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1.
Nat Commun ; 10(1): 4439, 2019 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-31570731

RESUMO

Adeno-associated virus (AAV) vectors have shown promising results in preclinical models, but the genomic consequences of transduction with AAV vectors encoding CRISPR-Cas nucleases is still being examined. In this study, we observe high levels of AAV integration (up to 47%) into Cas9-induced double-strand breaks (DSBs) in therapeutically relevant genes in cultured murine neurons, mouse brain, muscle and cochlea. Genome-wide AAV mapping in mouse brain shows no overall increase of AAV integration except at the CRISPR/Cas9 target site. To allow detailed characterization of integration events we engineer a miniature AAV encoding a 465 bp lambda bacteriophage DNA (AAV-λ465), enabling sequencing of the entire integrated vector genome. The integration profile of AAV-465λ in cultured cells display both full-length and fragmented AAV genomes at Cas9 on-target sites. Our data indicate that AAV integration should be recognized as a common outcome for applications that utilize AAV for genome editing.


Assuntos
Sistemas CRISPR-Cas , Quebras de DNA , Dependovirus/genética , Edição de Genes/métodos , Vetores Genéticos , Integração Viral/genética , Animais , Bacteriófago lambda/genética , Encéfalo , Linhagem Celular , Mapeamento Cromossômico , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Cóclea , Endonucleases , Marcação de Genes/métodos , Terapia Genética/métodos , Genoma , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Músculos , Neurônios/virologia , Reparo Gênico Alvo-Dirigido/métodos , Resultado do Tratamento
2.
Int J Mol Sci ; 20(15)2019 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-31366028

RESUMO

The CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeat-associated protein 9) is a powerful genome-editing tool in animals, plants, and humans. This system has some advantages, such as a high on-target mutation rate (targeting efficiency), less cost, simplicity, and high-efficiency multiplex loci editing, over conventional genome editing tools, including meganucleases, transcription activator-like effector nucleases (TALENs), and zinc finger nucleases (ZFNs). One of the crucial shortcomings of this system is unwanted mutations at off-target sites. We summarize and discuss different approaches, such as dCas9 and Cas9 paired nickase, to decrease the off-target effects in plants. According to studies, the most effective method to reduce unintended mutations is the use of ligand-dependent ribozymes called aptazymes. The single guide RNA (sgRNA)/ligand-dependent aptazyme strategy has helped researchers avoid unwanted mutations in human cells and can be used in plants as an alternative method to dramatically decrease the frequency of off-target mutations. We hope our concept provides a new, simple, and fast gene transformation and genome-editing approach, with advantages including reduced time and energy consumption, the avoidance of unwanted mutations, increased frequency of on-target changes, and no need for external forces or expensive equipment.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Melhoramento Vegetal/métodos , Reparo Gênico Alvo-Dirigido/métodos , Edição de Genes/normas , Magnoliopsida/genética , RNA Guia/genética , Reparo Gênico Alvo-Dirigido/normas
3.
Medicina (Kaunas) ; 55(8)2019 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-31357735

RESUMO

The upgraded knowledge of tumor biology and microenviroment provides information on differences in neoplastic and normal cells. Thus, the need to target these differences led to the development of novel molecules (targeted therapy) active against the neoplastic cells' inner workings. There are several types of targeted agents, including Small Molecules Inhibitors (SMIs), monoclonal antibodies (mAbs), interfering RNA (iRNA) molecules and microRNA. In the clinical practice, these new medicines generate a multilayered step in pharmacokinetics (PK), which encompasses a broad individual PK variability, and unpredictable outcomes according to the pharmacogenetics (PG) profile of the patient (e.g., cytochrome P450 enzyme), and to patient characteristics such as adherence to treatment and environmental factors. This review focuses on the use of targeted agents in-human phase I/II/III clinical trials in cancer-hematology. Thus, it outlines the up-to-date anticancer drugs suitable for targeted therapies and the most recent finding in pharmacogenomics related to drug response. Besides, a summary assessment of the genotyping costs has been discussed. Targeted therapy seems to be an effective and less toxic therapeutic approach in onco-hematology. The identification of individual PG profile should be a new resource for oncologists to make treatment decisions for the patients to minimize the toxicity and or inefficacy of therapy. This could allow the clinicians to evaluate benefits and restrictions, regarding costs and applicability, of the most suitable pharmacological approach for performing a tailor-made therapy.


Assuntos
Antineoplásicos/uso terapêutico , Reparo Gênico Alvo-Dirigido/métodos , Antineoplásicos/farmacologia , Antineoplásicos Imunológicos/farmacologia , Antineoplásicos Imunológicos/uso terapêutico , Humanos , MicroRNAs/farmacologia , MicroRNAs/uso terapêutico , Vírus Oncolíticos , RNA Interferente Pequeno/farmacologia , RNA Interferente Pequeno/uso terapêutico , Literatura de Revisão como Assunto , Reparo Gênico Alvo-Dirigido/estatística & dados numéricos
4.
Genomics ; 111(4): 560-566, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-29605634

RESUMO

The ability to direct the CRISPR/Cas9 nuclease to a unique target site within a genome would have broad use in targeted genome engineering. However, CRISPR RNA is reported to bind to other genomic locations that differ from the intended target site by a few nucleotides, demonstrating significant off-target activity. We have developed the CRISPcut tool that screens the off-targets using various parameters and predicts the ideal genomic target for -guide RNAs in human cell lines. sgRNAs for four different types of Cas9 nucleases can be designed with an option for the user to work with different PAM sequences. Direct experimental measurement of genome-wide DNA accessibility is incorporated that effectively restricts the prediction of CRISPR targets to open chromatin. An option to predict target sites for paired CRISPR nickases is also provided. The tool has been validated using a dataset of experimentally used sgRNA and their identified off-targets. URL: http://web.iitd.ac.in/crispcut.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , RNA Guia/genética , Software , Reparo Gênico Alvo-Dirigido/métodos , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Cromatina/química , Humanos , Motivos de Nucleotídeos , RNA Guia/metabolismo
6.
Methods Mol Biol ; 1780: 497-523, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29856033

RESUMO

Advances in molecular biology and genetics have been used to elucidate the fundamental genetic mechanisms underlying central nervous system (CNS) diseases, yet disease-modifying therapies are currently unavailable for most CNS conditions. Antisense oligonucleotides (ASOs) are synthetic single stranded chains of nucleic acids that bind to a specific sequence on ribonucleic acid (RNA) and regulate posttranscriptional gene expression. Decreased gene expression with ASOs might be able to reduce production of the disease-causing protein underlying dominantly inherited neurodegenerative disorders. Huntington's disease (HD), which is caused by a CAG repeat expansion in exon 1 of the huntingtin (HTT) gene and leads to the pathogenic expansion of a polyglutamine (PolyQ ) tract in the N terminus of the huntingtin protein (Htt), is a prime candidate for ASO therapy.State-of-the art translational science techniques can be applied to the development of an ASO targeting HTT RNA, allowing for a data-driven, stepwise progression through the drug development process. A deep and wide-ranging understanding of the basic, preclinical, clinical, and epidemiologic components of drug development will improve the likelihood of success. This includes characterizing the natural history of the disease, including evolution of biomarkers indexing the underlying pathology; using predictive preclinical models to assess the putative gain-of-function of mutant Htt protein and any loss-of-function of the wild-type protein; characterizing toxicokinetic and pharmacodynamic effects of ASOs in predictive animal models; developing sensitive and reliable biomarkers to monitor target engagement and effects on pathology that translate from animal models to patients with HD; establishing a drug delivery method that ensures reliable distribution to relevant CNS tissue; and designing clinical trials that move expeditiously from proof of concept to proof of efficacy. This review focuses on the translational science techniques that allow for efficient and informed development of an ASO for the treatment of HD.


Assuntos
Proteína Huntingtina/genética , Doença de Huntington/terapia , Oligonucleotídeos Antissenso/uso terapêutico , Reparo Gênico Alvo-Dirigido/métodos , Pesquisa Médica Translacional/métodos , Animais , Encéfalo/patologia , Modelos Animais de Doenças , Desenvolvimento de Medicamentos , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Proteína Huntingtina/metabolismo , Doença de Huntington/genética , Doença de Huntington/patologia , Macaca fascicularis , Camundongos , Mutação , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/farmacologia , Precursores de RNA/genética , Ratos , Resultado do Tratamento
7.
Nat Commun ; 9(1): 2481, 2018 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-29946143

RESUMO

Genetic diseases can be diagnosed early during pregnancy, but many monogenic disorders continue to cause considerable neonatal and pediatric morbidity and mortality. Early intervention through intrauterine gene editing, however, could correct the genetic defect, potentially allowing for normal organ development, functional disease improvement, or cure. Here we demonstrate safe intravenous and intra-amniotic administration of polymeric nanoparticles to fetal mouse tissues at selected gestational ages with no effect on survival or postnatal growth. In utero introduction of nanoparticles containing peptide nucleic acids (PNAs) and donor DNAs corrects a disease-causing mutation in the ß-globin gene in a mouse model of human ß-thalassemia, yielding sustained postnatal elevation of blood hemoglobin levels into the normal range, reduced reticulocyte counts, reversal of splenomegaly, and improved survival, with no detected off-target mutations in partially homologous loci. This work may provide the basis for a safe and versatile method of fetal gene editing for human monogenic disorders.


Assuntos
Terapias Fetais/métodos , Edição de Genes/métodos , Doenças Genéticas Inatas/genética , Doenças Genéticas Inatas/terapia , Nanopartículas/administração & dosagem , Reparo Gênico Alvo-Dirigido/métodos , Animais , DNA de Cadeia Simples/administração & dosagem , DNA de Cadeia Simples/genética , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Ácidos Nucleicos Peptídicos/administração & dosagem , Ácidos Nucleicos Peptídicos/genética , Gravidez , Segurança , Útero , Globinas beta/genética , Talassemia beta/sangue , Talassemia beta/genética , Talassemia beta/terapia
8.
Sci Rep ; 8(1): 6937, 2018 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-29720600

RESUMO

Loss-of-function mutations in TREM2 cause Nasu-Hakola disease (NHD), a rare genetic disease characterized by early-onset dementia with leukoencephalopathy and bone cysts. An NHD-associated mutation, c.482 + 2 T > C, disrupts the splice donor site of intron 3 and causes aberrant skipping of exon 3, resulting in the loss of full-length TREM2 protein. Here, we examined the efficacy of artificial U1 and U7 small nuclear RNAs (snRNAs) designed to enhance exon 3 inclusion. Using mutant TREM2 minigenes, we found that some modified U1, but not U7, snRNAs enhanced exon 3 inclusion and restored TREM2 protein expression. Unexpectedly, we found that exon 3 of wild-type TREM2 is an alternative exon, whose skipping leads to reduced expression of the full-length protein. Indeed, TREM2 protein levels were modulated by modified snRNAs that either promoted or repressed exon 3 inclusion. The splice donor site flanking exon 3 was predicted to be weak, which may explain both the alternative splicing of exon 3 under normal conditions and complete exon skipping when the c.482 + 2 T > C mutation was present. Collectively, our snRNA-based approaches provide a potential therapeutic strategy for NHD-associated mis-splicing and novel insights into the post-transcriptional regulation of TREM2.


Assuntos
Processamento Alternativo , Glicoproteínas de Membrana/genética , Mutação , Processamento Pós-Transcricional do RNA , RNA Nuclear Pequeno/genética , Receptores Imunológicos/genética , Reparo Gênico Alvo-Dirigido , Sequência de Bases , Linhagem Celular , Éxons , Humanos , Glicoproteínas de Membrana/metabolismo , Modelos Biológicos , Sítios de Splice de RNA , Receptores Imunológicos/metabolismo , Reparo Gênico Alvo-Dirigido/métodos
9.
Plant Sci ; 271: 117-126, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29650149

RESUMO

A major research topic nowadays is to study and understand the functions of the increasing number of predicted genes that have been discovered through the complete genome sequencing of many plant species. With the aim of developing tools for rapid and convenient gene function analysis, we have developed a set of "pGate" vectors based on the principle of Golden gate and Gateway cloning approaches. These vectors combine the positive aspects of both Golden gate and Gateway cloning strategies. pGate vectors can not only be used as Golden gate recipient vectors to assemble multiple DNA fragments in a pre-defined order, but they can also work as an entry vector to transfer the assembled DNA fragment(s) to a large number of already-existing, functionally diverse, Gateway compatible destination vectors without adding additional nucleotides during cloning. We show the pGate vectors are effective and convenient in several major aspects of gene function analyses, including BiFC (Bimolecular fluorescence complementation) to analyze protein-protein interaction, amiRNA (artificial microRNA) candidate screening and as assembly of CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats, CRISPR-associated protein-9 nuclease) system elements together for genome editing. The pGate system is a practical and flexible tool which can facilitate plant gene function research.


Assuntos
Genes de Plantas/genética , Plantas/genética , Clonagem Molecular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes/métodos , Vetores Genéticos/genética , Microscopia Confocal , Reparo Gênico Alvo-Dirigido/métodos
10.
Nat Biotechnol ; 36(6): 536-539, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29702637

RESUMO

Adenine base editors (ABEs) composed of an engineered adenine deaminase and the Streptococcus pyogenes Cas9 nickase enable adenine-to-guanine (A-to-G) single-nucleotide substitutions in a guide RNA (gRNA)-dependent manner. Here we demonstrate application of this technology in mouse embryos and adult mice. We also show that long gRNAs enable adenine editing at positions one or two bases upstream of the window that is accessible with standard single guide RNAs (sgRNAs). We introduced the Himalayan point mutation in the Tyr gene by microinjecting ABE mRNA and an extended gRNA into mouse embryos, obtaining Tyr mutant mice with an albino phenotype. Furthermore, we delivered the split ABE gene, using trans-splicing adeno-associated viral vectors, to muscle cells in a mouse model of Duchenne muscular dystrophy to correct a nonsense mutation in the Dmd gene, demonstrating the therapeutic potential of base editing in adult animals.


Assuntos
Edição de Genes/métodos , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Adenina/química , Albinismo/embriologia , Albinismo/genética , Albinismo/terapia , Animais , Sequência de Bases , Biotecnologia , DNA/genética , Modelos Animais de Doenças , Distrofina/deficiência , Distrofina/genética , Terapia Genética/métodos , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , Camundongos Mutantes , Monofenol Mono-Oxigenase/genética , Reparo Gênico Alvo-Dirigido/métodos
11.
Curr Gene Ther ; 18(2): 115-124, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29473500

RESUMO

Genome editing mediated by Clustered Regularly Interspaced Palindromic Repeats (CRISPR) and its associated proteins (Cas) has recently been considered to be used as efficient, rapid and site-specific tool in the modification of endogenous genes in biomedically important cell types and whole organisms. It has become a predictable and precise method of choice for genome engineering by specifying a 20-nt targeting sequence within its guide RNA. Firstly, this review aims to describe the biology of CRISPR system. Next, the applications of CRISPR-Cas9 in various ways, such as efficient generation of a wide variety of biomedically important cellular models as well as those of animals, modifying epigenomes, conducting genome-wide screens, gene therapy, labelling specific genomic loci in living cells, metabolic engineering of yeast and bacteria and endogenous gene expression regulation by an altered version of this system were reviewed.


Assuntos
Biotecnologia , Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas/genética , Sistemas CRISPR-Cas/fisiologia , Edição de Genes/métodos , Animais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Farmacorresistência Bacteriana Múltipla/genética , Epigênese Genética , Regulação da Expressão Gênica , Terapia Genética , Humanos , Engenharia Metabólica , Camundongos , Neoplasias/genética , Neoplasias/terapia , Reparo Gênico Alvo-Dirigido/métodos , Viroses/genética , Viroses/terapia
12.
Med Hypotheses ; 110: 97-100, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29317080

RESUMO

Duchenne muscular dystrophy (DMD) is a lethal muscle wasting disease caused by a lack of dystrophin, which eventually leads to apoptosis of muscle cells and impaired muscle contractility. Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9 (CRISPR/Cas9) gene editing of induced pluripotent stem cells (IPSC) offers the potential to correct the DMD gene defect and create healthy IPSC for autologous cell transplantation without causing immune activation. However, IPSC carry a risk of tumor formation, which can potentially be mitigated by differentiation of IPSC into myogenic progenitor cells (MPC). We hypothesize that precise genetic editing in IPSC using CRISPR-Cas9 technology, coupled with MPC differentiation and autologous transplantation, can lead to safe and effective muscle repair. With future research, our hypothesis may provide an optimal autologous stem cell-based approach to treat the dystrophic pathology and improve the quality of life for patients with DMD.


Assuntos
Células-Tronco Pluripotentes Induzidas/transplante , Desenvolvimento Muscular/genética , Distrofia Muscular de Duchenne/terapia , Reparo Gênico Alvo-Dirigido/métodos , Autoenxertos , Sistemas CRISPR-Cas , Distrofina/genética , Edição de Genes/métodos , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Modelos Biológicos , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patologia , Regeneração/genética
14.
Methods Mol Biol ; 1715: 61-78, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29188506

RESUMO

Antisense oligonucleotides (AONs) are small molecules able to bind to the pre-mRNA and modulate splicing. The increasing amount of intronic mutations leading to pseudoexon insertion in genes underlying inherited retinal dystrophies (IRDs) has highlighted the potential of AONs as a therapeutic tool for these disorders. Here we describe how to design and test AON molecules in vitro in order to correct pre-mRNA splicing defects involved in IRDs.


Assuntos
Vetores Genéticos , Oligonucleotídeos Antissenso/genética , Precursores de RNA/genética , Processamento de RNA , Distrofias Retinianas/terapia , Reparo Gênico Alvo-Dirigido/métodos , Células HEK293 , Humanos , Técnicas In Vitro , Distrofias Retinianas/genética
17.
Yeast ; 34(12): 483-494, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28810289

RESUMO

The basidiomycetous yeast Pseudozyma antarctica is a remarkable producer of industrially valuable enzymes and extracellular glycolipids. In this study, we developed a method for targeted gene replacement in P. antarctica. In addition, transformation conditions were optimized using lithium acetate, single-stranded carrier DNA and polyethylene glycol (lithium acetate treatment), generally used for ascomycetous yeast transformation. In the rice-derived P. antarctica strain GB-4(0), PaURA3, a homologue of the Saccharomyces cerevisiae orotidine-5'-phosphate decarboxylase gene (URA3), was selected as the target locus. A disruption cassette was constructed by linking the nouseothricine resistance gene (natMX4) to homologous DNA fragments of PaURA3, then electroporated into the strain GB-4(0). We obtained strain PGB015 as one of the PaURA3 disruptants (Paura3Δ::natMX4). Then the PCR-amplified PaURA3 fragment was introduced into PGB015, and growth of transformant colonies but not background colonies was observed on selective media lacking uracil. The complementation of uracil-auxotrophy in PGB015 by introduction of PaURA3 was also performed using lithium acetate treatment, which resulted in a transformation efficiency of 985 CFU/6.8 µg DNA and a gene-targeting ratio of two among 30 transformants. Copyright © 2017 John Wiley & Sons, Ltd.


Assuntos
Acetatos/farmacologia , Proteínas Fúngicas/genética , Reparo Gênico Alvo-Dirigido/métodos , Transformação Genética , Ustilaginales/genética , Sequência de Aminoácidos , DNA Fúngico/genética , Farmacorresistência Fúngica/genética , Eletroporação , Temperatura Alta , Ácido Orótico/análogos & derivados , Ácido Orótico/farmacologia , Orotidina-5'-Fosfato Descarboxilase/química , Orotidina-5'-Fosfato Descarboxilase/genética , Plasmídeos/genética , Estreptotricinas/farmacologia , Árvores/microbiologia , Ustilaginales/efeitos dos fármacos , Ustilaginales/crescimento & desenvolvimento
18.
Mol Ther ; 25(9): 2117-2128, 2017 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-28629821

RESUMO

Glycogen storage disease type II (GSDII) is a lysosomal disorder caused by the deficient activity of acid alpha-glucosidase (GAA) enzyme, leading to the accumulation of glycogen within the lysosomes. The disease has been classified in infantile and late-onset forms. Most late-onset patients share a splicing mutation c.-32-13T > G in intron 1 of the GAA gene that prevents efficient recognition of exon 2 by the spliceosome. In this study, we have mapped the splicing silencers of GAA exon 2 and developed antisense morpholino oligonucleotides (AMOs) to inhibit those regions and rescue normal splicing in the presence of the c.-32-13T > G mutation. Using a minigene approach and patient fibroblasts, we successfully increased inclusion of exon 2 in the mRNA and GAA enzyme production by targeting a specific silencer with a combination of AMOs. Most importantly, the use of these AMOs in patient myotubes results in a decreased accumulation of glycogen. To our knowledge, this is the only therapeutic approach resulting in a decrease of glycogen accumulation in patient tissues beside enzyme replacement therapy (ERT) and TFEB overexpression. As a result, it may represent a highly novel and promising therapeutic line for GSDII.


Assuntos
Doença de Depósito de Glicogênio Tipo II/genética , Doença de Depósito de Glicogênio Tipo II/metabolismo , Glicogênio/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Oligonucleotídeos Antissenso/genética , Reparo Gênico Alvo-Dirigido , Alelos , Linhagem Celular , Éxons , Ordem dos Genes/genética , Vetores Genéticos/genética , Doença de Depósito de Glicogênio Tipo II/terapia , Humanos , Mutação , Oligonucleotídeos Antissenso/uso terapêutico , Ligação Proteica , Processamento de RNA , Fatores de Processamento de RNA/metabolismo , Elementos Silenciadores Transcricionais , Reparo Gênico Alvo-Dirigido/métodos , alfa-Glucosidases/genética
19.
J Cell Biochem ; 118(12): 4152-4162, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28467679

RESUMO

The revolutionary technology for genome editing known as the clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein 9 (Cas9) system has sparked advancements in biological and biomedical research. The scientific breakthrough of the development of CRISPR-Cas9 technology has allowed us to recapitulate human diseases by generating animal models of interest ranging from zebrafish to non-human primates. The CRISPR-Cas9 system can also be used to delineate the mechanisms underlying the development of human disorders and to precisely correct disease-causing mutations. Repurposing this technology enables wider applications in transcriptome and epigenome manipulation and holds promise to reach the clinic. In this review, we highlight the latest advances of the CRISPR-Cas9 system in different platforms and discuss the hurdles and challenges this technology is facing. J. Cell. Biochem. 118: 4152-4162, 2017. © 2017 Wiley Periodicals, Inc.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Reparo Gênico Alvo-Dirigido/métodos , Animais , Modelos Animais de Doenças , Humanos
20.
Gene Ther ; 24(7): 425-432, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28530652

RESUMO

In humans, mutations in the ß-globin gene (HBB) have two important clinical manifestations: ß-thalassemia and sickle cell disease. The progress in genome editing and stem cell research may be relevant to the treatment of ß-globin-related diseases. In this work, we employed zinc-finger nuclease (ZFN)-mediated gene integration of synthetic ß-globin cDNA into HBB loci, thus correcting almost all ß-globin mutations. The integration was achieved in both HEK 293 cells and isolated dental pulp stem cell (DPSCs). We also showed that DPSCs with an artificial gene knock-in were capable of generating stable six-cell clones and were expandable at least 108-fold; therefore, they may serve as a personalized stem cell factory. In addition, transfection with non-integrated pCAG-hOct4 and culturing in a conditioned medium converted the genome-edited DPSCs to CD34+ HSC-like cells. We believe that this approach may be useful for the treatment of ß-globin-related diseases, especially the severe form of ß-thalassemia.


Assuntos
Células-Tronco Adultas/metabolismo , Polpa Dentária/citologia , Reparo Gênico Alvo-Dirigido/métodos , Globinas beta/genética , Antígenos CD34/genética , Antígenos CD34/metabolismo , Células Cultivadas , Endodesoxirribonucleases/química , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Células HEK293 , Humanos , Dedos de Zinco , Globinas beta/metabolismo
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