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Assay for Quantitative Evaluation of Target Site Cleavage by One Or More Crispr-Cas Guide Sequences

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(19) *EP003011035B1* (11) EP 3 011 035 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C12N 15/63 (2006.01) C12N 15/10 (2006.01) (2006.01) (2006.01) 13.05.2020 Bulletin 2020/20 C40B 40/08 C12N 9/22 (21) Application number: 14738672.6 (86) International application number: PCT/US2014/041790 (22) Date of filing: 10.06.2014 (87) International publication number: WO 2014/204723 (24.12.2014 Gazette 2014/52) (54) ASSAY FOR QUANTITATIVE EVALUATION OF TARGET SITE CLEAVAGE BY ONE OR MORE CRISPR-CAS GUIDE SEQUENCES TEST ZUR QUANTITATIVEN BEWERTUNG DER ZIELSTELLENSPALTUNG DURCH EINE ODER MEHRERE CRISPR-CAS FÜHRUNGSSEQUENZEN TEST POUR L’ÉVALUATION QUANTITATIVE DU CLIVAGE DE SITES CIBLES PAR UNE OU PLUSIEURS SÉQUENCES GUIDES DU SYSTÈME CRISPR-CAS (84) Designated Contracting States: (56) References cited: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB • GAJ THOMAS ET AL: "ZFN, TALEN, and GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO CRISPR/Cas-based methods for genome PL PT RO RS SE SI SK SM TR engineering", TRENDS IN BIOTECHNOLOGY, ELSEVIER PUBLICATIONS, CAMBRIDGE, GB, (30) Priority: 17.06.2013 US 201361836123 P vol. 31, no. 7, 9 May 2013 (2013-05-09), pages 12.12.2013 US 201361915397 P 397-405, XP028571313, ISSN: 0167-7799, DOI: 10.1016/J.TIBTECH.2013.04.004 (43) Date of publication of application: • JANSSEN K P ET AL: "Mouse models of 27.04.2016 Bulletin 2016/17 K-ras-initiated carcinogenesis", BBA - REVIEWS ON CANCER, ELSEVIER SCIENCE BV, (73) Proprietors: AMSTERDAM, NL, vol. 1756, no. 2, 25 November • The Broad Institute, Inc. 2005 (2005-11-25), pages 145-154, XP027829577, Cambridge, MA 02142 (US) ISSN: 0304-419X [retrieved on 2005-11-25] • Massachusetts Institute of Technology • B. C. LEWIS: "The c-myc and PyMT oncogenes Cambridge, MA 02139 (US) induce different tumor types in a somatic mouse • The Brigham and Women’s Hospital, Inc. model for pancreatic cancer", GENES & Boston, MA 02115 (US) DEVELOPMENT, vol. 17, no. 24, 15 December 2003 (2003-12-15), pages 3127-3138, XP55049590, (72) Inventors: ISSN: 0890-9369, DOI: 10.1101/gad.1140403 • ZHANG, Feng • L. CONG ET AL: "Multiplex Genome Engineering Cambrige, MA 02139 (US) Using CRISPR/Cas Systems", SCIENCE, vol. 339, • EBERT, Benjamin, Levine no. 6121, 3 January 2013 (2013-01-03), pages Brookline, MA 02445 (US) 819-823, XP055102030, ISSN: 0036-8075, DOI: • HECKL, Dirk 10.1126/science.1231143 29352 Adelheidsdorf (DE) • P. MALI ET AL: "RNA-Guided Human Genome Engineering via Cas9", SCIENCE, vol. 339, no. (74) Representative: Icely, Dominic Michael 6121, 3 January 2013 (2013-01-03), pages 823-826, The IP Asset Partnership Limited XP055111247, ISSN: 0036-8075, DOI: Prama House 10.1126/science.1232033 267 Banbury Road Oxford OX2 7HT (GB) Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 3 011 035 B1 Printed by Jouve, 75001 PARIS (FR) (Cont. next page) EP 3 011 035 B1 • LOMBARDO ANGELO ET AL: "Gene editing in • LUKE A. GILBERT ET AL: "CRISPR-Mediated human stem cells using zinc finger nucleases and Modular RNA-Guided Regulation of integrase-defective lentiviral vector delivery", Transcription in Eukaryotes", CELL, vol. 154, no. NATURE BIOTECHNOLOGY, NATURE 2, 1 July 2013 (2013-07-01), pages 442-451, PUBLISHING GROUP, NEW YORK, NY, US, vol. XP055115843, ISSN: 0092-8674, DOI: 25, no. 11, 1 November 2007 (2007-11-01), pages 10.1016/j.cell.2013.06.044 1298-1306, XP002465562, ISSN: 1087-0156, DOI: • MALINA ABBA ET AL: "Repurposing 10.1038/NBT1353 CRISPR/Cas9 for in situ functional assays", 1 • L. CONG ET AL: "Supplementary Material to : December 2013 (2013-12-01), GENES & Multiplex Genome Engineering Using DEVELOPMENT, VOL. 27, NR. 23, PAGE(S) CRISPR/Cas Systems", SCIENCE, vol. 339, no. 2602-2614 ISSN: 0890-9369(print) 6121, 3 January 2013 (2013-01-03), pages 1-25, • N. Manjunath ET AL: "Newer Gene Editing XP002730884, ISSN: 0036-8075, DOI: Technologies toward HIV Gene Therapy", 10.1126/science.1231143 Viruses, vol. 5, no. 11, 14 November 2013 • WANG HAOYI ET AL: "One-Step Generation of (2013-11-14), pages 2748-2766, XP055110267, Mice Carrying Mutations in Multiple Genes by DOI: 10.3390/v5112748 CRISPR/Cas-Mediated Genome Engineering", • Hirotaka Ebina ET AL: "Harnessing the CELL, CELL PRESS, US, vol. 153, no. 4, 2 May CRISPR/Cas9 system to disrupt latent HIV-1 2013 (2013-05-02), pages 910-918, XP028538358, provirus", Scientific Reports, vol. 3, 26 August ISSN: 0092-8674, DOI: 2013 (2013-08-26), XP055110157, DOI: 10.1016/J.CELL.2013.04.025 10.1038/srep02510 • SHALEM OPHIR ET AL: "Genome-Scale • MANNELL HANNA ET AL: "Targeted Endothelial CRISPR-Cas9 Knockout Screening in Human Gene Delivery by Ultrasonic Destruction of Cells", January 2014 (2014-01), SCIENCE Magnetic Microbubbles Carrying Lentiviral (WASHINGTON D C), VOL. 343, NR. 6166, Vectors", PHARMACEUTICAL RESEARCH PAGE(S) 84-87, XP002723432, ISSN: (DORDRECHT), vol. 29, no. 5, May 2012 (2012-05), 0036-8075(print) the whole document pages 1282-1294, ISSN: 0724-8741(print) • HSU PATRICK D ET AL: "Development and Applications of CRISPR-Cas9 for Genome Engineering", CELL, vol. 157, no. 6, 5 June 2014 (2014-06-05), pages 1262-1278, XP028849523, ISSN: 0092-8674, DOI: 10.1016/J.CELL.2014.05.010 2 EP 3 011 035 B1 Description FIELD OF THE INVENTION 5 [0001] The present disclosure generally relates to the delivery, engineering, optimization and therapeutic applications of systems, methods, compositions and vectors used for the control of gene expression involving sequence targeting, such as genome perturbation or gene-editing, that relate to Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and components thereof. 10 STATEMENT AS TO FEDERALLY SPONSORED RESEARCH [0002] This invention was made with government support under the NIH Pioneer Award (1DP1MH100706) and NIH (P01 CA108631) grant awarded by the National Institutes of Health. The government has certain rights in the invention. 15 BACKGROUND OF THE INVENTION [0003] Recent advances in genome sequencing techniques and analysis methods have significantly accelerated the ability to catalog and map genetic factors associated with a diverse range of biological functions and diseases. Precise genome targeting technologies are needed to enable systematic reverse engineering of causal genetic variations by 20 allowing selective perturbation of individual genetic elements, as well as to advance synthetic biology, biotechnological, and medical applications. Although genome-editing techniques such as designer zinc fingers, transcription activator- like effectors (TALEs), or homing meganucleases are available for producing targeted genome perturbations, there remains a need for new genome engineering technologies that are affordable, easy to set up, scalable, and amenable to targeting multiple positions within the eukaryotic genome. 25 SUMMARY OF THE INVENTION [0004] The CRISPR-Cas system does not require the generation of customized proteins to target specific sequences but rather a single Cas enzyme can be programmed by a short RNA molecule to recognize a specific DNA target. Adding 30 the CRISPR-Cas system to the repertoire of genome sequencing techniques and analysis methods may significantly simplify the methodology and accelerate the ability to catalog and map genetic factors associated with a diverse range of biological functions and diseases. To utilize the CRISPR-Cas system effectively for genome editing without deleterious effects, it is critical to understand aspects of engineering, optimization and tissue/organ specific delivery of these genome engineering tools, which are aspects of the claimed invention. 35 [0005] There exists a pressing need for alternative and robust systems and techniques for sequence targeting with a wide array of applications. Aspects of this invention address this need and provides related advantages. An exemplary CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence hybridized to a target sequence within the target polynucleotide. The guide sequence is linked to a tracr mate sequence, which in turn hybridizes to a tracr sequence. 40 [0006] In one aspect, the disclosure provides methods for using one or more elements of a CRISPR system. The CRISPR complex provides an effective means for modifying a target polynucleotide. The CRISPR complex has a wide variety of utilities including modifying (e.g., deleting, inserting, translocating, inactivating, activating) a target polynucle- otide in a multiplicity of cell types in various tissues and organs. As such the CRISPR complex has a broad spectrum of applications in, e.g., gene or genome editing, gene therapy, generation of cell and animal model systems, drug 45 discovery, drug screening, disease diagnosis, and prognosis. [0007] Aspects of the disclosure relate to the CRISPR-Cas system having the utility of engineering primary cells that recapitulate the genetic complexity of human disease. This complexity arises from the interaction of multiple genetic lesions, which is particularly true in the case of proliferative disorders like cancer. [0008] The disclosure comprehends a method of inducing a proliferative condition in a an organism comprising: isolating 50 a first population of cells from the organism, transducing the first population of cells with a non-naturally occurring or engineered composition comprising a vector system comprising one or more vectors comprising I. a first regulatory element operably linked to a CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide sequence, wherein the poly- nucleotide sequence comprises (a) three or more guide sequences capable of hybridizing to three or more target se- quences in genome of the organism, (b) a tracr mate sequence, and (c) a tracr sequence, and II.
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    www.oncotarget.com Oncotarget, 2019, Vol. 10, (No. 24), pp: 2384-2396 Research Paper Clinical validation of the tempus xT next-generation targeted oncology sequencing assay Nike Beaubier1, Robert Tell1, Denise Lau1, Jerod R. Parsons1, Stephen Bush1, Jason Perera1, Shelly Sorrells1, Timothy Baker1, Alan Chang1, Jackson Michuda1, Catherine Iguartua1, Shelley MacNeil1, Kaanan Shah1, Philip Ellis1, Kimberly Yeatts1, Brett Mahon1, Timothy Taxter1, Martin Bontrager1, Aly Khan1, Robert Huether1, Eric Lefkofsky1 and Kevin P. White1 1Tempus Labs Inc., Chicago, IL 60654, USA Correspondence to: Nike Beaubier, email: [email protected] Kevin P. White, email: [email protected] Keywords: tumor profiling, next-generation sequencing assay validation Received: August 03, 2018 Accepted: February 03, 2019 Published: March 22, 2019 Copyright: Beaubier et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ABSTRACT We developed and clinically validated a hybrid capture next generation sequencing assay to detect somatic alterations and microsatellite instability in solid tumors and hematologic malignancies. This targeted oncology assay utilizes tumor- normal matched samples for highly accurate somatic alteration calling and whole transcriptome RNA sequencing for unbiased identification of gene fusion events. The assay was validated with a combination of clinical specimens and cell lines, and recorded a sensitivity of 99.1% for single nucleotide variants, 98.1% for indels, 99.9% for gene rearrangements, 98.4% for copy number variations, and 99.9% for microsatellite instability detection. This assay presents a wide array of data for clinical management and clinical trial enrollment while conserving limited tissue.
  • Transcriptomic and Proteomic Profiling Provides Insight Into

    Transcriptomic and Proteomic Profiling Provides Insight Into

    BASIC RESEARCH www.jasn.org Transcriptomic and Proteomic Profiling Provides Insight into Mesangial Cell Function in IgA Nephropathy † † ‡ Peidi Liu,* Emelie Lassén,* Viji Nair, Celine C. Berthier, Miyuki Suguro, Carina Sihlbom,§ † | † Matthias Kretzler, Christer Betsholtz, ¶ Börje Haraldsson,* Wenjun Ju, Kerstin Ebefors,* and Jenny Nyström* *Department of Physiology, Institute of Neuroscience and Physiology, §Proteomics Core Facility at University of Gothenburg, University of Gothenburg, Gothenburg, Sweden; †Division of Nephrology, Department of Internal Medicine and Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, Michigan; ‡Division of Molecular Medicine, Aichi Cancer Center Research Institute, Nagoya, Japan; |Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden; and ¶Integrated Cardio Metabolic Centre, Karolinska Institutet Novum, Huddinge, Sweden ABSTRACT IgA nephropathy (IgAN), the most common GN worldwide, is characterized by circulating galactose-deficient IgA (gd-IgA) that forms immune complexes. The immune complexes are deposited in the glomerular mesangium, leading to inflammation and loss of renal function, but the complete pathophysiology of the disease is not understood. Using an integrated global transcriptomic and proteomic profiling approach, we investigated the role of the mesangium in the onset and progression of IgAN. Global gene expression was investigated by microarray analysis of the glomerular compartment of renal biopsy specimens from patients with IgAN (n=19) and controls (n=22). Using curated glomerular cell type–specific genes from the published literature, we found differential expression of a much higher percentage of mesangial cell–positive standard genes than podocyte-positive standard genes in IgAN. Principal coordinate analysis of expression data revealed clear separation of patient and control samples on the basis of mesangial but not podocyte cell–positive standard genes.