocytic (Mk) fates branch off directly from CD90-/49f− multipotent progenitors (MPPs). Recently, a new study suggested that separation between multipotent (Ery/My/Ly) long-term repopulating cells (Subset1, defined as CLEC9AhighCD34low) and cells with only My/Ly and no Ery potential (Subset2, defined as CLEC9AlowCD34high)occurs within the phenotypic HSC/MPP and CD49f+ HSCs compartment. Aims A general perturbed and stress condition is present in the thalassemic BM microenvironment. Since its impact on the hematopoietic cell subpopulations is mostly unknown, we will investigate which model of hematopoiesis/erythropoiesis occurs in Bthal. Moreover, since Beta-Thalassemia is an erythropoietic disorder, it could be considered as a disease model to study the 'erythroid branching' in the hematopoietic hierarchy. Methods We defined by immunophenotype and functional analysis the lineage commitment of most primitive HSC/MPP cells in patients affected by this pathology compared to healthy donors (HDs). Furthermore, in order to delineate the transcriptional networks governing hematopoiesis in Beta-thalassemia, RNAseq analysis was performed on sorted hematopoietic subpopulations from BM of Bthal patients and HDs. By droplet digital PCR on RNA purified from mesenchymal stromal cells of Bthal patients, we evaluated the expression levels of some niche factors involved in the regulation of hematopoiesis and erythropoiesis. Moreover, the protein levels in the BM plasma were analyzed by performing ELISA. Results Differences in the primitive compartment were observed with an increased proportion of multipotent progenitors in Bthal patients compared to HDs. The Subset1 compartment is actually endowed with an enhanced Ery potential. Focusing on progenitors (CD34+ CD38+) and using a new sorting scheme that efficiently resolved My, Ery, and Mk lineage fates, we quantified the new My (CD71-BAH1-/+) and Ery (CD71+ BAH1-/+) subsets and found a reduction of Ery subset in Bthal samples. We can hypothesize that the erythroid-enriched subsets are more prone to differentiate quickly due to the higher sensitivity to Epo stimuli or other bone marrow niche signals. Gene set enrichment analysis, perfomed on RNAseq data, showed that Bthal HSC/MPP presented negative enrichment of several pathways related to stemness and quiescence. Cellular processes involved in erythropoiesis were found altered in Bthal HSC. Moreover, some master erythroid transcription factors involved were overrepresented in Bthal across the hematopoietic cascade. We identified the niche factors which affect molecular pathways and the lineage commitment of Bthal HSCs. Summary/Conclusions Overall, these data indicate that Bthal HSCs are more cycling cells which egress from the quiescent state probably towards an erythroid differentiation, probably in response to a chronic BM stimulation. On the other hand,some evidences support our hypothesis of an 'erythroid branching' already present in the HSC pool, exacerbated by the pathophysiology of the disease. Disclosures No relevant conflicts of interest to declare.

AHSP Knockdown in Human Erythroleukemia Cell Line and Human Hematopoietic Stem Cells Results in Alpha Hemoglobin Chain Precipitation, Decreased Hemoglobin Levels and Increased Cell Death.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1778-1778
Author(s):  
Flavia O. Pinho ◽  
Dulcineia M. Albuquerque ◽  
Sara T.O Saad ◽  
Fernando F. Costa
Keyword(s):  
Stem Cells ◽  
Cell Death ◽  
Hematopoietic Stem Cells ◽  
Cell Line ◽  
K562 Cells ◽  
Erythroid Differentiation ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Erythroleukemia Cell ◽  
Human Red Cells

Abstract Alpha Hemoglobin Stabilizing Protein (AHSP) binds alpha hemoglobin chain (αHb), avoiding its precipitation and its pro-oxidant activity. In the presence of beta hemoglobin chain (βHb), the αHb-AHSP complex is dismembered and βHb displaces AHSP to generate the quaternary structure of hemoglobin. These data have been obtained in vitro and in mouse cells, but strongly suggest the importance of AHSP for normal hemoglobin synthesis in humans. To the best of our knowledge, the relationship between hemoglobin formation and alterations in AHSP expression has not yet been described in human red cells. Hence, to investigate the consequences of a reduced AHSP synthesis in human red cells, we established the RNA interference-mediated knockdown of AHSP expression in human erythroleukemia cell line (K562 cells) and human hematopoietic stem cells (CD34+ cells) induced to erythroid differentiation, and analyzed the consequent cellular and molecular aspects of AHSP knockdown in these cells. shRNA expression vectors, aimed at the AHSP mRNA target sequence, were cloned and transfected into K562 and CD34+ cells using a non-liposomal lipid reagent. Following transfection, K562 cells that stably expressed AHSP-shRNA and CD34+ cells that transiently expressed AHSP-shRNA were selected. K562 and CD34+ cells were stimulated to erythroid differentiation by hemin and erythropoietin (EPO) respectively. The cells were examined in terms of gene expression using quantitative real-time PCR; production of reactive oxygen species (ROS), apoptosis and hemoglobin production through flow cytometry assays; and immunofluorescence assays for globin chains. AHSP-shRNA hemin-induced K562 cells and AHSP-shRNA EPO-induced CD34+ cells presented 71% and 75% decreases in AHSP expression levels, respectively. The RNAi-mediated knockdown of AHSP expression resulted in a considerable αHb precipitation, as well as in a significant decrease in fetal hemoglobin formation. In addition, AHSP-knockdown cells demonstrated an increased ROS production and increased rate of apoptosis. These findings strengthen the hypothesis that AHSP stabilizes the alpha hemoglobin chain, avoiding its precipitation and its ability to generate ROS which implicate in cell death. Moreover, data indicate that AHSP may be highly significant for human hemoglobin formation and suggest that AHSP is a key chaperone protein during human erythropoiesis.

scholarly journals In VitroLarge Scale Production of Human Mature Red Blood Cells from Hematopoietic Stem Cells by Coculturing with Human Fetal Liver Stromal Cells

2013 ◽  
Vol 2013 ◽  
pp. 1-12 ◽  
Author(s):  
Jiafei Xi ◽  
Yanhua Li ◽  
Ruoyong Wang ◽  
Yunfang Wang ◽  
Xue Nan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Cord Blood ◽  
Red Blood Cells ◽  
Stromal Cells ◽  
Blood Cells ◽  
Fetal Liver ◽  
Erythroid Differentiation ◽  
Erythroid Cells ◽  
Hematopoietic Stem

In vitromodels of human erythropoiesis are useful in studying the mechanisms of erythroid differentiation in normal and pathological conditions. Here we describe an erythroid liquid culture system starting from cord blood derived hematopoietic stem cells (HSCs). HSCs were cultured for more than 50 days in erythroid differentiation conditions and resulted in a more than 109-fold expansion within 50 days under optimal conditions. Homogeneous erythroid cells were characterized by cell morphology, flow cytometry, and hematopoietic colony assays. Furthermore, terminal erythroid maturation was improved by cosculturing with human fetal liver stromal cells. Cocultured erythroid cells underwent multiple maturation events, including decrease in size, increase in glycophorin A expression, and nuclear condensation. This process resulted in extrusion of the pycnotic nuclei in up to 80% of the cells. Importantly, they possessed the capacity to express the adult definitiveβ-globin chain upon further maturation. We also show that the oxygen equilibrium curves of the cord blood-differentiated red blood cells (RBCs) are comparable to normal RBCs. The large number and purity of erythroid cells and RBCs produced from cord blood make this method useful for fundamental research in erythroid development, and they also provide a basis for future production of available RBCs for transfusion.

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