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Zhang T, Wang Z, Liu Q, Zhao D. Genetic Improvement of rice Grain size Using the CRISPR/Cas9 System. RICE (NEW YORK, N.Y.) 2025; 18:3. [PMID: 39865189 PMCID: PMC11769925 DOI: 10.1186/s12284-025-00758-8] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/25/2024] [Accepted: 01/18/2025] [Indexed: 01/28/2025]
Abstract
Rice grain size influences both grain yield and quality, making it a significant target for rice genetic improvement. In recent years, numerous genes related to grain size with differential effects have been cloned. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing system is a convenient tool for modifying genes. The use of the CRISPR/Cas9 tool for the genetic improvement of grain size-related genes is worth exploring. This paper summarizes the known grain size-related genes and the use of CRISPR/Cas9 for grain size modification and discusses the potential applications of CRISPR/Cas9 for improving rice grain size.
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Affiliation(s)
- Tao Zhang
- Key Laboratory of Plant Functional Genomics of the Ministry of Education / Jiangsu Key Laboratory of Crop Genomics and Molecular Breeding, Zhongshan Biological Breeding Laboratory, College of Agriculture, Yangzhou University, Yangzhou, 225009, China
| | - Zhengwei Wang
- Key Laboratory of Plant Functional Genomics of the Ministry of Education / Jiangsu Key Laboratory of Crop Genomics and Molecular Breeding, Zhongshan Biological Breeding Laboratory, College of Agriculture, Yangzhou University, Yangzhou, 225009, China
| | - Qiaoquan Liu
- Key Laboratory of Plant Functional Genomics of the Ministry of Education / Jiangsu Key Laboratory of Crop Genomics and Molecular Breeding, Zhongshan Biological Breeding Laboratory, College of Agriculture, Yangzhou University, Yangzhou, 225009, China
| | - Dongsheng Zhao
- Key Laboratory of Plant Functional Genomics of the Ministry of Education / Jiangsu Key Laboratory of Crop Genomics and Molecular Breeding, Zhongshan Biological Breeding Laboratory, College of Agriculture, Yangzhou University, Yangzhou, 225009, China.
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2
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Deshpande P, Chen CY, Chimata AV, Li JC, Sarkar A, Yeates C, Chen CH, Kango-Singh M, Singh A. miR-277 targets the proapoptotic gene-hid to ameliorate Aβ42-mediated neurodegeneration in Alzheimer's model. Cell Death Dis 2024; 15:71. [PMID: 38238337 PMCID: PMC10796706 DOI: 10.1038/s41419-023-06361-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/15/2023] [Revised: 11/28/2023] [Accepted: 11/30/2023] [Indexed: 01/22/2024]
Abstract
Alzheimer's disease (AD), an age-related progressive neurodegenerative disorder, exhibits reduced cognitive function with no cure to date. One of the reasons for AD is the accumulation of Amyloid-beta 42 (Aβ42) plaque(s) that trigger aberrant gene expression and signaling, which results in neuronal cell death by an unknown mechanism(s). Misexpression of human Aβ42 in the developing retina of Drosophila exhibits AD-like neuropathology. Small non-coding RNAs, microRNAs (miRNAs), post-transcriptionally regulate the expression of their target genes and thereby regulate different signaling pathways. In a forward genetic screen, we identified miR-277 (human ortholog is hsa-miR-3660) as a genetic modifier of Aβ42-mediated neurodegeneration. Loss-of-function of miR-277 enhances the Aβ42-mediated neurodegeneration. Whereas gain-of-function of miR-277 in the GMR > Aβ42 background downregulates cell death to maintain the number of neurons and thereby restores the retinal axonal targeting defects indicating the functional rescue. In addition, gain-of-function of miR-277 rescues the eclosion- and climbing assays defects observed in GMR > Aβ42 background. Thus, gain-of-function of miR-277 rescues both structurally as well as functionally the Aβ42-mediated neurodegeneration. Furthermore, we identified head involution defective (hid), an evolutionarily conserved proapoptotic gene, as one of the targets of miR-277 and validated these results using luciferase- and qPCR -assays. In the GMR > Aβ42 background, the gain-of-function of miR-277 results in the reduction of hid transcript levels to one-third of its levels as compared to GMR > Aβ42 background alone. Here, we provide a novel molecular mechanism where miR-277 targets and downregulates proapoptotic gene, hid, transcript levels to rescue Aβ42-mediated neurodegeneration by blocking cell death. These studies shed light on molecular mechanism(s) that mediate cell death response following Aβ42 accumulation seen in neurodegenerative disorders in humans and provide new therapeutic targets for neurodegeneration.
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Affiliation(s)
| | - Chao-Yi Chen
- Institution of Molecular and Cellular Biology, National Taiwan University, Taipei, Taiwan
| | | | - Jian-Chiuan Li
- Institution of Molecular and Cellular Biology, National Taiwan University, Taipei, Taiwan
- National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Miaoli, Taiwan
| | - Ankita Sarkar
- Department of Biology, University of Dayton, Dayton, OH, 45469, USA
| | - Catherine Yeates
- Department of Biology, University of Dayton, Dayton, OH, 45469, USA
| | - Chun-Hong Chen
- Institution of Molecular and Cellular Biology, National Taiwan University, Taipei, Taiwan.
- National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Miaoli, Taiwan.
| | - Madhuri Kango-Singh
- Department of Biology, University of Dayton, Dayton, OH, 45469, USA.
- Premedical Program, University of Dayton, Dayton, OH, USA.
- Integrative Science and Engineering (ISE), University of Dayton, Dayton, OH, USA.
| | - Amit Singh
- Department of Biology, University of Dayton, Dayton, OH, 45469, USA.
- Premedical Program, University of Dayton, Dayton, OH, USA.
- Integrative Science and Engineering (ISE), University of Dayton, Dayton, OH, USA.
- Center for Genomic Advocacy (TCGA), Indiana State University, Terre Haute, IN, USA.
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3
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Gulati P, Singh CV. The Crucial Role of Molecular Biology in Cancer Therapy: A Comprehensive Review. Cureus 2024; 16:e52246. [PMID: 38352075 PMCID: PMC10863367 DOI: 10.7759/cureus.52246] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/06/2023] [Accepted: 01/14/2024] [Indexed: 02/16/2024] Open
Abstract
Molecular biology shines a light of hope amid the complex terrain of cancer, bringing revolutionary approaches to cancer treatment. Instead of providing a synopsis, this review presents an engaging story that sheds light on the genetic nuances controlling the course of cancer. This review goes beyond just listing genetic alterations to examine the complex interactions that lead to oncogene activation, exploring particular triggers such as viral infections or proto-oncogene mutations. A comprehensive grasp of the significant influence of oncogenes is possible through the classification and clarification of their function in various types of cancer. Furthermore, the role of tumor suppressor genes in controlling cell division and preventing tumor growth is fully explained, providing concrete examples and case studies to ground the conversation and create a stronger story. This study highlights the practical applications of molecular biology and provides a comprehensive overview of various detection and treatment modalities. It emphasizes the effectiveness of RNA analysis, immunohistochemistry, and next-generation sequencing (NGS) in cancer diagnosis and prognosis prediction. Examples include the individualized classification of breast cancers through RNA profiling, the use of NGS to identify actionable mutations such as epidermal growth factor receptor and anaplastic lymphoma kinase in lung cancer, and the use of immunohistochemical staining for proteins such as Kirsten rat sarcoma viral oncogene to guide treatment decisions in colorectal cancer. This paper carefully examines how molecular biology is essential to creating new strategies to fight this difficult and widespread illness. It highlights the exciting array of available therapeutic approaches, offering concrete instances of how clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (CRISPR-Cas9), targeted pharmaceuticals, immunotherapy, and treatments that induce apoptosis are driving a paradigm shift in cancer care. The revolutionary CRISPR-Cas9 system takes center stage, showcasing how precise gene editing could transform cancer therapy. This study concludes by fervently highlighting the critical role that molecular biology plays in reducing the complexity of cancer and changing the treatment landscape. It lists accomplishments but also thoughtfully examines cases and findings that progress our search for more precisely customized and effective cancer therapies.
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Affiliation(s)
- Prisha Gulati
- Medicine and Surgery, Jawaharlal Nehru Medical College, Datta Meghe Institute of Higher Education and Research, Wardha, IND
| | - Chandra Veer Singh
- Otolaryngology - Head and Neck Surgery, Jawaharlal Nehru Medical College, Datta Meghe Institute of Higher Education and Research, Wardha, IND
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Richard I. Basic notions about gene therapy from the nucleic acid perspective and applications in a pediatric disease: Duchenne muscular dystrophy. Arch Pediatr 2023; 30:8S2-8S11. [PMID: 38043979 DOI: 10.1016/s0929-693x(23)00221-x] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/05/2023]
Abstract
Gene therapy involves the introduction of genetic material into cells as a therapeutic molecule to cure a disease. Through the transfer of specific nucleic acid to the target tissue, gene expression can be downregulated, augmented, or corrected thanks to the nucleic acid sequence as a support of gene expression. This is achieved through molecular interactions according to the sequence arrangement or the secondary structure of the molecules or through their catalytic properties. Over the past two decades, the rapid advances of knowledge and technologies in gene therapy have led to the development of different strategies and to the extension of its use to numerous indications, including certain cancers. Major success has been achieved in clinical trials and the field of gene therapy is booming. Several gene therapy products are now on the market in Europe, the United States, and China. In this review, we cover the basic principles of gene therapy and the characteristics of the main vectors used to transfer genetic material into the cell. As an example of applications, we address the various strategies applied to a rare pediatric muscle disease: Duchenne muscular dystrophy. © 2023 Published by Elsevier Masson SAS on behalf of French Society of Pediatrics.
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Affiliation(s)
- Isabelle Richard
- Genethon, 91000, Evry, France; Université Paris-Saclay, Univ. Evry, Inserm, Integrare research unit UMR_S951, 91000, Evry-Courcouronnes, France; Atamyo Therapeutics, 1, bis rue de l'internationale, Evry, France.
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Zhang Z, Bao X, Lin CP. Progress and Prospects of Gene Editing in Pluripotent Stem Cells. Biomedicines 2023; 11:2168. [PMID: 37626665 PMCID: PMC10452926 DOI: 10.3390/biomedicines11082168] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2023] [Revised: 07/16/2023] [Accepted: 07/18/2023] [Indexed: 08/27/2023] Open
Abstract
Applying programmable nucleases in gene editing has greatly shaped current research in basic biology and clinical translation. Gene editing in human pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), is highly relevant to clinical cell therapy and thus should be examined with particular caution. First, since all mutations in PSCs will be carried to all their progenies, off-target edits of editors will be amplified. Second, due to the hypersensitivity of PSCs to DNA damage, double-strand breaks (DSBs) made by gene editing could lead to low editing efficiency and the enrichment of cell populations with defective genomic safeguards. In this regard, DSB-independent gene editing tools, such as base editors and prime editors, are favored due to their nature to avoid these consequences. With more understanding of the microbial world, new systems, such as Cas-related nucleases, transposons, and recombinases, are also expanding the toolbox for gene editing. In this review, we discuss current applications of programmable nucleases in PSCs for gene editing, the efforts researchers have made to optimize these systems, as well as new tools that can be potentially employed for differentiation modeling and therapeutic applications.
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Affiliation(s)
| | | | - Chao-Po Lin
- School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China; (Z.Z.); (X.B.)
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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: 9] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [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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Braun T, Schrader A. Education and Empowering Special Forces to Eradicate Secret Defectors: Immune System-Based Treatment Approaches for Mature T- and NK-Cell Malignancies. Cancers (Basel) 2023; 15:cancers15092532. [PMID: 37173999 PMCID: PMC10177197 DOI: 10.3390/cancers15092532] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/29/2023] [Revised: 04/23/2023] [Accepted: 04/25/2023] [Indexed: 05/15/2023] Open
Abstract
Mature T- and NK-cell leukemia/lymphoma (MTCL/L) constitute a heterogeneous group of, currently, 30 distinct neoplastic entities that are overall rare, and all present with a challenging molecular markup. Thus, so far, the use of first-line cancer treatment modalities, including chemotherapies, achieve only limited clinical responses associated with discouraging prognoses. Recently, cancer immunotherapy has evolved rapidly, allowing us to help patients with, e.g., solid tumors and also relapsed/refractory B-cell malignancies to achieve durable clinical responses. In this review, we systematically unveiled the distinct immunotherapeutic approaches available, emphasizing the special impediments faced when trying to employ immune system defense mechanisms to target 'one of their own-gone mad'. We summarized the preclinical and clinical efforts made to employ the various platforms of cancer immunotherapies including antibody-drug conjugates, monoclonal as well as bispecific antibodies, immune-checkpoint blockades, and CAR T cell therapies. We emphasized the challenges to, but also the goals of, what needs to be done to achieve similar successes as seen for B-cell entities.
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Affiliation(s)
- Till Braun
- Department I of Internal Medicine, Center for Integrated Oncology, Aachen-Bonn-Cologne-Duesseldorf, Excellence Cluster for Cellular Stress Response and Aging-Associated Diseases, Center for Molecular Medicine Cologne, University of Cologne, 50937 Cologne, Germany
| | - Alexandra Schrader
- Department I of Internal Medicine, Center for Integrated Oncology, Aachen-Bonn-Cologne-Duesseldorf, Excellence Cluster for Cellular Stress Response and Aging-Associated Diseases, Center for Molecular Medicine Cologne, University of Cologne, 50937 Cologne, Germany
- Lymphoma Immuno Biology Team, Equipe Labellisée LIGUE 2023, Centre International de Recherche en Infectiologie, INSERM U1111-CNRS UMR5308, Faculté de Médecine Lyon-Sud, Hospices Civils de Lyon, Université Claude Bernard Lyon I-ENS de Lyon, 69921 Lyon, France
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8
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The challenge of dissecting gene function in model organisms: Tools to characterize genetic mutants and assess transcriptional adaptation in zebrafish. Methods Cell Biol 2023; 176:1-25. [PMID: 37164532 DOI: 10.1016/bs.mcb.2022.12.019] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/29/2023]
Abstract
Genome editing technologies including the CRISPR/Cas9 system have greatly improved our knowledge of gene function and biological processes, however, these approaches have also brought new challenges to determining genotype-phenotype correlations. In this chapter, we briefly review gene-editing technologies used in zebrafish and discuss the differences in phenotypes that can arise when gene expression is inhibited by anti-sense or by gene editing techniques. We outline possible explanations for why knockout phenotypes are milder, tissue-restricted, or even absent, compared with severe knockdown phenotypes. One proposed explanation is transcriptional adaptation, a form of genetic robustness that is induced by deleterious mutations but not gene knockdowns. Although much is unknown about what triggers this process, its relevance in shaping genome expression has been shown in multiple animal models. We recently explored if transcriptional adaptation could explain genotype-phenotype discrepancies seen between two zebrafish models of the centrosomal protein Cep290 deficiency. We compared cilia-related phenotypes in knockdown (anti-sense) and knockout (mutation) Cep290 models and showed that only cep290 gene mutation induces the upregulation of genes encoding the cilia-associated small GTPases Arl3, Arl13b, and Unc119b. Importantly, the ectopic expression of Arl3, Arl13b, and Unc119b in cep290 morphant zebrafish embryos rescued cilia defects. Here we provide protocols and experimental approaches that can be used to explore if transcriptional adaptation may be modulating gene expression in a zebrafish ciliary mutant model.
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Mitra S, Anand U, Ghorai M, Kant N, Kumar M, Radha, Jha NK, Swamy MK, Proćków J, de la Lastra JMP, Dey A. Genome editing technologies, mechanisms and improved production of therapeutic phytochemicals: Opportunities and prospects. Biotechnol Bioeng 2023; 120:82-94. [PMID: 36224758 PMCID: PMC10091730 DOI: 10.1002/bit.28260] [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: 05/18/2022] [Revised: 09/10/2022] [Accepted: 10/08/2022] [Indexed: 11/09/2022]
Abstract
Plants produce a large number of secondary metabolites, known as phytometabolites that may be employed as medicines, dyes, poisons, and insecticides in the field of medicine, agriculture, and industrial use, respectively. The rise of genome management approaches has promised a factual revolution in genetic engineering. Targeted genome editing in living entities permits the understanding of the biological systems very clearly, and also sanctions to address a wide-ranging objective in the direction of improving features of plant and their yields. The last few years have introduced a number of unique genome editing systems, including transcription activator-like effector nucleases, zinc finger nucleases, and miRNA-regulated clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9). Genome editing systems have helped in the transformation of metabolic engineering, allowing researchers to modify biosynthetic pathways of different secondary metabolites. Given the growing relevance of editing genomes in plant research, the exciting novel methods are briefly reviewed in this chapter. Also, this chapter highlights recent discoveries on the CRISPR-based modification of natural products in different medicinal plants.
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Affiliation(s)
- Sicon Mitra
- Department of Biotechnology, School of Engineering & TechnologySharda UniversityGreater NoidaUttar PradeshIndia
| | | | - Mimosa Ghorai
- Department of Life SciencesPresidency UniversityKolkataWest BengalIndia
| | - Nishi Kant
- Department of Chemical EngineeringIndian Institute of Technology DelhiDelhiNew DelhiIndia
| | - Manoj Kumar
- Chemical and Biochemical Processing DivisionICAR‐Central Institute for Research on Cotton TechnologyMumbaiMaharashtraIndia
| | - Radha
- School of Biological and Environmental SciencesShoolini University of Biotechnology and Management SciencesSolanHimachal PradeshIndia
| | - Niraj K. Jha
- Department of Biotechnology, School of Engineering & TechnologySharda UniversityGreater NoidaUttar PradeshIndia
- Department of Biotechnology Engineering and Food TechnologyChandigarh UniversityMohaliPunjabIndia
- Department of Biotechnology, School of Applied & Life SciencesUttaranchal UniversityDehradunUttarakhandIndia
| | - Mallappa K. Swamy
- Department of BiotechnologyEast West First Grade College of ScienceBengaluruKarnatakaIndia
| | - Jarosław Proćków
- Department of Plant Biology, Institute of Environmental BiologyWrocław University of Environmental and Life SciencesWrocławPoland
| | - José M. Pérez de la Lastra
- Biotechnology of Macromolecules Research Group, Department of Life and Earth SciencesInstituto de Productos Naturales y Agrobiología‐Consejo Superior de Investigaciones Científicas, (IPNA‐CSIC)San Cristóbal de La LagunaTenerifeSpain
| | - Abhijit Dey
- Department of Life SciencesPresidency UniversityKolkataWest BengalIndia
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10
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Tsai HC, Pietrobon V, Peng M, Wang S, Zhao L, Marincola FM, Cai Q. Current strategies employed in the manipulation of gene expression for clinical purposes. J Transl Med 2022; 20:535. [PMID: 36401279 PMCID: PMC9673226 DOI: 10.1186/s12967-022-03747-3] [Citation(s) in RCA: 11] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2022] [Accepted: 09/29/2022] [Indexed: 11/19/2022] Open
Abstract
Abnormal gene expression level or expression of genes containing deleterious mutations are two of the main determinants which lead to genetic disease. To obtain a therapeutic effect and thus to cure genetic diseases, it is crucial to regulate the host's gene expression and restore it to physiological conditions. With this purpose, several molecular tools have been developed and are currently tested in clinical trials. Genome editing nucleases are a class of molecular tools routinely used in laboratories to rewire host's gene expression. Genome editing nucleases include different categories of enzymes: meganucleses (MNs), zinc finger nucleases (ZFNs), clustered regularly interspaced short palindromic repeats (CRISPR)- CRISPR associated protein (Cas) and transcription activator-like effector nuclease (TALENs). Transposable elements are also a category of molecular tools which includes different members, for example Sleeping Beauty (SB), PiggyBac (PB), Tol2 and TcBuster. Transposons have been used for genetic studies and can serve as gene delivery tools. Molecular tools to rewire host's gene expression also include episomes, which are divided into different categories depending on their molecular structure. Finally, RNA interference is commonly used to regulate gene expression through the administration of small interfering RNA (siRNA), short hairpin RNA (shRNA) and bi-functional shRNA molecules. In this review, we will describe the different molecular tools that can be used to regulate gene expression and discuss their potential for clinical applications. These molecular tools are delivered into the host's cells in the form of DNA, RNA or protein using vectors that can be grouped into physical or biochemical categories. In this review we will also illustrate the different types of payloads that can be used, and we will discuss recent developments in viral and non-viral vector technology.
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Affiliation(s)
| | | | - Maoyu Peng
- Kite Pharma Inc, Santa Monica, CA, 90404, USA
| | - Suning Wang
- Kite Pharma Inc, Santa Monica, CA, 90404, USA
| | - Lihong Zhao
- Kite Pharma Inc, Santa Monica, CA, 90404, USA
| | | | - Qi Cai
- Kite Pharma Inc, Santa Monica, CA, 90404, USA.
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11
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Naseem A, Steinberg Z, Cavazza A. Genome editing for primary immunodeficiencies: A therapeutic perspective on Wiskott-Aldrich syndrome. Front Immunol 2022; 13:966084. [PMID: 36059471 PMCID: PMC9433875 DOI: 10.3389/fimmu.2022.966084] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2022] [Accepted: 07/26/2022] [Indexed: 11/13/2022] Open
Abstract
Primary immunodeficiency diseases (PIDs) are a group of rare inherited disorders affecting the immune system that can be conventionally treated with allogeneic hematopoietic stem cell transplantation and with experimental autologous gene therapy. With both approaches still facing important challenges, gene editing has recently emerged as a potential valuable alternative for the treatment of genetic disorders and within a relatively short period from its initial development, has already entered some landmark clinical trials aimed at tackling several life-threatening diseases. In this review, we discuss the progress made towards the development of gene editing-based therapeutic strategies for PIDs with a special focus on Wiskott - Aldrich syndrome and outline their main challenges as well as future directions with respect to already established treatments.
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Jamehdor S, Pajouhanfar S, Saba S, Uzan G, Teimoori A, Naserian S. Principles and Applications of CRISPR Toolkit in Virus Manipulation, Diagnosis, and Virus-Host Interactions. Cells 2022; 11:999. [PMID: 35326449 PMCID: PMC8946942 DOI: 10.3390/cells11060999] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2021] [Revised: 03/09/2022] [Accepted: 03/11/2022] [Indexed: 02/04/2023] Open
Abstract
Viruses are one of the most important concerns for human health, and overcoming viral infections is a worldwide challenge. However, researchers have been trying to manipulate viral genomes to overcome various disorders, including cancer, for vaccine development purposes. CRISPR (clustered regularly interspaced short palindromic repeats) is becoming one of the most functional and widely used tools for RNA and DNA manipulation in multiple organisms. This approach has provided an unprecedented opportunity for creating simple, inexpensive, specific, targeted, accurate, and practical manipulations of viruses, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), human immunodeficiency virus-1 (HIV-1), and vaccinia virus. Furthermore, this method can be used to make an effective and precise diagnosis of viral infections. Nevertheless, a valid and scientifically designed CRISPR system is critical to make more effective and accurate changes in viruses. In this review, we have focused on the best and the most effective ways to design sgRNA, gene knock-in(s), and gene knock-out(s) for virus-targeted manipulation. Furthermore, we have emphasized the application of CRISPR technology in virus diagnosis and in finding significant genes involved in virus-host interactions.
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Affiliation(s)
- Saleh Jamehdor
- Cellular and Molecular Research Center, Zahedan University of Medical Sciences, Zahedan 989155432609, Iran;
| | - Sara Pajouhanfar
- Department of Pediatrics, Washington University School of Medicine, St. Louis, MO 63110, USA;
| | - Sadaf Saba
- Center for Molecular Medicine & Genetics, Wayne State University School of Medicine, Detroit, MI 48201, USA;
| | - Georges Uzan
- INSERM UMR-S-MD 1197, Hôpital Paul Brousse, 94800 Villejuif, France;
- Paris-Saclay University, 94800 Villejuif, France
| | - Ali Teimoori
- Department of Virology, Faculty of Medicine, Hamadan University of Medical Sciences, Hamadan 6517838738, Iran
| | - Sina Naserian
- INSERM UMR-S-MD 1197, Hôpital Paul Brousse, 94800 Villejuif, France;
- Paris-Saclay University, 94800 Villejuif, France
- CellMedEx, 94100 Saint Maur Des Fossés, France
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Yamaguchi N. The epigenetic mechanisms regulating floral hub genes and their potential for manipulation. JOURNAL OF EXPERIMENTAL BOTANY 2022; 73:1277-1287. [PMID: 34752611 DOI: 10.1093/jxb/erab490] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/26/2021] [Accepted: 11/05/2021] [Indexed: 06/13/2023]
Abstract
Gene regulatory networks formed by transcription factors play essential roles in the regulation of gene expression during plant reproductive development. These networks integrate endogenous, phytohormonal, and environmental cues. Molecular genetic, biochemical, and chemical analyses performed mainly in Arabidopsis have identified network hub genes and revealed the contributions of individual components to these networks. Here, I outline current understanding of key epigenetic regulatory circuits identified by research on plant reproduction, and highlight significant recent examples of genetic engineering and chemical applications to modulate the epigenetic regulation of gene expression. Furthermore, I discuss future prospects for applying basic plant science to engineer useful floral traits in a predictable manner as well as the potential side effects.
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Affiliation(s)
- Nobutoshi Yamaguchi
- Division of Biological Science, Graduate School of Science and Technology, Nara Institute of Science and Technology, 8916-5, Takayama, Ikoma, Nara, 630-0192, Japan
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14
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Li Z, You L, Zhang Q, Yu Y, Tan A. A Targeted In-Fusion Expression System for Recombinant Protein Production in Bombyx mori. Front Genet 2022; 12:816075. [PMID: 35058975 PMCID: PMC8763709 DOI: 10.3389/fgene.2021.816075] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2021] [Accepted: 11/30/2021] [Indexed: 11/25/2022] Open
Abstract
The domesticated silkworm, Bombyx mori, is an economically important insect that synthesizes large amounts of silk proteins in its silk gland to make cocoons. In recent years, germline transformation strategies advanced the bioengineering of the silk gland as an ideal bioreactor for mass production of recombinant proteins. However, the yield of exogenous proteins varied largely due to the random insertion and gene drift caused by canonical transposon-based transformation, calling for site-specific and stable expression systems. In the current study, we established a targeted in-fusion expression system by using the transcription activator-like effector nuclease (TALEN)-mediated targeted insertion to target genomic locus of sericin, one of the major silk proteins. We successfully generated chimeric Sericin1-EGFP (Ser-2A-EGFP) transformant, producing up to 3.1% (w/w) of EGFP protein in the cocoon shell. With this strategy, we further expressed the medically important human epidermal growth factor (hEGF) and the protein yield in both middle silk glands, and cocoon shells reached to more than 15-fold higher than the canonical piggyBac-based transgenesis. This natural Sericin1 expression system provides a new strategy for producing recombinant proteins by using the silkworm silk gland as the bioreactor.
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Affiliation(s)
- Zhiqian Li
- Jiangsu Key Laboratory of Sericultural Biology and Biotechnology, School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, China.,Key Laboratory of Silkworm and Mulberry Genetic Improvement, Ministry of Agriculture, The Sericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang, China.,Key Laboratory of Insect Developmental and Evolutionary Biology, CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, China
| | - Lang You
- Jiangsu Key Laboratory of Sericultural Biology and Biotechnology, School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, China.,Key Laboratory of Silkworm and Mulberry Genetic Improvement, Ministry of Agriculture, The Sericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang, China
| | - Qichao Zhang
- Jiangsu Key Laboratory of Sericultural Biology and Biotechnology, School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, China.,Key Laboratory of Silkworm and Mulberry Genetic Improvement, Ministry of Agriculture, The Sericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang, China
| | - Ye Yu
- Jiangsu Key Laboratory of Sericultural Biology and Biotechnology, School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, China.,Key Laboratory of Silkworm and Mulberry Genetic Improvement, Ministry of Agriculture, The Sericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang, China
| | - Anjiang Tan
- Jiangsu Key Laboratory of Sericultural Biology and Biotechnology, School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, China.,Key Laboratory of Silkworm and Mulberry Genetic Improvement, Ministry of Agriculture, The Sericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang, China
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15
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Crispo M, Chenouard V, Dos Santos-Neto P, Tesson L, Souza-Neves M, Heslan JM, Cuadro F, Anegón I, Menchaca A. Generation of a Human Deafness Sheep Model Using the CRISPR/Cas System. Methods Mol Biol 2022; 2495:233-244. [PMID: 35696036 DOI: 10.1007/978-1-0716-2301-5_12] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/27/2023]
Abstract
CRISPR/Cas9 system is a promising method for the generation of human disease models by genome editing in non-conventional experimental animals. Medium/large-sized animals like sheep have several advantages to study human diseases and medicine. Here, we present a protocol that describes the generation of an otoferlin edited sheep model via CRISPR-assisted single-stranded oligodinucleotide-mediated Homology-Directed Repair (HDR), through direct cytoplasmic microinjection in in vitro produced zygotes.Otoferlin is a protein expressed in the cochlear inner hair cells, with different mutations at the OTOF gene being the major cause of nonsyndromic recessive auditory neuropathy spectrum disorder in humans. By using this protocol, we reported for the first time an OTOF KI model in sheep with 17.8% edited lambs showing indel mutations, and 61.5% of them bearing knock-in mutations by HDR . The reported method establishes the bases to produce a deafness model to test novel therapies in human disorders related to OTOF mutations.
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Affiliation(s)
- Martina Crispo
- Laboratory Animal Biotechnology Unit, Institut Pasteur de Montevideo, Montevideo, Uruguay
| | - Vanessa Chenouard
- INSERM Centre de Recherche en Transplantation et Immunologie UMR 1064, Transgenesis Rat ImmunoPhenomic Facility (TRIP), Nantes, France
| | | | - Laurent Tesson
- INSERM Centre de Recherche en Transplantation et Immunologie UMR 1064, Transgenesis Rat ImmunoPhenomic Facility (TRIP), Nantes, France
| | - Marcela Souza-Neves
- Instituto de Reproducción Animal Uruguay, Fundación IRAUy, Montevideo, Uruguay
| | - Jean-Marie Heslan
- INSERM Centre de Recherche en Transplantation et Immunologie UMR 1064, Transgenesis Rat ImmunoPhenomic Facility (TRIP), Nantes, France
| | - Federico Cuadro
- Instituto de Reproducción Animal Uruguay, Fundación IRAUy, Montevideo, Uruguay
| | - Ignacio Anegón
- INSERM Centre de Recherche en Transplantation et Immunologie UMR 1064, Transgenesis Rat ImmunoPhenomic Facility (TRIP), Nantes, France
| | - Alejo Menchaca
- Instituto de Reproducción Animal Uruguay, Fundación IRAUy, Montevideo, Uruguay.
- Instituto Nacional de Investigación Agropecuaria (INIA), Montevideo, Uruguay.
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16
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Chae K, Valentin C, Jakes E, Myles KM, Adelman ZN. Novel synthetic 3'-untranslated regions for controlling transgene expression in transgenic Aedes aegypti mosquitoes. RNA Biol 2021; 18:223-231. [PMID: 34464234 DOI: 10.1080/15476286.2021.1971440] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/20/2022] Open
Abstract
Transgenic technology for mosquitoes is now more than two decades old, and a wide array of control sequences have been described for regulating gene expression in various life stages or specific tissues. Despite this, comparatively little attention has been paid to the development and validation of other transgene-regulating elements, especially 3'-untranslated regions (3'UTRs). As a consequence, the same regulatory sequences are often used multiple times in a single transgene array, potentially leading to instability of transgenic effector genes. To increase the repertoire of characterized 3'UTRs available for genetics-based mosquito control, we generated fifteen synthetic sequences based on the base composition of the widely used SV40 3'UTR sequence, and tested their ability to contribute to the expression of reporter genes EGFP or luciferase. Transient transfection in mosquito cells identified nine candidate 3'UTRs that conferred moderate to strong gene expression. Two of these were engineered into the mosquito genome through CRISPR/Cas9-mediated site-specific insertion and compared to the original SV40 3'UTR. Both synthetic 3'UTRs were shown to successfully promote transgene expression in all mosquito life stages (larva, pupa and adults), similar to the SV40 3'UTR, albeit with differences in intensity. Thus, the synthetic 3'UTR elements described here are suitable for regulating transgene expression in Ae. aegypti, and provide valuable alternatives in the design of multi-gene cassettes. Additionally, the synthetic-scramble approach we validate here could be used to generate additional functional 3'UTR elements in this or other organisms.
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Affiliation(s)
- Keun Chae
- Department of Entomology, Texas A&M University, College Station, TX, USA
| | - Collin Valentin
- Department of Entomology, Texas A&M University, College Station, TX, USA
| | - Emma Jakes
- Department of Entomology, Texas A&M University, College Station, TX, USA
| | - Kevin M Myles
- Department of Entomology, Texas A&M University, College Station, TX, USA
| | - Zach N Adelman
- Department of Entomology, Texas A&M University, College Station, TX, USA
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17
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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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18
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Fritsche E, Haarmann-Stemmann T, Kapr J, Galanjuk S, Hartmann J, Mertens PR, Kämpfer AAM, Schins RPF, Tigges J, Koch K. Stem Cells for Next Level Toxicity Testing in the 21st Century. SMALL (WEINHEIM AN DER BERGSTRASSE, GERMANY) 2021; 17:e2006252. [PMID: 33354870 DOI: 10.1002/smll.202006252] [Citation(s) in RCA: 40] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/08/2020] [Revised: 11/13/2020] [Indexed: 06/12/2023]
Abstract
The call for a paradigm change in toxicology from the United States National Research Council in 2007 initiates awareness for the invention and use of human-relevant alternative methods for toxicological hazard assessment. Simple 2D in vitro systems may serve as first screening tools, however, recent developments infer the need for more complex, multicellular organotypic models, which are superior in mimicking the complexity of human organs. In this review article most critical organs for toxicity assessment, i.e., skin, brain, thyroid system, lung, heart, liver, kidney, and intestine are discussed with regards to their functions in health and disease. Embracing the manifold modes-of-action how xenobiotic compounds can interfere with physiological organ functions and cause toxicity, the need for translation of such multifaceted organ features into the dish seems obvious. Currently used in vitro methods for toxicological applications and ongoing developments not yet arrived in toxicity testing are discussed, especially highlighting the potential of models based on embryonic stem cells and induced pluripotent stem cells of human origin. Finally, the application of innovative technologies like organs-on-a-chip and genome editing point toward a toxicological paradigm change moves into action.
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Affiliation(s)
- Ellen Fritsche
- IUF - Leibniz Research Institute for Environmental Medicine, Düsseldorf, 40225, Germany
- Medical Faculty, Heinrich-Heine University Düsseldorf, Düsseldorf, 40225, Germany
| | | | - Julia Kapr
- IUF - Leibniz Research Institute for Environmental Medicine, Düsseldorf, 40225, Germany
| | - Saskia Galanjuk
- IUF - Leibniz Research Institute for Environmental Medicine, Düsseldorf, 40225, Germany
| | - Julia Hartmann
- IUF - Leibniz Research Institute for Environmental Medicine, Düsseldorf, 40225, Germany
| | - Peter R Mertens
- Department of Nephrology and Hypertension, Diabetes and Endocrinology, Otto-von-Guericke-University Magdeburg, Magdeburg, 39106, Germany
| | - Angela A M Kämpfer
- IUF - Leibniz Research Institute for Environmental Medicine, Düsseldorf, 40225, Germany
| | - Roel P F Schins
- IUF - Leibniz Research Institute for Environmental Medicine, Düsseldorf, 40225, Germany
| | - Julia Tigges
- IUF - Leibniz Research Institute for Environmental Medicine, Düsseldorf, 40225, Germany
| | - Katharina Koch
- IUF - Leibniz Research Institute for Environmental Medicine, Düsseldorf, 40225, Germany
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19
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Su Y, Bayarjargal M, Hale TK, Filichev VV. DNA with zwitterionic and negatively charged phosphate modifications: Formation of DNA triplexes, duplexes and cell uptake studies. Beilstein J Org Chem 2021; 17:749-761. [PMID: 33828619 PMCID: PMC8022206 DOI: 10.3762/bjoc.17.65] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/08/2020] [Accepted: 03/11/2021] [Indexed: 12/15/2022] Open
Abstract
Two phosphate modifications were introduced into the DNA backbone using the Staudinger reaction between the 3’,5’-dinucleoside β-cyanoethyl phosphite triester formed during DNA synthesis and sulfonyl azides, 4-(azidosulfonyl)-N,N,N-trimethylbutan-1-aminium iodide (N+ azide) or p-toluenesulfonyl (tosyl or Ts) azide, to provide either a zwitterionic phosphoramidate with N+ modification or a negatively charged phosphoramidate for Ts modification in the DNA sequence. The incorporation of these N+ and Ts modifications led to the formation of thermally stable parallel DNA triplexes, regardless of the number of modifications incorporated into the oligodeoxynucleotides (ONs). For both N+ and Ts-modified ONs, the antiparallel duplexes formed with complementary RNA were more stable than those formed with complementary DNA (except for ONs with modification in the middle of the sequence). Additionally, the incorporation of N+ modifications led to the formation of duplexes with a thermal stability that was less dependent on the ionic strength than native DNA duplexes. The thermodynamic analysis of the melting curves revealed that it is the reduction in unfavourable entropy, despite the decrease in favourable enthalpy, which is responsible for the stabilisation of duplexes with N+ modification. N+ONs also demonstrated greater resistance to nuclease digestion by snake venom phosphodiesterase I than the corresponding Ts-ONs. Cell uptake studies showed that Ts-ONs can enter the nucleus of mouse fibroblast NIH3T3 cells without any transfection reagent, whereas, N+ONs remain concentrated in vesicles within the cytoplasm. These results indicate that both N+ and Ts-modified ONs are promising for various in vivo applications.
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Affiliation(s)
- Yongdong Su
- School of Fundamental Sciences, Massey University, Private Bag 11-222, 4442 Palmerston North, New Zealand
| | - Maitsetseg Bayarjargal
- School of Fundamental Sciences, Massey University, Private Bag 11-222, 4442 Palmerston North, New Zealand
| | - Tracy K Hale
- School of Fundamental Sciences, Massey University, Private Bag 11-222, 4442 Palmerston North, New Zealand.,Maurice Wilkins Centre for Molecular Biodiscovery, Auckland 1142, New Zealand
| | - Vyacheslav V Filichev
- School of Fundamental Sciences, Massey University, Private Bag 11-222, 4442 Palmerston North, New Zealand.,Maurice Wilkins Centre for Molecular Biodiscovery, Auckland 1142, New Zealand
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20
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Ballios BG, Pierce EA, Huckfeldt RM. Gene editing technology: Towards precision medicine in inherited retinal diseases. Semin Ophthalmol 2021; 36:176-184. [PMID: 33621144 DOI: 10.1080/08820538.2021.1887903] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
Abstract
Purpose: To review preclinical and clinical advances in gene therapy, with a focus on gene editing technologies, and application to inherited retinal disease.Methods: A narrative overview of the literature, summarizing the state-of-the-art in clinical gene therapy for inherited retinal disease, as well as the science and application of new gene editing technology.Results: The last three years has seen the first FDA approval of an in vivo gene replacement therapy for a hereditary blinding eye disease and, recently, the first clinical application of an in vivo gene editing technique. Limitations and challenges in this evolving field are highlighted, as well as new technologies developed to address the multitude of molecular mechanisms of disease.Conclusion: Genetic therapy for the treatment of inherited retinal disease is a rapidly expanding area of ophthalmology. New technologies have revolutionized the field of genome engineering and rekindled an interest in precision medicines for these conditions.
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Affiliation(s)
- Brian G Ballios
- Ocular Genomics Institute, Department of Ophthalmology, Massachusetts Eye and Ear, Harvard Medical School, Boston, Massachusetts, USA
| | - Eric A Pierce
- Ocular Genomics Institute, Department of Ophthalmology, Massachusetts Eye and Ear, Harvard Medical School, Boston, Massachusetts, USA
| | - Rachel M Huckfeldt
- Ocular Genomics Institute, Department of Ophthalmology, Massachusetts Eye and Ear, Harvard Medical School, Boston, Massachusetts, USA
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21
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Long Y, Cech TR. Targeted mutagenesis in human iPSCs using CRISPR genome-editing tools. Methods 2021; 191:44-58. [PMID: 33444739 DOI: 10.1016/j.ymeth.2021.01.002] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/26/2020] [Revised: 11/27/2020] [Accepted: 01/02/2021] [Indexed: 12/27/2022] Open
Abstract
Mutagenesis studies have rapidly evolved in the era of CRISPR genome editing. Precise manipulation of genes in human induced pluripotent stem cells (iPSCs) allows biomedical researchers to study the physiological functions of individual genes during development. Furthermore, such genetic manipulation applied to patient-specific iPSCs allows disease modeling, drug screening and development of therapeutics. Although various genome-editing methods have been developed to introduce or remove mutations in human iPSCs, comprehensive strategic designs taking account of the potential side effects of CRISPR editing are needed. Here we present several novel and highly efficient strategies to introduce point mutations, insertions and deletions in human iPSCs, including step-by-step experimental protocols. These approaches involve the application of drug selection for effortless clone screening and the generation of a wild type control strain along with the mutant. We also present several examples of application of these strategies in human iPSCs and show that they are highly efficient and could be applied to other cell types.
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Affiliation(s)
- Yicheng Long
- Department of Biochemistry and BioFrontiers Institute, University of Colorado, Boulder, CO 80309, United States
| | - Thomas R Cech
- Department of Biochemistry and BioFrontiers Institute, University of Colorado, Boulder, CO 80309, United States; Howard Hughes Medical Institute, University of Colorado, Boulder, CO 80309, United States.
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22
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Azangou-Khyavy M, Ghasemi M, Khanali J, Boroomand-Saboor M, Jamalkhah M, Soleimani M, Kiani J. CRISPR/Cas: From Tumor Gene Editing to T Cell-Based Immunotherapy of Cancer. Front Immunol 2020; 11:2062. [PMID: 33117331 PMCID: PMC7553049 DOI: 10.3389/fimmu.2020.02062] [Citation(s) in RCA: 46] [Impact Index Per Article: 9.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/05/2020] [Accepted: 07/29/2020] [Indexed: 12/26/2022] Open
Abstract
The clustered regularly interspaced short palindromic repeats system has demonstrated considerable advantages over other nuclease-based genome editing tools due to its high accuracy, efficiency, and strong specificity. Given that cancer is caused by an excessive accumulation of mutations that lead to the activation of oncogenes and inactivation of tumor suppressor genes, the CRISPR/Cas9 system is a therapy of choice for tumor genome editing and treatment. In defining its superior use, we have reviewed the novel applications of the CRISPR genome editing tool in discovering, sorting, and prioritizing targets for subsequent interventions, and passing different hurdles of cancer treatment such as epigenetic alterations and drug resistance. Moreover, we have reviewed the breakthroughs precipitated by the CRISPR system in the field of cancer immunotherapy, such as identification of immune system-tumor interplay, production of universal Chimeric Antigen Receptor T cells, inhibition of immune checkpoint inhibitors, and Oncolytic Virotherapy. The existing challenges and limitations, as well as the prospects of CRISPR based systems, are also discussed.
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Affiliation(s)
| | - Mobina Ghasemi
- Student Research Committee, Shahid Beheshti University of Medical Sciences, Tehran, Iran
| | - Javad Khanali
- Student Research Committee, Shahid Beheshti University of Medical Sciences, Tehran, Iran
| | | | - Monire Jamalkhah
- Department of Biotechnology, College of Science, University of Tehran, Tehran, Iran
| | - Masoud Soleimani
- Hematology Department, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
| | - Jafar Kiani
- Oncopathology Research Center, Iran University of Medical Sciences, Tehran, Iran
- Department of Molecular Medicine, Faculty of Advanced Technologies in Medicine, Iran University of Medical Sciences, Tehran, Iran
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23
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Sensing through Non-Sensing Ocular Ion Channels. Int J Mol Sci 2020; 21:ijms21186925. [PMID: 32967234 PMCID: PMC7554890 DOI: 10.3390/ijms21186925] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/03/2020] [Revised: 09/17/2020] [Accepted: 09/18/2020] [Indexed: 12/12/2022] Open
Abstract
Ion channels are membrane-spanning integral proteins expressed in multiple organs, including the eye. In the eye, ion channels are involved in various physiological processes, like signal transmission and visual processing. A wide range of mutations have been reported in the corresponding genes and their interacting subunit coding genes, which contribute significantly to an array of blindness, termed ocular channelopathies. These mutations result in either a loss- or gain-of channel functions affecting the structure, assembly, trafficking, and localization of channel proteins. A dominant-negative effect is caused in a few channels formed by the assembly of several subunits that exist as homo- or heteromeric proteins. Here, we review the role of different mutations in switching a “sensing” ion channel to “non-sensing,” leading to ocular channelopathies like Leber’s congenital amaurosis 16 (LCA16), cone dystrophy, congenital stationary night blindness (CSNB), achromatopsia, bestrophinopathies, retinitis pigmentosa, etc. We also discuss the various in vitro and in vivo disease models available to investigate the impact of mutations on channel properties, to dissect the disease mechanism, and understand the pathophysiology. Innovating the potential pharmacological and therapeutic approaches and their efficient delivery to the eye for reversing a “non-sensing” channel to “sensing” would be life-changing.
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24
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Bacman SR, Gammage PA, Minczuk M, Moraes CT. Manipulation of mitochondrial genes and mtDNA heteroplasmy. Methods Cell Biol 2020; 155:441-487. [PMID: 32183972 DOI: 10.1016/bs.mcb.2019.12.004] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Most patients with mitochondrial DNA (mtDNA) mutations have a mixture of mutant and wild-type mtDNA in their cells. This phenomenon, known as mtDNA heteroplasmy, provides an opportunity to develop therapies by selectively eliminating the mutant fraction. In the last decade, several enzyme-based gene editing platforms were developed to cleave specific DNA sequences. We have taken advantage of these enzymes to develop reagents to selectively eliminate mutant mtDNA. The replication of intact mitochondrial genomes normalizes mtDNA levels and consequently mitochondrial function. In this chapter, we describe the methodology used to design and express these nucleases in mammalian cells in culture and in vivo.
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Affiliation(s)
- Sandra R Bacman
- Department of Neurology, University of Miami School of Medicine, Miami, FL, United States
| | - P A Gammage
- CRUK Beatson Institute for Cancer Research, Glasgow, United Kingdom
| | - M Minczuk
- MRC Mitochondrial Biology Unit, University of Cambridge, Cambridge, United Kingdom.
| | - Carlos T Moraes
- Department of Neurology, University of Miami School of Medicine, Miami, FL, United States.
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25
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Drumm ML. Gene Editing for CF. Respir Med 2020. [DOI: 10.1007/978-3-030-42382-7_25] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
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26
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Humanized Mouse Model of HIV-1 Latency with Enrichment of Latent Virus in PD-1 + and TIGIT + CD4 T Cells. J Virol 2019; 93:JVI.02086-18. [PMID: 30842333 DOI: 10.1128/jvi.02086-18] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/21/2018] [Accepted: 03/01/2019] [Indexed: 12/18/2022] Open
Abstract
Combination anti-retroviral drug therapy (ART) potently suppresses HIV-1 replication but does not result in virus eradication or a cure. A major contributing factor is the long-term persistence of a reservoir of latently infected cells. To study this reservoir, we established a humanized mouse model of HIV-1 infection and ART suppression based on an oral ART regimen. Similar to humans, HIV-1 levels in the blood of ART-treated animals were frequently suppressed below the limits of detection. However, the limited timeframe of the mouse model and the small volume of available samples makes it a challenging model with which to achieve full viral suppression and to investigate the latent reservoir. We therefore used an ex vivo latency reactivation assay that allows a semiquantitative measure of the latent reservoir that establishes in individual animals, regardless of whether they are treated with ART. Using this assay, we found that latently infected human CD4 T cells can be readily detected in mouse lymphoid tissues and that latent HIV-1 was enriched in populations expressing markers of T cell exhaustion, PD-1 and TIGIT. In addition, we were able to use the ex vivo latency reactivation assay to demonstrate that HIV-specific TALENs can reduce the fraction of reactivatable virus in the latently infected cell population that establishes in vivo, supporting the use of targeted nuclease-based approaches for an HIV-1 cure.IMPORTANCE HIV-1 can establish latent infections that are not cleared by current antiretroviral drugs or the body's immune responses and therefore represent a major barrier to curing HIV-infected individuals. However, the lack of expression of viral antigens on latently infected cells makes them difficult to identify or study. Here, we describe a humanized mouse model that can be used to detect latent but reactivatable HIV-1 in both untreated mice and those on ART and therefore provides a simple system with which to study the latent HIV-1 reservoir and the impact of interventions aimed at reducing it.
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Targeted deletion of BCL11A gene by CRISPR-Cas9 system for fetal hemoglobin reactivation: A promising approach for gene therapy of beta thalassemia disease. Eur J Pharmacol 2019; 854:398-405. [PMID: 31039344 DOI: 10.1016/j.ejphar.2019.04.042] [Citation(s) in RCA: 49] [Impact Index Per Article: 8.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/12/2019] [Revised: 04/18/2019] [Accepted: 04/26/2019] [Indexed: 11/24/2022]
Abstract
Hemoglobinopathies, such as β-thalassemia, and sickle cell disease (SCD) are caused by abnormal structure or reduced production of β-chains and affect millions of people worldwide. Hereditary persistence of fetal hemoglobin (HPFH) is a condition which is naturally occurring and characterized by a considerable elevation of fetal hemoglobin (HbF) in adult red blood cells. Individuals with compound heterozygous β-thalassemia or SCD and HPFH have milder clinical symptoms. So, HbF reactivation has long been sought as an approach to mitigate the clinical symptoms of β-thalassemia and SCD. Using CRISPR-Cas9 genome-editing strategy, we deleted a 200bp genomic region within the human erythroid-specific BCL11A (B-cell lymphoma/leukemia 11A) enhancer in KU-812, KG-1, and K562 cell lines. In our study, deletion of 200bp of BCL11A erythroid enhancer including GATAA motif leads to strong induction of γ-hemoglobin expression in K562 cells, but not in KU-812 and KG-1 cells. Altogether, our findings highlight the therapeutic potential of CRISPR-Cas9 as a precision genome editing tool for treating β-thalassemia. In addition, our data indicate that KU-812 and KG-1 cell lines are not good models for studying HbF reactivation through inactivation of BCL11A silencing pathway.
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Logan S, Arzua T, Canfield SG, Seminary ER, Sison SL, Ebert AD, Bai X. Studying Human Neurological Disorders Using Induced Pluripotent Stem Cells: From 2D Monolayer to 3D Organoid and Blood Brain Barrier Models. Compr Physiol 2019; 9:565-611. [PMID: 30873582 PMCID: PMC6705133 DOI: 10.1002/cphy.c180025] [Citation(s) in RCA: 88] [Impact Index Per Article: 14.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
Neurological disorders have emerged as a predominant healthcare concern in recent years due to their severe consequences on quality of life and prevalence throughout the world. Understanding the underlying mechanisms of these diseases and the interactions between different brain cell types is essential for the development of new therapeutics. Induced pluripotent stem cells (iPSCs) are invaluable tools for neurological disease modeling, as they have unlimited self-renewal and differentiation capacity. Mounting evidence shows: (i) various brain cells can be generated from iPSCs in two-dimensional (2D) monolayer cultures; and (ii) further advances in 3D culture systems have led to the differentiation of iPSCs into organoids with multiple brain cell types and specific brain regions. These 3D organoids have gained widespread attention as in vitro tools to recapitulate complex features of the brain, and (iii) complex interactions between iPSC-derived brain cell types can recapitulate physiological and pathological conditions of blood-brain barrier (BBB). As iPSCs can be generated from diverse patient populations, researchers have effectively applied 2D, 3D, and BBB models to recapitulate genetically complex neurological disorders and reveal novel insights into molecular and genetic mechanisms of neurological disorders. In this review, we describe recent progress in the generation of 2D, 3D, and BBB models from iPSCs and further discuss their limitations, advantages, and future ventures. This review also covers the current status of applications of 2D, 3D, and BBB models in drug screening, precision medicine, and modeling a wide range of neurological diseases (e.g., neurodegenerative diseases, neurodevelopmental disorders, brain injury, and neuropsychiatric disorders). © 2019 American Physiological Society. Compr Physiol 9:565-611, 2019.
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Affiliation(s)
- Sarah Logan
- Department of Cell Biology, Neurobiology & Anatomy, Medical College of Wisconsin, Milwaukee, WI, USA
- Department of Physiology, Medical College of Wisconsin, Milwaukee, WI, USA
| | - Thiago Arzua
- Department of Cell Biology, Neurobiology & Anatomy, Medical College of Wisconsin, Milwaukee, WI, USA
- Department of Physiology, Medical College of Wisconsin, Milwaukee, WI, USA
| | - Scott G. Canfield
- Department of Cellular & Integrative Physiology, IU School of Medicine-Terre Haute, Terre Haute, IN, USA
| | - Emily R. Seminary
- Department of Cell Biology, Neurobiology & Anatomy, Medical College of Wisconsin, Milwaukee, WI, USA
| | - Samantha L. Sison
- Department of Cell Biology, Neurobiology & Anatomy, Medical College of Wisconsin, Milwaukee, WI, USA
| | - Allison D. Ebert
- Department of Cell Biology, Neurobiology & Anatomy, Medical College of Wisconsin, Milwaukee, WI, USA
| | - Xiaowen Bai
- Department of Cell Biology, Neurobiology & Anatomy, Medical College of Wisconsin, Milwaukee, WI, USA
- Department of Physiology, Medical College of Wisconsin, Milwaukee, WI, USA
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Czerwińska P, Mazurek S, Kołodziejczak I, Wiznerowicz M. Gene delivery methods and genome editing of human pluripotent stem cells. Rep Pract Oncol Radiother 2019; 24:180-187. [PMID: 30820192 DOI: 10.1016/j.rpor.2019.01.007] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/15/2018] [Revised: 11/21/2018] [Accepted: 01/27/2019] [Indexed: 12/24/2022] Open
Abstract
Induced pluripotent stem cells derived from normal somatic cells could be utilized to study tumorigenesis through overexpression of specific oncogenes, downregulation of tumor suppressors and dysregulation of other factors thought to promote tumorigenesis. Therefore, effective approaches that provide direct modifications of induced pluripotent stem cell genome are extremely needed. Emerging strategies are expected to provide the ability to more effectively introduce diverse genetic alterations, from as small as single-nucleotide modifications to whole gene amplification or deletion, all with a high degree of target specificity. To date, several techniques have been applied in stem cell studies to directly edit cell genome (ZFNs, TALENs or CRISPR/Cas9). In this review, we summarize specific gene delivery strategies that were applied to stem cell studies together with genome editing techniques, which enable a direct modification of endogenous DNA sequences in the context of cancer studies.
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Affiliation(s)
- Patrycja Czerwińska
- Laboratory of Gene Therapy, Department of Diagnostics and Cancer Immunology, Greater Poland Cancer Centre, Poznan, Poland.,Department of Cancer Immunology, Chair of Medical Biotechnology, Poznan University of Medical Sciences, Poznan, Poland
| | - Sylwia Mazurek
- Laboratory of Gene Therapy, Department of Diagnostics and Cancer Immunology, Greater Poland Cancer Centre, Poznan, Poland.,Department of Cancer Immunology, Chair of Medical Biotechnology, Poznan University of Medical Sciences, Poznan, Poland.,Postgraduate School of Molecular Medicine, Medical University of Warsaw, Warsaw, Poland
| | - Iga Kołodziejczak
- Department of Cancer Immunology, Chair of Medical Biotechnology, Poznan University of Medical Sciences, Poznan, Poland
| | - Maciej Wiznerowicz
- Laboratory of Gene Therapy, Department of Diagnostics and Cancer Immunology, Greater Poland Cancer Centre, Poznan, Poland.,Department of Cancer Immunology, Chair of Medical Biotechnology, Poznan University of Medical Sciences, Poznan, Poland
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30
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Şişli HB, Hayal TB, Seçkin S, Şenkal S, Kıratlı B, Şahin F, Doğan A. Gene Editing in Human Pluripotent Stem Cells: Recent Advances for Clinical Therapies. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2019; 1237:17-28. [PMID: 31728915 DOI: 10.1007/5584_2019_439] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
Abstract
The identification of human embryonic stem cells and reprogramming technology to obtain induced pluripotent stem cells from adult somatic cells have provided unique opportunity to create human disease models, gene editing strategies and cell therapy options.Development of pluripotent stem cells from somatic cells and genomic manipulation tools enabled to use site specific nucleases in the cell therapy research. Identification of efficient gene manipulation, safe differentiation and use will provide a novel strategy to treat many diseases in the near future. Current available registered clinical trials clearly indicate the need for pluripotent stem cell and gene therapy treatment options. Although gene editing based pluripotent stem cell research is a popular field for research worldwide, improvement of clinical approaches for treatment still remains to be investigated. In this review, we summarized the current situation of gene editing based pluripotent cell therapy developments and applications in clinics.
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Affiliation(s)
- Hatice Burcu Şişli
- Department of Genetics and Bioengineering, Faculty of Engineering, Yeditepe University, Istanbul, Turkey
| | - Taha Bartu Hayal
- Department of Genetics and Bioengineering, Faculty of Engineering, Yeditepe University, Istanbul, Turkey
| | - Selin Seçkin
- Department of Genetics and Bioengineering, Faculty of Engineering, Yeditepe University, Istanbul, Turkey
| | - Selinay Şenkal
- Department of Genetics and Bioengineering, Faculty of Engineering, Yeditepe University, Istanbul, Turkey
| | - Binnur Kıratlı
- Department of Genetics and Bioengineering, Faculty of Engineering, Yeditepe University, Istanbul, Turkey
| | - Fikrettin Şahin
- Department of Genetics and Bioengineering, Faculty of Engineering, Yeditepe University, Istanbul, Turkey
| | - Ayşegül Doğan
- Department of Genetics and Bioengineering, Faculty of Engineering, Yeditepe University, Istanbul, Turkey.
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31
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Jiang Z, Cui W, Prasad P, Touve MA, Gianneschi NC, Mager J, Thayumanavan S. Bait-and-Switch Supramolecular Strategy To Generate Noncationic RNA-Polymer Complexes for RNA Delivery. Biomacromolecules 2018; 20:435-442. [PMID: 30525500 DOI: 10.1021/acs.biomac.8b01321] [Citation(s) in RCA: 26] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Abstract
RNA interference (RNAi) requires the intracellular delivery of RNA molecules to initiate the neutralization of targeted mRNA molecules, inhibiting the expression or translation of the targeted gene. Current polymers and lipids that are used to deliver RNA molecules are generally required to be positively charged, to achieve complexation with RNA and the cellular internalization. However, positive surface charge has been implicated as the reason for toxicity in many of these systems. Herein, we report a novel strategy to generate noncationic RNA-polymer complexes for RNA delivery with low cytotoxicity. We use an in situ electrostatic complexation using a methylated pyridinium group, which is simultaneously removed during the RNA binding step. The resultant complexes demonstrate successful knockdown in preimplantation mammalian embryos, thus providing a new approach for nucleic acid delivery.
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Affiliation(s)
| | | | | | - Mollie A Touve
- Department of Chemistry , Northwestern University , Evanston , Illinois 60208 , United States.,Department of Chemistry and Biochemistry , University of California San Diego , La Jolla , California 92093 , United States
| | - Nathan C Gianneschi
- Department of Chemistry , Northwestern University , Evanston , Illinois 60208 , United States.,Department of Chemistry and Biochemistry , University of California San Diego , La Jolla , California 92093 , United States
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32
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Kou J, Kuang YQ. Mutations in chemokine receptors and AIDS. PROGRESS IN MOLECULAR BIOLOGY AND TRANSLATIONAL SCIENCE 2018; 161:113-124. [PMID: 30711024 DOI: 10.1016/bs.pmbts.2018.10.001] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
Chemokines are a class of chemotactic small molecule peptides whose receptors CCR5 and CXCR4 play important role in the entry of human immunodeficiency virus (HIV-1) into immune cells. Chemokines belong to G protein-coupled receptor superfamily containing seven hydrophobic transmembrane helices, causing physiological effects such as chemotaxis, immune regulation, antiviral immunity, regulation of hematopoiesis and angiogenesis, as well as cell growth and metabolism, through certain signaling pathways. Earlier studies have shown that HIV infects the human immune cells by binding to the CD4 receptor. Soon, it was discovered that HIV-1 enters into human immune cells by binding to another receptor, chemokine receptor, which acts as co-receptor for CD4 during the invasion of HIV-1 into cells. Since complex receptor binding is important for HIV-1 invasion, antagonizing the binding has become an attractive and rational drug design goal. Early studies sought to block the interaction between virus and the receptors by chemically modifying the CCR5 and CXCR4 ligands. Although drug treatment is widely used, drug treatment cannot cure AIDS; it can only inhibit the replication of the virus, and HIV/AIDS patients need to take drugs for life. In addition, anti-AIDS drugs also produce side effects such as diseases of the cardiovascular system, nervous system, and metabolic system. In 2006, the emergence of "Berlin patient" led researchers to focus on gene therapy in chemokine receptors. In 2006 and 2007, the attending physician of "Berlin patient" cured his AIDS by transplantation of the stem cells from a donor who was homozygous for the CCR5 Δ32 mutation. This review summarizes the research progress in the mutation in chemokine receptor of HIV/AIDS.
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Affiliation(s)
- Jing Kou
- International Education College, Henan University, Kaifeng, China
| | - Yi-Qun Kuang
- Center for Translational Medicine, Huaihe Clinical College, Huaihe Hospital of Henan University, Kaifeng, China.
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33
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McElvaney OJ, Gunaratnam C, McElvaney OF, Bagwe I, Reeves EP, McElvaney NG. Emerging pharmacotherapies in cystic fibrosis. Expert Rev Respir Med 2018; 12:843-855. [DOI: 10.1080/17476348.2018.1512409] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/22/2023]
Affiliation(s)
- Oliver J McElvaney
- Irish Centre for Genetic Lung Disease, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin, Ireland
| | - Cedric Gunaratnam
- Irish Centre for Genetic Lung Disease, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin, Ireland
| | - Oisin Fiachra McElvaney
- Irish Centre for Genetic Lung Disease, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin, Ireland
| | - Isha Bagwe
- Irish Centre for Genetic Lung Disease, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin, Ireland
| | - Emer P Reeves
- Irish Centre for Genetic Lung Disease, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin, Ireland
| | - Noel G McElvaney
- Irish Centre for Genetic Lung Disease, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin, Ireland
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34
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Lu GA, Zhao Y, Yang H, Lan A, Shi S, Liufu Z, Huang Y, Tang T, Xu J, Shen X, Wu CI. Death of new microRNA genes in Drosophila via gradual loss of fitness advantages. Genome Res 2018; 28:1309-1318. [PMID: 30049791 PMCID: PMC6120634 DOI: 10.1101/gr.233809.117] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2017] [Accepted: 07/20/2018] [Indexed: 01/23/2023]
Abstract
The prevalence of de novo coding genes is controversial due to length and coding constraints. Noncoding genes, especially small ones, are freer to evolve de novo by comparison. The best examples are microRNAs (miRNAs), a large class of regulatory molecules ∼22 nt in length. Here, we study six de novo miRNAs in Drosophila, which, like most new genes, are testis-specific. We ask how and why de novo genes die because gene death must be sufficiently frequent to balance the many new births. By knocking out each miRNA gene, we analyzed their contributions to the nine components of male fitness (sperm production, length, and competitiveness, among others). To our surprise, the knockout mutants often perform better than the wild type in some components, and slightly worse in others. When two of the younger miRNAs are assayed in long-term laboratory populations, their total fitness contributions are found to be essentially zero. These results collectively suggest that adaptive de novo genes die regularly, not due to the loss of functionality, but due to the canceling out of positive and negative fitness effects, which may be characterized as "quasi-neutrality." Since de novo genes often emerge adaptively and become lost later, they reveal ongoing period-specific adaptations, reminiscent of the "Red-Queen" metaphor for long-term evolution.
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Affiliation(s)
- Guang-An Lu
- State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, Guangdong, China
| | - Yixin Zhao
- State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, Guangdong, China
| | - Hao Yang
- State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, Guangdong, China
| | - Ao Lan
- State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, Guangdong, China
| | - Suhua Shi
- State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, Guangdong, China
| | - Zhongqi Liufu
- State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, Guangdong, China
| | - Yumei Huang
- State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, Guangdong, China
| | - Tian Tang
- State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, Guangdong, China
| | - Jin Xu
- State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, Guangdong, China
- Center for Personal Dynamic Regulomes, Stanford University, Stanford, California 94305, USA
| | - Xu Shen
- State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, Guangdong, China
| | - Chung-I Wu
- State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, Guangdong, China
- Department of Ecology and Evolution, University of Chicago, Chicago, Illinois 60637, USA
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35
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Mesuraca M, Amodio N, Chiarella E, Scicchitano S, Aloisio A, Codispoti B, Lucchino V, Montalcini Y, Bond HM, Morrone G. Turning Stem Cells Bad: Generation of Clinically Relevant Models of Human Acute Myeloid Leukemia through Gene Delivery- or Genome Editing-Based Approaches. Molecules 2018; 23:E2060. [PMID: 30126100 PMCID: PMC6222541 DOI: 10.3390/molecules23082060] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/16/2018] [Revised: 08/09/2018] [Accepted: 08/14/2018] [Indexed: 02/07/2023] Open
Abstract
Acute myeloid leukemia (AML), the most common acute leukemia in the adult, is believed to arise as a consequence of multiple molecular events that confer on primitive hematopoietic progenitors unlimited self-renewal potential and cause defective differentiation. A number of genetic aberrations, among which a variety of gene fusions, have been implicated in the development of a transformed phenotype through the generation of dysfunctional molecules that disrupt key regulatory mechanisms controlling survival, proliferation, and differentiation in normal stem and progenitor cells. Such genetic aberrations can be recreated experimentally to a large extent, to render normal hematopoietic stem cells "bad", analogous to the leukemic stem cells. Here, we wish to provide a brief outline of the complementary experimental approaches, largely based on gene delivery and more recently on gene editing, employed over the last two decades to gain insights into the molecular mechanisms underlying AML development and progression and on the prospects that their applications offer for the discovery and validation of innovative therapies.
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Affiliation(s)
- Maria Mesuraca
- Laboratory of Molecular Haematopoiesis and Stem Cell Biology, Department of Experimental and Clinical Medicine, University Magna Græcia, 88100 Catanzaro, Italy.
| | - Nicola Amodio
- Laboratory of Medical Oncology, Department of Experimental and Clinical Medicine, University Magna Græcia, 88100 Catanzaro, Italy.
| | - Emanuela Chiarella
- Laboratory of Molecular Haematopoiesis and Stem Cell Biology, Department of Experimental and Clinical Medicine, University Magna Græcia, 88100 Catanzaro, Italy.
| | - Stefania Scicchitano
- Laboratory of Molecular Haematopoiesis and Stem Cell Biology, Department of Experimental and Clinical Medicine, University Magna Græcia, 88100 Catanzaro, Italy.
| | - Annamaria Aloisio
- Laboratory of Molecular Haematopoiesis and Stem Cell Biology, Department of Experimental and Clinical Medicine, University Magna Græcia, 88100 Catanzaro, Italy.
| | - Bruna Codispoti
- Tecnologica Research Institute-Marrelli Hospital, 88900 Crotone, Italy.
| | - Valeria Lucchino
- Laboratory of Molecular Haematopoiesis and Stem Cell Biology, Department of Experimental and Clinical Medicine, University Magna Græcia, 88100 Catanzaro, Italy.
- German Center for Neurodegenerative Diseases (DZNE), 53127 Bonn, Germany.
| | - Ylenia Montalcini
- Laboratory of Molecular Haematopoiesis and Stem Cell Biology, Department of Experimental and Clinical Medicine, University Magna Græcia, 88100 Catanzaro, Italy.
| | - Heather M Bond
- Laboratory of Molecular Haematopoiesis and Stem Cell Biology, Department of Experimental and Clinical Medicine, University Magna Græcia, 88100 Catanzaro, Italy.
| | - Giovanni Morrone
- Laboratory of Molecular Haematopoiesis and Stem Cell Biology, Department of Experimental and Clinical Medicine, University Magna Græcia, 88100 Catanzaro, Italy.
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Shrestha A, Khan A, Dey N. cis-trans Engineering: Advances and Perspectives on Customized Transcriptional Regulation in Plants. MOLECULAR PLANT 2018; 11:886-898. [PMID: 29859265 DOI: 10.1016/j.molp.2018.05.008] [Citation(s) in RCA: 32] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/26/2018] [Revised: 05/23/2018] [Accepted: 05/23/2018] [Indexed: 05/03/2023]
Abstract
Coordinated transcriptional control employing synthetic promoters and transcription factors (TFs) can be used to achieve customized regulation of gene expression in planta. Synthetic promoter technology has yielded a series of promoters with modified cis-regulatory elements that provide useful tools for efficient modulation of gene expression. In addition, the use of zinc fingers (ZFs), transcription activator-like effectors (TALEs), and catalytically inactive clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (dCas9) has made it feasible to engineer TFs that can produce targeted gene expression regulation; these approaches are particularly effective when artificial TFs are coupled with transcriptional activators or repressors. This review focuses on strategies used to engineer both promoters and TFs in the context of targeted transcriptional regulation. We also discuss the creation of synthetic inducible platforms, which can be used to impart stress tolerance to plants. We propose that combinatorial "cis-trans engineering" using a CRISPR-dCas9-based bipartite module could be used to regulate the expression of multiple target genes. This approach provides an attractive tool for introduction of specific qualitative traits into plants, thus enhancing their overall environmental adaptability.
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Affiliation(s)
- Ankita Shrestha
- Division of Microbial and Plant Biotechnology, Institute of Life Sciences, Department of Biotechnology, Government of India, Chandrasekharpur, Bhubaneswar, Odisha, India
| | - Ahamed Khan
- Division of Microbial and Plant Biotechnology, Institute of Life Sciences, Department of Biotechnology, Government of India, Chandrasekharpur, Bhubaneswar, Odisha, India
| | - Nrisingha Dey
- Division of Microbial and Plant Biotechnology, Institute of Life Sciences, Department of Biotechnology, Government of India, Chandrasekharpur, Bhubaneswar, Odisha, India.
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Laux H, Romand S, Nuciforo S, Farady CJ, Tapparel J, Buechmann‐Moeller S, Sommer B, Oakeley EJ, Bodendorf U. Degradation of recombinant proteins by Chinese hamster ovary host cell proteases is prevented by matriptase‐1 knockout. Biotechnol Bioeng 2018; 115:2530-2540. [DOI: 10.1002/bit.26731] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2018] [Revised: 04/23/2018] [Accepted: 05/17/2018] [Indexed: 12/18/2022]
Affiliation(s)
- Holger Laux
- Integrated Biologics Profiling UnitNovartis Pharma AGBasel Switzerland
| | - Sandrine Romand
- Integrated Biologics Profiling UnitNovartis Pharma AGBasel Switzerland
| | - Sandro Nuciforo
- Integrated Biologics Profiling UnitNovartis Pharma AGBasel Switzerland
- Department of BiomedicineUniversity Hospital Basel, University of BaselBasel Switzerland
| | - Christopher J. Farady
- Autoimmunity, Transplantation & Inflammatory DiseaseNovartis Institutes for Biomedical ResearchBasel Switzerland
| | - Joel Tapparel
- Early Phase DevelopmentNovartis Pharma AGBasel Switzerland
| | - Stine Buechmann‐Moeller
- Autoimmunity, Transplantation & Inflammatory DiseaseNovartis Institutes for Biomedical ResearchBasel Switzerland
| | | | - Edward J. Oakeley
- Autoimmunity, Transplantation & Inflammatory DiseaseNovartis Institutes for Biomedical ResearchBasel Switzerland
| | - Ursula Bodendorf
- Autoimmunity, Transplantation & Inflammatory DiseaseNovartis Institutes for Biomedical ResearchBasel Switzerland
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38
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Chandrasekaran AP, Song M, Kim KS, Ramakrishna S. Different Methods of Delivering CRISPR/Cas9 Into Cells. PROGRESS IN MOLECULAR BIOLOGY AND TRANSLATIONAL SCIENCE 2018; 159:157-176. [PMID: 30340786 DOI: 10.1016/bs.pmbts.2018.05.001] [Citation(s) in RCA: 40] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system (Cas) is comprised of repetitive bases followed by short fragments of DNA from a previously invading organism that provide immunity to the most prokaryotic organisms. An RNA-dependent spacer is required for CRISPR/Cas9 to recognize the target DNA. Delivery of the CRISPR/Cas9-guide RNA (gRNA) complex to any cell results in modification of the target sequence. The CRISPR/Cas9-mediated genome editing technique is currently in the spotlight and has several research interests, including molecular medicine and agriculture. There are several factors that hinder the delivery of this complex, such as the large size of the plasmid or high dosage of the chemical agent. There are several methods available to deliver CRISPR/Cas9 and its components to the target cells. It includes viral, non-viral and physical methods to deliver plasmid or ribonucleoprotein (RNP) of CRISPR components. But in vivo CRISPR/Cas9 delivery remains challenging to the researchers due to insertional mutagenesis, targeted delivery, immunogenicity, and off-targets. However, studies suggesting that the CRISPR/Cas9-RNP delivery can overcome these hurdles. Here, we review the various methods for delivery of CRISPR/Cas9 and gRNA to several cell lines, highlighting the limitations of each approach, and suggest possible alternative methods.
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Affiliation(s)
| | - Minjung Song
- Department of Food Biotechnology, College of Medical and Life Science, Silla University, Seoul, South Korea
| | - Kye-Seong Kim
- Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul, South Korea; College of Medicine, Hanyang University, Seoul, South Korea.
| | - Suresh Ramakrishna
- Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul, South Korea; College of Medicine, Hanyang University, Seoul, South Korea.
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39
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Lu GA, Zhao Y, Liufu Z, Wu CI. On the possibility of death of new genes - evidence from the deletion of de novo microRNAs. BMC Genomics 2018; 19:388. [PMID: 29792159 PMCID: PMC5966946 DOI: 10.1186/s12864-018-4755-1] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/28/2017] [Accepted: 05/02/2018] [Indexed: 01/21/2023] Open
Abstract
Background New genes are constantly formed, sometimes from non-genic sequences, creating what is referred to as de novo genes. Since the total number of genes remains relatively steady, gene deaths likely balance out new births. In metazoan genomes, microRNAs (miRs) genes, small and non-coding, account for the bulk of functional de novo genes and are particularly suited to the investigation of gene death. Results In this study, we discover a Drosophila-specific de novo miRNA (mir-977) that may be facing impending death. Strikingly, after this testis-specific gene is deleted from D. melanogaster, most components of male fitness increase, rather than decrease as had been expected. These components include male viability, fertility and males’ ability to repress female re-mating. Given that mir-977 has a negative fitness effect in D. melanogaster, this de novo gene with an adaptive history for over 60 Myrs may be facing elimination. In some other species where mir-977 is not found, gene death may have already happened. Conclusion The surprising result suggests that de novo genes, constantly rising and falling during evolution, may often be transiently adaptive and then purged from the genome. Electronic supplementary material The online version of this article (10.1186/s12864-018-4755-1) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Guang-An Lu
- State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275, Guangdong, China
| | - Yixin Zhao
- State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275, Guangdong, China
| | - Zhongqi Liufu
- State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275, Guangdong, China
| | - Chung-I Wu
- State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275, Guangdong, China. .,Department of Ecology and Evolution, University of Chicago, Chicago, Illinois, 60637, USA.
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40
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Affiliation(s)
- Ianis G Matsoukas
- Faculty of Health and Wellbeing, School of Sport and Biomedical Sciences, University of Bolton, Bolton, United Kingdom
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41
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Hu M, Guo G, Huang Q, Cheng C, Xu R, Li A, Liu N, Liu S. The harsh microenvironment in infarcted heart accelerates transplanted bone marrow mesenchymal stem cells injury: the role of injured cardiomyocytes-derived exosomes. Cell Death Dis 2018; 9:357. [PMID: 29500342 PMCID: PMC5834521 DOI: 10.1038/s41419-018-0392-5] [Citation(s) in RCA: 69] [Impact Index Per Article: 9.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/08/2017] [Revised: 02/08/2018] [Accepted: 02/09/2018] [Indexed: 12/11/2022]
Abstract
Stem cell therapy can be used to repair and regenerate damaged hearts tissue; nevertheless, the low survival rate of transplanted cells limits their therapeutic efficacy. Recently, it has been proposed that exosomes regulate multiple cellular processes by mediating cell survival and communication among cells. The following study investigates whether injured cardiomyocytes-derived exosomes (cardiac exosomes) affect the survival of transplanted bone marrow mesenchymal stem cells (BMSCs) in infarcted heart. To mimic the harsh microenvironment in infarcted heart that the cardiomyocytes or transplanted BMSCs encounter in vivo, cardiomyocytes conditioned medium and cardiac exosomes collected from H2O2-treated cardiomyocytes culture medium were cultured with BMSCs under oxidative stress in vitro. Cardiomyocytes conditioned medium and cardiac exosomes significantly accelerated the injury of BMSCs induced by H2O2; increased cleaved caspase-3/caspase-3 and apoptotic percentage, and decreased the ratio of Bcl-2/Bax and cell viability in those cells. Next, we explored the role of cardiac exosomes in the survival of transplanted BMSCs in vivo by constructing a Rab27a knockout (KO) mice model by a transcription activator-like effector nuclease (TALEN) genome-editing technique; Rab27a is a family of GTPases, which has critical role in secretion of exosomes. Male mouse GFP-modified BMSCs were implanted into the viable myocardium bordering the infarction in Rab27a KO and wild-type female mice. The obtained results showed that the transplanted BMSCs survival in infarcted heart was increased in Rab27a KO mice by the higher level of Y-chromosome Sry DNA, GFP mRNA, and the GFP fluorescence signal intensity. To sum up, these findings revealed that the injured cardiomyocytes-derived exosomes accelerate transplanted BMSCs injury in infarcted heart, thus highlighting a new mechanism underlying the survival of transplanted cells after myocardial infarction.
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Affiliation(s)
- Ming Hu
- Guangzhou Institute of Cardiovascular Disease, the Second Affiliated Hospital of Guangzhou Medical University, Guangzhou, 510260, Guangdong, China
| | - Guixian Guo
- Guangzhou Institute of Cardiovascular Disease, the Second Affiliated Hospital of Guangzhou Medical University, Guangzhou, 510260, Guangdong, China
| | - Qiang Huang
- Guangzhou Institute of Cardiovascular Disease, the Second Affiliated Hospital of Guangzhou Medical University, Guangzhou, 510260, Guangdong, China
| | - Chuanfang Cheng
- Guangzhou Institute of Cardiovascular Disease, the Second Affiliated Hospital of Guangzhou Medical University, Guangzhou, 510260, Guangdong, China
| | - Ruqin Xu
- Guangzhou Institute of Cardiovascular Disease, the Second Affiliated Hospital of Guangzhou Medical University, Guangzhou, 510260, Guangdong, China
| | - Aiqun Li
- Guangzhou Institute of Cardiovascular Disease, the Second Affiliated Hospital of Guangzhou Medical University, Guangzhou, 510260, Guangdong, China
| | - Ningning Liu
- Guangzhou Institute of Cardiovascular Disease, the Second Affiliated Hospital of Guangzhou Medical University, Guangzhou, 510260, Guangdong, China.
| | - Shiming Liu
- Guangzhou Institute of Cardiovascular Disease, the Second Affiliated Hospital of Guangzhou Medical University, Guangzhou, 510260, Guangdong, China.
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Lupoli F, Vannocci T, Longo G, Niccolai N, Pastore A. The role of oxidative stress in Friedreich's ataxia. FEBS Lett 2018; 592:718-727. [PMID: 29197070 PMCID: PMC5887922 DOI: 10.1002/1873-3468.12928] [Citation(s) in RCA: 73] [Impact Index Per Article: 10.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/07/2017] [Revised: 11/24/2017] [Accepted: 11/27/2017] [Indexed: 12/12/2022]
Abstract
Oxidative stress and an increase in the levels of free radicals are important markers associated with several pathologies, including Alzheimer's disease, cancer and diabetes. Friedreich's ataxia (FRDA) is an excellent paradigmatic example of a disease in which oxidative stress plays an important, albeit incompletely understood, role. FRDA is a rare genetic neurodegenerative disease that involves the partial silencing of frataxin, a small mitochondrial protein that was completely overlooked before being linked to FRDA. More than 20 years later, we now know how important this protein is in terms of being an essential and vital part of the machinery that produces iron-sulfur clusters in the cell. In this review, we revisit the most important steps that have brought us to our current understanding of the function of frataxin and its role in disease. We discuss the current hypotheses on the role of oxidative stress in FRDA and review some of the existing animal and cellular models. We also evaluate new techniques that can assist in the study of the disease mechanisms, as well as in our understanding of the interplay between primary and secondary phenotypes.
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Affiliation(s)
- Federica Lupoli
- Department of Biotechnology, Chemistry and PharmacyUniversity of SienaItaly
| | - Tommaso Vannocci
- The Maurice Wohl InstituteDementia Research CentreKing's College LondonUK
| | | | - Neri Niccolai
- Department of Biotechnology, Chemistry and PharmacyUniversity of SienaItaly
| | - Annalisa Pastore
- The Maurice Wohl InstituteDementia Research CentreKing's College LondonUK
- Department of Molecular MedicineUniversity of PaviaItaly
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43
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The current landscape for the treatment of mitochondrial disorders. J Genet Genomics 2018; 45:71-77. [DOI: 10.1016/j.jgg.2017.11.008] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/26/2017] [Revised: 10/29/2017] [Accepted: 11/18/2017] [Indexed: 12/14/2022]
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Zaboikin M, Freter C, Srinivasakumar N. Gaussian decomposition of high-resolution melt curve derivatives for measuring genome-editing efficiency. PLoS One 2018; 13:e0190192. [PMID: 29300734 PMCID: PMC5754072 DOI: 10.1371/journal.pone.0190192] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/19/2017] [Accepted: 12/08/2017] [Indexed: 11/19/2022] Open
Abstract
We describe a method for measuring genome editing efficiency from in silico analysis of high-resolution melt curve data. The melt curve data derived from amplicons of genome-edited or unmodified target sites were processed to remove the background fluorescent signal emanating from free fluorophore and then corrected for temperature-dependent quenching of fluorescence of double-stranded DNA-bound fluorophore. Corrected data were normalized and numerically differentiated to obtain the first derivatives of the melt curves. These were then mathematically modeled as a sum or superposition of minimal number of Gaussian components. Using Gaussian parameters determined by modeling of melt curve derivatives of unedited samples, we were able to model melt curve derivatives from genetically altered target sites where the mutant population could be accommodated using an additional Gaussian component. From this, the proportion contributed by the mutant component in the target region amplicon could be accurately determined. Mutant component computations compared well with the mutant frequency determination from next generation sequencing data. The results were also consistent with our earlier studies that used difference curve areas from high-resolution melt curves for determining the efficiency of genome-editing reagents. The advantage of the described method is that it does not require calibration curves to estimate proportion of mutants in amplicons of genome-edited target sites.
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Affiliation(s)
- Michail Zaboikin
- Division of Hematology-Oncology, Department of Internal Medicine, Saint Louis University, Saint Louis, Missouri, United States of America
| | - Carl Freter
- Division of Hematology-Oncology, Department of Internal Medicine, Saint Louis University, Saint Louis, Missouri, United States of America
| | - Narasimhachar Srinivasakumar
- Division of Hematology-Oncology, Department of Internal Medicine, Saint Louis University, Saint Louis, Missouri, United States of America
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45
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Williams DA. Principles of Cell-Based Genetic Therapies. Hematology 2018. [DOI: 10.1016/b978-0-323-35762-3.00098-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/18/2022] Open
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46
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White MK, Khalili K. CRISPR/Cas9 and cancer targets: future possibilities and present challenges. Oncotarget 2017; 7:12305-17. [PMID: 26840090 PMCID: PMC4914286 DOI: 10.18632/oncotarget.7104] [Citation(s) in RCA: 34] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2015] [Accepted: 01/23/2016] [Indexed: 02/07/2023] Open
Abstract
All cancers have multiple mutations that can largely be grouped into certain classes depending on the function of the gene in which they lie and these include oncogenic changes that enhance cellular proliferation, loss of function of tumor suppressors that regulate cell growth potential and induction of metabolic enzymes that confer resistance to chemotherapeutic agents. Thus the ability to correct such mutations is an important goal in cancer treatment. Recent research has led to the developments of reagents which specifically target nucleotide sequences within the cellular genome and these have a huge potential for expanding our anticancer armamentarium. One such a reagent is the clustered regulatory interspaced short palindromic repeat (CRISPR)-associated 9 (Cas9) system, a powerful, highly specific and adaptable tool that provides unparalleled control for editing the cellular genome. In this short review, we discuss the potential of CRISPR/Cas9 against human cancers and the current difficulties in translating this for novel therapeutic approaches.
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Affiliation(s)
- Martyn K White
- Department of Neuroscience, Center for Neurovirology and Comprehensive Neuroaids Center, Temple University School of Medicine, Philadelphia, PA, USA
| | - Kamel Khalili
- Department of Neuroscience, Center for Neurovirology and Comprehensive Neuroaids Center, Temple University School of Medicine, Philadelphia, PA, USA
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Shin WH, Christoffer CW, Kihara D. In silico structure-based approaches to discover protein-protein interaction-targeting drugs. Methods 2017; 131:22-32. [PMID: 28802714 PMCID: PMC5683929 DOI: 10.1016/j.ymeth.2017.08.006] [Citation(s) in RCA: 49] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/12/2017] [Revised: 08/08/2017] [Accepted: 08/08/2017] [Indexed: 02/07/2023] Open
Abstract
A core concept behind modern drug discovery is finding a small molecule that modulates a function of a target protein. This concept has been successfully applied since the mid-1970s. However, the efficiency of drug discovery is decreasing because the druggable target space in the human proteome is limited. Recently, protein-protein interaction (PPI) has been identified asan emerging target space for drug discovery. PPI plays a pivotal role in biological pathways including diseases. Current human interactome research suggests that the number of PPIs is between 130,000 and 650,000, and only a small number of them have been targeted as drug targets. For traditional drug targets, in silico structure-based methods have been successful in many cases. However, their performance suffers on PPI interfaces because PPI interfaces are different in five major aspects: From a geometric standpoint, they have relatively large interface regions, flat geometry, and the interface surface shape tends to fluctuate upon binding. Also, their interactions are dominated by hydrophobic atoms, which is different from traditional binding-pocket-targeted drugs. Finally, PPI targets usually lack natural molecules that bind to the target PPI interface. Here, we first summarize characteristics of PPI interfaces and their known binders. Then, we will review existing in silico structure-based approaches for discovering small molecules that bind to PPI interfaces.
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Affiliation(s)
- Woong-Hee Shin
- Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA
| | | | - Daisuke Kihara
- Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA; Department of Computer Science, Purdue University, West Lafayette, IN 47907, USA.
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48
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Roesch EA, Drumm ML. Powerful tools for genetic modification: Advances in gene editing. Pediatr Pulmonol 2017; 52:S15-S20. [PMID: 28960896 DOI: 10.1002/ppul.23791] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/31/2017] [Accepted: 07/31/2017] [Indexed: 12/27/2022]
Abstract
Recent discoveries and technical advances in genetic engineering, methods called gene or genome editing, provide hope for repairing genes that cause diseases like cystic fibrosis (CF) or otherwise altering a gene for therapeutic benefit. There are both hopes and hurdles with these technologies, with new ideas emerging almost daily. Initial studies using intestinal organoid cultures carrying the common, F508del mutation have shown that gene editing by CRISPR/Cas9 can convert cells lacking CFTR function to cells with normal channel function, providing a precedent that this technology can be harnessed for CF. While this is an important precedent, the challenges that remain are not trivial. A logistical issue for this and many other genetic diseases is genetic heterogeneity. Approximately, 2000 mutations associated with CF have been found in CFTR, the gene responsible for CF, and thus a feasible strategy that would encompass all individuals affected by the disease is particularly difficult to envision. However, single strategies that would be applicable to all subjects affected by CF have been conceived and are being investigated. With all of these approaches, efficiency (the proportion of cells edited), accuracy (how often other sites in the genome are affected), and delivery of the gene editing components to the desired cells are perhaps the most significant, impending hurdles. Our understanding of each of these areas is increasing rapidly, and while it is impossible to predict when a successful strategy will reach the clinic, there is every reason to believe it is a question of "when" and not "if."
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Affiliation(s)
- Erica A Roesch
- Department of Pediatrics, Case Western Reserve University, Cleveland, Ohio
| | - Mitchell L Drumm
- Department of Pediatrics, Department of Genetics and Genome Sciences, Research Institute for Children's Health, Case Western Reserve University, Cleveland, Ohio
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IMRAN A, QAMAR HY, ALI Q, NAEEM H, RIAZ M, AMIN S, KANWAL N, ALI F, SABAR * MF, NASIR IA. Role of Molecular Biology in Cancer Treatment: A Review Article. IRANIAN JOURNAL OF PUBLIC HEALTH 2017; 46:1475-1485. [PMID: 29167765 PMCID: PMC5696686 DOI: pmid/29167765] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 11/16/2016] [Accepted: 05/19/2017] [Indexed: 02/08/2023]
Abstract
BACKGROUND Cancer is a genetic disease and mainly arises due to a number of reasons include activation of onco-genes, malfunction of tumor suppressor genes or mutagenesis due to external factors. METHODS This article was written from the data collected from PubMed, Nature, Science Direct, Springer and Elsevier groups of journals. RESULTS Oncogenes are deregulated form of normal proto-oncogenes required for cell division, differentiation and regulation. The conversion of proto-oncogene to oncogene is caused due to translocation, rearrangement of chromosomes or mutation in gene due to addition, deletion, duplication or viral infection. These oncogenes are targeted by drugs or RNAi system to prevent proliferation of cancerous cells. There have been developed different techniques of molecular biology used to diagnose and treat cancer, including retroviral therapy, silencing of oncogenes and mutations in tumor suppressor genes. CONCLUSION Among all the techniques used, RNAi, zinc finger nucleases and CRISPR hold a brighter future towards creating a Cancer Free World.
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Affiliation(s)
- Aman IMRAN
- Center of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan
| | - Hafiza Yasara QAMAR
- Center of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan
| | - Qurban ALI
- Center of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan
- Institute of Molecular Biology and Biotechnology, University of Lahore, Lahore, Pakistan
| | - Hafsa NAEEM
- Center of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan
| | - Mariam RIAZ
- Center of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan
| | - Saima AMIN
- Center of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan
| | - Naila KANWAL
- Dept. of Plant Breeding and Genetics, University of Agriculture, Faisalabad, Pakistan
| | - Fawad ALI
- Dept. of Plant Breeding and Genetics, University of Agriculture, Faisalabad, Pakistan
- Southern Cross Plant Science, Southern Cross University, Lismore, Australia
| | | | - Idrees Ahmad NASIR
- Center of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan
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Affiliation(s)
| | | | - Markus Hartung
- Berufskolleg Hilden des Kreises Mettmann - Europaschule; Am Holterhöfchen 34 40724 Hilden
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