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https://www.readbyqxmd.com/read/29776993/tumor-immune-evasion-arises-through-loss-of-tnf-sensitivity
#1
Conor J Kearney, Stephin J Vervoort, Simon J Hogg, Kelly M Ramsbottom, Andrew J Freeman, Najoua Lalaoui, Lizzy Pijpers, Jessica Michie, Kristin K Brown, Deborah A Knight, Vivien Sutton, Paul A Beavis, Ilia Voskoboinik, Phil K Darcy, John Silke, Joseph A Trapani, Ricky W Johnstone, Jane Oliaro
Immunotherapy has revolutionized outcomes for cancer patients, but the mechanisms of resistance remain poorly defined. We used a series of whole-genome clustered regularly interspaced short palindromic repeat (CRISPR)-based screens performed in vitro and in vivo to identify mechanisms of tumor immune evasion from cytotoxic lymphocytes [CD8+ T cells and natural killer (NK) cells]. Deletion of key genes within the tumor necrosis factor (TNF) signaling, interferon-γ (IFN-γ) signaling, and antigen presentation pathways provided protection of tumor cells from CD8+ T cell-mediated killing and blunted antitumor immune responses in vivo...
May 18, 2018: Science Immunology
https://www.readbyqxmd.com/read/29764210/curative-ex-vivo-hepatocyte-directed-gene-editing-in-a-mouse-model-of-hereditary-tyrosinemia-type-1
#2
Caitlin VanLith, Rebekah Guthman, Clara T Nicolas, Kari Allen, Zeji Du, Dong Jin Joo, Scott L Nyberg, Joseph B Lillegard, Raymond Daniel Hickey
Hereditary tyrosinemia type 1 (HT1) is an autosomal recessive disorder caused by deficiency of fumarylacetoacetate hydrolase (FAH). It has been previously shown that ex vivo hepatocyte-directed gene therapy using an integrating lentiviral vector to replace the defective Fah gene can cure liver disease in small and large animal models of HT1. In this study, we hypothesized that ex vivo hepatocyte-directed gene editing using CRISPR-Cas9 could be used to correct a mouse model of HT1, in which a single point mutation results in loss of FAH function...
May 15, 2018: Human Gene Therapy
https://www.readbyqxmd.com/read/29760953/hyperglycemia-potentiates-a-shift-from-apoptosis-to-rip1-dependent-necroptosis
#3
William D McCaig, Payal S Patel, Sergey A Sosunov, Nicole L Shakerley, Tori A Smiraglia, Miranda M Craft, Katharine M Walker, Matthew A Deragon, Vadim S Ten, Timothy J LaRocca
Apoptosis and necroptosis are the primary modes of eukaryotic cell death, with apoptosis being non-inflammatory while necroptosis is highly inflammatory. We previously demonstrated that, once activated, necroptosis is enhanced by hyperglycemia in several cell types. Here, we determine if hyperglycemia affects apoptosis similarly. We show that hyperglycemia does not enhance extrinsic apoptosis but potentiates a shift to RIP1-dependent necroptosis. This is due to increased levels and activity of RIP1, RIP3, and MLKL, as well as decreased levels and activity of executioner caspases under hyperglycemic conditions following stimulation of apoptosis...
2018: Cell Death Discovery
https://www.readbyqxmd.com/read/29743177/hypomorphic-rag1-mutations-alter-the-pre-immune-repertoire-at-early-stages-of-lymphoid-development
#4
Lisa M Ott de Bruin, Marita Bosticardo, Alessandro Barbieri, Sherry G Lin, Jared H Rowe, Pietro L Poliani, Kimberly Ching, Daniel Eriksson, Nils Landegren, Olle Kämpe, John P Manis, Luigi D Notarangelo
Hypomorphic RAG1 mutations allowing residual T and B cell development have been found in patients presenting with delayed-onset combined immune deficiency with granulomas and/or autoimmunity (CID-G/AI) and abnormalities of the peripheral T and B cell repertoire. To examine how hypomorphic Rag1 mutations affect the earliest stages of lymphocyte development, we used CRISPR/Cas9 to generate mouse models with equivalent mutations found in patients with CID-G/AI. Immunological characterization showed partial development of T and B lymphocytes, with persistence of naïve cells, preserved serum immunoglobulin, but impaired antibody responses and presence of autoantibodies, thereby recapitulating the phenotype seen in patients with CID-G/AI...
May 9, 2018: Blood
https://www.readbyqxmd.com/read/29709474/keratinocyte-growth-factor-protects-endometrial-cells-from-oxygen-glucose-deprivation-re-oxygenation-via-activating-nrf2-signaling
#5
Xuting Shi, Hia-Yan Liu, Shu-Ping Li, Hong-Bin Xu
Oxygen and glucose deprivation (OGD)-re-oxygenation (OGDR) exposure to endometrial cells mimics ischemia-reperfusion injury. The present study tests the potential effect of keratinocyte growth factor (KGF) on the process. We show that KGF receptor KGFR is expressed in human endometrial T-HESC cells and primary murine endometrial cells. KGF pre-treatment protected endometrial cells from OGDR, inhibiting cell viability reduction and cell death. KGF attenuated OGDR-induced programmed necrosis in endometrial cells...
April 27, 2018: Biochemical and Biophysical Research Communications
https://www.readbyqxmd.com/read/29706119/nucleofection-with-plasmid-dna-for-crispr-cas9-mediated-inactivation-of-pd-1-in-cd133-specific-car-t-cells
#6
Bian Hu, Yan Zou, Linlin Zhang, Jiaxing Tang, Gabriele Niedermann, Elke Firat, Xingxu Huang, Xuekai Zhu
CRISPR/Cas9-mediated PD-1 disruption in chimeric antigen receptor (CAR) T cells could be an appealing choice to improve the therapeutic efficacy of CAR T cells in an immunosuppressive tumor microenvironment. In most of the reported cases, Cas9 was delivered into T cells by way of electroporation with RNA or protein. However, transient expression of Cas9 by transfection with a plasmid encoding its gene is apparently simpler, as it avoids the steps of <i>in vitro</i> transcription of DNA or protein production...
April 28, 2018: Human Gene Therapy
https://www.readbyqxmd.com/read/29565806/crispr-cas9-based-cellular-engineering-for-targeted-gene-overexpression
#7
Mark J Osborn, Christopher J Lees, Amber N McElroy, Sarah C Merkel, Cindy R Eide, Wendy Mathews, Colby J Feser, Madison Tschann, Ron T McElmury, Beau R Webber, Chong Jai Kim, Bruce R Blazar, Jakub Tolar
Gene and cellular therapies hold tremendous promise as agents for treating genetic disorders. However, the effective delivery of genes, particularly large ones, and expression at therapeutic levels can be challenging in cells of clinical relevance. To address this engineering hurdle, we sought to employ the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system to insert powerful regulatory elements upstream of an endogenous gene. We achieved robust activation of the COL7A1 gene in primary human umbilical cord blood CD34⁺ hematopoietic stem cells and peripheral blood T-cells...
March 22, 2018: International Journal of Molecular Sciences
https://www.readbyqxmd.com/read/29507424/crispr-cas9-screens-in-human-cells-and-primary-neurons-identify-modifiers-of-c9orf72-dipeptide-repeat-protein-toxicity
#8
Nicholas J Kramer, Michael S Haney, David W Morgens, Ana Jovičić, Julien Couthouis, Amy Li, James Ousey, Rosanna Ma, Gregor Bieri, C Kimberly Tsui, Yingxiao Shi, Nicholas T Hertz, Marc Tessier-Lavigne, Justin K Ichida, Michael C Bassik, Aaron D Gitler
Hexanucleotide-repeat expansions in the C9ORF72 gene are the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (c9ALS/FTD). The nucleotide-repeat expansions are translated into dipeptide-repeat (DPR) proteins, which are aggregation prone and may contribute to neurodegeneration. We used the CRISPR-Cas9 system to perform genome-wide gene-knockout screens for suppressors and enhancers of C9ORF72 DPR toxicity in human cells. We validated hits by performing secondary CRISPR-Cas9 screens in primary mouse neurons...
April 2018: Nature Genetics
https://www.readbyqxmd.com/read/29483708/an-off-the-shelf-fratricide-resistant-car-t-for-the-treatment-of-t-cell-hematologic-malignancies
#9
Matthew L Cooper, Jaebok Choi, Karl Staser, Julie K Ritchey, Jessica M Devenport, Kayla Eckardt, Michael P Rettig, Bing Wang, Linda G Eissenberg, Armin Ghobadi, Leah N Gehrs, Julie L Prior, Samuel Achilefu, Christopher A Miller, Catrina C Fronick, Julie O'Neal, Feng Gao, David M Weinstock, Alejandro Gutierrez, Robert S Fulton, John F DiPersio
T cell malignancies represent a group of hematologic cancers with high rates of relapse and mortality in patients for whom no effective targeted therapies exist. The shared expression of target antigens between chimeric antigen receptor (CAR) T cells and malignant T cells has limited the development of CAR-T because of unintended CAR-T fratricide and an inability to harvest sufficient autologous T cells. Here, we describe a fratricide-resistant "off-the-shelf" CAR-T (or UCART7) that targets CD7+ T cell malignancies and, through CRISPR/Cas9 gene editing, lacks both CD7 and T cell receptor alpha chain (TRAC) expression...
February 20, 2018: Leukemia: Official Journal of the Leukemia Society of America, Leukemia Research Fund, U.K
https://www.readbyqxmd.com/read/29480524/transgenic-mouse-lines-expressing-the-3xflag-dcas9-protein-for-enchip-analysis
#10
Toshitsugu Fujita, Fusako Kitaura, Asami Oji, Naoki Tanigawa, Miyuki Yuno, Masahito Ikawa, Ichiro Taniuchi, Hodaka Fujii
We developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technology to isolate specific genomic regions while retaining their molecular interactions. In enChIP, the locus of interest is tagged with an engineered DNA-binding molecule, such as a modified form of the clustered regularly interspaced short palindromic repeats (CRISPR) system containing a guide RNA (gRNA) and a catalytically inactive form of Cas9 (dCas9). The locus is then affinity-purified to enable identification of associated molecules...
April 2018: Genes to Cells: Devoted to Molecular & Cellular Mechanisms
https://www.readbyqxmd.com/read/29472270/crispr-rnas-trigger-innate-immune-responses-in-human-cells
#11
Sojung Kim, Taeyoung Koo, Hyeon-Gun Jee, Hee-Yeon Cho, Gyeorae Lee, Dong-Gyun Lim, Hyoung Shik Shin, Jin-Soo Kim
Here, we report that CRISPR guide RNAs (gRNAs) with a 5'-triphosphate group (5'-ppp gRNAs) produced via in vitro transcription trigger RNA-sensing innate immune responses in human and murine cells, leading to cytotoxicity. 5'-ppp gRNAs in the cytosol are recognized by DDX58, which in turn activates type I interferon responses, causing up to ∼80% cell death. We show that the triphosphate group can be removed by a phosphatase in vitro and that the resulting 5'-hydroxyl gRNAs in complex with Cas9 or Cpf1 avoid innate immune responses and can achieve targeted mutagenesis at a frequency of 95% in primary human CD4+ T cells...
February 22, 2018: Genome Research
https://www.readbyqxmd.com/read/29459677/a-crispr-based-screen-for-hedgehog-signaling-provides-insights-into-ciliary-function-and-ciliopathies
#12
David K Breslow, Sascha Hoogendoorn, Adam R Kopp, David W Morgens, Brandon K Vu, Margaret C Kennedy, Kyuho Han, Amy Li, Gaelen T Hess, Michael C Bassik, James K Chen, Maxence V Nachury
Primary cilia organize Hedgehog signaling and shape embryonic development, and their dysregulation is the unifying cause of ciliopathies. We conducted a functional genomic screen for Hedgehog signaling by engineering antibiotic-based selection of Hedgehog-responsive cells and applying genome-wide CRISPR-mediated gene disruption. The screen can robustly identify factors required for ciliary signaling with few false positives or false negatives. Characterization of hit genes uncovered novel components of several ciliary structures, including a protein complex that contains δ-tubulin and ε-tubulin and is required for centriole maintenance...
March 2018: Nature Genetics
https://www.readbyqxmd.com/read/29445007/highly-efficient-and-versatile-plasmid-based-gene-editing-in-primary-t-cells
#13
Mara Kornete, Romina Marone, Lukas T Jeker
Adoptive cell transfer is an important approach for basic research and emerges as an effective treatment for various diseases, including infections and blood cancers. Direct genetic manipulation of primary immune cells opens up unprecedented research opportunities and could be applied to enhance cellular therapeutic products. In this article, we report highly efficient genome engineering in primary murine T cells using a plasmid-based RNA-guided CRISPR system. We developed a straightforward approach to ablate genes in up to 90% of cells and to introduce precisely targeted single nucleotide polymorphisms in up to 25% of the transfected primary T cells...
April 1, 2018: Journal of Immunology: Official Journal of the American Association of Immunologists
https://www.readbyqxmd.com/read/29436394/optimized-rnp-transfection-for-highly-efficient-crispr-cas9-mediated-gene-knockout-in-primary-t-cells
#14
Akiko Seki, Sascha Rutz
CRISPR (clustered, regularly interspaced, short palindromic repeats)/Cas9 (CRISPR-associated protein 9) has become the tool of choice for generating gene knockouts across a variety of species. The ability for efficient gene editing in primary T cells not only represents a valuable research tool to study gene function but also holds great promise for T cell-based immunotherapies, such as next-generation chimeric antigen receptor (CAR) T cells. Previous attempts to apply CRIPSR/Cas9 for gene editing in primary T cells have resulted in highly variable knockout efficiency and required T cell receptor (TCR) stimulation, thus largely precluding the study of genes involved in T cell activation or differentiation...
March 5, 2018: Journal of Experimental Medicine
https://www.readbyqxmd.com/read/29399409/development-of-chimeric-antigen-receptors-targeting-t-cell-malignancies-using-two-structurally-different-anti-cd5-antigen-binding-domains-in-nk-and-crispr-edited-t-cell-lines
#15
Sunil S Raikar, Lauren C Fleischer, Robert Moot, Andrew Fedanov, Na Yoon Paik, Kristopher A Knight, Christopher B Doering, H Trent Spencer
Relapsed T-cell malignancies have poor outcomes when treated with chemotherapy, but survival after allogeneic bone marrow transplantation (BMT) approaches 50%. A limitation to BMT is the difficulty of achieving remission prior to transplant. Chimeric antigen receptor (CAR) T-cell therapy has shown successes in B-cell malignancies. This approach is difficult to adapt for the treatment of T-cell disease due to lack of a T-lymphoblast specific antigen and the fratricide of CAR T cells that occurs with T-cell antigen targeting...
2018: Oncoimmunology
https://www.readbyqxmd.com/read/29352001/-keap1-editing-using-crispr-cas9-for-therapeutic-nrf2-activation-in-primary-human-t-lymphocytes
#16
Sanjeev Noel, Sul A Lee, Mohanraj Sadasivam, Abdel R A Hamad, Hamid Rabb
Oxidant stress modifies T lymphocyte activation and function. Previous work demonstrated that murine T cell-specific kelch like-ECH-associated protein 1 ( Keap1 ) deletion enhances antioxidant capacity and protects from experimental acute kidney injury. In this study, we used CRISPR technology to develop clinically translatable human T cell-specific KEAP1 deletion. Delivery of KEAP1 exon 2 specific Cas9:guide RNA in Jurkat T cells led to significant (∼70%) editing and upregulation of NRF2-regulated antioxidant genes NADPH dehydrogenase quinone 1 ( NQO1 ) (up to 11-fold), heme oxygenase 1 ( HO1 ) (up to 11-fold), and GCLM (up to 2-fold)...
March 1, 2018: Journal of Immunology: Official Journal of the American Association of Immunologists
https://www.readbyqxmd.com/read/29321333/microrna-130a-regulates-both-hepatitis-c-virus-and-hepatitis-b-virus-replication-through-a-central-metabolic-pathway
#17
Xiaoqiong Duan, Shilin Li, Jacinta A Holmes, Zeng Tu, Yujia Li, Dachuan Cai, Xiao Liu, Wenting Li, Chunhui Yang, Baihai Jiao, Esperance A Schaefer, Dahlene N Fusco, Shadi Salloum, Limin Chen, Wenyu Lin, Raymond T Chung
Hepatitis C virus (HCV) infection has been shown to regulate microRNA 130a (miR-130a) in patient biopsy specimens and in cultured cells. We sought to identify miR-130a target genes and to explore the mechanisms by which miR-130a regulates HCV and hepatitis B virus (HBV) replication. We used bioinformatics software, including miRanda, TargetScan, PITA, and RNAhybrid, to predict potential miR-130a target genes. miR-130a and its target genes were overexpressed or were knocked down by use of small interfering RNA (siRNA) or clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 guide RNA (gRNA)...
April 1, 2018: Journal of Virology
https://www.readbyqxmd.com/read/29245982/endothelial-derived-interleukin-6-induces-cancer-stem-cell-motility-by-generating-a-chemotactic-gradient-towards-blood-vessels
#18
Hong Sun Kim, Yu-Chih Chen, Felipe Nör, Kristy A Warner, April Andrews, Vivian P Wagner, Zhaocheng Zhang, Zhixiong Zhang, Manoela D Martins, Alexander T Pearson, Euisik Yoon, Jacques E Nör
Recent evidence suggests that the metastatic spread of head and neck squamous cell carcinomas (HNSCC) requires the function of cancer stem cells endowed with multipotency, self-renewal, and high tumorigenic potential. We demonstrated that cancer stem cells reside in perivascular niches and are characterized by high aldehyde dehydrogenase (ALDH) activity and high CD44 expression (ALDHhigh CD44high ) in HNSCC. Here, we hypothesize that endothelial cell-secreted interleukin-6 (IL-6) contributes to tumor progression by enhancing the migratory phenotype and survival of cancer stem cells...
November 21, 2017: Oncotarget
https://www.readbyqxmd.com/read/29236701/anti-leukemic-activity-of-bortezomib-and-carfilzomib-on-b-cell-precursor-all-cell-lines
#19
Kazuya Takahashi, Takeshi Inukai, Toshihiko Imamura, Mio Yano, Chihiro Tomoyasu, David M Lucas, Atsushi Nemoto, Hiroki Sato, Meixian Huang, Masako Abe, Keiko Kagami, Tamao Shinohara, Atsushi Watanabe, Shinpei Somazu, Hiroko Oshiro, Koshi Akahane, Kumiko Goi, Jiro Kikuchi, Yusuke Furukawa, Hiroaki Goto, Masayoshi Minegishi, Shotaro Iwamoto, Kanji Sugita
Prognosis of childhood acute lymphoblastic leukemia (ALL) has been dramatically improved. However, prognosis of the cases refractory to primary therapy is still poor. Recent phase 2 study on the efficacy of combination chemotherapy with bortezomib (BTZ), a proteasome inhibitor, for refractory childhood ALL demonstrated favorable clinical outcomes. However, septic death was observed in over 10% of patients, indicating the necessity of biomarkers that could predict BTZ sensitivity. We investigated in vitro BTZ sensitivity in a large panel of ALL cell lines that acted as a model system for refractory ALL, and found that Philadelphia chromosome-positive (Ph+) ALL, IKZF1 deletion, and biallelic loss of CDKN2A were associated with favorable response...
2017: PloS One
https://www.readbyqxmd.com/read/29158345/stable-oxidative-cytosine-modifications-accumulate-in-cardiac-mesenchymal-cells-from-type2-diabetes-patients-rescue-by-%C3%AE-ketoglutarate-and-tet-tdg-functional-reactivation
#20
Francesco Spallotta, Chiara Cencioni, Sandra Atlante, Davide Garella, Mattia Cocco, Mattia Mori, Raffaella Mastrocola, Carsten Kuenne, Stefan Guenther, Simona Nanni, Valerio Azzimato, Sven Zukunft, Angela Kornberger, Duran Sürün, Frank Schnütgen, Harald von Melchner, Antonella Di Stilo, Manuela Aragno, Maarten Braspenning, Wim van Criekinge, Miles J De Blasio, Rebecca H Ritchie, Germana Zaccagnini, Fabio Martelli, Antonella Farsetti, Ingrid Fleming, Thomas Braun, Andres Beiras-Fernandez, Bruno Botta, Massimo Collino, Massimo Bertinaria, Andreas M Zeiher, Carlo Gaetano
RATIONALE: Human cardiac mesenchymal cells (CMSCs) are a therapeutically relevant primary cell population. Diabetes mellitus compromises CMSC function as consequence of metabolic alterations and incorporation of stable epigenetic changes. OBJECTIVE: To investigate the role of α-ketoglutarate (αKG) in the epimetabolic control of DNA demethylation in CMSCs. METHODS AND RESULTS: Quantitative global analysis, methylated and hydroxymethylated DNA sequencing, and gene-specific GC methylation detection revealed an accumulation of 5-methylcytosine, 5-hydroxymethylcytosine, and 5-formylcytosine in the genomic DNA of human CMSCs isolated from diabetic donors...
January 5, 2018: Circulation Research
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