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Ball J, Bradley A, Le A, Tisdale JF, Uchida N. Current and future treatments for sickle cell disease: From hematopoietic stem cell transplantation to in vivo gene therapy. Mol Ther 2025; 33:2172-2191. [PMID: 40083162 DOI: 10.1016/j.ymthe.2025.03.016] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2025] [Revised: 03/04/2025] [Accepted: 03/07/2025] [Indexed: 03/16/2025] Open
Abstract
Sickle cell disease (SCD) is a single-gene disorder caused by a point mutation of the β-globin gene, resulting in hemolytic anemia, acute pain, multiorgan damage, and early mortality. Hydroxyurea is a first-line drug therapy that switches sickle-globin to non-pathogenic γ-globin; however, it requires lifelong oral administration. Allogeneic hematopoietic stem cell (HSC) transplantation allows for a one-time cure for SCD, albeit with histocompatibility limitations. Therefore, autologous HSC gene therapy was developed to cure SCD in a single treatment, without HSC donors. Current HSC gene therapy is based on the ex vivo culture of patients' HSCs with lentiviral gene addition and gene editing, followed by autologous transplantation back to the patient. However, the complexity of the treatment process and high costs hinder the universal application of ex vivo gene therapy. Therefore, the development of in vivo HSC gene therapy, where gene therapy tools are directly administered to patients, is desirable to provide a more accessible, cost-effective solution that can cure SCD worldwide. In this review, we discuss current treatments, including drug therapies, HSC transplantation, and ex vivo gene therapy; the development of gene therapy tools; and progress toward curative in vivo gene therapy in SCD.
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Affiliation(s)
- Julia Ball
- Cellular and Molecular Therapeutics Branch, National Heart, Lung, and Blood Institute (NHLBI), National Institutes of Health (NIH), Bethesda, MD 20892, USA
| | - Avery Bradley
- Cellular and Molecular Therapeutics Branch, National Heart, Lung, and Blood Institute (NHLBI), National Institutes of Health (NIH), Bethesda, MD 20892, USA
| | - Anh Le
- Cellular and Molecular Therapeutics Branch, National Heart, Lung, and Blood Institute (NHLBI), National Institutes of Health (NIH), Bethesda, MD 20892, USA
| | - John F Tisdale
- Cellular and Molecular Therapeutics Branch, National Heart, Lung, and Blood Institute (NHLBI), National Institutes of Health (NIH), Bethesda, MD 20892, USA
| | - Naoya Uchida
- Cellular and Molecular Therapeutics Branch, National Heart, Lung, and Blood Institute (NHLBI), National Institutes of Health (NIH), Bethesda, MD 20892, USA.
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2
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Chen L, Liu D, Hong W, Xu L, Cheng L, Luo Y, Xu H, Liang J, Fang J, Li X. Creating New Cis-Regulatory Elements of HBD to Reactivate Delta-Globin. Hum Gene Ther 2025; 36:765-773. [PMID: 40114594 DOI: 10.1089/hum.2024.186] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 03/22/2025] Open
Abstract
β-thalassemia and sickle cell disease (SCD) are global monogenic blood system disorders, and reactivated δ-globin is expected to replace missing or abnormal β-globin. With the development of gene editing technology, activating γ-globin for treating β-thalassemia and SCD has been highly successful. However, δ-globin, as another important potential therapeutic target, has few related studies. Gene editing technology introduced cis-acting elements, including NF-Y, KLF1, GATA1, and TAL1, into the regulatory region of HBD, successfully activating the expression of δ-globin. It was confirmed that the activation effect of δ-globin was closely related to the location of the introduced cis-acting elements. In this study, the mutation creates a de novo binding site for KLF1 at -85∼93 bp upstream of the transcription start site of the HBD gene, as well as the site for TAL1 and GATA1 cobinding motifs at -59 to ∼-78 bp, which could effectively activate δ-globin.
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Affiliation(s)
- Lini Chen
- Department of Pediatrics, Sun Yat-Sen Memorial Hospital of Sun Yat-Sen University, Guangzhou, People's Republic of China
- Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, People's Republic of China
| | - Diandian Liu
- Department of Pediatrics, Sun Yat-Sen Memorial Hospital of Sun Yat-Sen University, Guangzhou, People's Republic of China
- Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, People's Republic of China
| | - Weicong Hong
- Department of Pediatrics, Sun Yat-Sen Memorial Hospital of Sun Yat-Sen University, Guangzhou, People's Republic of China
- Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, People's Republic of China
| | - Luhong Xu
- Department of Pediatrics, Sun Yat-Sen Memorial Hospital of Sun Yat-Sen University, Guangzhou, People's Republic of China
- Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, People's Republic of China
| | - Lin Cheng
- Reforgene Medicine, Guangzhou, People's Republic of China
| | - Ying Luo
- Reforgene Medicine, Guangzhou, People's Republic of China
| | - Hui Xu
- Reforgene Medicine, Guangzhou, People's Republic of China
| | - Junbin Liang
- Reforgene Medicine, Guangzhou, People's Republic of China
| | - Jianpei Fang
- Department of Pediatrics, Sun Yat-Sen Memorial Hospital of Sun Yat-Sen University, Guangzhou, People's Republic of China
- Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, People's Republic of China
| | - Xinyu Li
- Department of Pediatrics, Sun Yat-Sen Memorial Hospital of Sun Yat-Sen University, Guangzhou, People's Republic of China
- Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, People's Republic of China
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Hardouin G, Miccio A, Brusson M. Gene therapy for β-thalassemia: current and future options. Trends Mol Med 2025; 31:344-358. [PMID: 39794177 DOI: 10.1016/j.molmed.2024.12.001] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2024] [Revised: 11/28/2024] [Accepted: 12/02/2024] [Indexed: 01/13/2025]
Abstract
Beta-thalassemia is a severe, hereditary blood disorder characterized by anemia, transfusion dependence, reduced life expectancy, and poor quality of life. Allogeneic transplantation of hematopoietic stem cells (HSCs) is the only curative treatment for transfusion-dependent β-thalassemia, but a lack of compatible donors prevents the use of this approach for most patients. Over the past 20 years, the rise of gene therapy and the development of lentiviral vectors and genome-editing tools has extended curative options to a broader range of patients. Here, we review breakthroughs in gene addition- and genome-editing-based therapies for β-thalassemia, the clinical outcomes enabling approval by regulatory agencies, and perspectives for further development.
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Affiliation(s)
- Giulia Hardouin
- Université Paris Cité, Imagine Institute, Laboratory of chromatin and gene regulation during development, INSERM UMR 1163, 75015, Paris, France.
| | - Annarita Miccio
- Université Paris Cité, Imagine Institute, Laboratory of chromatin and gene regulation during development, INSERM UMR 1163, 75015, Paris, France.
| | - Megane Brusson
- Université Paris Cité, Imagine Institute, Laboratory of chromatin and gene regulation during development, INSERM UMR 1163, 75015, Paris, France
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Hoffmann M, Vaz T, Chhatrala S, Hennighausen L. Data-driven projections of candidate enhancer-activating SNPs in immune regulation. BMC Genomics 2025; 26:197. [PMID: 40011812 PMCID: PMC11863423 DOI: 10.1186/s12864-025-11374-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2024] [Accepted: 02/17/2025] [Indexed: 02/28/2025] Open
Abstract
BACKGROUND Millions of single nucleotide polymorphisms (SNPs) have been identified in humans, but the functionality of almost all SNPs remains unclear. While current research focuses primarily on SNPs altering one amino acid to another one, the majority of SNPs are located in intergenic spaces. Some of these SNPs can be found in candidate cis-regulatory elements (CREs) such as promoters and enhancers, potentially destroying or creating DNA-binding motifs for transcription factors (TFs) and, hence, deregulating the expression of nearby genes. These aspects are understudied due to the sheer number of SNPs and TF binding motifs, making it challenging to identify SNPs that yield phenotypic changes or altered gene expression. RESULTS We developed a data-driven computational protocol to prioritize high-potential SNPs informed from former knowledge for experimental validation. We evaluated the protocol by investigating SNPs in CREs in the Janus kinase (JAK) - Signal Transducer and Activator of Transcription (-STAT) signaling pathway, which is activated by a plethora of cytokines and crucial in controlling immune responses and has been implicated in diseases like cancer, autoimmune disorders, and responses to viral infections. The protocol involves scanning the entire human genome (hg38) to pinpoint DNA sequences that deviate by only one nucleotide from the canonical binding sites (TTCnnnGAA) for STAT TFs. We narrowed down from an initial pool of 3,301,512 SNPs across 17,039,967 nearly complete STAT motifs and identified six potential gain-of-function SNPs in regions likely to influence regulation within the JAK-STAT pathway. This selection was guided by publicly available open chromatin and gene expression data and further refined by filtering for proximity to immune response genes and conservation between the mouse and human genomes. CONCLUSION Our findings highlight the value of combining genomic, epigenomic, and cross-species conservation data to effectively narrow down millions of SNPs to a smaller number with a high potential to induce interferon regulation of nearby genes. These SNPs can finally be reviewed manually, laying the groundwork for a more focused and efficient exploration of regulatory SNPs in an experimental setting.
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Affiliation(s)
- Markus Hoffmann
- Section of Genetics and Physiology, Digestive and Kidney Diseases, National Institute of Diabetes, National Institutes of Health, Bethesda, MD, 20892, USA.
| | - Tiago Vaz
- Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, 20892, USA
| | - Shreeti Chhatrala
- Section of Genetics and Physiology, Digestive and Kidney Diseases, National Institute of Diabetes, National Institutes of Health, Bethesda, MD, 20892, USA
- Department of Biochemistry and Molecular & Cellular Biology, Georgetown University Medical Center, Washington, D.C., 20007, USA
| | - Lothar Hennighausen
- Section of Genetics and Physiology, Digestive and Kidney Diseases, National Institute of Diabetes, National Institutes of Health, Bethesda, MD, 20892, USA.
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Luo H, Wang J, Qin L, Zhang X, Liu H, Niu C, Song M, Shao C, Xu P, Yu M, Zhang H, Ye Y, Xu X. Activation of γ-globin expression by a common variant disrupting IKAROS-binding motif in β-thalassemia. J Genet Genomics 2025; 52:157-167. [PMID: 39521044 DOI: 10.1016/j.jgg.2024.10.015] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/04/2024] [Revised: 10/27/2024] [Accepted: 10/28/2024] [Indexed: 11/16/2024]
Abstract
Programmed silencing of γ-globin genes in adult erythropoiesis is mediated by several chromatin remodeling complexes, which determine the stage-specific genome architecture in this region. Identification of cis- or trans-acting mutations contributing to the diverse extent of fetal hemoglobin (Hb F) might illustrate the underlying mechanism of γ-β-globin switching. Here, we recruit a cohort of 1142 β-thalassemia patients and dissect the natural variants in the whole β-globin gene cluster through a targeted next-generation sequencing panel. A previously unreported SNP rs7948668, predicted to disrupt the binding motif of IKAROS as a key component of chromatin remodeling complexes, is identified to be significantly associated with higher levels of Hb F and age at onset. Gene-editing on this SNP leads to the elevation of Hb F in both HUDEP-2 and primary CD34+ cells while the extent of elevation is amplified in the context of β-thalassemia mutations, indicating epistasis effects of the SNP in the regulation of Hb F. Finally, we perform ChIP-qPCR and 4C assays to prove that this variant disrupts the binding motif of IKAROS, leading to enhanced competitiveness of HBG promoters to locus control regions. This study highlights the significance of common regulatory SNPs and provides potential targets for treating β-hemoglobinopathy.
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Affiliation(s)
- Hualei Luo
- Innovation Center for Diagnostics and Treatment of Thalassemia, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong 510515, China; Department of Medical Genetics, School of Basic Medical Sciences, Southern Medical University, Guangzhou, Guangdong 510515, China
| | - Jueheng Wang
- State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai 201114, China
| | - Lang Qin
- Innovation Center for Diagnostics and Treatment of Thalassemia, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong 510515, China; Department of Medical Genetics, School of Basic Medical Sciences, Southern Medical University, Guangzhou, Guangdong 510515, China
| | - Xinhua Zhang
- Department of Pediatrics, 923rd Hospital of the People's Liberation Army, Nanning, Guangxi 530021, China
| | - Hailiang Liu
- Innovation Center for Diagnostics and Treatment of Thalassemia, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong 510515, China; Department of Medical Genetics, School of Basic Medical Sciences, Southern Medical University, Guangzhou, Guangdong 510515, China
| | - Chao Niu
- Innovation Center for Diagnostics and Treatment of Thalassemia, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong 510515, China; Department of Medical Genetics, School of Basic Medical Sciences, Southern Medical University, Guangzhou, Guangdong 510515, China
| | - Mengyang Song
- Innovation Center for Diagnostics and Treatment of Thalassemia, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong 510515, China; Department of Medical Genetics, School of Basic Medical Sciences, Southern Medical University, Guangzhou, Guangdong 510515, China
| | - Congwen Shao
- Innovation Center for Diagnostics and Treatment of Thalassemia, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong 510515, China; Department of Medical Genetics, School of Basic Medical Sciences, Southern Medical University, Guangzhou, Guangdong 510515, China
| | - Peng Xu
- Cyrus Tang Medical Institute, National Clinical Research Centre for Hematologic Diseases, Collaborative Innovation Centre of Hematology, State Key Laboratory of Radiation Medicine and Protection, Soochow University Suzhou, Suzhou, Jiangsu 215031, China
| | - Miao Yu
- State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai 201114, China
| | - Haokun Zhang
- State Key Laboratory of Genetic Engineering, MOE Engineering Research Center of Gene Technology, School of Life Sciences, Fudan University, Shanghai 200438, China
| | - Yuhua Ye
- Innovation Center for Diagnostics and Treatment of Thalassemia, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong 510515, China; Department of Medical Genetics, School of Basic Medical Sciences, Southern Medical University, Guangzhou, Guangdong 510515, China.
| | - Xiangmin Xu
- Innovation Center for Diagnostics and Treatment of Thalassemia, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong 510515, China; Department of Medical Genetics, School of Basic Medical Sciences, Southern Medical University, Guangzhou, Guangdong 510515, China.
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Allisha J, Das J, Dunnigan T, Sharfstein ST, Datta P. Stipulations of cell and gene therapy and the ties to biomanufacturing. Biotechnol Prog 2025:e3521. [PMID: 39846483 DOI: 10.1002/btpr.3521] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2024] [Revised: 11/15/2024] [Accepted: 11/20/2024] [Indexed: 01/24/2025]
Abstract
Cell and gene therapy (CGT) products are emerging and innovative biopharmaceuticals that hold promise for treating diseases that are otherwise beyond the scope of conventional medicines. The evolution of CGT from a research idea to a promising therapeutic product is due to the complementary advancements across various scientific disciplines. First, the innovations and advancements in gene editing and delivery technology have provided fundamental tools to manipulate genes and cells for therapeutic pursuits. Second, advancements in applied and translational research, including how clinical trials are designed, performed, evaluated, and analyzed, have transformed the technology into a potential therapeutic product. Third, advancements in scaling up the production of CGT products have been critical in delivering the product for preclinical studies, clinical trials, and approved treatments. In parallel, regulatory requirements have continuously evolved, with lessons learned from translational studies and biomanufacturing. These combined efforts have transformed CGT products from a promising concept into a reality with the potential to treat a wide range of diseases. However, continued R&D and regulatory oversight are crucial to further improve the safety, efficacy, and accessibility of CGT products.
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Affiliation(s)
- Justin Allisha
- Department of Life Sciences, Albany College of Pharmacy and Health Sciences, Albany, New York, USA
| | - Juthika Das
- Department of Life Sciences, Albany College of Pharmacy and Health Sciences, Albany, New York, USA
| | - Thomas Dunnigan
- Department of Life Sciences, Albany College of Pharmacy and Health Sciences, Albany, New York, USA
| | - Susan T Sharfstein
- Department of Nanoscale Science and Engineering and The RNA Institute, University at Albany, State University of New York, Albany, New York, USA
| | - Payel Datta
- Department of Life Sciences, Albany College of Pharmacy and Health Sciences, Albany, New York, USA
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Chandraprabha PB, Azhagiri MKK, Venkatesan V, Magis W, Prasad K, Suresh S, Pai AA, Marepally S, Srivastava A, Mohankumar KM, Martin DIK, Thangavel S. Enhanced fetal hemoglobin production via dual-beneficial mutation editing of the HBG promoter in hematopoietic stem and progenitor cells for β-hemoglobinopathies. Stem Cell Res Ther 2024; 15:504. [PMID: 39736768 DOI: 10.1186/s13287-024-04117-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/12/2024] [Accepted: 12/11/2024] [Indexed: 01/01/2025] Open
Abstract
BACKGROUND Sickle cell disease (SCD) and β-thalassemia patients with elevated gamma globin (HBG1/G2) levels exhibit mild or no symptoms. To recapitulate this natural phenomenon, the most coveted gene therapy approach is to edit the regulatory sequences of HBG1/G2 to reactivate them. By editing more than one regulatory sequence in the HBG promoter, the production of fetal hemoglobin (HbF) can be significantly increased. However, achieving this goal requires precise nucleotide conversions in hematopoietic stem and progenitor cells (HSPCs) at therapeutic efficiency, which remains a challenge. METHODS We employed Cas9 RNP-ssODN-mediated homology-directed repair (HDR) gene editing to mimic two naturally occurring HBG promoter point mutations; -175T > C, associated with high HbF levels, and -158 C > T, a common polymorphism in the Indian population that induces HbF under erythropoietic stress, in HSPCs. RESULTS Asymmetric, nontarget ssODN induced high rates of complete HDR conversions, with at least 15% of HSPCs exhibiting both the -175T > C and -158 C > T mutations. Optimized conditions and treatment with the small molecule AZD-7648 increased this rate, with up to 57% of long-term engrafting human HSPCs in NBSGW mice containing at least one beneficial mutation. Functionally, in vivo erythroblasts exhibited high levels of HbF, which was sufficient to reverse the cellular phenotype of β-thalassemia. Further support through bone marrow MSC co-culture boosted complete HDR conversion rates to exceed 80%, with minimal InDels, improved cell viability, and induced fetal hemoglobin levels similar to those of Cas9 RNP-mediated indels at BCL11A enhancer and HBG promoter. CONCLUSIONS Cas9 RNP-ssODN-based nucleotide conversion at the HBG promoter offers a promising gene therapy approach to ameliorate the phenotypes of β-thalassemia and SCD. The developed approach can simplify and broaden applications that require the cointroduction of multiple nucleotide modifications in HSPCs.
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Affiliation(s)
- Prathibha Babu Chandraprabha
- Centre for Stem Cell Research (CSCR), A Unit of InStem Bengaluru, Christian Medical College Campus, Vellore, Tamil Nadu, 632002, India
- Manipal Academy of Higher Education, Manipal, Karnataka, 576104, India
| | - Manoj Kumar K Azhagiri
- Centre for Stem Cell Research (CSCR), A Unit of InStem Bengaluru, Christian Medical College Campus, Vellore, Tamil Nadu, 632002, India
- Manipal Academy of Higher Education, Manipal, Karnataka, 576104, India
| | - Vigneshwaran Venkatesan
- Centre for Stem Cell Research (CSCR), A Unit of InStem Bengaluru, Christian Medical College Campus, Vellore, Tamil Nadu, 632002, India
- Manipal Academy of Higher Education, Manipal, Karnataka, 576104, India
| | - Wendy Magis
- Children's Hospital Oakland Research Institute, UCSF Benioff Children's Hospital Oakland, Oakland, CA, 94609, USA
| | - Kirti Prasad
- Centre for Stem Cell Research (CSCR), A Unit of InStem Bengaluru, Christian Medical College Campus, Vellore, Tamil Nadu, 632002, India
- Manipal Academy of Higher Education, Manipal, Karnataka, 576104, India
| | - Sevanthy Suresh
- Centre for Stem Cell Research (CSCR), A Unit of InStem Bengaluru, Christian Medical College Campus, Vellore, Tamil Nadu, 632002, India
- Manipal Academy of Higher Education, Manipal, Karnataka, 576104, India
| | - Aswin Anand Pai
- Department of Hematology, Christian Medical College, Vellore, Tamil Nadu, 632004, India
| | - Srujan Marepally
- Centre for Stem Cell Research (CSCR), A Unit of InStem Bengaluru, Christian Medical College Campus, Vellore, Tamil Nadu, 632002, India
| | - Alok Srivastava
- Centre for Stem Cell Research (CSCR), A Unit of InStem Bengaluru, Christian Medical College Campus, Vellore, Tamil Nadu, 632002, India
- Department of Hematology, Christian Medical College, Vellore, Tamil Nadu, 632004, India
| | | | - David I K Martin
- Children's Hospital Oakland Research Institute, UCSF Benioff Children's Hospital Oakland, Oakland, CA, 94609, USA
| | - Saravanabhavan Thangavel
- Centre for Stem Cell Research (CSCR), A Unit of InStem Bengaluru, Christian Medical College Campus, Vellore, Tamil Nadu, 632002, India.
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Diamantidis MD, Ikonomou G, Argyrakouli I, Pantelidou D, Delicou S. Genetic Modifiers of Hemoglobin Expression from a Clinical Perspective in Hemoglobinopathy Patients with Beta Thalassemia and Sickle Cell Disease. Int J Mol Sci 2024; 25:11886. [PMID: 39595957 PMCID: PMC11593634 DOI: 10.3390/ijms252211886] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2024] [Revised: 10/30/2024] [Accepted: 11/04/2024] [Indexed: 11/28/2024] Open
Abstract
Hemoglobinopathies, namely β-thalassemia and sickle cell disease (SCD), are hereditary diseases, characterized by molecular genetic aberrations in the beta chains of hemoglobin. These defects affect the normal production of hemoglobin with severe anemia due to less or no amount of beta globins in patients with β-thalassemia (quantitative disorder), while SCD is a serious disease in which a mutated form of hemoglobin distorts the red blood cells into a crescent shape at low oxygen levels (qualitative disorder). Despite the revolutionary progress in recent years with the approval of gene therapy and gene editing for specific patients, there is an unmet need for highlighting the mechanisms influencing hemoglobin production and for the development of novel drugs and targeted therapies. The identification of the transcription factors and other genetic modifiers of hemoglobin expression is of utmost importance for discovering novel therapeutic approaches for patients with hemoglobinopathies. The aim of this review is to describe these complex molecular mechanisms and pathways affecting hemoglobin expression and to highlight the relevant investigational approaches or pharmaceutical interventions focusing on restoring the hemoglobin normal function by linking the molecular background of the disease with the clinical perspective. All the associated drugs increasing the hemoglobin expression in patients with hemoglobinopathies, along with gene therapy and gene editing, are also discussed.
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Affiliation(s)
- Michael D. Diamantidis
- Department of Hematology, Thalassemia and Sickle Cell Disease Unit, General Hospital of Larissa, 41221 Larissa, Greece;
| | - Georgia Ikonomou
- Thalassemia and Sickle Cell Disease Prevention Unit, General Hospital of Larissa, 41221 Larissa, Greece;
| | - Ioanna Argyrakouli
- Department of Hematology, Thalassemia and Sickle Cell Disease Unit, General Hospital of Larissa, 41221 Larissa, Greece;
| | - Despoina Pantelidou
- Thalassemia and Sickle Cell Disease Unit, AHEPA University General Hospital, 41221 Thessaloniki, Greece;
| | - Sophia Delicou
- Center of Expertise in Hemoglobinopathies and Their Complications, Thalassemia and Sickle Cell Disease Unit, Hippokration General Hospital, 41221 Athens, Greece;
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9
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Khandros E, Blobel GA. Elevating fetal hemoglobin: recently discovered regulators and mechanisms. Blood 2024; 144:845-852. [PMID: 38728575 PMCID: PMC11830979 DOI: 10.1182/blood.2023022190] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2023] [Revised: 04/30/2024] [Accepted: 04/30/2024] [Indexed: 05/12/2024] Open
Abstract
ABSTRACT It has been known for over half a century that throughout ontogeny, humans produce different forms of hemoglobin, a tetramer of α- and β-like hemoglobin chains. The switch from fetal to adult hemoglobin occurs around the time of birth when erythropoiesis shifts from the fetal liver to the bone marrow. Naturally, diseases caused by defective adult β-globin genes, such as sickle cell disease and β-thalassemia, manifest themselves as the production of fetal hemoglobin fades. Reversal of this developmental switch has been a major goal to treat these diseases and has been a driving force to understand its underlying molecular biology. Several review articles have illustrated the long and at times arduous paths that led to the discovery of the first transcriptional regulators involved in this process. Here, we survey recent developments spurred by the discovery of CRISPR tools that enabled for the first time high-throughput genetic screens for new molecules that impact the fetal-to-adult hemoglobin switch. Numerous opportunities for therapeutic intervention have thus come to light, offering hope for effective pharmacologic intervention for patients for whom gene therapy is out of reach.
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Affiliation(s)
- Eugene Khandros
- Division of Hematology, Children’s Hospital of Philadelphia, Philadelphia, PA
- Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA
| | - Gerd A. Blobel
- Division of Hematology, Children’s Hospital of Philadelphia, Philadelphia, PA
- Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA
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Jiao L, Zhao J, Wang C, Liu X, Liu F, Li L, Shang R, Li Y, Ma W, Yang S. Nature-Inspired Intelligent Computing: A Comprehensive Survey. RESEARCH (WASHINGTON, D.C.) 2024; 7:0442. [PMID: 39156658 PMCID: PMC11327401 DOI: 10.34133/research.0442] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 05/21/2024] [Accepted: 07/14/2024] [Indexed: 08/20/2024]
Abstract
Nature, with its numerous surprising rules, serves as a rich source of creativity for the development of artificial intelligence, inspiring researchers to create several nature-inspired intelligent computing paradigms based on natural mechanisms. Over the past decades, these paradigms have revealed effective and flexible solutions to practical and complex problems. This paper summarizes the natural mechanisms of diverse advanced nature-inspired intelligent computing paradigms, which provide valuable lessons for building general-purpose machines capable of adapting to the environment autonomously. According to the natural mechanisms, we classify nature-inspired intelligent computing paradigms into 4 types: evolutionary-based, biological-based, social-cultural-based, and science-based. Moreover, this paper also illustrates the interrelationship between these paradigms and natural mechanisms, as well as their real-world applications, offering a comprehensive algorithmic foundation for mitigating unreasonable metaphors. Finally, based on the detailed analysis of natural mechanisms, the challenges of current nature-inspired paradigms and promising future research directions are presented.
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Affiliation(s)
- Licheng Jiao
- School of Artificial Intelligence, Xidian University, Xi’an, China
| | - Jiaxuan Zhao
- School of Artificial Intelligence, Xidian University, Xi’an, China
| | - Chao Wang
- School of Artificial Intelligence, Xidian University, Xi’an, China
| | - Xu Liu
- School of Artificial Intelligence, Xidian University, Xi’an, China
| | - Fang Liu
- School of Artificial Intelligence, Xidian University, Xi’an, China
| | - Lingling Li
- School of Artificial Intelligence, Xidian University, Xi’an, China
| | - Ronghua Shang
- School of Artificial Intelligence, Xidian University, Xi’an, China
| | - Yangyang Li
- School of Artificial Intelligence, Xidian University, Xi’an, China
| | - Wenping Ma
- School of Artificial Intelligence, Xidian University, Xi’an, China
| | - Shuyuan Yang
- School of Artificial Intelligence, Xidian University, Xi’an, China
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11
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Zhang X, Xia F, Zhang X, Blumenthal RM, Cheng X. C2H2 Zinc Finger Transcription Factors Associated with Hemoglobinopathies. J Mol Biol 2024; 436:168343. [PMID: 37924864 PMCID: PMC11185177 DOI: 10.1016/j.jmb.2023.168343] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/04/2023] [Revised: 10/23/2023] [Accepted: 10/30/2023] [Indexed: 11/06/2023]
Abstract
In humans, specific aberrations in β-globin results in sickle cell disease and β-thalassemia, symptoms of which can be ameliorated by increased expression of fetal globin (HbF). Two recent CRISPR-Cas9 screens, centered on ∼1500 annotated sequence-specific DNA binding proteins and performed in a human erythroid cell line that expresses adult hemoglobin, uncovered four groups of candidate regulators of HbF gene expression. They are (1) members of the nucleosome remodeling and deacetylase (NuRD) complex proteins that are already known for HbF control; (2) seven C2H2 zinc finger (ZF) proteins, including some (ZBTB7A and BCL11A) already known for directly silencing the fetal γ-globin genes in adult human erythroid cells; (3) a few other transcription factors of different structural classes that might indirectly influence HbF gene expression; and (4) DNA methyltransferase 1 (DNMT1) that maintains the DNA methylation marks that attract the MBD2-associated NuRD complex to DNA as well as associated histone H3 lysine 9 methylation. Here we briefly discuss the effects of these regulators, particularly C2H2 ZFs, in inducing HbF expression for treating β-hemoglobin disorders, together with recent advances in developing safe and effective small-molecule therapeutics for the regulation of this well-conserved hemoglobin switch.
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Affiliation(s)
- Xing Zhang
- Department of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.
| | - Fangfang Xia
- Department of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA
| | - Xiaotian Zhang
- Department of Biochemistry and Molecular Biology, The University of Texas Health Science Center Houston, McGovern Medical School, Houston, TX 77030, USA
| | - Robert M Blumenthal
- Department of Medical Microbiology and Immunology, and Program in Bioinformatics, The University of Toledo College of Medicine and Life Sciences, Toledo, OH 43614, USA
| | - Xiaodong Cheng
- Department of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.
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12
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Wu Q, Carlos AR, Braza F, Bergman ML, Kitoko JZ, Bastos-Amador P, Cuadrado E, Martins R, Oliveira BS, Martins VC, Scicluna BP, Landry JJ, Jung FE, Ademolue TW, Peitzsch M, Almeida-Santos J, Thompson J, Cardoso S, Ventura P, Slot M, Rontogianni S, Ribeiro V, Domingues VDS, Cabral IA, Weis S, Groth M, Ameneiro C, Fidalgo M, Wang F, Demengeot J, Amsen D, Soares MP. Ferritin heavy chain supports stability and function of the regulatory T cell lineage. EMBO J 2024; 43:1445-1483. [PMID: 38499786 PMCID: PMC11021483 DOI: 10.1038/s44318-024-00064-x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2023] [Revised: 02/15/2024] [Accepted: 02/20/2024] [Indexed: 03/20/2024] Open
Abstract
Regulatory T (TREG) cells develop via a program orchestrated by the transcription factor forkhead box protein P3 (FOXP3). Maintenance of the TREG cell lineage relies on sustained FOXP3 transcription via a mechanism involving demethylation of cytosine-phosphate-guanine (CpG)-rich elements at conserved non-coding sequences (CNS) in the FOXP3 locus. This cytosine demethylation is catalyzed by the ten-eleven translocation (TET) family of dioxygenases, and it involves a redox reaction that uses iron (Fe) as an essential cofactor. Here, we establish that human and mouse TREG cells express Fe-regulatory genes, including that encoding ferritin heavy chain (FTH), at relatively high levels compared to conventional T helper cells. We show that FTH expression in TREG cells is essential for immune homeostasis. Mechanistically, FTH supports TET-catalyzed demethylation of CpG-rich sequences CNS1 and 2 in the FOXP3 locus, thereby promoting FOXP3 transcription and TREG cell stability. This process, which is essential for TREG lineage stability and function, limits the severity of autoimmune neuroinflammation and infectious diseases, and favors tumor progression. These findings suggest that the regulation of intracellular iron by FTH is a stable property of TREG cells that supports immune homeostasis and limits the pathological outcomes of immune-mediated inflammation.
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Affiliation(s)
- Qian Wu
- Instituto Gulbenkian de Ciência, Oeiras, Portugal
- International Institutes of Medicine, the Fourth Affiliated Hospital of Zhejiang University, School of Medicine, Yiwu, Zhejiang, China
| | - Ana Rita Carlos
- Instituto Gulbenkian de Ciência, Oeiras, Portugal
- Departamento de Biologia Animal, Centro de Ecologia, Evolução e Alterações Ambientais, Faculdade de Ciências, Universidade de Lisboa, Lisboa, Portugal
| | - Faouzi Braza
- Instituto Gulbenkian de Ciência, Oeiras, Portugal
| | | | | | | | - Eloy Cuadrado
- Department of Hematopoiesis and Department of Immunopathology, Sanquin Research and Landsteiner Laboratory, Amsterdam, The Netherlands
| | - Rui Martins
- Instituto Gulbenkian de Ciência, Oeiras, Portugal
| | | | | | - Brendon P Scicluna
- Department of Applied Biomedical Science, Faculty of Health Sciences, Mater Dei Hospital, and Centre for Molecular Medicine and Biobanking, University of Malta, Msida, Malta
| | - Jonathan Jm Landry
- Genomic Core Facility, European Molecular Biology Laboratory, Heidelberg, Germany
| | - Ferris E Jung
- Genomic Core Facility, European Molecular Biology Laboratory, Heidelberg, Germany
| | | | - Mirko Peitzsch
- Institute for Clinical Chemistry and Laboratory Medicine, University Clinic Carl Gustav Carus, TU Dresden, Dresden, Germany
| | | | | | | | | | - Manon Slot
- Department of Hematopoiesis and Department of Immunopathology, Sanquin Research and Landsteiner Laboratory, Amsterdam, The Netherlands
| | - Stamatia Rontogianni
- Department of Hematopoiesis and Department of Immunopathology, Sanquin Research and Landsteiner Laboratory, Amsterdam, The Netherlands
| | - Vanessa Ribeiro
- Departamento de Biologia Animal, Centro de Ecologia, Evolução e Alterações Ambientais, Faculdade de Ciências, Universidade de Lisboa, Lisboa, Portugal
| | | | | | - Sebastian Weis
- Department for Anesthesiology and Intensive Care Medicine, Jena University Hospital, Friedrich-Schiller University, Jena, Germany
- Institute for Infectious Disease and Infection Control, Jena University Hospital, Friedrich-Schiller University, Jena, Germany
- Leibniz Institute for Natural Product Research and Infection Biology, Hans-Knöll Institute-HKI, Jena, Germany
| | - Marco Groth
- Leibniz Institute on Aging-Fritz Lipmann Institute, Jena, Germany
| | - Cristina Ameneiro
- Center for Research in Molecular Medicine and Chronic Diseases (CiMUS), Universidade de Santiago de Compostela-Health Research Institute (IDIS), Santiago de Compostela, Spain
| | - Miguel Fidalgo
- Center for Research in Molecular Medicine and Chronic Diseases (CiMUS), Universidade de Santiago de Compostela-Health Research Institute (IDIS), Santiago de Compostela, Spain
| | - Fudi Wang
- The Second Affiliated Hospital, School of Public Health, Zhejiang University School of Medicine, Hangzhou, 310058, China
| | | | - Derk Amsen
- Department of Hematopoiesis and Department of Immunopathology, Sanquin Research and Landsteiner Laboratory, Amsterdam, The Netherlands
- Department of Experimental Immunology, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands
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13
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Mayuranathan T, Newby GA, Feng R, Yao Y, Mayberry KD, Lazzarotto CR, Li Y, Levine RM, Nimmagadda N, Dempsey E, Kang G, Porter SN, Doerfler PA, Zhang J, Jang Y, Chen J, Bell HW, Crossley M, Bhoopalan SV, Sharma A, Tisdale JF, Pruett-Miller SM, Cheng Y, Tsai SQ, Liu DR, Weiss MJ, Yen JS. Potent and uniform fetal hemoglobin induction via base editing. Nat Genet 2023; 55:1210-1220. [PMID: 37400614 PMCID: PMC10722557 DOI: 10.1038/s41588-023-01434-7] [Citation(s) in RCA: 27] [Impact Index Per Article: 13.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/11/2022] [Accepted: 05/23/2023] [Indexed: 07/05/2023]
Abstract
Inducing fetal hemoglobin (HbF) in red blood cells can alleviate β-thalassemia and sickle cell disease. We compared five strategies in CD34+ hematopoietic stem and progenitor cells, using either Cas9 nuclease or adenine base editors. The most potent modification was adenine base editor generation of γ-globin -175A>G. Homozygous -175A>G edited erythroid colonies expressed 81 ± 7% HbF versus 17 ± 11% in unedited controls, whereas HbF levels were lower and more variable for two Cas9 strategies targeting a BCL11A binding motif in the γ-globin promoter or a BCL11A erythroid enhancer. The -175A>G base edit also induced HbF more potently than a Cas9 approach in red blood cells generated after transplantation of CD34+ hematopoietic stem and progenitor cells into mice. Our data suggest a strategy for potent, uniform induction of HbF and provide insights into γ-globin gene regulation. More generally, we demonstrate that diverse indels generated by Cas9 can cause unexpected phenotypic variation that can be circumvented by base editing.
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Affiliation(s)
| | - Gregory A Newby
- Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, MA, USA
- Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA
- Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA
| | - Ruopeng Feng
- Department of Hematology, St. Jude Children's Research Hospital, Memphis, TN, USA
| | - Yu Yao
- Department of Hematology, St. Jude Children's Research Hospital, Memphis, TN, USA
| | - Kalin D Mayberry
- Department of Hematology, St. Jude Children's Research Hospital, Memphis, TN, USA
| | - Cicera R Lazzarotto
- Department of Hematology, St. Jude Children's Research Hospital, Memphis, TN, USA
| | - Yichao Li
- Department of Hematology, St. Jude Children's Research Hospital, Memphis, TN, USA
| | - Rachel M Levine
- Department of Hematology, St. Jude Children's Research Hospital, Memphis, TN, USA
| | - Nikitha Nimmagadda
- Department of Hematology, St. Jude Children's Research Hospital, Memphis, TN, USA
| | - Erin Dempsey
- Department of Hematology, St. Jude Children's Research Hospital, Memphis, TN, USA
| | - Guolian Kang
- Department of Biostatistics, St. Jude Children's Research Hospital, Memphis, TN, USA
| | - Shaina N Porter
- Department of Cell and Molecular Biology, St. Jude Children's Research Hospital, Memphis, TN, USA
| | - Phillip A Doerfler
- Department of Hematology, St. Jude Children's Research Hospital, Memphis, TN, USA
| | - Jingjing Zhang
- Department of Hematology, St. Jude Children's Research Hospital, Memphis, TN, USA
| | - Yoonjeong Jang
- Department of Hematology, St. Jude Children's Research Hospital, Memphis, TN, USA
| | - Jingjing Chen
- Department of Hematology, St. Jude Children's Research Hospital, Memphis, TN, USA
| | - Henry W Bell
- School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, New South Wales, Australia
| | - Merlin Crossley
- School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, New South Wales, Australia
| | | | - Akshay Sharma
- Department of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children's Research Hospital, Memphis, TN, USA
| | - John F Tisdale
- Cellular and Molecular Therapeutics Branch, National Heart, Lung, and Blood Institute and National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD, USA
| | - Shondra M Pruett-Miller
- Department of Cell and Molecular Biology, St. Jude Children's Research Hospital, Memphis, TN, USA
| | - Yong Cheng
- Department of Hematology, St. Jude Children's Research Hospital, Memphis, TN, USA
| | - Shengdar Q Tsai
- Department of Hematology, St. Jude Children's Research Hospital, Memphis, TN, USA
| | - David R Liu
- Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, MA, USA.
- Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA.
- Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA.
| | - Mitchell J Weiss
- Department of Hematology, St. Jude Children's Research Hospital, Memphis, TN, USA.
| | - Jonathan S Yen
- Department of Hematology, St. Jude Children's Research Hospital, Memphis, TN, USA.
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14
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Paschoudi K, Yannaki E, Psatha N. Precision Editing as a Therapeutic Approach for β-Hemoglobinopathies. Int J Mol Sci 2023; 24:9527. [PMID: 37298481 PMCID: PMC10253463 DOI: 10.3390/ijms24119527] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/21/2023] [Revised: 05/19/2023] [Accepted: 05/26/2023] [Indexed: 06/12/2023] Open
Abstract
Beta-hemoglobinopathies are the most common genetic disorders worldwide, caused by a wide spectrum of mutations in the β-globin locus, and associated with morbidity and early mortality in case of patient non-adherence to supportive treatment. Allogeneic transplantation of hematopoietic stem cells (allo-HSCT) used to be the only curative option, although the indispensable need for an HLA-matched donor markedly restricted its universal application. The evolution of gene therapy approaches made possible the ex vivo delivery of a therapeutic β- or γ- globin gene into patient-derived hematopoietic stem cells followed by the transplantation of corrected cells into myeloablated patients, having led to high rates of transfusion independence (thalassemia) or complete resolution of painful crises (sickle cell disease-SCD). Hereditary persistence of fetal hemoglobin (HPFH), a syndrome characterized by increased γ-globin levels, when co-inherited with β-thalassemia or SCD, converts hemoglobinopathies to a benign condition with mild clinical phenotype. The rapid development of precise genome editing tools (ZFN, TALENs, CRISPR/Cas9) over the last decade has allowed the targeted introduction of mutations, resulting in disease-modifying outcomes. In this context, genome editing tools have successfully been used for the introduction of HPFH-like mutations both in HBG1/HBG2 promoters or/and in the erythroid enhancer of BCL11A to increase HbF expression as an alternative curative approach for β-hemoglobinopathies. The current investigation of new HbF modulators, such as ZBTB7A, KLF-1, SOX6, and ZNF410, further expands the range of possible genome editing targets. Importantly, genome editing approaches have recently reached clinical translation in trials investigating HbF reactivation in both SCD and thalassemic patients. Showing promising outcomes, these approaches are yet to be confirmed in long-term follow-up studies.
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Affiliation(s)
- Kiriaki Paschoudi
- Department of Genetics, Development and Molecular Biology, School of Biology, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece;
- Gene and Cell Therapy Center, Hematology Clinic, George Papanikolaou Hospital, Exokhi, 57010 Thessaloniki, Greece;
| | - Evangelia Yannaki
- Gene and Cell Therapy Center, Hematology Clinic, George Papanikolaou Hospital, Exokhi, 57010 Thessaloniki, Greece;
- Department of Hematology, School of Medicine, University of Washington, Seattle, WA 98195, USA
| | - Nikoletta Psatha
- Department of Genetics, Development and Molecular Biology, School of Biology, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece;
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15
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Martin-Rufino JD, Castano N, Pang M, Grody EI, Joubran S, Caulier A, Wahlster L, Li T, Qiu X, Riera-Escandell AM, Newby GA, Al'Khafaji A, Chaudhary S, Black S, Weng C, Munson G, Liu DR, Wlodarski MW, Sims K, Oakley JH, Fasano RM, Xavier RJ, Lander ES, Klein DE, Sankaran VG. Massively parallel base editing to map variant effects in human hematopoiesis. Cell 2023; 186:2456-2474.e24. [PMID: 37137305 PMCID: PMC10225359 DOI: 10.1016/j.cell.2023.03.035] [Citation(s) in RCA: 66] [Impact Index Per Article: 33.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/13/2022] [Revised: 02/26/2023] [Accepted: 03/30/2023] [Indexed: 05/05/2023]
Abstract
Systematic evaluation of the impact of genetic variants is critical for the study and treatment of human physiology and disease. While specific mutations can be introduced by genome engineering, we still lack scalable approaches that are applicable to the important setting of primary cells, such as blood and immune cells. Here, we describe the development of massively parallel base-editing screens in human hematopoietic stem and progenitor cells. Such approaches enable functional screens for variant effects across any hematopoietic differentiation state. Moreover, they allow for rich phenotyping through single-cell RNA sequencing readouts and separately for characterization of editing outcomes through pooled single-cell genotyping. We efficiently design improved leukemia immunotherapy approaches, comprehensively identify non-coding variants modulating fetal hemoglobin expression, define mechanisms regulating hematopoietic differentiation, and probe the pathogenicity of uncharacterized disease-associated variants. These strategies will advance effective and high-throughput variant-to-function mapping in human hematopoiesis to identify the causes of diverse diseases.
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Affiliation(s)
- Jorge D Martin-Rufino
- Division of Hematology/Oncology, Boston Children's Hospital and Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; PhD Program in Biological and Biomedical Sciences, Harvard Medical School, Boston, MA 02115, USA
| | - Nicole Castano
- Division of Hematology/Oncology, Boston Children's Hospital and Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA
| | - Michael Pang
- Division of Hematology/Oncology, Boston Children's Hospital and Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Harvard-MIT Health Sciences and Technology, Harvard Medical School, Boston, MA 02115, USA
| | | | - Samantha Joubran
- Division of Hematology/Oncology, Boston Children's Hospital and Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Chemical Biology PhD Program, Harvard Medical School, Boston, MA 02115, USA
| | - Alexis Caulier
- Division of Hematology/Oncology, Boston Children's Hospital and Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA
| | - Lara Wahlster
- Division of Hematology/Oncology, Boston Children's Hospital and Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA
| | - Tongqing Li
- Department of Pharmacology and Yale Cancer Biology Institute, Yale University School of Medicine, New Haven, CT 06510, USA
| | - Xiaojie Qiu
- Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA
| | | | - Gregory A Newby
- Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA; Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138, USA
| | - Aziz Al'Khafaji
- Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA
| | | | - Susan Black
- Division of Hematology/Oncology, Boston Children's Hospital and Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA
| | - Chen Weng
- Division of Hematology/Oncology, Boston Children's Hospital and Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA
| | - Glen Munson
- Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA
| | - David R Liu
- Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA; Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138, USA
| | - Marcin W Wlodarski
- Department of Hematology, St Jude Children's Research Hospital, Memphis, TN 38105, USA
| | - Kacie Sims
- St. Jude Affiliate Clinic at Our Lady of the Lake Children's Health, Baton Rouge, LA 70809, USA
| | - Jamie H Oakley
- Aflac Cancer and Blood Disorders Center, Children's Healthcare of Atlanta and Emory University, Atlanta, GA 30322, USA
| | - Ross M Fasano
- Aflac Cancer and Blood Disorders Center, Children's Healthcare of Atlanta and Emory University, Atlanta, GA 30322, USA
| | - Ramnik J Xavier
- Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Center for Computational and Integrative Biology, Department of Molecular Biology, and Center for the Study of Inflammatory Bowel Disease, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA
| | - Eric S Lander
- Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA
| | - Daryl E Klein
- Department of Pharmacology and Yale Cancer Biology Institute, Yale University School of Medicine, New Haven, CT 06510, USA
| | - Vijay G Sankaran
- Division of Hematology/Oncology, Boston Children's Hospital and Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Harvard Stem Cell Institute, Cambridge, MA 02138, USA.
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16
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Tognon M, Giugno R, Pinello L. A survey on algorithms to characterize transcription factor binding sites. Brief Bioinform 2023; 24:bbad156. [PMID: 37099664 PMCID: PMC10422928 DOI: 10.1093/bib/bbad156] [Citation(s) in RCA: 9] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/13/2023] [Revised: 03/27/2023] [Accepted: 04/01/2023] [Indexed: 04/28/2023] Open
Abstract
Transcription factors (TFs) are key regulatory proteins that control the transcriptional rate of cells by binding short DNA sequences called transcription factor binding sites (TFBS) or motifs. Identifying and characterizing TFBS is fundamental to understanding the regulatory mechanisms governing the transcriptional state of cells. During the last decades, several experimental methods have been developed to recover DNA sequences containing TFBS. In parallel, computational methods have been proposed to discover and identify TFBS motifs based on these DNA sequences. This is one of the most widely investigated problems in bioinformatics and is referred to as the motif discovery problem. In this manuscript, we review classical and novel experimental and computational methods developed to discover and characterize TFBS motifs in DNA sequences, highlighting their advantages and drawbacks. We also discuss open challenges and future perspectives that could fill the remaining gaps in the field.
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Affiliation(s)
- Manuel Tognon
- Computer Science Department, University of Verona, Verona, Italy
- Molecular Pathology Unit, Center for Computational and Integrative Biology and Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts, United States of America
- Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States of America
| | - Rosalba Giugno
- Computer Science Department, University of Verona, Verona, Italy
| | - Luca Pinello
- Molecular Pathology Unit, Center for Computational and Integrative Biology and Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts, United States of America
- Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States of America
- Department of Pathology, Harvard Medical School, Boston, Massachusetts, United States of America
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17
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Arif T, Farooq A, Ahmad FJ, Akhtar M, Choudhery MS. Prime editing: A potential treatment option for β-thalassemia. Cell Biol Int 2023; 47:699-713. [PMID: 36480796 DOI: 10.1002/cbin.11972] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/26/2022] [Accepted: 11/22/2022] [Indexed: 12/13/2022]
Abstract
The potential to therapeutically alter the genome is one of the remarkable scientific developments in recent years. Genome editing technologies have provided an opportunity to precisely alter genomic sequence(s) in eukaryotic cells as a treatment option for various genetic disorders. These technologies allow the correction of harmful mutations in patients by precise nucleotide editing. Genome editing technologies such as CRISPR (clustered regularly interspaced short palindromic repeat) and base editors have greatly contributed to the practical applications of gene editing. However, these technologies have certain limitations, including imperfect editing, undesirable mutations, off-target effects, and lack of potential to simultaneously edit multiple loci. Recently, prime editing (PE) has emerged as a new gene editing technology with the potential to overcome the above-mentioned limitations. Interestingly, PE not only has higher specificity but also does not require double-strand breaks. In addition, a minimum possibility of potential off-target mutant sites makes PE a preferred choice for therapeutic gene editing. Furthermore, PE has the potential to introduce insertion and deletions of all 12 single-base mutations at target sequences. Considering its potential, PE has been applied as a treatment option for genetic diseases including hemoglobinopathies. β-Thalassemia, for example, one of the most significant blood disorders characterized by reduced levels of functional hemoglobin, could potentially be treated using PE. Therapeutic reactivation of the γ-globin gene in adult β-thalassemia patients through PE technology is considered a promising therapeutic strategy. The current review aims to briefly discuss the genome editing strategies and potential applications of PE for the treatment of β-thalassemia. In addition, the review will also focus on challenges associated with the use of PE.
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Affiliation(s)
- Taqdees Arif
- Department of Human Genetics and Molecular Biology, University of Health Sciences Lahore, Lahore, Punjab, Pakistan
| | - Aroosa Farooq
- Department of Human Genetics and Molecular Biology, University of Health Sciences Lahore, Lahore, Punjab, Pakistan
| | - Fridoon Jawad Ahmad
- Department of Human Genetics and Molecular Biology, University of Health Sciences Lahore, Lahore, Punjab, Pakistan
| | - Muhammad Akhtar
- School of Biological Sciences, University of Punjab Lahore, Lahore, Punjab, Pakistan
| | - Mahmood S Choudhery
- Department of Human Genetics and Molecular Biology, University of Health Sciences Lahore, Lahore, Punjab, Pakistan
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18
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Abstract
Thalassemia syndromes are common monogenic disorders and represent a significant health issue worldwide. In this review, the authors elaborate on fundamental genetic knowledge about thalassemias, including the structure and location of globin genes, the production of hemoglobin during development, the molecular lesions causing α-, β-, and other thalassemia syndromes, the genotype-phenotype correlation, and the genetic modifiers of these conditions. In addition, they briefly discuss the molecular techniques applied for diagnosis and innovative cell and gene therapy strategies to cure these conditions.
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Affiliation(s)
- Nicolò Tesio
- Department of Clinical and Biological Sciences, San Luigi Gonzaga University Hospital, University of Torino, Regione Gonzole, 10, 10043 Orbassano, Turin, Italy. https://twitter.com/nicolotesio
| | - Daniel E Bauer
- Division of Hematology/Oncology, Boston Children's Hospital, Boston, MA, USA; Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, USA; Department of Pediatrics, Harvard Stem Cell Institute, Broad Institute, Harvard Medical School, Boston, MA, USA.
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19
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Peslak SA, Demirci S, Chandra V, Ryu B, Bhardwaj SK, Jiang J, Rupon JW, Throm RE, Uchida N, Leonard A, Essawi K, Bonifacino AC, Krouse AE, Linde NS, Donahue RE, Ferrara F, Wielgosz M, Abdulmalik O, Hamagami N, Germino-Watnick P, Le A, Chu R, Hinds M, Weiss MJ, Tong W, Tisdale JF, Blobel GA. Forced enhancer-promoter rewiring to alter gene expression in animal models. MOLECULAR THERAPY. NUCLEIC ACIDS 2023; 31:452-465. [PMID: 36852088 PMCID: PMC9958407 DOI: 10.1016/j.omtn.2023.01.016] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 06/20/2022] [Accepted: 01/25/2023] [Indexed: 02/01/2023]
Abstract
Transcriptional enhancers can be in physical proximity of their target genes via chromatin looping. The enhancer at the β-globin locus (locus control region [LCR]) contacts the fetal-type (HBG) and adult-type (HBB) β-globin genes during corresponding developmental stages. We have demonstrated previously that forcing proximity between the LCR and HBG genes in cultured adult-stage erythroid cells can activate HBG transcription. Activation of HBG expression in erythroid cells is of benefit to patients with sickle cell disease. Here, using the β-globin locus as a model, we provide proof of concept at the organismal level that forced enhancer rewiring might present a strategy to alter gene expression for therapeutic purposes. Hematopoietic stem and progenitor cells (HSPCs) from mice bearing human β-globin genes were transduced with lentiviral vectors expressing a synthetic transcription factor (ZF-Ldb1) that fosters LCR-HBG contacts. When engrafted into host animals, HSPCs gave rise to adult-type erythroid cells with elevated HBG expression. Vectors containing ZF-Ldb1 were optimized for activity in cultured human and rhesus macaque erythroid cells. Upon transplantation into rhesus macaques, erythroid cells from HSPCs expressing ZF-Ldb1 displayed elevated HBG production. These findings in two animal models suggest that forced redirection of gene-regulatory elements may be used to alter gene expression to treat disease.
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Affiliation(s)
- Scott A. Peslak
- Division of Hematology/Oncology, Department of Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA
- Division of Hematology, The Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA
| | - Selami Demirci
- Cellular and Molecular Therapeutics Branch, National Heart, Lung, and Blood Institutes (NHLBI), National Institutes of Health (NIH), Bethesda, MD 20892, USA
| | - Vemika Chandra
- Division of Hematology, The Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA
| | - Byoung Ryu
- Department of Hematology, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA
| | - Saurabh K. Bhardwaj
- Division of Hematology, The Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA
| | - Jing Jiang
- Division of Hematology, The Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA
- CAS Engineering Laboratory for Nanozyme, Institute of Biophysics, Chinese Academy of Sciences, Beijing, People’s Republic of China
| | - Jeremy W. Rupon
- Division of Hematology, The Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA
| | - Robert E. Throm
- Department of Hematology, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA
| | - Naoya Uchida
- Cellular and Molecular Therapeutics Branch, National Heart, Lung, and Blood Institutes (NHLBI), National Institutes of Health (NIH), Bethesda, MD 20892, USA
- Division of Molecular and Medical Genetics, Center for Gene and Cell Therapy, The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo, Japan
| | - Alexis Leonard
- Cellular and Molecular Therapeutics Branch, National Heart, Lung, and Blood Institutes (NHLBI), National Institutes of Health (NIH), Bethesda, MD 20892, USA
| | - Khaled Essawi
- Cellular and Molecular Therapeutics Branch, National Heart, Lung, and Blood Institutes (NHLBI), National Institutes of Health (NIH), Bethesda, MD 20892, USA
- Department of Medical Laboratory Science, College of Applied Medical Sciences, Jazan University, Jazan, Saudi Arabia
| | | | - Allen E. Krouse
- Translational Stem Cell Biology Branch, NHLBI, NIH, Bethesda, MD 20814, USA
| | - Nathaniel S. Linde
- Translational Stem Cell Biology Branch, NHLBI, NIH, Bethesda, MD 20814, USA
| | - Robert E. Donahue
- Cellular and Molecular Therapeutics Branch, National Heart, Lung, and Blood Institutes (NHLBI), National Institutes of Health (NIH), Bethesda, MD 20892, USA
| | - Francesca Ferrara
- Department of Hematology, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA
| | - Matthew Wielgosz
- Department of Hematology, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA
| | - Osheiza Abdulmalik
- Division of Hematology, The Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA
| | - Nicole Hamagami
- Division of Hematology, The Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA
| | - Paula Germino-Watnick
- Cellular and Molecular Therapeutics Branch, National Heart, Lung, and Blood Institutes (NHLBI), National Institutes of Health (NIH), Bethesda, MD 20892, USA
| | - Anh Le
- Cellular and Molecular Therapeutics Branch, National Heart, Lung, and Blood Institutes (NHLBI), National Institutes of Health (NIH), Bethesda, MD 20892, USA
| | - Rebecca Chu
- Cellular and Molecular Therapeutics Branch, National Heart, Lung, and Blood Institutes (NHLBI), National Institutes of Health (NIH), Bethesda, MD 20892, USA
| | - Malikiya Hinds
- Cellular and Molecular Therapeutics Branch, National Heart, Lung, and Blood Institutes (NHLBI), National Institutes of Health (NIH), Bethesda, MD 20892, USA
| | - Mitchell J. Weiss
- Department of Hematology, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA
| | - Wei Tong
- Division of Hematology, The Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA
| | - John F. Tisdale
- Cellular and Molecular Therapeutics Branch, National Heart, Lung, and Blood Institutes (NHLBI), National Institutes of Health (NIH), Bethesda, MD 20892, USA
| | - Gerd A. Blobel
- Division of Hematology, The Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA
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20
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Antoniou P, Hardouin G, Martinucci P, Frati G, Felix T, Chalumeau A, Fontana L, Martin J, Masson C, Brusson M, Maule G, Rosello M, Giovannangeli C, Abramowski V, de Villartay JP, Concordet JP, Del Bene F, El Nemer W, Amendola M, Cavazzana M, Cereseto A, Romano O, Miccio A. Base-editing-mediated dissection of a γ-globin cis-regulatory element for the therapeutic reactivation of fetal hemoglobin expression. Nat Commun 2022; 13:6618. [PMID: 36333351 PMCID: PMC9636226 DOI: 10.1038/s41467-022-34493-1] [Citation(s) in RCA: 31] [Impact Index Per Article: 10.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/06/2022] [Accepted: 10/20/2022] [Indexed: 11/06/2022] Open
Abstract
Sickle cell disease and β-thalassemia affect the production of the adult β-hemoglobin chain. The clinical severity is lessened by mutations that cause fetal γ-globin expression in adult life (i.e., the hereditary persistence of fetal hemoglobin). Mutations clustering ~200 nucleotides upstream of the HBG transcriptional start sites either reduce binding of the LRF repressor or recruit the KLF1 activator. Here, we use base editing to generate a variety of mutations in the -200 region of the HBG promoters, including potent combinations of four to eight γ-globin-inducing mutations. Editing of patient hematopoietic stem/progenitor cells is safe, leads to fetal hemoglobin reactivation and rescues the pathological phenotype. Creation of a KLF1 activator binding site is the most potent strategy - even in long-term repopulating hematopoietic stem/progenitor cells. Compared with a Cas9-nuclease approach, base editing avoids the generation of insertions, deletions and large genomic rearrangements and results in higher γ-globin levels. Our results demonstrate that base editing of HBG promoters is a safe, universal strategy for treating β-hemoglobinopathies.
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Affiliation(s)
- Panagiotis Antoniou
- Université Paris Cité, Imagine Institute, Laboratory of chromatin and gene regulation during development, INSERM UMR 1163, 75015, Paris, France
| | - Giulia Hardouin
- Université Paris Cité, Imagine Institute, Laboratory of chromatin and gene regulation during development, INSERM UMR 1163, 75015, Paris, France
- Université Paris Cité, Imagine Institute, Laboratory of Human Lymphohematopoiesis, INSERM UMR 1163, 75015, Paris, France
- Biotherapy Department and Clinical Investigation Center, Assistance Publique Hopitaux de Paris, INSERM, 75015, Paris, France
| | - Pierre Martinucci
- Université Paris Cité, Imagine Institute, Laboratory of chromatin and gene regulation during development, INSERM UMR 1163, 75015, Paris, France
| | - Giacomo Frati
- Université Paris Cité, Imagine Institute, Laboratory of chromatin and gene regulation during development, INSERM UMR 1163, 75015, Paris, France
| | - Tristan Felix
- Université Paris Cité, Imagine Institute, Laboratory of chromatin and gene regulation during development, INSERM UMR 1163, 75015, Paris, France
| | - Anne Chalumeau
- Université Paris Cité, Imagine Institute, Laboratory of chromatin and gene regulation during development, INSERM UMR 1163, 75015, Paris, France
| | - Letizia Fontana
- Université Paris Cité, Imagine Institute, Laboratory of chromatin and gene regulation during development, INSERM UMR 1163, 75015, Paris, France
| | - Jeanne Martin
- Université Paris Cité, Imagine Institute, Laboratory of chromatin and gene regulation during development, INSERM UMR 1163, 75015, Paris, France
| | - Cecile Masson
- Bioinformatics Platform, Imagine Institute, 75015, Paris, France
| | - Megane Brusson
- Université Paris Cité, Imagine Institute, Laboratory of chromatin and gene regulation during development, INSERM UMR 1163, 75015, Paris, France
| | - Giulia Maule
- CIBIO, University of Trento, 38100, Trento, Italy
| | - Marion Rosello
- Sorbonne Université, INSERM, CNRS, Institut de la Vision, 75015, Paris, France
| | | | - Vincent Abramowski
- Université Paris Cité, Imagine Institute, Laboratory of genome dynamics in the immune system, INSERM UMR 1163, 75015, Paris, France
| | - Jean-Pierre de Villartay
- Université Paris Cité, Imagine Institute, Laboratory of genome dynamics in the immune system, INSERM UMR 1163, 75015, Paris, France
| | - Jean-Paul Concordet
- INSERM U1154, CNRS UMR7196, Museum National d'Histoire Naturelle, Paris, France
| | - Filippo Del Bene
- Sorbonne Université, INSERM, CNRS, Institut de la Vision, 75015, Paris, France
| | - Wassim El Nemer
- Établissement Français du Sang, UMR 7268, 13005, Marseille, France
- Laboratoire d'Excellence GR-Ex, 75015, Paris, France
| | - Mario Amendola
- Genethon, 91000, Evry, France
- Université Paris-Saclay, Univ Evry, Inserm, Genethon, Integrare research unit UMR_S951, 91000, Evry, France
| | - Marina Cavazzana
- Biotherapy Department and Clinical Investigation Center, Assistance Publique Hopitaux de Paris, INSERM, 75015, Paris, France
- Université Paris Cité, 75015, Paris, France
- Imagine Institute, 75015, Paris, France
| | | | - Oriana Romano
- Department of Life Sciences, University of Modena and Reggio Emilia, 41125, Modena, Italy
| | - Annarita Miccio
- Université Paris Cité, Imagine Institute, Laboratory of chromatin and gene regulation during development, INSERM UMR 1163, 75015, Paris, France.
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21
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Shash H. Non-Transfusion-Dependent Thalassemia: A Panoramic Review. MEDICINA (KAUNAS, LITHUANIA) 2022; 58:medicina58101496. [PMID: 36295656 PMCID: PMC9608723 DOI: 10.3390/medicina58101496] [Citation(s) in RCA: 11] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 08/20/2022] [Revised: 10/08/2022] [Accepted: 10/18/2022] [Indexed: 11/06/2022]
Abstract
Non-transfusion-dependent thalassemia (NTDT) has been considered less severe than its transfusion-dependent variants. The most common forms of NTDT include β-thalassemia intermedia, hemoglobin E/beta thalassemia, and hemoglobin H disease. Patients with NTDT develop several clinical complications, despite their regular transfusion independence. Ineffective erythropoiesis, iron overload, and hypercoagulability are pathophysiological factors that lead to morbidities in these patients. Therefore, an early and accurate diagnosis of NTDT is essential to ascertaining early interventions. Currently, several conventional management options are available, with guidelines suggested by the Thalassemia International Federation, and novel therapies are being developed in light of the advancement of the understanding of this disease. This review aimed to increase clinicians’ awareness of NTDT, from its basic medical definition and genetics to its pathophysiology. Specific complications to NTDT were reviewed, along with the risk factors for its development. The indications of different therapeutic options were outlined, and recent advancements were reviewed.
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Affiliation(s)
- Hwazen Shash
- College of Medicine, Imam Abdulrahman Bin Faisal University, Dammam 31441, Saudi Arabia;
- Department of Pediatrics, King Fahad Hospital of the University, Al-Khobar 31952, Saudi Arabia
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22
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Krüppel-Like Factor 1: A Pivotal Gene Regulator in Erythropoiesis. Cells 2022; 11:cells11193069. [PMID: 36231031 PMCID: PMC9561966 DOI: 10.3390/cells11193069] [Citation(s) in RCA: 16] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2022] [Revised: 09/21/2022] [Accepted: 09/26/2022] [Indexed: 11/17/2022] Open
Abstract
Krüppel-like factor 1 (KLF1) plays a crucial role in erythropoiesis. In-depth studies conducted on mice and humans have highlighted its importance in erythroid lineage commitment, terminal erythropoiesis progression and the switching of globin genes from γ to β. The role of KLF1 in haemoglobin switching is exerted by the direct activation of β-globin gene and by the silencing of γ-globin through activation of BCL11A, an important γ-globin gene repressor. The link between KLF1 and γ-globin silencing identifies this transcription factor as a possible therapeutic target for β-hemoglobinopathies. Moreover, several mutations have been identified in the human genes that are responsible for various benign phenotypes and erythroid disorders. The study of the phenotype associated with each mutation has greatly contributed to the current understanding of the complex role of KLF1 in erythropoiesis. This review will focus on some of the principal functions of KLF1 on erythroid cell commitment and differentiation, spanning from primitive to definitive erythropoiesis. The fundamental role of KLF1 in haemoglobin switching will be also highlighted. Finally, an overview of the principal human mutations and relative phenotypes and disorders will be described.
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23
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Rahimmanesh I, Boshtam M, Kouhpayeh S, Khanahmad H, Dabiri A, Ahangarzadeh S, Esmaeili Y, Bidram E, Vaseghi G, Haghjooy Javanmard S, Shariati L, Zarrabi A, Varma RS. Gene Editing-Based Technologies for Beta-hemoglobinopathies Treatment. BIOLOGY 2022; 11:biology11060862. [PMID: 35741383 PMCID: PMC9219845 DOI: 10.3390/biology11060862] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/10/2022] [Revised: 05/19/2022] [Accepted: 05/31/2022] [Indexed: 06/12/2023]
Abstract
Beta (β)-thalassemia is a group of human inherited abnormalities caused by various molecular defects, which involves a decrease or cessation in the balanced synthesis of the β-globin chains in hemoglobin structure. Traditional treatment for β-thalassemia major is allogeneic bone marrow transplantation (BMT) from a completely matched donor. The limited number of human leukocyte antigen (HLA)-matched donors, long-term use of immunosuppressive regimen and higher risk of immunological complications have limited the application of this therapeutic approach. Furthermore, despite improvements in transfusion practices and chelation treatment, many lingering challenges have encouraged researchers to develop newer therapeutic strategies such as nanomedicine and gene editing. One of the most powerful arms of genetic manipulation is gene editing tools, including transcription activator-like effector nucleases, zinc-finger nucleases, and clustered regularly interspaced short palindromic repeat-Cas-associated nucleases. These tools have concentrated on γ- or β-globin addition, regulating the transcription factors involved in expression of endogenous γ-globin such as KLF1, silencing of γ-globin inhibitors including BCL11A, SOX6, and LRF/ZBTB7A, and gene repair strategies. In this review article, we present a systematic overview of the appliances of gene editing tools for β-thalassemia treatment and paving the way for patients' therapy.
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Affiliation(s)
- Ilnaz Rahimmanesh
- Applied Physiology Research Center, Cardiovascular Research Institute, Isfahan University of Medical Sciences, Isfahan 73461-81746, Iran
| | - Maryam Boshtam
- Isfahan Cardiovascular Research Center, Cardiovascular Research Institute, Isfahan University of Medical Sciences, Isfahan 81583-88994, Iran
| | - Shirin Kouhpayeh
- Erythron Genetics and Pathobiology Laboratory, Department of Immunology, Isfahan 76351-81647, Iran
| | - Hossein Khanahmad
- Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan 73461-81746, Iran
| | - Arezou Dabiri
- Applied Physiology Research Center, Cardiovascular Research Institute, Isfahan University of Medical Sciences, Isfahan 73461-81746, Iran
| | - Shahrzad Ahangarzadeh
- Infectious Diseases and Tropical Medicine Research Center, Isfahan University of Medical Sciences, Isfahan 73461-81746, Iran
| | - Yasaman Esmaeili
- Biosensor Research Center, School of Advanced Technologies in Medicine, Isfahan University of Medical Sciences, Isfahan 73461-81746, Iran
| | - Elham Bidram
- Biosensor Research Center, School of Advanced Technologies in Medicine, Isfahan University of Medical Sciences, Isfahan 73461-81746, Iran
- Department of Biomaterials, Nanotechnology and Tissue Engineering, School of Advanced Technologies in Medicine, Isfahan University of Medical Sciences, Isfahan 73461-81746, Iran
| | - Golnaz Vaseghi
- Isfahan Cardiovascular Research Center, Cardiovascular Research Institute, Isfahan University of Medical Sciences, Isfahan 81583-88994, Iran
| | - Shaghayegh Haghjooy Javanmard
- Applied Physiology Research Center, Cardiovascular Research Institute, Isfahan University of Medical Sciences, Isfahan 73461-81746, Iran
| | - Laleh Shariati
- Department of Biomaterials, Nanotechnology and Tissue Engineering, School of Advanced Technologies in Medicine, Isfahan University of Medical Sciences, Isfahan 73461-81746, Iran
- Cancer Prevention Research, Isfahan University of Medical Sciences, Isfahan 73461-81746, Iran
| | - Ali Zarrabi
- Department of Biomedical Engineering, Faculty of Engineering and Natural Sciences, Istinye University, Sariyer, Istanbul 34396, Turkey
| | - Rajender S Varma
- Regional Centre of Advanced Technologies and Materials, Department of Physical Chemistry, Faculty of Science, Palacky University, Šlechtitelů 27, 783 71 Olomouc, Czech Republic
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24
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Mitra S, Sarker J, Mojumder A, Shibbir TB, Das R, Emran TB, Tallei TE, Nainu F, Alshahrani AM, Chidambaram K, Simal-Gandara J. Genome editing and cancer: How far has research moved forward on CRISPR/Cas9? Biomed Pharmacother 2022; 150:113011. [PMID: 35483191 DOI: 10.1016/j.biopha.2022.113011] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/26/2022] [Revised: 04/16/2022] [Accepted: 04/19/2022] [Indexed: 11/02/2022] Open
Abstract
Cancer accounted for almost ten million deaths worldwide in 2020. Metastasis, characterized by cancer cell invasion to other parts of the body, is the main cause of cancer morbidity and mortality. Therefore, understanding the molecular mechanisms of tumor formation and discovery of potential drug targets are of great importance. Gene editing techniques can be used to find novel drug targets and study molecular mechanisms. In this review, we describe how popular gene-editing methods such as CRISPR/Cas9, TALEN and ZFNs work, and, by comparing them, we demonstrate that CRISPR/Cas9 has superior efficiency and precision. We further provide an overview of the recent applications of CRISPR/Cas9 to cancer research, focusing on the most common cancers such as breast cancer, lung cancer, colorectal cancer, and prostate cancer. We describe how these applications will shape future research and treatment of cancer, and propose new ways to overcome current challenges.
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Affiliation(s)
- Saikat Mitra
- Department of Pharmacy, Faculty of Pharmacy, University of Dhaka, Dhaka 1000, Bangladesh
| | - Joyatry Sarker
- Department of Genetic Engineering and Biotechnology, University of Dhaka, Dhaka 1000, Bangladesh
| | - Anik Mojumder
- Department of Genetic Engineering and Biotechnology, University of Dhaka, Dhaka 1000, Bangladesh
| | - Tasmim Bintae Shibbir
- Department of Genetic Engineering and Biotechnology, University of Dhaka, Dhaka 1000, Bangladesh
| | - Rajib Das
- Department of Pharmacy, Faculty of Pharmacy, University of Dhaka, Dhaka 1000, Bangladesh
| | - Talha Bin Emran
- Department of Pharmacy, BGC Trust University Bangladesh, Chittagong 4381, Bangladesh.
| | - Trina Ekawati Tallei
- Department of Biology, Faculty of Mathematics and Natural Sciences, Sam Ratulangi University, Manado 95115, North Sulawesi, Indonesia
| | - Firzan Nainu
- Faculty of Pharmacy, Hasanuddin University, Makassar 90245, Sulawesi Selatan, Indonesia
| | - Asma M Alshahrani
- Department of Clinical Pharmacy, College of Pharmacy, King Khalid University, Abha 61441, Saudi Arabia
| | - Kumarappan Chidambaram
- Department of Pharmacology and Toxicology, College of Pharmacy, King Khalid University, Abha 61421, Saudi Arabia
| | - Jesus Simal-Gandara
- Universidade de Vigo, Nutrition and Bromatology Group, Department of Analytical Chemistry and Food Science, Faculty of Science, E32004 Ourense, Spain.
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25
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Abstract
Enhancers control the establishment of spatiotemporal gene expression patterns throughout development. Over the past decade, the development of new technologies has improved our capacity to link enhancers with their target genes based on their colocalization within the same topological domains. However, the mechanisms that regulate how enhancers specifically activate some genes but not others within a given domain remain unclear. In this Review, we discuss recent insights into the factors controlling enhancer specificity, including the genetic composition of enhancers and promoters, the linear and 3D distance between enhancers and their target genes, and cell-type specific chromatin landscapes. We also discuss how elucidating the molecular principles of enhancer specificity might help us to better understand and predict the pathological consequences of human genetic, epigenetic and structural variants.
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Affiliation(s)
- Tomás Pachano
- Institute of Biomedicine and Biotechnology of Cantabria (IBBTEC), CSIC/Universidad de Cantabria/SODERCAN, Albert Einstein 22, 39011 Santander, Spain
| | - Endika Haro
- Institute of Biomedicine and Biotechnology of Cantabria (IBBTEC), CSIC/Universidad de Cantabria/SODERCAN, Albert Einstein 22, 39011 Santander, Spain
| | - Alvaro Rada-Iglesias
- Institute of Biomedicine and Biotechnology of Cantabria (IBBTEC), CSIC/Universidad de Cantabria/SODERCAN, Albert Einstein 22, 39011 Santander, Spain
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26
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Lu D, Xu Z, Peng Z, Yang Y, Song B, Xiong Z, Ma Z, Guan H, Chen B, Nakamura Y, Zeng J, Liu N, Sun X, Chen D. Induction of Fetal Hemoglobin by Introducing Natural Hereditary Persistence of Fetal Hemoglobin Mutations in the γ-Globin Gene Promoters for Genome Editing Therapies for β-Thalassemia. Front Genet 2022; 13:881937. [PMID: 35656314 PMCID: PMC9152165 DOI: 10.3389/fgene.2022.881937] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2022] [Accepted: 04/26/2022] [Indexed: 11/13/2022] Open
Abstract
Reactivation of γ-globin expression is a promising therapeutic approach for β-hemoglobinopathies. Here, we propose a novel Cas9/AAV6-mediated genome editing strategy for the treatment of β-thalassemia: Natural HPFH mutations -113A > G, -114C > T, -117G>A, -175T > C, -195C > G, and -198T > C were introduced by homologous recombination following disruption of BCL11A binding sites in HBG1/HBG2 promoters. Precise on-target editing and significantly increased γ-globin expression during erythroid differentiation were observed in both HUDEP-2 cells and primary HSPCs from β-thalassemia major patients. Moreover, edited HSPCs maintained the capacity for long-term hematopoietic reconstitution in B-NDG hTHPO mice. This study provides evidence of the effectiveness of introducing naturally occurring HPFH mutations as a genetic therapy for β-thalassemia.
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Affiliation(s)
- Dian Lu
- Department of Obstetrics and Gynecology, Center for Reproductive Medicine/Department of Fetal Medicine and Prenatal Diagnosis/BioResource Research Center, Key Laboratory for Major Obstetric Diseases of Guangdong Province, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, China
| | - Zhiliang Xu
- Department of Obstetrics and Gynecology, Center for Reproductive Medicine/Department of Fetal Medicine and Prenatal Diagnosis/BioResource Research Center, Key Laboratory for Major Obstetric Diseases of Guangdong Province, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, China
| | - Zhiyong Peng
- Nanfang-Chunfu Children’s Institute of Hematology, Taixin Hospital, Dongguan, China
| | - Yinghong Yang
- Department of Obstetrics and Gynecology, Center for Reproductive Medicine/Department of Fetal Medicine and Prenatal Diagnosis/BioResource Research Center, Key Laboratory for Major Obstetric Diseases of Guangdong Province, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, China
| | - Bing Song
- Department of Obstetrics and Gynecology, Center for Reproductive Medicine/Department of Fetal Medicine and Prenatal Diagnosis/BioResource Research Center, Key Laboratory for Major Obstetric Diseases of Guangdong Province, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, China
| | - Zeyu Xiong
- Department of Obstetrics and Gynecology, Center for Reproductive Medicine/Department of Fetal Medicine and Prenatal Diagnosis/BioResource Research Center, Key Laboratory for Major Obstetric Diseases of Guangdong Province, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, China
| | - Zhirui Ma
- Nanfang-Chunfu Children’s Institute of Hematology, Taixin Hospital, Dongguan, China
| | - Hongmei Guan
- Department of Obstetrics and Gynecology, Center for Reproductive Medicine/Department of Fetal Medicine and Prenatal Diagnosis/BioResource Research Center, Key Laboratory for Major Obstetric Diseases of Guangdong Province, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, China
| | - Bangzhu Chen
- Department of Obstetrics and Gynecology, Center for Reproductive Medicine/Department of Fetal Medicine and Prenatal Diagnosis/BioResource Research Center, Key Laboratory for Major Obstetric Diseases of Guangdong Province, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, China
| | - Yukio Nakamura
- Cell Engineering Division, RIKEN BioResource Center, Tsukuba, Japan
| | - Juan Zeng
- Department of Obstetrics and Gynecology, Center for Reproductive Medicine/Department of Fetal Medicine and Prenatal Diagnosis/BioResource Research Center, Key Laboratory for Major Obstetric Diseases of Guangdong Province, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, China
| | - Nengqing Liu
- Department of Obstetrics and Gynecology, Center for Reproductive Medicine/Department of Fetal Medicine and Prenatal Diagnosis/BioResource Research Center, Key Laboratory for Major Obstetric Diseases of Guangdong Province, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, China
| | - Xiaofang Sun
- Department of Obstetrics and Gynecology, Center for Reproductive Medicine/Department of Fetal Medicine and Prenatal Diagnosis/BioResource Research Center, Key Laboratory for Major Obstetric Diseases of Guangdong Province, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, China
| | - Diyu Chen
- Department of Obstetrics and Gynecology, Center for Reproductive Medicine/Department of Fetal Medicine and Prenatal Diagnosis/BioResource Research Center, Key Laboratory for Major Obstetric Diseases of Guangdong Province, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, China
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Topfer SK, Feng R, Huang P, Ly LC, Martyn GE, Blobel GA, Weiss MJ, Quinlan KGR, Crossley M. Disrupting the adult globin promoter alleviates promoter competition and reactivates fetal globin gene expression. Blood 2022; 139:2107-2118. [PMID: 35090172 PMCID: PMC8990374 DOI: 10.1182/blood.2021014205] [Citation(s) in RCA: 27] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/22/2021] [Accepted: 01/18/2022] [Indexed: 12/16/2022] Open
Abstract
The benign condition hereditary persistence of fetal hemoglobin (HPFH) is known to ameliorate symptoms of co-inherited β-hemoglobinopathies, such as sickle cell disease and β-thalassemia. The condition is sometimes associated with point mutations in the fetal globin promoters that disrupt the binding of the repressors BCL11A or ZBTB7A/LRF, which have been extensively studied. HPFH is also associated with a range of deletions within the β-globin locus that all reside downstream of the fetal HBG2 gene. These deletional forms of HPFH are poorly understood and are the focus of this study. Numerous different mechanisms have been proposed to explain how downstream deletions can boost the expression of the fetal globin genes, including the deletion of silencer elements, of genes encoding noncoding RNA, and bringing downstream enhancer elements into proximity with the fetal globin gene promoters. Here we systematically analyze the deletions associated with both HPFH and a related condition known as δβ-thalassemia and propose a unifying mechanism. In all cases where fetal globin is upregulated, the proximal adult β-globin (HBB) promoter is deleted. We use clustered regularly interspaced short palindromic repeats-mediated gene editing to delete or disrupt elements within the promoter and find that virtually all mutations that reduce ΗΒΒ promoter activity result in elevated fetal globin expression. These results fit with previous models where the fetal and adult globin genes compete for the distal locus control region and suggest that targeting the ΗΒΒ promoter might be explored to elevate fetal globin and reduce sickle globin expression as a treatment of β-hemoglobinopathies.
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Affiliation(s)
- Sarah K Topfer
- School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, NSW, Australia
| | - Ruopeng Feng
- Department of Hematology, St. Jude Children's Research Hospital, Memphis, TN
| | - Peng Huang
- Division of Hematology, The Children's Hospital of Philadelphia, Philadelphia, PA; and
| | - Lana C Ly
- School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, NSW, Australia
| | - Gabriella E Martyn
- School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, NSW, Australia
| | - Gerd A Blobel
- Division of Hematology, The Children's Hospital of Philadelphia, Philadelphia, PA; and
- Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA
| | - Mitchell J Weiss
- Department of Hematology, St. Jude Children's Research Hospital, Memphis, TN
| | - Kate G R Quinlan
- School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, NSW, Australia
| | - Merlin Crossley
- School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, NSW, Australia
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Ravi NS, Wienert B, Wyman SK, Bell HW, George A, Mahalingam G, Vu JT, Prasad K, Bandlamudi BP, Devaraju N, Rajendiran V, Syedbasha N, Pai AA, Nakamura Y, Kurita R, Narayanasamy M, Balasubramanian P, Thangavel S, Marepally S, Velayudhan SR, Srivastava A, DeWitt MA, Crossley M, Corn JE, Mohankumar KM. Identification of novel HPFH-like mutations by CRISPR base editing that elevate the expression of fetal hemoglobin. eLife 2022; 11:e65421. [PMID: 35147495 PMCID: PMC8865852 DOI: 10.7554/elife.65421] [Citation(s) in RCA: 36] [Impact Index Per Article: 12.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2020] [Accepted: 02/11/2022] [Indexed: 11/29/2022] Open
Abstract
Naturally occurring point mutations in the HBG promoter switch hemoglobin synthesis from defective adult beta-globin to fetal gamma-globin in sickle cell patients with hereditary persistence of fetal hemoglobin (HPFH) and ameliorate the clinical severity. Inspired by this natural phenomenon, we tiled the highly homologous HBG proximal promoters using adenine and cytosine base editors that avoid the generation of large deletions and identified novel regulatory regions including a cluster at the -123 region. Base editing at -123 and -124 bp of HBG promoter induced fetal hemoglobin (HbF) to a higher level than disruption of well-known BCL11A binding site in erythroblasts derived from human CD34+ hematopoietic stem and progenitor cells (HSPC). We further demonstrated in vitro that the introduction of -123T > C and -124T > C HPFH-like mutations drives gamma-globin expression by creating a de novo binding site for KLF1. Overall, our findings shed light on so far unknown regulatory elements within the HBG promoter and identified additional targets for therapeutic upregulation of fetal hemoglobin.
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Affiliation(s)
- Nithin Sam Ravi
- Centre for Stem Cell Research (a Unit of inStem, Bengaluru), Christian Medical College CampusVelloreIndia
- Sree Chitra Tirunal Institute for Medical Sciences and TechnologyThiruvananthapuramIndia
| | - Beeke Wienert
- Innovative Genomics Institute, University of California, BerkeleyBerkeleyUnited States
- Institute of Data Science and Biotechnology, Gladstone InstitutesSan FranciscoUnited States
- School of Biotechnology and Biomolecular Sciences, University of New South WalesSydneyAustralia
| | - Stacia K Wyman
- Innovative Genomics Institute, University of California, BerkeleyBerkeleyUnited States
| | - Henry William Bell
- School of Biotechnology and Biomolecular Sciences, University of New South WalesSydneyAustralia
| | - Anila George
- Centre for Stem Cell Research (a Unit of inStem, Bengaluru), Christian Medical College CampusVelloreIndia
- Sree Chitra Tirunal Institute for Medical Sciences and TechnologyThiruvananthapuramIndia
| | - Gokulnath Mahalingam
- Centre for Stem Cell Research (a Unit of inStem, Bengaluru), Christian Medical College CampusVelloreIndia
| | - Jonathan T Vu
- Innovative Genomics Institute, University of California, BerkeleyBerkeleyUnited States
| | - Kirti Prasad
- Centre for Stem Cell Research (a Unit of inStem, Bengaluru), Christian Medical College CampusVelloreIndia
- Manipal Academy of Higher EducationKarnatakaIndia
| | - Bhanu Prasad Bandlamudi
- Centre for Stem Cell Research (a Unit of inStem, Bengaluru), Christian Medical College CampusVelloreIndia
| | - Nivedhitha Devaraju
- Centre for Stem Cell Research (a Unit of inStem, Bengaluru), Christian Medical College CampusVelloreIndia
- Manipal Academy of Higher EducationKarnatakaIndia
| | - Vignesh Rajendiran
- Centre for Stem Cell Research (a Unit of inStem, Bengaluru), Christian Medical College CampusVelloreIndia
- Sree Chitra Tirunal Institute for Medical Sciences and TechnologyThiruvananthapuramIndia
| | - Nazar Syedbasha
- Centre for Stem Cell Research (a Unit of inStem, Bengaluru), Christian Medical College CampusVelloreIndia
| | - Aswin Anand Pai
- Sree Chitra Tirunal Institute for Medical Sciences and TechnologyThiruvananthapuramIndia
- Department of Haematology, Christian Medical College & HospitalVelloreIndia
| | - Yukio Nakamura
- Cell Engineering Division, RIKEN BioResource CenterIbarakiJapan
| | - Ryo Kurita
- Research and Development Department, Central Blood Institute Blood Service Headquarters, Japanese Red Cross Society, JapanTokyoJapan
| | - Muthuraman Narayanasamy
- Centre for Stem Cell Research (a Unit of inStem, Bengaluru), Christian Medical College CampusVelloreIndia
- Department of Biochemistry, Christian Medical CollegeVelloreIndia
| | - Poonkuzhali Balasubramanian
- Sree Chitra Tirunal Institute for Medical Sciences and TechnologyThiruvananthapuramIndia
- Department of Haematology, Christian Medical College & HospitalVelloreIndia
| | - Saravanabhavan Thangavel
- Centre for Stem Cell Research (a Unit of inStem, Bengaluru), Christian Medical College CampusVelloreIndia
| | - Srujan Marepally
- Centre for Stem Cell Research (a Unit of inStem, Bengaluru), Christian Medical College CampusVelloreIndia
| | - Shaji R Velayudhan
- Centre for Stem Cell Research (a Unit of inStem, Bengaluru), Christian Medical College CampusVelloreIndia
- Sree Chitra Tirunal Institute for Medical Sciences and TechnologyThiruvananthapuramIndia
- Department of Haematology, Christian Medical College & HospitalVelloreIndia
| | - Alok Srivastava
- Centre for Stem Cell Research (a Unit of inStem, Bengaluru), Christian Medical College CampusVelloreIndia
- Sree Chitra Tirunal Institute for Medical Sciences and TechnologyThiruvananthapuramIndia
- Department of Haematology, Christian Medical College & HospitalVelloreIndia
| | - Mark A DeWitt
- Innovative Genomics Institute, University of California, BerkeleyBerkeleyUnited States
- Department of Microbiology, Immunology and Molecular Genetics, University of California, Los AngelesLos AngelesUnited States
| | - Merlin Crossley
- School of Biotechnology and Biomolecular Sciences, University of New South WalesSydneyAustralia
| | - Jacob E Corn
- Innovative Genomics Institute, University of California, BerkeleyBerkeleyUnited States
- Institute of Molecular Health Sciences, Department of BiologyZurichSwitzerland
| | - Kumarasamypet M Mohankumar
- Centre for Stem Cell Research (a Unit of inStem, Bengaluru), Christian Medical College CampusVelloreIndia
- Sree Chitra Tirunal Institute for Medical Sciences and TechnologyThiruvananthapuramIndia
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29
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Barbarani G, Łabedz A, Ronchi AE. β-Hemoglobinopathies: The Test Bench for Genome Editing-Based Therapeutic Strategies. Front Genome Ed 2021; 2:571239. [PMID: 34713219 PMCID: PMC8525389 DOI: 10.3389/fgeed.2020.571239] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2020] [Accepted: 10/29/2020] [Indexed: 12/26/2022] Open
Abstract
Hemoglobin is a tetrameric protein composed of two α and two β chains, each containing a heme group that reversibly binds oxygen. The composition of hemoglobin changes during development in order to fulfill the need of the growing organism, stably maintaining a balanced production of α-like and β-like chains in a 1:1 ratio. Adult hemoglobin (HbA) is composed of two α and two β subunits (α2β2 tetramer), whereas fetal hemoglobin (HbF) is composed of two γ and two α subunits (α2γ2 tetramer). Qualitative or quantitative defects in β-globin production cause two of the most common monogenic-inherited disorders: β-thalassemia and sickle cell disease. The high frequency of these diseases and the relative accessibility of hematopoietic stem cells make them an ideal candidate for therapeutic interventions based on genome editing. These strategies move in two directions: the correction of the disease-causing mutation and the reactivation of the expression of HbF in adult cells, in the attempt to recreate the effect of hereditary persistence of fetal hemoglobin (HPFH) natural mutations, which mitigate the severity of β-hemoglobinopathies. Both lines of research rely on the knowledge gained so far on the regulatory mechanisms controlling the differential expression of globin genes during development.
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Affiliation(s)
- Gloria Barbarani
- Dipartimento di Biotecnologie e Bioscienze, Università di Milano-Bicocca, Milan, Italy
| | - Agata Łabedz
- Dipartimento di Biotecnologie e Bioscienze, Università di Milano-Bicocca, Milan, Italy
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Li X, Chen M, Liu B, Lu P, Lv X, Zhao X, Cui S, Xu P, Nakamura Y, Kurita R, Chen B, Huang DCS, Liu DP, Liu M, Zhao Q. Transcriptional silencing of fetal hemoglobin expression by NonO. Nucleic Acids Res 2021; 49:9711-9723. [PMID: 34379783 PMCID: PMC8464040 DOI: 10.1093/nar/gkab671] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2021] [Revised: 07/19/2021] [Accepted: 07/23/2021] [Indexed: 12/21/2022] Open
Abstract
Human fetal globin (γ-globin) genes are developmentally silenced after birth, and reactivation of γ-globin expression in adulthood ameliorates symptoms of hemoglobin disorders, such as sickle cell disease (SCD) and β-thalassemia. However, the mechanisms by which γ-globin expression is precisely regulated are still incompletely understood. Here, we found that NonO (non-POU domain-containing octamer-binding protein) interacted directly with SOX6, and repressed the expression of γ-globin gene in human erythroid cells. We showed that NonO bound to the octamer binding motif, ATGCAAAT, of the γ-globin proximal promoter, resulting in inhibition of γ-globin transcription. Depletion of NonO resulted in significant activation of γ-globin expression in K562, HUDEP-2, and primary human erythroid progenitor cells. To confirm the role of NonO in vivo, we further generated a conditional knockout of NonO by using IFN-inducible Mx1-Cre transgenic mice. We found that induced NonO deletion reactivated murine embryonic globin and human γ-globin gene expression in adult β-YAC mice, suggesting a conserved role for NonO during mammalian evolution. Thus, our data indicate that NonO acts as a novel transcriptional repressor of γ-globin gene expression through direct promoter binding, and is essential for γ-globin gene silencing.
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Affiliation(s)
- Xinyu Li
- The State Key Laboratory of Pharmaceutical Biotechnology, Department of Hematology and Urology, the Affiliated Drum Tower Hospital of Nanjing University Medical School, China-Australia Institute of Translational Medicine, School of Life Sciences, Nanjing University, Nanjing, China
| | - Mengxia Chen
- The State Key Laboratory of Pharmaceutical Biotechnology, Department of Hematology and Urology, the Affiliated Drum Tower Hospital of Nanjing University Medical School, China-Australia Institute of Translational Medicine, School of Life Sciences, Nanjing University, Nanjing, China
| | - Biru Liu
- The State Key Laboratory of Pharmaceutical Biotechnology, Department of Hematology and Urology, the Affiliated Drum Tower Hospital of Nanjing University Medical School, China-Australia Institute of Translational Medicine, School of Life Sciences, Nanjing University, Nanjing, China
| | - Peifen Lu
- The State Key Laboratory of Pharmaceutical Biotechnology, Department of Hematology and Urology, the Affiliated Drum Tower Hospital of Nanjing University Medical School, China-Australia Institute of Translational Medicine, School of Life Sciences, Nanjing University, Nanjing, China
| | - Xiang Lv
- State Key Laboratory of Medical Molecular Biology, Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
| | - Xiang Zhao
- State Key Laboratory of Medical Molecular Biology, Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
| | - Shuaiying Cui
- Section of Hematology-Medical Oncology, Department of Medicine, Boston University School of Medicine, Boston, MA, USA
| | - Peipei Xu
- The State Key Laboratory of Pharmaceutical Biotechnology, Department of Hematology and Urology, the Affiliated Drum Tower Hospital of Nanjing University Medical School, China-Australia Institute of Translational Medicine, School of Life Sciences, Nanjing University, Nanjing, China
| | - Yukio Nakamura
- Cell Engineering Division, RIKEN BioResource Center, Tsukuba, Ibaraki 305-0074, Japan
| | - Ryo Kurita
- Department of Research and Development, Central Blood Institute, Japanese Red Cross Society, Tokyo, Japan
| | - Bing Chen
- The State Key Laboratory of Pharmaceutical Biotechnology, Department of Hematology and Urology, the Affiliated Drum Tower Hospital of Nanjing University Medical School, China-Australia Institute of Translational Medicine, School of Life Sciences, Nanjing University, Nanjing, China
| | - David C S Huang
- The Walter and Eliza Hall Institute of Medical Research, Department of Medical Biology, University of Melbourne, Melbourne, VIC, Australia
| | - De-Pei Liu
- State Key Laboratory of Medical Molecular Biology, Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
| | - Ming Liu
- The State Key Laboratory of Pharmaceutical Biotechnology, Department of Hematology and Urology, the Affiliated Drum Tower Hospital of Nanjing University Medical School, China-Australia Institute of Translational Medicine, School of Life Sciences, Nanjing University, Nanjing, China
| | - Quan Zhao
- The State Key Laboratory of Pharmaceutical Biotechnology, Department of Hematology and Urology, the Affiliated Drum Tower Hospital of Nanjing University Medical School, China-Australia Institute of Translational Medicine, School of Life Sciences, Nanjing University, Nanjing, China
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31
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Azhagiri MKK, Babu P, Venkatesan V, Thangavel S. Homology-directed gene-editing approaches for hematopoietic stem and progenitor cell gene therapy. Stem Cell Res Ther 2021; 12:500. [PMID: 34503562 PMCID: PMC8428126 DOI: 10.1186/s13287-021-02565-6] [Citation(s) in RCA: 20] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2021] [Accepted: 08/19/2021] [Indexed: 12/26/2022] Open
Abstract
The advent of next-generation genome engineering tools like CRISPR-Cas9 has transformed the field of gene therapy, rendering targeted treatment for several incurable diseases. Hematopoietic stem and progenitor cells (HSPCs) continue to be the ideal target cells for gene manipulation due to their long-term repopulation potential. Among the gene manipulation strategies such as lentiviral gene augmentation, non-homologous end joining (NHEJ)-mediated gene editing, base editing and prime editing, only the homology-directed repair (HDR)-mediated gene editing provides the option of inserting a large transgene under its endogenous promoter or any desired locus. In addition, HDR-mediated gene editing can be applied for the gene knock-out, correction of point mutations and introduction of beneficial mutations. HSPC gene therapy studies involving lentiviral vectors and NHEJ-based gene-editing studies have exhibited substantial clinical progress. However, studies involving HDR-mediated HSPC gene editing have not yet progressed to the clinical testing. This suggests the existence of unique challenges in exploiting HDR pathway for HSPC gene therapy. Our review summarizes the mechanism, recent progresses, challenges, and the scope of HDR-based gene editing for the HSPC gene therapy.
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Affiliation(s)
- Manoj Kumar K Azhagiri
- Centre for Stem Cell Research (CSCR), a Unit of InStem Bengaluru, Christian Medical College Campus, Vellore, Tamil Nadu, India
- Manipal Academy of Higher Education, Manipal, Karnataka, India
| | - Prathibha Babu
- Centre for Stem Cell Research (CSCR), a Unit of InStem Bengaluru, Christian Medical College Campus, Vellore, Tamil Nadu, India
- Manipal Academy of Higher Education, Manipal, Karnataka, India
| | - Vigneshwaran Venkatesan
- Centre for Stem Cell Research (CSCR), a Unit of InStem Bengaluru, Christian Medical College Campus, Vellore, Tamil Nadu, India
- Manipal Academy of Higher Education, Manipal, Karnataka, India
| | - Saravanabhavan Thangavel
- Centre for Stem Cell Research (CSCR), a Unit of InStem Bengaluru, Christian Medical College Campus, Vellore, Tamil Nadu, India.
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32
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Yao Q, Ferragina P, Reshef Y, Lettre G, Bauer DE, Pinello L. Motif-Raptor: a cell type-specific and transcription factor centric approach for post-GWAS prioritization of causal regulators. Bioinformatics 2021; 37:2103-2111. [PMID: 33532840 PMCID: PMC11025460 DOI: 10.1093/bioinformatics/btab072] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/27/2020] [Revised: 11/30/2020] [Accepted: 01/28/2021] [Indexed: 11/14/2022] Open
Abstract
MOTIVATION Genome-wide association studies (GWASs) have identified thousands of common trait-associated genetic variants but interpretation of their function remains challenging. These genetic variants can overlap the binding sites of transcription factors (TFs) and therefore could alter gene expression. However, we currently lack a systematic understanding on how this mechanism contributes to phenotype. RESULTS We present Motif-Raptor, a TF-centric computational tool that integrates sequence-based predictive models, chromatin accessibility, gene expression datasets and GWAS summary statistics to systematically investigate how TF function is affected by genetic variants. Given trait-associated non-coding variants, Motif-Raptor can recover relevant cell types and critical TFs to drive hypotheses regarding their mechanism of action. We tested Motif-Raptor on complex traits such as rheumatoid arthritis and red blood cell count and demonstrated its ability to prioritize relevant cell types, potential regulatory TFs and non-coding SNPs which have been previously characterized and validated. AVAILABILITY AND IMPLEMENTATION Motif-Raptor is freely available as a Python package at: https://github.com/pinellolab/MotifRaptor. SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.
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Affiliation(s)
- Qiuming Yao
- Department of Pathology, Massachusetts General Hospital, Charlestown, MA 02129, USA
- Division of Hematology/Oncology, Boston Children’s Hospital, Boston, MA 02115, USA
- Harvard Medical School, Boston, MA 02115, USA
| | - Paolo Ferragina
- Department of Computer Science, University of Pisa, Pisa 56128, Italy
| | - Yakir Reshef
- Department of Computer Science, Harvard University, Cambridge, MA 02138, USA
- Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA
| | - Guillaume Lettre
- Faculty of Medicine, Université de Montréal, Montreal, Quebec H3C3J7, Canada
- Montreal Heart Institute, Montreal, Quebec H1T1C8, Canada
| | - Daniel E Bauer
- Division of Hematology/Oncology, Boston Children’s Hospital, Boston, MA 02115, USA
- Harvard Medical School, Boston, MA 02115, USA
- Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA
- Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA 02115, USA
| | - Luca Pinello
- Department of Pathology, Massachusetts General Hospital, Charlestown, MA 02129, USA
- Harvard Medical School, Boston, MA 02115, USA
- Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA
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33
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Activation of γ-globin gene expression by GATA1 and NF-Y in hereditary persistence of fetal hemoglobin. Nat Genet 2021; 53:1177-1186. [PMID: 34341563 PMCID: PMC8610173 DOI: 10.1038/s41588-021-00904-0] [Citation(s) in RCA: 20] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/15/2020] [Accepted: 06/25/2021] [Indexed: 11/30/2022]
Abstract
Hereditary persistence of fetal hemoglobin (HPFH) ameliorates β-hemoglobinopathies by inhibiting the developmental switch from γ-globin (HBG1/HBG2) to β-globin (HBB) gene expression. Some forms of HPFH are associated with γ-globin promoter variants that either disrupt binding motifs for transcriptional repressors or create new motifs for transcriptional activators. How these variants sustain γ-globin gene expression postnatally remains undefined. We mapped γ-globin promoter sequences functionally in erythroid cells harboring different HPFH variants. Those that disrupt a BCL11A repressor binding element induce γ-globin expression by facilitating the recruitment of transcription factors NF-Y to a nearby proximal CCAAT box and GATA1 to an upstream motif. The proximal CCAAT element becomes dispensable for HPFH variants that generate new binding motifs for activators NF-Y or KLF1, but GATA1 recruitment remains essential. Our findings define distinct mechanisms through which transcription factors and their cis-regulatory elements activate γ-globin expression in different forms of HPFH, some of which are being recreated by therapeutic genome editing.
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34
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Eksi YE, Sanlioglu AD, Akkaya B, Ozturk BE, Sanlioglu S. Genome engineering and disease modeling via programmable nucleases for insulin gene therapy; promises of CRISPR/Cas9 technology. World J Stem Cells 2021; 13:485-502. [PMID: 34249224 PMCID: PMC8246254 DOI: 10.4252/wjsc.v13.i6.485] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/27/2021] [Revised: 04/02/2021] [Accepted: 06/16/2021] [Indexed: 02/06/2023] Open
Abstract
Targeted genome editing is a continually evolving technology employing programmable nucleases to specifically change, insert, or remove a genomic sequence of interest. These advanced molecular tools include meganucleases, zinc finger nucleases, transcription activator-like effector nucleases and RNA-guided engineered nucleases (RGENs), which create double-strand breaks at specific target sites in the genome, and repair DNA either by homologous recombination in the presence of donor DNA or via the error-prone non-homologous end-joining mechanism. A recently discovered group of RGENs known as CRISPR/Cas9 gene-editing systems allowed precise genome manipulation revealing a causal association between disease genotype and phenotype, without the need for the reengineering of the specific enzyme when targeting different sequences. CRISPR/Cas9 has been successfully employed as an ex vivo gene-editing tool in embryonic stem cells and patient-derived stem cells to understand pancreatic beta-cell development and function. RNA-guided nucleases also open the way for the generation of novel animal models for diabetes and allow testing the efficiency of various therapeutic approaches in diabetes, as summarized and exemplified in this manuscript.
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Affiliation(s)
- Yunus E Eksi
- Department of Gene and Cell Therapy, Akdeniz University Faculty of Medicine, Antalya 07058, Turkey
| | - Ahter D Sanlioglu
- Department of Gene and Cell Therapy, Akdeniz University Faculty of Medicine, Antalya 07058, Turkey
| | - Bahar Akkaya
- Department of Gene and Cell Therapy, Akdeniz University Faculty of Medicine, Antalya 07058, Turkey
| | - Bilge Esin Ozturk
- Department of Ophthalmology, University of Pittsburgh, Pittsburgh, PA 15213, United States
| | - Salih Sanlioglu
- Department of Gene and Cell Therapy, Akdeniz University Faculty of Medicine, Antalya 07058, Turkey.
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35
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A gain-of-function single nucleotide variant creates a new promoter which acts as an orientation-dependent enhancer-blocker. Nat Commun 2021; 12:3806. [PMID: 34155213 PMCID: PMC8217497 DOI: 10.1038/s41467-021-23980-6] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/05/2020] [Accepted: 05/19/2021] [Indexed: 02/08/2023] Open
Abstract
Many single nucleotide variants (SNVs) associated with human traits and genetic diseases are thought to alter the activity of existing regulatory elements. Some SNVs may also create entirely new regulatory elements which change gene expression, but the mechanism by which they do so is largely unknown. Here we show that a single base change in an otherwise unremarkable region of the human α-globin cluster creates an entirely new promoter and an associated unidirectional transcript. This SNV downregulates α-globin expression causing α-thalassaemia. Of note, the new promoter lying between the α-globin genes and their associated super-enhancer disrupts their interaction in an orientation-dependent manner. Together these observations show how both the order and orientation of the fundamental elements of the genome determine patterns of gene expression and support the concept that active genes may act to disrupt enhancer-promoter interactions in mammals as in Drosophila. Finally, these findings should prompt others to fully evaluate SNVs lying outside of known regulatory elements as causing changes in gene expression by creating new regulatory elements. The role of promoters as potential insulator elements has been largely unexplored in mammals. Here the authors show that a single nucleotide variant in the α-globin locus forms a new promoter and acts as an orientation-dependent enhancer-blocking insulator element.
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Karamperis K, Tsoumpeli MT, Kounelis F, Koromina M, Mitropoulou C, Moutinho C, Patrinos GP. Genome-based therapeutic interventions for β-type hemoglobinopathies. Hum Genomics 2021; 15:32. [PMID: 34090531 PMCID: PMC8178887 DOI: 10.1186/s40246-021-00329-0] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/06/2021] [Accepted: 04/28/2021] [Indexed: 12/18/2022] Open
Abstract
For decades, various strategies have been proposed to solve the enigma of hemoglobinopathies, especially severe cases. However, most of them seem to be lagging in terms of effectiveness and safety. So far, the most prevalent and promising treatment options for patients with β-types hemoglobinopathies, among others, predominantly include drug treatment and gene therapy. Despite the significant improvements of such interventions to the patient's quality of life, a variable response has been demonstrated among different groups of patients and populations. This is essentially due to the complexity of the disease and other genetic factors. In recent years, a more in-depth understanding of the molecular basis of the β-type hemoglobinopathies has led to significant upgrades to the current technologies, as well as the addition of new ones attempting to elucidate these barriers. Therefore, the purpose of this article is to shed light on pharmacogenomics, gene addition, and genome editing technologies, and consequently, their potential use as direct and indirect genome-based interventions, in different strategies, referring to drug and gene therapy. Furthermore, all the latest progress, updates, and scientific achievements for patients with β-type hemoglobinopathies will be described in detail.
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Affiliation(s)
- Kariofyllis Karamperis
- Department of Pharmacy, School of Health Sciences, Laboratory of Pharmacogenomics and Individualized Therapy, University of Patras, Patras, Greece
- The Golden Helix Foundation, London, UK
| | - Maria T Tsoumpeli
- School of Veterinary Medicine and Science, University of Nottingham, Nottingham, UK
| | - Fotios Kounelis
- Department of Computing, Group of Large-Scale Data & Systems, Imperial College London, London, UK
| | - Maria Koromina
- Department of Pharmacy, School of Health Sciences, Laboratory of Pharmacogenomics and Individualized Therapy, University of Patras, Patras, Greece
| | | | - Catia Moutinho
- Garvan-Weizmann Centre for Cellular Genomics, Garvan Institute of Medical Research, Darlinghurst, Sydney, Australia
| | - George P Patrinos
- Department of Pharmacy, School of Health Sciences, Laboratory of Pharmacogenomics and Individualized Therapy, University of Patras, Patras, Greece.
- College of Medicine and Health Sciences, Department of Pathology, United Arab Emirates University, Al-Ain, United Arab Emirates.
- Zayed Center of Health Sciences, United Arab Emirates University, Al-Ain, United Arab Emirates.
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37
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Garg H, Tatiossian KJ, Peppel K, Kato GJ, Herzog E. Gene therapy as the new frontier for Sickle Cell Disease. Curr Med Chem 2021; 29:453-466. [PMID: 34047257 DOI: 10.2174/0929867328666210527092456] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2020] [Revised: 03/28/2021] [Accepted: 04/11/2021] [Indexed: 11/22/2022]
Abstract
Sickle Cell Disease (SCD) is one of the most common monogenic disorders caused by a point mutation in the β-globin gene. This mutation results in polymerization of hemoglobin (Hb) under reduced oxygenation conditions, causing rigid sickle-shaped RBCs and hemolytic anemia. This clearly defined fundamental molecular mechanism makes SCD a prototypical target for precision therapy. Both the mutant β-globin protein and its downstream pathophysiology are pharmacological targets of intensive research. SCD also is a disease well-suited for biological interventions like gene therapy. Recent advances in hematopoietic stem cell (HSC) transplantation and gene therapy platforms, like Lentiviral vectors and gene editing strategies, expand the potentially curative options for patients with SCD. This review discusses the recent advances in precision therapy for SCD and the preclinical and clinical advances in autologous HSC gene therapy for SCD.
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Affiliation(s)
- Himanshu Garg
- CSL Behring, 1020 1St Ave, King of Prussia, PA 19406, United States
| | | | - Karsten Peppel
- CSL Behring, 1020 1St Ave, King of Prussia, PA 19406, United States
| | - Gregory J Kato
- CSL Behring, 1020 1St Ave, King of Prussia, PA 19406, United States
| | - Eva Herzog
- CSL Behring, 1020 1St Ave, King of Prussia, PA 19406, United States
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38
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Zhang D, Lam J, Blobel GA. Engineering three-dimensional genome folding. Nat Genet 2021; 53:602-611. [PMID: 33958782 DOI: 10.1038/s41588-021-00860-9] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2020] [Accepted: 03/29/2021] [Indexed: 02/02/2023]
Abstract
Animal genomes are partitioned and folded at various scales that contribute distinctly to nuclear processes. While structural features have been disrupted either globally or at select loci in loss-of-function studies, gain-of-function studies that probe the role of genome architecture have lagged behind. Here we examine recent advances in experimentally creating chromatin loops, contact domains, boundaries and compartments. Furthermore, we explore parallels between this emerging theme and natural evolution of mammalian genomes with increasing architectural complexity. Finally, we provide a perspective on how insights arising from recent gain-of-function studies may inform future endeavors toward engineering the three-dimensional genome.
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Affiliation(s)
- Di Zhang
- Division of Hematology, The Children's Hospital of Philadelphia, Philadelphia, PA, USA.,Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
| | - Jessica Lam
- Division of Hematology, The Children's Hospital of Philadelphia, Philadelphia, PA, USA.,Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
| | - Gerd A Blobel
- Division of Hematology, The Children's Hospital of Philadelphia, Philadelphia, PA, USA. .,Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
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Barbarani G, Labedz A, Stucchi S, Abbiati A, Ronchi AE. Physiological and Aberrant γ-Globin Transcription During Development. Front Cell Dev Biol 2021; 9:640060. [PMID: 33869190 PMCID: PMC8047207 DOI: 10.3389/fcell.2021.640060] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/10/2020] [Accepted: 02/23/2021] [Indexed: 12/24/2022] Open
Abstract
The expression of the fetal Gγ- and Aγ-globin genes in normal development is confined to the fetal period, where two γ-globin chains assemble with two α-globin chains to form α2γ2 tetramers (HbF). HbF sustains oxygen delivery to tissues until birth, when β-globin replaces γ-globin, leading to the formation of α2β2 tetramers (HbA). However, in different benign and pathological conditions, HbF is expressed in adult cells, as it happens in the hereditary persistence of fetal hemoglobin, in anemias and in some leukemias. The molecular basis of γ-globin differential expression in the fetus and of its inappropriate activation in adult cells is largely unknown, although in recent years, a few transcription factors involved in this process have been identified. The recent discovery that fetal cells can persist to adulthood and contribute to disease raises the possibility that postnatal γ-globin expression could, in some cases, represent the signature of the fetal cellular origin.
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Affiliation(s)
- Gloria Barbarani
- Dipartimento di Biotecnologie e Bioscienze, Università di Milano-Bicocca, Milano, Italy
| | - Agata Labedz
- Dipartimento di Biotecnologie e Bioscienze, Università di Milano-Bicocca, Milano, Italy
| | - Sarah Stucchi
- Dipartimento di Biotecnologie e Bioscienze, Università di Milano-Bicocca, Milano, Italy
| | - Alessia Abbiati
- Dipartimento di Biotecnologie e Bioscienze, Università di Milano-Bicocca, Milano, Italy
| | - Antonella E Ronchi
- Dipartimento di Biotecnologie e Bioscienze, Università di Milano-Bicocca, Milano, Italy
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40
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Bao X, Zhang X, Wang L, Wang Z, Huang J, Zhang Q, Ye Y, Liu Y, Chen D, Zuo Y, Liu Q, Xu P, Huang B, Fang J, Lao J, Feng X, Li Y, Kurita R, Nakamura Y, Yu W, Ju C, Huang C, Mohandas N, Li D, Zhao C, Xu X. Epigenetic inactivation of ERF reactivates γ-globin expression in β-thalassemia. Am J Hum Genet 2021; 108:709-721. [PMID: 33735615 DOI: 10.1016/j.ajhg.2021.03.005] [Citation(s) in RCA: 18] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/27/2020] [Accepted: 03/01/2021] [Indexed: 12/16/2022] Open
Abstract
The fetal-to-adult hemoglobin switch is regulated in a developmental stage-specific manner and reactivation of fetal hemoglobin (HbF) has therapeutic implications for treatment of β-thalassemia and sickle cell anemia, two major global health problems. Although significant progress has been made in our understanding of the molecular mechanism of the fetal-to-adult hemoglobin switch, the mechanism of epigenetic regulation of HbF silencing remains to be fully defined. Here, we performed whole-genome bisulfite sequencing and RNA sequencing analysis of the bone marrow-derived GYPA+ erythroid cells from β-thalassemia-affected individuals with widely varying levels of HbF groups (HbF ≥ 95th percentile or HbF ≤ 5th percentile) to screen epigenetic modulators of HbF and phenotypic diversity of β-thalassemia. We identified an ETS2 repressor factor encoded by ERF, whose promoter hypermethylation and mRNA downregulation are associated with high HbF levels in β-thalassemia. We further observed that hypermethylation of the ERF promoter mediated by enrichment of DNMT3A leads to demethylation of γ-globin genes and attenuation of binding of ERF on the HBG promoter and eventually re-activation of HbF in β-thalassemia. We demonstrated that ERF depletion markedly increased HbF production in human CD34+ erythroid progenitor cells, HUDEP-2 cell lines, and transplanted NCG-Kit-V831M mice. ERF represses γ-globin expression by directly binding to two consensus motifs regulating γ-globin gene expression. Importantly, ERF depletion did not affect maturation of erythroid cells. Identification of alterations in DNA methylation of ERF as a modulator of HbF synthesis opens up therapeutic targets for β-hemoglobinopathies.
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CRISPR-Cas systems for genome editing of mammalian cells. PROGRESS IN MOLECULAR BIOLOGY AND TRANSLATIONAL SCIENCE 2021; 181:15-30. [PMID: 34127192 DOI: 10.1016/bs.pmbts.2021.01.011] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
In the past decade, ZFNs and TALENs have been used for targeted genome engineering and have gained scientific attention. It has demonstrated huge potential for gene knockout, knock-in, and indels in desired locations of genomes to understand molecular mechanism of diseases and also discover therapy. However, both the genome engineering techniques are still suffering from design, screening and validation in cell and higher organisms. CRISPR-Cas9 is a rapid, simple, specific, and versatile technology and it has been applied in many organisms including mammalian cells. CRISPR-Cas9 has been used for animal models to modify animal cells for understanding human disease for novel drug discovery and therapy. Additionally, base editing has also been discussed herewith for conversion of C/G-to-T/A or A/T-to-G/C without DNA cleavage or donor DNA templates for correcting mutations or altering gene functions. In this chapter, we highlight CRISPR-Cas9 and base editing for desired genome editing in mammalian cells for a better understanding of molecular mechanisms, and biotechnological and therapeutic applications.
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Insight of fetal to adult hemoglobin switch: Genetic modulators and therapeutic targets. Blood Rev 2021; 49:100823. [PMID: 33726930 DOI: 10.1016/j.blre.2021.100823] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/19/2020] [Revised: 02/08/2021] [Accepted: 03/03/2021] [Indexed: 01/31/2023]
Abstract
The clinical heterogeneity of β-hemoglobinopathies is so variable that it prompted the researchers to identify the genetic modulators of these diseases. Though the primary modulator is the type of β-globin mutation which affects the degree of β-globin chain synthesis, the co-inheritance of α-thalassemia and the fetal hemoglobin (HbF) levels also act as potent secondary genetic modifiers. As elevated HbF levels ameliorate the severity of hemoglobinopathies, in this review, the genetic modulators lying within and outside the β-globin gene cluster with their plausible role in governing the HbF levels have been summarised, which in future may act as potential therapeutic targets.
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Brusson M, Miccio A. Genome editing approaches to β-hemoglobinopathies. PROGRESS IN MOLECULAR BIOLOGY AND TRANSLATIONAL SCIENCE 2021; 182:153-183. [PMID: 34175041 DOI: 10.1016/bs.pmbts.2021.01.025] [Citation(s) in RCA: 17] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
Abstract
β-hemoglobinopathies are the most common monogenic disorders worldwide and are caused by mutations in the β-globin locus altering the production of adult hemoglobin (HbA). Transplantation of autologous hematopoietic stem cells (HSCs) corrected by lentiviral vector-mediated addition of a functional β-like globin raised new hopes to treat sickle cell disease and β-thalassemia patients; however, the low expression of the therapeutic gene per vector copy is often not sufficient to fully correct the patients with a severe clinical phenotype. Recent advances in the genome editing field brought new possibilities to cure β-hemoglobinopathies by allowing the direct modification of specific endogenous loci. Double-strand breaks (DSBs)-inducing nucleases (i.e., ZFNs, TALENs and CRISPR-Cas9) or DSB-free tools (i.e., base and prime editing) have been used to directly correct the disease-causing mutations, restoring HbA expression, or to reactivate the expression of the fetal hemoglobin (HbF), which is known to alleviate clinical symptoms of β-hemoglobinopathy patients. Here, we describe the different genome editing tools, their application to develop therapeutic approaches to β-hemoglobinopathies and ongoing clinical trials using genome editing strategies.
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Affiliation(s)
- Mégane Brusson
- Université de Paris, Imagine Institute, Laboratory of Chromatin and Gene Regulation During Development, INSERM UMR 1163, Paris, France.
| | - Annarita Miccio
- Université de Paris, Imagine Institute, Laboratory of Chromatin and Gene Regulation During Development, INSERM UMR 1163, Paris, France.
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44
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Singh V. An introduction to CRISPR-Cas systems for reprogramming the genome of mammalian cells. PROGRESS IN MOLECULAR BIOLOGY AND TRANSLATIONAL SCIENCE 2021; 181:1-13. [PMID: 34127190 DOI: 10.1016/bs.pmbts.2021.01.010] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
In the past few decades, it has been possible to introduce unprecedented mutations in genes of the mammalian cells owing to the development of advanced technologies/methods/assays. Sometimes, these mutations occurring at the cellular level may even cost the life of organisms. A number of diseases in mammals have shown to leave some serious impact on their lives. There are no drugs or medicines available in market for the correction or repair of these mutated genes in order to reverse gene function. A pressing need therefore arises to develop a next generation technology that cannot just corrects gene mutations but also restores gene function. Recent advances in CRISPR-Cas9 technology play a key role for correction of defective genes in wide range of mammalian cells. This chapter highlights CRISPR-Cas systems for basic, biomedical, biotechnological and therapeutic applications.
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Affiliation(s)
- Vijai Singh
- Department of Biosciences, School of Science, Indrashil University, Rajpur, Mehsana, Gujarat, India.
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45
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Ma Y, Liu S, Gao J, Chen C, Zhang X, Yuan H, Chen Z, Yin X, Sun C, Mao Y, Zhou F, Shao Y, Liu Q, Xu J, Cheng L, Yu D, Li P, Yi P, He J, Geng G, Guo Q, Si Y, Zhao H, Li H, Banes GL, Liu H, Nakamura Y, Kurita R, Huang Y, Wang X, Wang F, Fang G, Engel JD, Shi L, Zhang YE, Yu J. Genome-wide analysis of pseudogenes reveals HBBP1's human-specific essentiality in erythropoiesis and implication in β-thalassemia. Dev Cell 2021; 56:478-493.e11. [PMID: 33476555 DOI: 10.1016/j.devcel.2020.12.019] [Citation(s) in RCA: 28] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/28/2020] [Revised: 11/16/2020] [Accepted: 12/28/2020] [Indexed: 02/05/2023]
Abstract
The human genome harbors 14,000 duplicated or retroposed pseudogenes. Given their functionality as regulatory RNAs and low conservation, we hypothesized that pseudogenes could shape human-specific phenotypes. To test this, we performed co-expression analyses and found that pseudogene exhibited tissue-specific expression, especially in the bone marrow. By incorporating genetic data, we identified a bone-marrow-specific duplicated pseudogene, HBBP1 (η-globin), which has been implicated in β-thalassemia. Extensive functional assays demonstrated that HBBP1 is essential for erythropoiesis by binding the RNA-binding protein (RBP), HNRNPA1, to upregulate TAL1, a key regulator of erythropoiesis. The HBBP1/TAL1 interaction contributes to a milder symptom in β-thalassemia patients. Comparative studies further indicated that the HBBP1/TAL1 interaction is human-specific. Genome-wide analyses showed that duplicated pseudogenes are often bound by RBPs and less commonly bound by microRNAs compared with retropseudogenes. Taken together, we not only demonstrate that pseudogenes can drive human evolution but also provide insights on their functional landscapes.
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Affiliation(s)
- Yanni Ma
- State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Science, Chinese Academy of Medical Sciences (CAMS) & School of Basic Medicine, Peking Union Medical College (PUMC), Beijing 100005, China; Key Laboratory of RNA and Hematopoietic Regulation, Chinese Academy of Medical Sciences, Beijing 100005, China.
| | - Siqi Liu
- State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Science, Chinese Academy of Medical Sciences (CAMS) & School of Basic Medicine, Peking Union Medical College (PUMC), Beijing 100005, China; Key Laboratory of RNA and Hematopoietic Regulation, Chinese Academy of Medical Sciences, Beijing 100005, China
| | - Jie Gao
- State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, China
| | - Chunyan Chen
- Key Laboratory of Zoological Systematics and Evolution & State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China
| | - Xin Zhang
- Laboratory of Molecular Cardiology & Medical Molecular Imaging, First Affiliated Hospital of Shantou University Medical College, Shantou 515041, China
| | - Hao Yuan
- Key Laboratory of Zoological Systematics and Evolution & State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China
| | - Zhongyang Chen
- State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Science, Chinese Academy of Medical Sciences (CAMS) & School of Basic Medicine, Peking Union Medical College (PUMC), Beijing 100005, China; Key Laboratory of RNA and Hematopoietic Regulation, Chinese Academy of Medical Sciences, Beijing 100005, China
| | - Xiaolin Yin
- 923rd Hospital of the Joint Logistics Support Force of the Chinese People's Liberation Army, Guangxi 530021, China
| | - Chenguang Sun
- State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Science, Chinese Academy of Medical Sciences (CAMS) & School of Basic Medicine, Peking Union Medical College (PUMC), Beijing 100005, China; Key Laboratory of RNA and Hematopoietic Regulation, Chinese Academy of Medical Sciences, Beijing 100005, China
| | - Yanan Mao
- Key Laboratory of Zoological Systematics and Evolution & State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China
| | - Fanqi Zhou
- State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Science, Chinese Academy of Medical Sciences (CAMS) & School of Basic Medicine, Peking Union Medical College (PUMC), Beijing 100005, China; Key Laboratory of RNA and Hematopoietic Regulation, Chinese Academy of Medical Sciences, Beijing 100005, China
| | - Yi Shao
- Key Laboratory of Zoological Systematics and Evolution & State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China
| | - Qian Liu
- Shantou University Medical College, Shantou 515041, China
| | - Jiayue Xu
- State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Science, Chinese Academy of Medical Sciences (CAMS) & School of Basic Medicine, Peking Union Medical College (PUMC), Beijing 100005, China; Key Laboratory of RNA and Hematopoietic Regulation, Chinese Academy of Medical Sciences, Beijing 100005, China
| | - Li Cheng
- State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Science, Chinese Academy of Medical Sciences (CAMS) & School of Basic Medicine, Peking Union Medical College (PUMC), Beijing 100005, China
| | - Daqi Yu
- Key Laboratory of Zoological Systematics and Evolution & State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China
| | - Pingping Li
- 923rd Hospital of the Joint Logistics Support Force of the Chinese People's Liberation Army, Guangxi 530021, China
| | - Ping Yi
- Department of Obstetrics and Gynecology, the Third Affiliated Hospital of Chongqing Medical University (General Hospital), Chongqing 401120, China
| | - Jiahuan He
- State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Science, Chinese Academy of Medical Sciences (CAMS) & School of Basic Medicine, Peking Union Medical College (PUMC), Beijing 100005, China; Key Laboratory of RNA and Hematopoietic Regulation, Chinese Academy of Medical Sciences, Beijing 100005, China
| | - Guangfeng Geng
- State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, China
| | - Qing Guo
- State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, China
| | - Yanmin Si
- State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Science, Chinese Academy of Medical Sciences (CAMS) & School of Basic Medicine, Peking Union Medical College (PUMC), Beijing 100005, China; Key Laboratory of RNA and Hematopoietic Regulation, Chinese Academy of Medical Sciences, Beijing 100005, China
| | - Hualu Zhao
- State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Science, Chinese Academy of Medical Sciences (CAMS) & School of Basic Medicine, Peking Union Medical College (PUMC), Beijing 100005, China; Key Laboratory of RNA and Hematopoietic Regulation, Chinese Academy of Medical Sciences, Beijing 100005, China
| | - Haipeng Li
- Chinese Academy of Sciences Key Laboratory of Computational Biology, Shanghai Institute of Nutrition and Health, Chinese Academy of Sciences, Shanghai 200031, China; CAS Center for Excellence in Animal Evolution and Genetics, Chinese Academy of Sciences, Kunming 650223, China
| | - Graham L Banes
- Chinese Academy of Sciences Key Laboratory of Computational Biology, Shanghai Institute of Nutrition and Health, Chinese Academy of Sciences, Shanghai 200031, China; Wisconsin National Primate Research Center, University of Wisconsin Madison, 1220 Capitol Court, Madison, WI 53715, USA
| | - He Liu
- Beijing Key Laboratory of Captive Wildlife Technology, Beijing Zoo, Beijing 100044, China
| | - Yukio Nakamura
- Cell Engineering Division, RIKEN BioResource Research Center, Ibaraki 305-0074, Japan
| | - Ryo Kurita
- Department of Research and Development, Central Blood Institute, Japanese Red Cross Society, Tokyo 105-8521, Japan
| | - Yue Huang
- State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Science, Chinese Academy of Medical Sciences (CAMS) & School of Basic Medicine, Peking Union Medical College (PUMC), Beijing 100005, China
| | - Xiaoshuang Wang
- State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Science, Chinese Academy of Medical Sciences (CAMS) & School of Basic Medicine, Peking Union Medical College (PUMC), Beijing 100005, China; Key Laboratory of RNA and Hematopoietic Regulation, Chinese Academy of Medical Sciences, Beijing 100005, China
| | - Fang Wang
- State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Science, Chinese Academy of Medical Sciences (CAMS) & School of Basic Medicine, Peking Union Medical College (PUMC), Beijing 100005, China; Key Laboratory of RNA and Hematopoietic Regulation, Chinese Academy of Medical Sciences, Beijing 100005, China
| | - Gang Fang
- NYU Shanghai, 1555 Century Avenue, Shanghai 20012, China; Department of Biology, 1009 Silver Center, New York University, New York, NY 10003, USA; School of Computer Science and Software Engineering, East China Normal University, Shanghai 200062, China
| | - James Douglas Engel
- Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI 48109, USA
| | - Lihong Shi
- State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, China.
| | - Yong E Zhang
- Key Laboratory of Zoological Systematics and Evolution & State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China; CAS Center for Excellence in Animal Evolution and Genetics, Chinese Academy of Sciences, Kunming 650223, China; Chinese Institute for Brain Research, Beijing 102206, China.
| | - Jia Yu
- State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Science, Chinese Academy of Medical Sciences (CAMS) & School of Basic Medicine, Peking Union Medical College (PUMC), Beijing 100005, China; Key Laboratory of RNA and Hematopoietic Regulation, Chinese Academy of Medical Sciences, Beijing 100005, China; State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, China.
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Zittersteijn HA, Harteveld CL, Klaver-Flores S, Lankester AC, Hoeben RC, Staal FJT, Gonçalves MAFV. A Small Key for a Heavy Door: Genetic Therapies for the Treatment of Hemoglobinopathies. Front Genome Ed 2021; 2:617780. [PMID: 34713239 PMCID: PMC8525365 DOI: 10.3389/fgeed.2020.617780] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/15/2020] [Accepted: 12/14/2020] [Indexed: 12/26/2022] Open
Abstract
Throughout the past decades, the search for a treatment for severe hemoglobinopathies has gained increased interest within the scientific community. The discovery that ɤ-globin expression from intact HBG alleles complements defective HBB alleles underlying β-thalassemia and sickle cell disease, has provided a promising opening for research directed at relieving ɤ-globin repression mechanisms and, thereby, improve clinical outcomes for patients. Various gene editing strategies aim to reverse the fetal-to-adult hemoglobin switch to up-regulate ɤ-globin expression through disabling either HBG repressor genes or repressor binding sites in the HBG promoter regions. In addition to these HBB mutation-independent strategies involving fetal hemoglobin (HbF) synthesis de-repression, the expanding genome editing toolkit is providing increased accuracy to HBB mutation-specific strategies encompassing adult hemoglobin (HbA) restoration for a personalized treatment of hemoglobinopathies. Moreover, besides genome editing, more conventional gene addition strategies continue under investigation to restore HbA expression. Together, this research makes hemoglobinopathies a fertile ground for testing various innovative genetic therapies with high translational potential. Indeed, the progressive understanding of the molecular clockwork underlying the hemoglobin switch together with the ongoing optimization of genome editing tools heightens the prospect for the development of effective and safe treatments for hemoglobinopathies. In this context, clinical genetics plays an equally crucial role by shedding light on the complexity of the disease and the role of ameliorating genetic modifiers. Here, we cover the most recent insights on the molecular mechanisms underlying hemoglobin biology and hemoglobinopathies while providing an overview of state-of-the-art gene editing platforms. Additionally, current genetic therapies under development, are equally discussed.
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Affiliation(s)
- Hidde A. Zittersteijn
- Department of Cell and Chemical Biology, Leiden University Medical Center, Leiden, Netherlands
| | - Cornelis L. Harteveld
- Department of Human and Clinical Genetics, The Hemoglobinopathies Laboratory, Leiden University Medical Center, Leiden, Netherlands
| | | | - Arjan C. Lankester
- Department of Pediatrics, Stem Cell Transplantation Program, Willem-Alexander Children's Hospital, Leiden University Medical Center, Leiden, Netherlands
| | - Rob C. Hoeben
- Department of Cell and Chemical Biology, Leiden University Medical Center, Leiden, Netherlands
| | - Frank J. T. Staal
- Department of Immunology, Leiden University Medical Center, Leiden, Netherlands
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47
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Li C, Wang H, Georgakopoulou A, Gil S, Yannaki E, Lieber A. In Vivo HSC Gene Therapy Using a Bi-modular HDAd5/35++ Vector Cures Sickle Cell Disease in a Mouse Model. Mol Ther 2021; 29:822-837. [PMID: 32949495 PMCID: PMC7854285 DOI: 10.1016/j.ymthe.2020.09.001] [Citation(s) in RCA: 44] [Impact Index Per Article: 11.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2020] [Revised: 08/21/2020] [Accepted: 09/01/2020] [Indexed: 12/26/2022] Open
Abstract
We have recently reported that, after in vivo hematopoietic stem cell/progenitor (HSPC) transduction with HDAd5/35++ vectors, SB100x transposase-mediated γ-globin gene addition achieved 10%-15% γ-globin of adult mouse globin, resulting in significant but incomplete phenotypic correction in a thalassemia intermedia mouse model. Furthermore, genome editing of a γ-globin repressor binding site within the γ-globin promoter by CRISPR-Cas9 results in efficient reactivation of endogenous γ-globin. Here, we aimed to combine these two mechanisms to obtain curative levels of γ-globin after in vivo HSPC transduction. We generated a HDAd5/35++ adenovirus vector (HDAd-combo) containing both modules and tested it in vitro and after in vivo HSPC transduction in healthy CD46/β-YAC mice and in a sickle cell disease mouse model (CD46/Townes). Compared to HDAd vectors containing either the γ-globin addition or the CRISPR-Cas9 reactivation units alone, in vivo HSC transduction of CD46/Townes mice with the HDAd-combo resulted in significantly higher γ-globin in red blood cells, reaching 30% of that of adult human α and βS chains and a complete phenotypic correction of sickle cell disease.
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Affiliation(s)
- Chang Li
- Department of Medicine, Division of Medical Genetics, University of Washington, Seattle, WA 98195, USA
| | - Hongjie Wang
- Department of Medicine, Division of Medical Genetics, University of Washington, Seattle, WA 98195, USA
| | - Aphrodite Georgakopoulou
- Department of Medicine, Division of Medical Genetics, University of Washington, Seattle, WA 98195, USA; Gene and Cell Therapy Center, Hematology Department, George Papanicolaou Hospital, Thessaloniki 57010, Greece
| | - Sucheol Gil
- Department of Medicine, Division of Medical Genetics, University of Washington, Seattle, WA 98195, USA
| | - Evangelia Yannaki
- Gene and Cell Therapy Center, Hematology Department, George Papanicolaou Hospital, Thessaloniki 57010, Greece
| | - André Lieber
- Department of Medicine, Division of Medical Genetics, University of Washington, Seattle, WA 98195, USA; Department of Pathology, University of Washington, Seattle, WA 98195, USA.
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48
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Abstract
PURPOSE OF REVIEW In this work we briefly summarize the key features and currently available conventional therapies for the two main β-hemoglobinopathies, sickle cell disease (SCD) and β-thalassemia, and review the rapidly evolving field of novel and emerging genetic therapies to cure the disease. RECENT FINDINGS Gene therapy using viral vectors or designer nuclease-based gene editing is a relatively new field of medicine that uses the patient's own genetically modified cells to treat his or her own disease. Multiple different approaches are currently in development, and some have entered phase I clinical studies, including innovative therapies aiming at induction of fetal hemoglobin. SUMMARY Early short-term therapeutic benefit has been reported for some of the ongoing clinical trials, but confirmation of long-term safety and efficacy remains to be shown. Future therapies aiming at the targeted correction of specific disease-causing DNA mutations are emerging and will likely enter clinical testing in the near future.
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Papizan JB, Porter SN, Sharma A, Pruett-Miller SM. Therapeutic gene editing strategies using CRISPR-Cas9 for the β-hemoglobinopathies. J Biomed Res 2021; 35:115-134. [PMID: 33349624 PMCID: PMC8038529 DOI: 10.7555/jbr.34.20200096] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022] Open
Abstract
With advancements in gene editing technologies, our ability to make precise and efficient modifications to the genome is increasing at a remarkable rate, paving the way for scientists and clinicians to uniquely treat a multitude of previously irremediable diseases. CRISPR-Cas9, short for clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9, is a gene editing platform with the ability to alter the nucleotide sequence of the genome in living cells. This technology is increasing the number and pace at which new gene editing treatments for genetic disorders are moving toward the clinic. The β-hemoglobinopathies are a group of monogenic diseases, which despite their high prevalence and chronic debilitating nature, continue to have few therapeutic options available. In this review, we will discuss our existing comprehension of the genetics and current state of treatment for β-hemoglobinopathies, consider potential genome editing therapeutic strategies, and provide an overview of the current state of clinical trials using CRISPR-Cas9 gene editing.
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Affiliation(s)
- James B Papizan
- Department of Cellular and Molecular Biology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.,Center for Advanced Genome Engineering, St. Jude Children's Research Hospital, Memphis, TN 38105, USA
| | - Shaina N Porter
- Department of Cellular and Molecular Biology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.,Center for Advanced Genome Engineering, St. Jude Children's Research Hospital, Memphis, TN 38105, USA
| | - Akshay Sharma
- Department of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children's Research Hospital, Memphis, TN 38105, USA
| | - Shondra M Pruett-Miller
- Department of Cellular and Molecular Biology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.,Center for Advanced Genome Engineering, St. Jude Children's Research Hospital, Memphis, TN 38105, USA
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50
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Venkatesan V, Srinivasan S, Babu P, Thangavel S. Manipulation of Developmental Gamma-Globin Gene Expression: an Approach for Healing Hemoglobinopathies. Mol Cell Biol 2020; 41:e00253-20. [PMID: 33077498 PMCID: PMC7849396 DOI: 10.1128/mcb.00253-20] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022] Open
Abstract
β-Hemoglobinopathies are the most common monogenic disorders, and a century of research has provided us with a better understanding of the attributes of these diseases. Allogenic stem cell transplantation was the only potentially curative option available for these diseases until the discovery of gene therapy. The findings on the protective nature of fetal hemoglobin in sickle cell disease (SCD) and thalassemia patients carrying hereditary persistence of fetal hemoglobin (HPFH) mutations has given us the best evidence that the cure for β-hemoglobinopathies remains hidden in the hemoglobin locus. The detailed understanding of the developmental gene regulation of gamma-globin (γ-globin) and the emergence of gene manipulation strategies offer us the opportunity for developing a γ-globin gene-modified autologous stem cell transplantation therapy. In this review, we summarize different therapeutic strategies that reactivate fetal hemoglobin for the gene therapy of β-hemoglobinopathies.
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Affiliation(s)
- Vigneshwaran Venkatesan
- Centre for Stem Cell Research (CSCR), InStem Bengaluru, Christian Medical College, Vellore, Tamil Nadu, India
- Manipal Academy of Higher Education, Manipal, Karnataka, India
| | - Saranya Srinivasan
- Centre for Stem Cell Research (CSCR), InStem Bengaluru, Christian Medical College, Vellore, Tamil Nadu, India
| | - Prathibha Babu
- Centre for Stem Cell Research (CSCR), InStem Bengaluru, Christian Medical College, Vellore, Tamil Nadu, India
- Manipal Academy of Higher Education, Manipal, Karnataka, India
| | - Saravanabhavan Thangavel
- Centre for Stem Cell Research (CSCR), InStem Bengaluru, Christian Medical College, Vellore, Tamil Nadu, India
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