The Experts below are selected from a list of 1785 Experts worldwide ranked by ideXlab platform
Jeffrey C Miller - One of the best experts on this subject based on the ideXlab platform.
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An unbiased genome-wide analysis of Zinc-Finger Nuclease specificity
Nature biotechnology, 2011Co-Authors: Richard Gabriel, Jeffrey C Miller, Jianbin Wang, A. Lombardo, Anne Arens, Pietro Genovese, Christine Kaeppel, Ali Nowrouzi, Cynthia C. Bartholomae, Geoffrey FriedmanAbstract:Zinc-Finger Nucleases (ZFNs) allow gene editing in live cells by inducing a targeted DNA double-strand break (DSB) at a specific genomic locus. However, strategies for characterizing the genome-wide specificity of ZFNs remain limited. We show that nonhomologous end-joining captures integrase-defective lentiviral vectors at DSBs, tagging these transient events. Genome-wide integration site analysis mapped the actual in vivo cleavage activity of four ZFN pairs targeting CCR5 or IL2RG. Ranking loci with repeatedly detectable Nuclease activity by deep-sequencing allowed us to monitor the degree of ZFN specificity in vivo at these positions. Cleavage required binding of ZFNs in specific spatial arrangements on DNA bearing high homology to the intended target site and only tolerated mismatches at individual positions of the ZFN binding sites. Whereas the consensus binding sequence derived in vivo closely matched that obtained in biochemical experiments, the ranking of in vivo cleavage sites could not be predicted in silico. Comprehensive mapping of ZFN activity in vivo will facilitate the broad application of these reagents in translational research.
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enhancing Zinc Finger Nuclease activity with improved obligate heterodimeric architectures
Nature Methods, 2011Co-Authors: Yannick Doyon, Thuy D Vo, Matthew C Mendel, Shon G Greenberg, Jeffrey C Miller, Fyodor D Urnov, Philip D Gregory, Jianbin Wang, Michael C HolmesAbstract:Identification of residues critical for dimerization of the Fok1 Nuclease domain of Zinc-Finger Nucleases permits rational design of enzymes with improved cleavage activity and retained obligate heterodimerization.
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Zinc Finger Nuclease driven targeted integration into mammalian genomes using donors with limited chromosomal homology
Nucleic Acids Research, 2010Co-Authors: Salvatore Orlando, Yolanda Santiago, Russell Dekelver, Yevgeniy Freyvert, Elizabeth A Boydston, Erica A Moehle, Vivian M Choi, Sunita Gopalan, Jacqueline Lou, Jeffrey C MillerAbstract:We previously demonstrated high-frequency, targeted DNA addition mediated by the homology-directed DNA repair pathway. This method uses a Zinc-Finger Nuclease (ZFN) to create a site-specific double-strand break (DSB) that facilitates copying of genetic information into the chromosome from an exogenous donor molecule. Such donors typically contain two approximately 750 bp regions of chromosomal sequence required for homology-directed DNA repair. Here, we demonstrate that easily-generated linear donors with extremely short (50 bp) homology regions drive transgene integration into 5-10% of chromosomes. Moreover, we measure the overhangs produced by ZFN cleavage and find that oligonucleotide donors with single-stranded 5' overhangs complementary to those made by ZFNs are efficiently ligated in vivo to the DSB. Greater than 10% of all chromosomes directly incorporate this exogenous DNA via a process that is dependent upon and guided by complementary 5' overhangs on the donor DNA. Finally, we extend this non-homologous end-joining (NHEJ)-based technique by directly inserting donor DNA comprising recombinase sites into large deletions created by the simultaneous action of two separate ZFN pairs. Up to 50% of deletions contained a donor insertion. Targeted DNA addition via NHEJ complements our homology-directed targeted integration approaches, adding versatility to the manipulation of mammalian genomes.
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Zinc Finger Nuclease driven targeted integration into mammalian genomes using donors with limited chromosomal homology
Nucleic Acids Research, 2010Co-Authors: Salvatore Orlando, Yolanda Santiago, Russell Dekelver, Yevgeniy Freyvert, Elizabeth A Boydston, Erica A Moehle, Vivian M Choi, Sunita Gopalan, James Li, Jeffrey C MillerAbstract:We previously demonstrated high-frequency, targeted DNA addition mediated by the homology-directed DNA repair pathway. This method uses a Zinc-Finger Nuclease (ZFN) to create a site-specific double-strand break (DSB) that facilitates copying of genetic information into the chromosome from an exogenous donor molecule. Such donors typically contain two ∼750 bp regions of chromosomal sequence required for homology-directed DNA repair. Here, we demonstrate that easily-generated linear donors with extremely short (50 bp) homology regions drive transgene integration into 5–10% of chromosomes. Moreover, we measure the overhangs produced by ZFN cleavage and find that oligonucleotide donors with single-stranded 5′ overhangs complementary to those made by ZFNs are efficiently ligated in vivo to the DSB. Greater than 10% of all chromosomes directly incorporate this exogenous DNA via a process that is dependent upon and guided by complementary 5′ overhangs on the donor DNA. Finally, we extend this non-homologous end-joining (NHEJ)-based technique by directly inserting donor DNA comprising recombinase sites into large deletions created by the simultaneous action of two separate ZFN pairs. Up to 50% of deletions contained a donor insertion. Targeted DNA addition via NHEJ complements our homology-directed targeted integration approaches, adding versatility to the manipulation of mammalian genomes.
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BAK and BAX deletion using Zinc-Finger Nucleases yields apoptosis-resistant CHO cells.
Biotechnology and bioengineering, 2010Co-Authors: Gregory J Cost, Jeffrey C Miller, Yolanda Santiago, Yevgeniy Freyvert, Annamaria Vafiadis, Edward J. Rebar, Trevor Collingwood, Andrew Snowden, Philip D GregoryAbstract:Anoxic and metabolic stresses in large-scale cell culture during biopharmaceutical production can induce apoptosis. Strategies designed to ameliorate the problem of apoptosis in cell culture have focused on mRNA knockdown of pro-apoptotic proteins and over-expression of anti-apoptotic ones. Apoptosis in cell culture involves mitochondrial permeabilization by the pro-apoptotic Bak and Bax proteins; activity of either protein is sufficient to permit apoptosis. We demonstrate here the complete and permanent elimination of both the Bak and Bax proteins in combination in Chinese hamster ovary (CHO) cells using Zinc-Finger Nuclease-mediated gene disruption. Zinc-Finger Nuclease cleavage of BAX and BAK followed by inaccurate DNA repair resulted in knockout of both genes. Cells lacking Bax and Bak grow normally but fail to activate caspases in response to apoptotic stimuli. When grown using scale-down systems under conditions that mimic growth in large-scale bioreactors they are significantly more resistant to apoptosis induced by starvation, staurosporine, and sodium butyrate. When grown under starvation conditions, BAX- and BAK-deleted cells produce two- to fivefold more IgG than wild-type CHO cells. Under normal growth conditions in suspension culture in shake flasks, double-knockout cultures achieve equal or higher cell densities than unmodified wild-type cultures and reach viable cell densities relevant for large-scale industrial protein production.
Henrik Clausen - One of the best experts on this subject based on the ideXlab platform.
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corrigendum mining the o glycoproteome using Zinc Finger Nuclease glycoengineered simplecell lines
Nature Methods, 2015Co-Authors: Catharina Steentoft, Eric P Bennett, Sergey Y Vakhrushev, Yun Kong, Ulla Mandel, Steven B Levery, Katrine T Schjoldager, Hans H Wandall, Malene Bech Vesterchristensen, Henrik ClausenAbstract:Nat. Methods 8, 977–982 (2011); published online 9 October 2011; corrected after print 8 January 2015 In the version of this article initially published, the T3M4 cell line used for the experiments was mislabeled as Capan-1. The error has been corrected in the HTML and PDF versions of the article.
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glycoengineering of human cell lines using Zinc Finger Nuclease gene targeting simplecells with homogeneous galnac o glycosylation allow isolation of the o glycoproteome by one step lectin affinity chromatography
Methods of Molecular Biology, 2013Co-Authors: Catharina Steentoft, Eric P Bennett, Henrik ClausenAbstract:Lectin affinity chromatography is a powerful technique for isolation of glycoproteins carrying a specific glycan structure of interest. However, the enormous diversity of glycans present on the cell surface, as well as on individual proteins, makes it difficult to isolate an entire glycoproteome with one or even a series of lectins. Here we present a technique to generate cell lines with homogenous truncated O-glycans using Zinc Finger Nuclease gene targeting. Because of their simplified O-glycoproteome, the cells have been named SimpleCells. Glycoproteins from SimpleCells can be isolated in a single purification step by lectin chromatography performed on a long lectin column. This protocol describes Zinc Finger Nuclease gene targeting of human cells to simplify the glycoproteome, as well as lectin chromatography and isolation of glycopeptides from total cell lysates of SimpleCells.
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mining the o glycoproteome using Zinc Finger Nuclease glycoengineered simplecell lines
Nature Methods, 2011Co-Authors: Catharina Steentoft, Eric P Bennett, Sergey Y Vakhrushev, Yun Kong, Ulla Mandel, Steven B Levery, Katrine T Schjoldager, Hans H Wandall, Malene Bech Vesterchristensen, Henrik ClausenAbstract:Owing to a lack of tools and a lack of a consensus sequence for O-glycosylation, studies of the O-glycoproteome have been few and far between, despite the biological importance of O-glycosylation. This method to analyze O-glycan attachment sites to proteins using glycoengineered cell lines with simplified, homogenous O-glycoproteomes should facilitate future O-glycoproteomics studies.
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mining the o glycoproteome using Zinc Finger Nuclease glycoengineered simplecell lines
Nature Methods, 2011Co-Authors: Catharina Steentoft, Eric P Bennett, Sergey Y Vakhrushev, Yun Kong, Ulla Mandel, Steven B Levery, Katrine T Schjoldager, Hans H Wandall, Malene Bech Vesterchristensen, Henrik ClausenAbstract:Zinc-Finger Nuclease (ZFN) gene targeting is emerging as a versatile tool for engineering of multiallelic gene deficiencies. A longstanding obstacle for detailed analysis of glycoproteomes has been the extensive heterogeneities in glycan structures and attachment sites. Here we applied ZFN targeting to truncate the O-glycan elongation pathway in human cells, generating stable 'SimpleCell' lines with homogenous O-glycosylation. Three SimpleCell lines expressing only truncated GalNAcα or NeuAcα2-6GalNAcα O-glycans were produced, allowing straightforward isolation and sequencing of GalNAc O-glycopeptides from total cell lysates using lectin chromatography and nanoflow liquid chromatography-mass spectrometry (nLC-MS/MS) with electron transfer dissociation fragmentation. We identified >100 O-glycoproteins with >350 O-glycan sites (the great majority previously unidentified), including a GalNAc O-glycan linkage to a tyrosine residue. The SimpleCell method should facilitate analyses of important functions of protein glycosylation. The strategy is also applicable to other O-glycoproteomes.
Catharina Steentoft - One of the best experts on this subject based on the ideXlab platform.
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corrigendum mining the o glycoproteome using Zinc Finger Nuclease glycoengineered simplecell lines
Nature Methods, 2015Co-Authors: Catharina Steentoft, Eric P Bennett, Sergey Y Vakhrushev, Yun Kong, Ulla Mandel, Steven B Levery, Katrine T Schjoldager, Hans H Wandall, Malene Bech Vesterchristensen, Henrik ClausenAbstract:Nat. Methods 8, 977–982 (2011); published online 9 October 2011; corrected after print 8 January 2015 In the version of this article initially published, the T3M4 cell line used for the experiments was mislabeled as Capan-1. The error has been corrected in the HTML and PDF versions of the article.
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glycoengineering of human cell lines using Zinc Finger Nuclease gene targeting simplecells with homogeneous galnac o glycosylation allow isolation of the o glycoproteome by one step lectin affinity chromatography
Methods of Molecular Biology, 2013Co-Authors: Catharina Steentoft, Eric P Bennett, Henrik ClausenAbstract:Lectin affinity chromatography is a powerful technique for isolation of glycoproteins carrying a specific glycan structure of interest. However, the enormous diversity of glycans present on the cell surface, as well as on individual proteins, makes it difficult to isolate an entire glycoproteome with one or even a series of lectins. Here we present a technique to generate cell lines with homogenous truncated O-glycans using Zinc Finger Nuclease gene targeting. Because of their simplified O-glycoproteome, the cells have been named SimpleCells. Glycoproteins from SimpleCells can be isolated in a single purification step by lectin chromatography performed on a long lectin column. This protocol describes Zinc Finger Nuclease gene targeting of human cells to simplify the glycoproteome, as well as lectin chromatography and isolation of glycopeptides from total cell lysates of SimpleCells.
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probing isoform specific functions of polypeptide galnac transferases using Zinc Finger Nuclease glycoengineered simplecells
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Katrine T Schjoldager, Catharina Steentoft, Eric P Bennett, Sergey Y Vakhrushev, Yun Kong, Ulla Mandel, Aaron S Nudelman, Nis Borbye Pedersen, Hans H Wandall, Steven B LeveryAbstract:Our knowledge of the O-glycoproteome [N-acetylgalactosamine (GalNAc) type] is highly limited. The O-glycoproteome is differentially regulated in cells by dynamic expression of a subset of 20 polypeptide GalNAc-transferases (GalNAc-Ts), and methods to identify important functions of individual GalNAc-Ts are largely unavailable. We recently introduced SimpleCells, i.e., human cell lines made deficient in O-glycan extension by Zinc Finger Nuclease targeting of a key gene in O-glycan elongation (Cosmc), which allows for proteome-wide discovery of O-glycoproteins. Here we have extended the SimpleCell concept to include proteome-wide discovery of unique functions of individual GalNAc-Ts. We used the GalNAc-T2 isoform implicated in dyslipidemia and the human HepG2 liver cell line to demonstrate unique functions of this isoform. We confirm that GalNAc-T2–directed site-specific O-glycosylation inhibits proprotein activation of the lipase inhibitor ANGPTL3 in HepG2 cells and further identify eight O-glycoproteins exclusively glycosylated by T2 of which one, ApoC-III, is implicated in dyslipidemia. Our study supports an essential role for GalNAc-T2 in lipid metabolism, provides serum biomarkers for GalNAc-T2 enzyme function, and validates the use of GALNT gene targeting with SimpleCells for broad discovery of disease-causing deficiencies in O-glycosylation. The presented glycoengineering strategy opens the way for proteome-wide discovery of functions of GalNAc-T isoforms and their role in congenital diseases and disorders.
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mining the o glycoproteome using Zinc Finger Nuclease glycoengineered simplecell lines
Nature Methods, 2011Co-Authors: Catharina Steentoft, Eric P Bennett, Sergey Y Vakhrushev, Yun Kong, Ulla Mandel, Steven B Levery, Katrine T Schjoldager, Hans H Wandall, Malene Bech Vesterchristensen, Henrik ClausenAbstract:Owing to a lack of tools and a lack of a consensus sequence for O-glycosylation, studies of the O-glycoproteome have been few and far between, despite the biological importance of O-glycosylation. This method to analyze O-glycan attachment sites to proteins using glycoengineered cell lines with simplified, homogenous O-glycoproteomes should facilitate future O-glycoproteomics studies.
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mining the o glycoproteome using Zinc Finger Nuclease glycoengineered simplecell lines
Nature Methods, 2011Co-Authors: Catharina Steentoft, Eric P Bennett, Sergey Y Vakhrushev, Yun Kong, Ulla Mandel, Steven B Levery, Katrine T Schjoldager, Hans H Wandall, Malene Bech Vesterchristensen, Henrik ClausenAbstract:Zinc-Finger Nuclease (ZFN) gene targeting is emerging as a versatile tool for engineering of multiallelic gene deficiencies. A longstanding obstacle for detailed analysis of glycoproteomes has been the extensive heterogeneities in glycan structures and attachment sites. Here we applied ZFN targeting to truncate the O-glycan elongation pathway in human cells, generating stable 'SimpleCell' lines with homogenous O-glycosylation. Three SimpleCell lines expressing only truncated GalNAcα or NeuAcα2-6GalNAcα O-glycans were produced, allowing straightforward isolation and sequencing of GalNAc O-glycopeptides from total cell lysates using lectin chromatography and nanoflow liquid chromatography-mass spectrometry (nLC-MS/MS) with electron transfer dissociation fragmentation. We identified >100 O-glycoproteins with >350 O-glycan sites (the great majority previously unidentified), including a GalNAc O-glycan linkage to a tyrosine residue. The SimpleCell method should facilitate analyses of important functions of protein glycosylation. The strategy is also applicable to other O-glycoproteomes.
Michael C Holmes - One of the best experts on this subject based on the ideXlab platform.
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Disruption of the BCL11A Erythroid Enhancer Reactivates Fetal Hemoglobin in Erythroid Cells of Patients with β-Thalassemia Major
Elsevier, 2018Co-Authors: Nikoletta Psatha, Fyodor D Urnov, Michael C Holmes, Andreas Reik, Susan Phelps, Yuanyue Zhou, Demetri Dalas, Evangelia Yannaki, Dana N. Levasseur, Thalia PapayannopoulouAbstract:In the present report, we carried out clinical-scale editing in adult mobilized CD34+ hematopoietic stem and progenitor cells (HSPCs) using Zinc-Finger Nuclease-mediated disruption of BCL11a to upregulate the expression of γ-globin (fetal hemoglobin). In these cells, disruption of the erythroid-specific enhancer of the BCL11A gene increased endogenous γ-globin expression to levels that reached or exceeded those observed following knockout of the BCL11A coding region without negatively affecting survival or in vivo long-term proliferation of edited HSPCs and other lineages. In addition, BCL11A enhancer modification in mobilized CD34+ cells from patients with β-thalassemia major resulted in a readily detectable γ-globin increase with a preferential increase in G-gamma, leading to an improved phenotype and, likely, a survival advantage for maturing erythroid cells after editing. Furthermore, we documented that both normal and β-thalassemia HSPCs not only can be efficiently expanded ex vivo after editing but can also be successfully edited post-expansion, resulting in enhanced early in vivo engraftment compared with unexpanded cells. Overall, this work highlights a novel and effective treatment strategy for correcting the β-thalassemia phenotype by genome editing. Keywords: hemoglobinopathies, BCL11a, genome editing, thalassemia, HbF reactivation, Zinc Finger Nuclease
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484 in vivo Zinc Finger Nuclease mediated iduronate 2 sulfatase ids target gene insertion and correction of metabolic disease in a mouse model of mucopolysaccharidosis type ii mps ii
Molecular Therapy, 2016Co-Authors: Kanut Laoharawee, Russell Dekelver, Kelly M Podetzpetersen, Susan Tom, Robert Radeke, Michelle Rohde, Amy Manningbog, Scott Sproul, Kathleen Meyer, Michael C HolmesAbstract:Hunter syndrome (Mucopolysaccharidosis Type II, MPS II) is a rare X-linked lysosomal disorder caused by lack of functional iduronate-2 sulfatase (IDS) enzyme and subsequent accumulation of glycosaminoglycans (GAG) in affected individuals. Manifestations include skeleton dysplasia, splenohepatomegaly, cardiopulmonary obstruction, and shortened life expectancy. In severe cases there is also neurologic impairment. Enzyme replacement therapy (ERT) is currently the only FDA-approved treatment to manage disease progression; however, ERT does not affect neurological aspects of the disease and requires that patients receive long and costly infusions of replacement factor on a frequent basis. We have developed a Zinc-Finger Nuclease (ZFN) approach to insert the human IDS (hIDS) coding sequence into the albumin locus using AAV2/8 vectors. In this study IDS-deficient MPS II mice (n= 8-13 per group, age 7-8 weeks) were treated by intravenous infusion of a mixture of ZFN-encoding AAV vectors along with an AAV vector encoding the hIDS partial cDNA flanked by albumin sequence homology arms at three different vector doses. Wild-type littermates, untreated MPS II mice, and MPS II animals infused only with the hIDS donor vector (without ZFN-encoding vectors) were included as controls. Successful insertion of the hIDS coding sequence will result in hIDS expression regulated by the endogenous albumin promoter. Plasma and tissue IDS activities as well as urine and tissue GAG contents were monitored throughout the study to evaluate the effectiveness of the treatment. Sufficient animals were maintained for neurobehavioral testing at four-months post-injection to determine whether the treatment is neurologically beneficial. We found that IDS activities in the plasma of the treated groups were 10- to 100-fold higher than wild-type and stably expressed through the entire study duration in a dose-dependent fashion, while only very low levels of IDS activity were found in the animals infused with hIDS donor vector alone. At 4 weeks post-treatment IDS activities in peripheral tissues ranged from 1% to 200% wild-type in a dose-dependent fashion, while in the hIDS donor-only group enzyme activity was not detected in any tissue except liver (10% that of wild-type). We observed up to 2% of the wild-type IDS activity in the brains of animals administered the complete set of AAV vectors, while no IDS activity was observed in the brains of animals infused with the IDS donor vector alone. Urine GAGs were reduced in all of the ZFN + Donor treatment groups regardless of the vector dose. Tissue GAGs in the treatment groups were also decreased, but GAG content in the brain was not different from untreated MPS II litter mates at the initial analysis conducted four weeks post-treatment. No tissue GAG reduction was observed in animals infused with hIDS donor vector alone. Before conclusion of this study, animals from all six groups will be tested in the Barnes Maze as neurobehavioral assessment to determine the neurological effect of targeted hIDS expression in the liver. These results together with hIDS expression and GAG level data from final necropsy tissues will be presented. These results demonstrate that ZFN can be effectively used to mediate in vivo insertion of the hIDS coding sequence into the albumin locus with resultant stable and high-level hIDS enzyme expression and metabolic correction in MPS II.
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highly efficient homology driven genome editing in human t cells by combining Zinc Finger Nuclease mrna and aav6 donor delivery
Nucleic Acids Research, 2016Co-Authors: Jianbin Wang, Philip D Gregory, Joshua J Declercq, Samuel B Hayward, David A Shivak, Gary Lee, Michael C HolmesAbstract:The adoptive transfer of engineered T cells for the treatment of cancer, autoimmunity, and infectious disease is a rapidly growing field that has shown great promise in recent clinical trials. Nuclease-driven genome editing provides a method in which to precisely target genetic changes to further enhance T cell function in vivo. We describe the development of a highly efficient method to genome edit both primary human CD8 and CD4 T cells by homology-directed repair at a pre-defined site of the genome. Two different homology donor templates were evaluated, representing both minor gene editing events (restriction site insertion) to mimic gene correction, or the more significant insertion of a larger gene cassette. By combining Zinc Finger Nuclease mRNA delivery with AAV6 delivery of a homologous donor we could gene correct 41% of CCR5 or 55% of PPP1R12C (AAVS1) alleles in CD8(+) T cells and gene targeting of a GFP transgene cassette in >40% of CD8(+) and CD4(+) T cells at both the CCR5 and AAVS1 safe harbor locus, potentially providing a robust genome editing tool for T cell-based immunotherapy.
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Zinc Finger Nuclease editing of human cxcr4 promotes hiv 1 cd4 t cell resistance and enrichment
Molecular Therapy, 2012Co-Authors: Jinyun Yuan, Philip D Gregory, Michael C Holmes, Jianbin Wang, Karen Crain, Colleen Fearns, Kenneth Kim, Kevin L Hua, Bruce E TorbettAbstract:HIV-1-infected individuals can harbor viral isolates that can use CCR5, as well as CXCR4, for viral entry. To genetically engineer HIV-1 resistance in CD4+ T cells, we assessed whether transient, adenovirus delivered Zinc-Finger Nuclease (ZFN) disruption of genomic cxcr4 or stable lentiviral expression of short hairpin RNAs (shRNAs) targeting CXCR4 mRNAs provides durable resistance to HIV-1 challenge. ZFN-modification of cxcr4 in CD4+ T cells was found to be superior to cell integrated lentivirus-expressing CXCR4 targeting shRNAs when CD4+ T cells were challenged with HIV-1s that utilizes CXCR4 for entry. Cxcr4 disruption in CD4+ T cells was found to be stable, conferred resistance, and provided for continued cell enrichment during HIV-1 infection in tissue culture and, in vivo, in peripheral blood mononuclear cell transplanted NSG mice. Moreover, HIV-1-infected mice with engrafted cxcr4 ZFN-modified CD4+ T cells demonstrated lower viral levels in contrast to mice engrafted with unmodified CD4+ T cells. These findings provide evidence that ZFN-mediated disruption of cxcr4 provides a selective advantage to CD4+ T cells during HIV-1 infection.
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enhancing Zinc Finger Nuclease activity with improved obligate heterodimeric architectures
Nature Methods, 2011Co-Authors: Yannick Doyon, Thuy D Vo, Matthew C Mendel, Shon G Greenberg, Jeffrey C Miller, Fyodor D Urnov, Philip D Gregory, Jianbin Wang, Michael C HolmesAbstract:Identification of residues critical for dimerization of the Fok1 Nuclease domain of Zinc-Finger Nucleases permits rational design of enzymes with improved cleavage activity and retained obligate heterodimerization.
Steven B Levery - One of the best experts on this subject based on the ideXlab platform.
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corrigendum mining the o glycoproteome using Zinc Finger Nuclease glycoengineered simplecell lines
Nature Methods, 2015Co-Authors: Catharina Steentoft, Eric P Bennett, Sergey Y Vakhrushev, Yun Kong, Ulla Mandel, Steven B Levery, Katrine T Schjoldager, Hans H Wandall, Malene Bech Vesterchristensen, Henrik ClausenAbstract:Nat. Methods 8, 977–982 (2011); published online 9 October 2011; corrected after print 8 January 2015 In the version of this article initially published, the T3M4 cell line used for the experiments was mislabeled as Capan-1. The error has been corrected in the HTML and PDF versions of the article.
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probing isoform specific functions of polypeptide galnac transferases using Zinc Finger Nuclease glycoengineered simplecells
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Katrine T Schjoldager, Catharina Steentoft, Eric P Bennett, Sergey Y Vakhrushev, Yun Kong, Ulla Mandel, Aaron S Nudelman, Nis Borbye Pedersen, Hans H Wandall, Steven B LeveryAbstract:Our knowledge of the O-glycoproteome [N-acetylgalactosamine (GalNAc) type] is highly limited. The O-glycoproteome is differentially regulated in cells by dynamic expression of a subset of 20 polypeptide GalNAc-transferases (GalNAc-Ts), and methods to identify important functions of individual GalNAc-Ts are largely unavailable. We recently introduced SimpleCells, i.e., human cell lines made deficient in O-glycan extension by Zinc Finger Nuclease targeting of a key gene in O-glycan elongation (Cosmc), which allows for proteome-wide discovery of O-glycoproteins. Here we have extended the SimpleCell concept to include proteome-wide discovery of unique functions of individual GalNAc-Ts. We used the GalNAc-T2 isoform implicated in dyslipidemia and the human HepG2 liver cell line to demonstrate unique functions of this isoform. We confirm that GalNAc-T2–directed site-specific O-glycosylation inhibits proprotein activation of the lipase inhibitor ANGPTL3 in HepG2 cells and further identify eight O-glycoproteins exclusively glycosylated by T2 of which one, ApoC-III, is implicated in dyslipidemia. Our study supports an essential role for GalNAc-T2 in lipid metabolism, provides serum biomarkers for GalNAc-T2 enzyme function, and validates the use of GALNT gene targeting with SimpleCells for broad discovery of disease-causing deficiencies in O-glycosylation. The presented glycoengineering strategy opens the way for proteome-wide discovery of functions of GalNAc-T isoforms and their role in congenital diseases and disorders.
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mining the o glycoproteome using Zinc Finger Nuclease glycoengineered simplecell lines
Nature Methods, 2011Co-Authors: Catharina Steentoft, Eric P Bennett, Sergey Y Vakhrushev, Yun Kong, Ulla Mandel, Steven B Levery, Katrine T Schjoldager, Hans H Wandall, Malene Bech Vesterchristensen, Henrik ClausenAbstract:Owing to a lack of tools and a lack of a consensus sequence for O-glycosylation, studies of the O-glycoproteome have been few and far between, despite the biological importance of O-glycosylation. This method to analyze O-glycan attachment sites to proteins using glycoengineered cell lines with simplified, homogenous O-glycoproteomes should facilitate future O-glycoproteomics studies.
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mining the o glycoproteome using Zinc Finger Nuclease glycoengineered simplecell lines
Nature Methods, 2011Co-Authors: Catharina Steentoft, Eric P Bennett, Sergey Y Vakhrushev, Yun Kong, Ulla Mandel, Steven B Levery, Katrine T Schjoldager, Hans H Wandall, Malene Bech Vesterchristensen, Henrik ClausenAbstract:Zinc-Finger Nuclease (ZFN) gene targeting is emerging as a versatile tool for engineering of multiallelic gene deficiencies. A longstanding obstacle for detailed analysis of glycoproteomes has been the extensive heterogeneities in glycan structures and attachment sites. Here we applied ZFN targeting to truncate the O-glycan elongation pathway in human cells, generating stable 'SimpleCell' lines with homogenous O-glycosylation. Three SimpleCell lines expressing only truncated GalNAcα or NeuAcα2-6GalNAcα O-glycans were produced, allowing straightforward isolation and sequencing of GalNAc O-glycopeptides from total cell lysates using lectin chromatography and nanoflow liquid chromatography-mass spectrometry (nLC-MS/MS) with electron transfer dissociation fragmentation. We identified >100 O-glycoproteins with >350 O-glycan sites (the great majority previously unidentified), including a GalNAc O-glycan linkage to a tyrosine residue. The SimpleCell method should facilitate analyses of important functions of protein glycosylation. The strategy is also applicable to other O-glycoproteomes.
