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CRISPR-Cas9 AND Thalassemia

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https://www.readbyqxmd.com/read/30059493/an-apobec3a-cas9-base-editor-with-minimized-bystander-and-off-target-activities
#1
Jason M Gehrke, Oliver Cervantes, M Kendell Clement, Yuxuan Wu, Jing Zeng, Daniel E Bauer, Luca Pinello, J Keith Joung
Base editor technology, which uses CRISPR-Cas9 to direct cytidine deaminase enzymatic activity to specific genomic loci, enables the highly efficient introduction of precise cytidine-to-thymidine DNA alterations. However, existing base editors create unwanted C-to-T alterations when more than one C is present in the enzyme's five-base-pair editing window. Here we describe a strategy for reducing bystander mutations using an engineered human APOBEC3A (eA3A) domain, which preferentially deaminates cytidines in specific motifs according to a TCR>TCY>VCN hierarchy...
July 30, 2018: Nature Biotechnology
https://www.readbyqxmd.com/read/30038942/hdad5-35-adenovirus-vector-expressing-anti-crispr-peptides-decreases-crispr-cas9-toxicity-in-human-hematopoietic-stem-cells
#2
Chang Li, Nikoletta Psatha, Sucheol Gil, Hongjie Wang, Thalia Papayannopoulou, André Lieber
We generated helper-dependent HDAd5/35++ adenovirus vectors expressing CRISPR/Cas9 for potential hematopoietic stem cells (HSCs) gene therapy of β-thalassemia and sickle cell disease through re-activation of fetal γ-globin expression (HDAd-globin-CRISPR). The process of CRISPR/Cas9 gene transfer using these vectors was not associated with death of human CD34+ cells and did not affect their in vitro expansion and erythroid differentiation. However, functional assays for primitive HSCs, e.g., multi-lineage progenitor colony formation and engraftment in irradiated NOD/Shi-scid/interleukin-2 receptor γ (IL-2Rγ) null (NSG) mice, revealed toxicity of HDAd-globin-CRISPR vectors related to the prolonged expression and activity of CRISPR/Cas9...
June 15, 2018: Molecular Therapy. Methods & Clinical Development
https://www.readbyqxmd.com/read/30026227/domain-focused-crispr-screen-identifies-hri-as-a-fetal-hemoglobin-regulator-in-human-erythroid-cells
#3
Jeremy D Grevet, Xianjiang Lan, Nicole Hamagami, Christopher R Edwards, Laavanya Sankaranarayanan, Xinjun Ji, Saurabh K Bhardwaj, Carolyne J Face, David F Posocco, Osheiza Abdulmalik, Cheryl A Keller, Belinda Giardine, Simone Sidoli, Ben A Garcia, Stella T Chou, Stephen A Liebhaber, Ross C Hardison, Junwei Shi, Gerd A Blobel
Increasing fetal hemoglobin (HbF) levels in adult red blood cells provides clinical benefit to patients with sickle cell disease and some forms of β-thalassemia. To identify potentially druggable HbF regulators in adult human erythroid cells, we employed a protein kinase domain-focused CRISPR-Cas9-based genetic screen with a newly optimized single-guide RNA scaffold. The screen uncovered the heme-regulated inhibitor HRI (also known as EIF2AK1), an erythroid-specific kinase that controls protein translation, as an HbF repressor...
July 20, 2018: Science
https://www.readbyqxmd.com/read/30010219/disruption-of-sox6-gene-using-crispr-cas9-technology-for-gamma-globin-reactivation-an-approach-towards-gene-therapy-of-%C3%AE-thalassemia
#4
Laleh Shariati, Fattah Rohani, Nahid Heidari Hafshejani, Shirin Kouhpayeh, Maryam Boshtam, Mina Mirian, Ilnaz Rahimmanesh, Zahra Hejazi, Mehran Modarres, Ina Laura Pieper, Hossein Khanahmad
Elevation of Hemoglobin F ameliorates symptoms of β-thalassemia, a common autosomal recessive disorder. The transcription factor SOX6 plays a key role in the γ to β-globin gene switching. In the current investigation, a mutation was induced using the CRISPR/Cas9 technology in the binding domain region of SOX6 to reactivate γ-globin expression. Three CRISPR/Cas9 cassettes were provided, whose single-guide RNAs targeted different regions in the SOX6 gene-binding domain. After transfection of K562 cells with CRISPR a, b and c, and subsequent erythroid differentiation, the indel percentage of the cells was about 30%, 25%, and 24%, respectively...
July 16, 2018: Journal of Cellular Biochemistry
https://www.readbyqxmd.com/read/29789357/reactivation-of-%C3%AE-globin-in-adult-%C3%AE-yac-mice-after-ex-vivo-and-in-vivo-hematopoietic-stem-cell-genome-editing
#5
Chang Li, Nikoletta Psatha, Pavel Sova, Sucheol Gil, Hongjie Wang, Jiho Kim, Chandana Kulkarni, Cristina Valensisi, R David Hawkins, George Stamatoyannopoulos, André Lieber
Disorders involving β-globin gene mutations, primarily β-thalassemia and sickle cell disease, represent a major target for hematopoietic stem/progenitor cell (HSPC) gene therapy. This includes CRISPR/Cas9-mediated genome editing approaches in adult CD34+ cells aimed toward the reactivation of fetal γ-globin expression in red blood cells. Because models involving erythroid differentiation of CD34+ cells have limitations in assessing γ-globin reactivation, we focused on human β-globin locus-transgenic (β-YAC) mice...
June 28, 2018: Blood
https://www.readbyqxmd.com/read/29610478/natural-regulatory-mutations-elevate-the-fetal-globin-gene-via-disruption-of-bcl11a-or-zbtb7a-binding
#6
Gabriella E Martyn, Beeke Wienert, Lu Yang, Manan Shah, Laura J Norton, Jon Burdach, Ryo Kurita, Yukio Nakamura, Richard C M Pearson, Alister P W Funnell, Kate G R Quinlan, Merlin Crossley
β-hemoglobinopathies such as sickle cell disease (SCD) and β-thalassemia result from mutations in the adult HBB (β-globin) gene. Reactivating the developmentally silenced fetal HBG1 and HBG2 (γ-globin) genes is a therapeutic goal for treating SCD and β-thalassemia 1 . Some forms of hereditary persistence of fetal hemoglobin (HPFH), a rare benign condition in which individuals express the γ-globin gene throughout adulthood, are caused by point mutations in the γ-globin gene promoter at regions residing ~115 and 200 bp upstream of the transcription start site...
April 2018: Nature Genetics
https://www.readbyqxmd.com/read/29519807/induction-of-fetal-hemoglobin-synthesis-by-crispr-cas9-mediated-editing-of-the-human-%C3%AE-globin-locus
#7
Chiara Antoniani, Vasco Meneghini, Annalisa Lattanzi, Tristan Felix, Oriana Romano, Elisa Magrin, Leslie Weber, Giulia Pavani, Sara El Hoss, Ryo Kurita, Yukio Nakamura, Thomas J Cradick, Ante S Lundberg, Matthew Porteus, Mario Amendola, Wassim El Nemer, Marina Cavazzana, Fulvio Mavilio, Annarita Miccio
Naturally occurring, large deletions in the β-globin locus result in hereditary persistence of fetal hemoglobin, a condition that mitigates the clinical severity of sickle cell disease (SCD) and β-thalassemia. We designed a clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9) strategy to disrupt a 13.6-kb genomic region encompassing the δ- and β-globin genes and a putative γ-δ intergenic fetal hemoglobin (HbF) silencer. Disruption of just the putative HbF silencer results in a mild increase in γ-globin expression, whereas deletion or inversion of a 13...
April 26, 2018: Blood
https://www.readbyqxmd.com/read/29482624/one-step-genetic-correction-of-hemoglobin-e-beta-thalassemia-patient-derived-ipscs-by-the-crispr-cas9-system
#8
Methichit Wattanapanitch, Nattaya Damkham, Ponthip Potirat, Kongtana Trakarnsanga, Montira Janan, Yaowalak U-Pratya, Pakpoom Kheolamai, Nuttha Klincumhom, Surapol Issaragrisil
BACKGROUND: Thalassemia is the most common genetic disease worldwide; those with severe disease require lifelong blood transfusion and iron chelation therapy. The definitive cure for thalassemia is allogeneic hematopoietic stem cell transplantation, which is limited due to lack of HLA-matched donors and the risk of post-transplant complications. Induced pluripotent stem cell (iPSC) technology offers prospects for autologous cell-based therapy which could avoid the immunological problems...
February 26, 2018: Stem Cell Research & Therapy
https://www.readbyqxmd.com/read/29370156/crispr-cas9-genome-editing-in-human-hematopoietic-stem-cells
#9
Rasmus O Bak, Daniel P Dever, Matthew H Porteus
Genome editing via homologous recombination (HR) (gene targeting) in human hematopoietic stem cells (HSCs) has the power to reveal gene-function relationships and potentially transform curative hematological gene and cell therapies. However, there are no comprehensive and reproducible protocols for targeting HSCs for HR. Herein, we provide a detailed protocol for the production, enrichment, and in vitro and in vivo analyses of HR-targeted HSCs by combining CRISPR/Cas9 technology with the use of rAAV6 and flow cytometry...
February 2018: Nature Protocols
https://www.readbyqxmd.com/read/29164808/a-universal-approach-to-correct-various-hbb-gene-mutations-in-human-stem-cells-for-gene-therapy-of-beta-thalassemia-and-sickle-cell-disease
#10
Liuhong Cai, Hao Bai, Vasiliki Mahairaki, Yongxing Gao, Chaoxia He, Yanfei Wen, You-Chuan Jin, You Wang, Rachel L Pan, Armaan Qasba, Zhaohui Ye, Linzhao Cheng
Beta-thalassemia is one of the most common recessive genetic diseases, caused by mutations in the HBB gene. Over 200 different types of mutations in the HBB gene containing three exons have been identified in patients with β-thalassemia (β-thal) whereas a homozygous mutation in exon 1 causes sickle cell disease (SCD). Novel therapeutic strategies to permanently correct the HBB mutation in stem cells that are able to expand and differentiate into erythrocytes producing corrected HBB proteins are highly desirable...
January 2018: Stem Cells Translational Medicine
https://www.readbyqxmd.com/read/28871148/editing-an-%C3%AE-globin-enhancer-in-primary-human-hematopoietic-stem-cells-as-a-treatment-for-%C3%AE-thalassemia
#11
Sachith Mettananda, Chris A Fisher, Deborah Hay, Mohsin Badat, Lynn Quek, Kevin Clark, Philip Hublitz, Damien Downes, Jon Kerry, Matthew Gosden, Jelena Telenius, Jackie A Sloane-Stanley, Paula Faustino, Andreia Coelho, Jessica Doondeea, Batchimeg Usukhbayar, Paul Sopp, Jacqueline A Sharpe, Jim R Hughes, Paresh Vyas, Richard J Gibbons, Douglas R Higgs
β-Thalassemia is one of the most common inherited anemias, with no effective cure for most patients. The pathophysiology reflects an imbalance between α- and β-globin chains with an excess of free α-globin chains causing ineffective erythropoiesis and hemolysis. When α-thalassemia is co-inherited with β-thalassemia, excess free α-globin chains are reduced significantly ameliorating the clinical severity. Here we demonstrate the use of CRISPR/Cas9 genome editing of primary human hematopoietic stem/progenitor (CD34+) cells to emulate a natural mutation, which deletes the MCS-R2 α-globin enhancer and causes α-thalassemia...
September 4, 2017: Nature Communications
https://www.readbyqxmd.com/read/28639471/an-enhancer-haplotype-may-influence-bcl11a-expression-levels-and-the-response-to-hydroxyurea-in-%C3%AE-thalassemia-patients
#12
Nahal Maroofi, Azita Azarkeivan, Soosan Banihashemi, Saeid Mohammadparast, Ali Aghajanirefah, Mehdi Banan
AIM: To identify the BCL11A intron-2 enhancer linkage disequilibrium (LD) block, harboring two previously identified SNPs, associating with the hydroxyurea response in β-thalassemia patients and the functional significance of this region. MATERIALS & METHODS: Several neighboring SNPs were genotyped in our cohort. The associating LD block was identified, and its function studied in K562 erythroid cells via CRISPR/Cas9 genome editing. RESULTS: A haplotype harboring three tag SNPs correlated significantly with the HU-response and BCL11A transcript levels in the patients' reticulocytes...
July 2017: Pharmacogenomics
https://www.readbyqxmd.com/read/28325300/one-step-biallelic-and-scarless-correction-of-a-%C3%AE-thalassemia-mutation-in-patient-specific-ipscs-without-drug-selection
#13
Yali Liu, Yi Yang, Xiangjin Kang, Bin Lin, Qian Yu, Bing Song, Ge Gao, Yaoyong Chen, Xiaofang Sun, Xiaoping Li, Lei Bu, Yong Fan
Monogenic disorders (MGDs), which are caused by single gene mutations, have a serious effect on human health. Among these, β-thalassemia (β-thal) represents one of the most common hereditary hematological diseases caused by mutations in the human hemoglobin β (HBB) gene. The technologies of induced pluripotent stem cells (iPSCs) and genetic correction provide insights into the treatments for MGDs, including β-thal. However, traditional approaches for correcting mutations have a low efficiency and leave a residual footprint, which leads to some safety concerns in clinical applications...
March 17, 2017: Molecular Therapy. Nucleic Acids
https://www.readbyqxmd.com/read/27868048/ground-state-na%C3%A3-ve-pluripotent-stem-cells-and-crispr-cas9-gene-correction-for-%C3%AE-thalassemia
#14
EDITORIAL
Alessia Finotti, Monica Borgatti, Roberto Gambari
No abstract text is available yet for this article.
2016: Stem Cell Investigation
https://www.readbyqxmd.com/read/27736664/crispr-cas9-system-and-its-applications-in-human-hematopoietic-cells
#15
REVIEW
Xiaotang Hu
Since 2012, the CRISPR-Cas9 system has been quickly and successfully tested in a broad range of organisms and cells including hematopoietic cells. The application of CRISPR-Cas9 in human hematopoietic cells mainly involves the genes responsible for HIV infection, β-thalassemia and sickle cell disease (SCD). The successful disruption of CCR5 and CXCR4 genes in T cells by CRISPR-Cas9 promotes the prospect of the technology in the functional cure of HIV. More recently, eliminating CCR5 and CXCR4 in induced pluripotent stem cells (iPSCs) derived from patients and targeting the HIV genome have been successfully carried out in several laboratories...
November 2016: Blood Cells, Molecules & Diseases
https://www.readbyqxmd.com/read/27668420/genetic-disruption-of-the-klf1-gene-to-overexpress-the-%C3%AE-globin-gene-using-the-crispr-cas9-system
#16
Laleh Shariati, Hossein Khanahmad, Mansoor Salehi, Zahra Hejazi, Ilnaz Rahimmanesh, Mohammad Amin Tabatabaiefar, Mohammad Hossein Modarressi
BACKGROUND: β-thalassemia comprises a major group of human genetic disorders involving a decrease in or an end to the normal synthesis of the β-globin chains of hemoglobin. KLF1 is a key regulatory molecule involved in the γ- to β-globin gene switching process directly inducing the expression of the β-globin gene and indirectly repressing γ-globin. The present study aimed to investigate the ability of an engineered CRISPR/Cas9 system with respect to disrupting the KLF1 gene to inhibit the γ- to β-hemoglobin switching process in K562 cells...
October 2016: Journal of Gene Medicine
https://www.readbyqxmd.com/read/27601644/genome-editing-using-crispr-cas9-to-create-the-hpfh-genotype-in-hspcs-an-approach-for-treating-sickle-cell-disease-and-%C3%AE-thalassemia
#17
Lin Ye, Jiaming Wang, Yuting Tan, Ashley I Beyer, Fei Xie, Marcus O Muench, Yuet Wai Kan
Hereditary persistence of fetal hemoglobin (HPFH) is a condition in some individuals who have a high level of fetal hemoglobin throughout life. Individuals with compound heterozygous β-thalassemia or sickle cell disease (SCD) and HPFH have milder clinical manifestations. Using RNA-guided clustered regularly interspaced short palindromic repeats-associated Cas9 (CRISPR-Cas9) genome-editing technology, we deleted, in normal hematopoietic stem and progenitor cells (HSPCs), 13 kb of the β-globin locus to mimic the naturally occurring Sicilian HPFH mutation...
September 20, 2016: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/27581487/the-combination-of-crispr-cas9-and-ipsc-technologies-in-the-gene-therapy-of-human-%C3%AE-thalassemia-in-mice
#18
Zhanhui Ou, Xiaohua Niu, Wenyin He, Yuchang Chen, Bing Song, Yexing Xian, Di Fan, Daolin Tang, Xiaofang Sun
β-thalassemia results from point mutations or small deletions in the β-globin (HBB) gene that ultimately cause anemia. The generation of induced pluripotent stem cells (iPSCs) from the somatic cells of patients in combination with subsequent homologous recombination-based gene correction provides new approaches to cure this disease. CRISPR/Cas9 is a genome editing tool that is creating a buzz in the scientific community for treating human diseases, especially genetic disorders. Here, we reported that correction of β-thalassemia mutations in patient-specific iPSCs using the CRISPR/Cas9 tool promotes hematopoietic differentiation in vivo...
September 1, 2016: Scientific Reports
https://www.readbyqxmd.com/read/27578458/switch-telomerase-to-alt-mechanism-by-inducing-telomeric-dna-damages-and-dysfunction-of-atrx-and-daxx
#19
Yang Hu, Guang Shi, Laichen Zhang, Feng Li, Yuanling Jiang, Shuai Jiang, Wenbin Ma, Yong Zhao, Zhou Songyang, Junjiu Huang
Activation of telomerase or alternative lengthening of telomeres (ALT) is necessary for tumours to escape from dysfunctional telomere-mediated senescence. Anti-telomerase drugs might be effective in suppressing tumour growth in approximately 85-90% of telomerase-positive cancer cells. However, there are still chances for these cells to bypass drug treatment after switching to the ALT mechanism to maintain their telomere integrity. But the mechanism underlying this switch is unknown. In this study, we used telomerase-positive cancer cells (HTC75) to discover the mechanism of the telomerase-ALT switch by inducing telomere-specific DNA damage, alpha-thalassemia X-linked syndrome protein (ATRX) knockdown and deletion of death associated protein (DAXX)...
August 31, 2016: Scientific Reports
https://www.readbyqxmd.com/read/27525524/a-genome-editing-strategy-to-treat-%C3%AE-hemoglobinopathies-that-recapitulates-a-mutation-associated-with-a-benign-genetic-condition
#20
Elizabeth A Traxler, Yu Yao, Yong-Dong Wang, Kaitly J Woodard, Ryo Kurita, Yukio Nakamura, Jim R Hughes, Ross C Hardison, Gerd A Blobel, Chunliang Li, Mitchell J Weiss
Disorders resulting from mutations in the hemoglobin subunit beta gene (HBB; which encodes β-globin), mainly sickle cell disease (SCD) and β-thalassemia, become symptomatic postnatally as fetal γ-globin expression from two paralogous genes, hemoglobin subunit gamma 1 (HBG1) and HBG2, decreases and adult β-globin expression increases, thereby shifting red blood cell (RBC) hemoglobin from the fetal (referred to as HbF or α2γ2) to adult (referred to as HbA or α2β2) form. These disorders are alleviated when postnatal expression of fetal γ-globin is maintained...
September 2016: Nature Medicine
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