WO 2016/070129 Al 6 May 2016 (06.05.2016) W P O P C T

Total Page:16

File Type:pdf, Size:1020Kb

WO 2016/070129 Al 6 May 2016 (06.05.2016) W P O P C T (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2016/070129 Al 6 May 2016 (06.05.2016) W P O P C T (51) International Patent Classification: (74) Agent: BAKER, C , Hunter; Wolf, Greenfield & Sacks, A61K 9/00 (2006.01) C07K 14/435 (2006.01) P.C., 600 Atlantic Avenue, Boston, MA 02210-2206 (US). (21) International Application Number: (81) Designated States (unless otherwise indicated, for every PCT/US20 15/058479 kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, (22) International Filing Date: BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, 30 October 2015 (30.10.201 5) DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, (25) Filing Language: English HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, (26) Publication Language: English MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, (30) Priority Data: PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, 14/529,010 30 October 2014 (30. 10.2014) US SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (71) Applicant: PRESIDENT AND FELLOWS OF HAR¬ VARD COLLEGE [US/US]; 17 Quincy Street, Cam (84) Designated States (unless otherwise indicated, for every bridge, MA 02138 (US). kind of regional protection available): ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, (72) Inventors: LIU, David, R.; 3 Whitman Circle, Lexington, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, MA 02420 (US). THOMPSON, David, B.; 50 Follen TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, Street, # 11, Cambridge, MA 02138 (US). ZURIS, John, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, Anthony; 60 Wadsworth St., Apt. 22B, Cambridge, MA LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, 02142 (US). SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, KM, ML, MR, NE, SN, TD, TG). [Continued on next page] (54) Title: DELIVERY OF NEGATIVELY CHARGED PROTEINS USING CATIONIC LIPIDS (57) Abstract: Compositions, methods, strategies, kits, and systems for the delivery of negatively charged proteins, protein complexes, and fusion pro teins, using cationic polymers or lipids are provided. Delivery of proteins into cells can be effected in vivo, ex vivo, or in vitro. Proteins that can be de Net negatively Cationic lipids livered using the compositions, methods, strategies, kits, and systems charged protein provided herein include, without limitation, enzymes, transcription factors, genome editing proteins, Cas9 proteins, TALEs, TALENs, nucleases, binding Treatment of proteins (e.g., ligands, receptors, antibodies, antibody fragments; nucleic acid mammalian cells with cationic lipidanionic binding proteins, etc.), structural proteins, and therapeutic proteins (e.g., tu - protein complexes mor suppressor proteins, therapeutic enzymes, growth factors, growth factor receptors, transcription factors, proteases, etc.), as well as variants and fu sions of such proteins. Extracellularspace Cytoplasm \ \ Liposome endoc tosis Endosomai escape Endosome o FIG. 27B © v o o w o 2016/070129 Al II 11 II I 1 Illlll I II II III II III III II I II Published: DELIVERY OF NEGATIVELY CHARGED PROTEINS USING CATIONIC LIPIDS RELATED APPLICATION [0001] This application is claims priority to U.S. patent application, U.S.S.N. 14/529,010, filed October 30, 2014, which is a continuation-in-part of and claims priority under 35 U.S.C. § 120 to U.S. patent application, U.S.S.N. 14/462,189, filed August 18, 2014, to U.S. patent application, U.S.S.N. 14/462,163, filed August 18, 2014, and to International Application, PCT/US2014/05424735, filed September 5, 2014, which claims priority under U.S.C. § 365(c) to U.S. patent application, U.S.S.N. 14/462,189, filed August 18, 2014, and to U.S. patent application, U.S.S.N. 14/462,163, filed August 18, 2014, and also claims priority under 35 U.S.C. § 119(e) to U.S. provisional patent application, U.S.S.N. 61/874,746, filed September 6, 2013, the entire contents of each of which are incorporated herein by reference. BACKGROUND OF THE INVENTION [0002] Macromolecular delivery into mammalian cells is an attractive approach for cell manipulation, as it would allow modulation of gene expression and modification of the genome, which, in turn, would open new avenues for research and enable the therapeutic targeting of molecules currently viewed as "undruggable" by small molecules. In particular, recombinant nucleases targeting genes or alleles associated with disease have great potential as therapeutic agents. The current methods of macromolecular delivery include viral delivery of nucleic acid molecules, receptor-mediated delivery of nucleic acids or proteins, and the use of protein fusions with cell-penetrating peptides such as TAT, Arg9, or Penetratin for the delivery of proteins. Each of these delivery systems offers benefits for particular applications; in most cases, however, questions regarding efficacy, cytotoxicity, and ease of preparation remain. Easily prepared reagents capable of effectively delivering macromolecules (e.g. , functional effector proteins) to a variety of cell lines without significant cytotoxicity or other adverse side effect remain of considerable concern. [0003] Most proteins do not spontaneously enter mammalian cells and are thus naturally limited in their use as research tools and their potential as therapeutic agents. Techniques for the delivery of proteins into mammalian cells have been developed recently to address intracellular targets. These techniques include the use of lipid-based reagents (Zelphati et a , J. Biol. Chem. 276, 35103-351 10, 2001), nanoparticles (Hasadsri et al., J. Biol. Chem., 2009), vault ribonucleoprotein particles (Lai et al, ACS Nano 3, 691-699, 2009); genetic or chemical fusion to receptor ligands (Gabel et al, J. Cell Biol. 103, 1817-1827, 1986; Rizk et al, Proc. Natl. Acad. Sci. U.S.A. 106, 1101 1-1 1015, 2009); and fusion to cell-penetrating peptides (Wadia et al, Curr. Protein Pept. Sci. 4, 97-104, 2003; Zhou et al, Cell Stem Cell 4, 381-384, 2009). Perhaps the most common method for protein delivery is genetic fusion to protein transduction domains (PTDs) including the HIV-1 transactivator of transcription (Tat) peptide and polyarginine peptides. These cationic PTDs promote association with negatively charged cell-surface structures and subsequent endocytosis of exogenous proteins. Both Tat and polyarginine have been used to deliver a variety of macromolecules into cells both in vitro and in vivo (Wadia et al, Curr. Protein Pept. Sci. 4, 97-104, 2003; Zhou et al, Cell Stem Cell 4, 381-384, 2009; Myou et al, J. Immunol. 169, 2670-2676, 2002; Bae et al, Clin. Exp. Immunol. 157, 128-138, 2009; Schwarze et al, Science 285, 1569-1572, 1999). Despite these advances, intracellular targets remain difficult to affect using exogenous proteins, and even modest success can require toxic concentrations of the respective transduction agent due to the low efficiency with which proteins are functionally delivered into cells (Zhou et al, Cell Stem Cell 4, 381-384, 2009; Wang et al, Nat. Biotechnol. 26, 901-908, 2008). Therefore, there remains a need for better delivery systems for getting functional effector proteins into cells to target intracellular biomolecules. SUMMARY OF THE INVENTION [0004] The present disclosure provides systems, compositions, preparations, kits, and related methods for delivering proteins into cells using cationic polymers or cationic lipids. In some embodiments, the proteins to be delivered are negatively charged proteins, also referred to herein as anionic proteins, such as, for example, naturally occurring or engineered negatively charged proteins. In some embodiments, the proteins to be delivered are associated with a negatively charged protein, e.g., via covalent or non-covalent interactions, to form a protein complex. In some such embodiments, the complex comprising the protein to be delivered associated with the negatively charged proteins, e.g., a supernegatively charged protein or a naturally occurring negatively charged protein, has a net negative charge. In some embodiments, the proteins to be delivered bind a nucleic acid thus forming a protein:nucleic acid complex having a net negative charge. [0005] Some aspects of this disclosure are based on the discovery that negatively charged proteins can be associated with cationic polymers or cationic lipids and that such protein:lipid complexes are efficiently delivered into cells. This technology can be applied to naturally negatively charged proteins (e.g., the proteins listed in tables 3-6, Sirtl (-59, 86 kDa), PPARg (- 13, 54 kDa), PRDM16 (-23, 140 kDa), PGCla (-15, 9 1 kDa), TP53BP1 (-148, 213 kDa), Utrophin (-142, 394 kDa), Dystrophin (-89, 426 kDa), Bik (-17, 18 kDa), ΙκΒα (-29, 35 kDa), Von Hippel-Lindau disease tumor suppressor (-18, 24 kDa), E3 ubiquitin ligases, metal-binding proteins, VP64 transcriptional activators, the anionic 3xFLAG peptide tag, and fusions thereof), to engineered supernegatively charged proteins (e.g., supernegatively charged GFP or streptavidin variants), to proteins that bind to nucleic acids and form negatively charged protein:nucleic acid complexes (e.g., Cas9 proteins, and variants and fusions thereof), or to protein fusions in which a protein to be delivered is associated with a negatively
Recommended publications
  • Whole-Genome Landscape of Pancreatic Neuroendocrine Tumours
    Scarpa, A. et al. (2017) Whole-genome landscape of pancreatic neuroendocrine tumours. Nature, 543(7643), pp. 65-71. (doi:10.1038/nature21063) This is the author’s final accepted version. There may be differences between this version and the published version. You are advised to consult the publisher’s version if you wish to cite from it. http://eprints.gla.ac.uk/137698/ Deposited on: 12 December 2018 Enlighten – Research publications by members of the University of Glasgow http://eprints.gla.ac.uk Whole-genome landscape of pancreatic neuroendocrine tumours Aldo Scarpa1,2*§, David K. Chang3,4, 7,29,36* , Katia Nones5,6*, Vincenzo Corbo1,2*, Ann-Marie Patch5,6, Peter Bailey3,6, Rita T. Lawlor1,2, Amber L. Johns7, David K. Miller6, Andrea Mafficini1, Borislav Rusev1, Maria Scardoni2, Davide Antonello8, Stefano Barbi2, Katarzyna O. Sikora1, Sara Cingarlini9, Caterina Vicentini1, Skye McKay7, Michael C. J. Quinn5,6, Timothy J. C. Bruxner6, Angelika N. Christ6, Ivon Harliwong6, Senel Idrisoglu6, Suzanne McLean6, Craig Nourse3, 6, Ehsan Nourbakhsh6, Peter J. Wilson6, Matthew J. Anderson6, J. Lynn Fink6, Felicity Newell5,6, Nick Waddell6, Oliver Holmes5,6, Stephen H. Kazakoff5,6, Conrad Leonard5,6, Scott Wood5,6, Qinying Xu5,6, Shivashankar Hiriyur Nagaraj6, Eliana Amato1,2, Irene Dalai1,2, Samantha Bersani2, Ivana Cataldo1,2, Angelo P. Dei Tos10, Paola Capelli2, Maria Vittoria Davì11, Luca Landoni8, Anna Malpaga8, Marco Miotto8, Vicki L.J. Whitehall5,12,13, Barbara A. Leggett5,12,14, Janelle L. Harris5, Jonathan Harris15, Marc D. Jones3, Jeremy Humphris7, Lorraine A. Chantrill7, Venessa Chin7, Adnan M. Nagrial7, Marina Pajic7, Christopher J. Scarlett7,16, Andreia Pinho7, Ilse Rooman7†, Christopher Toon7, Jianmin Wu7,17, Mark Pinese7, Mark Cowley7, Andrew Barbour18, Amanda Mawson7†, Emily S.
    [Show full text]
  • Modulated in Intestinal Inflammation A
    BTNL2, a Butyrophilin/B7-Like Molecule, Is a Negative Costimulatory Molecule Modulated in Intestinal Inflammation This information is current as Heather A. Arnett, Sabine S. Escobar, Eva Gonzalez-Suarez, of September 28, 2021. Alison L. Budelsky, Lori A. Steffen, Norman Boiani, Ming Zhang, Gerald Siu, Avery W. Brewer and Joanne L. Viney J Immunol 2007; 178:1523-1533; ; doi: 10.4049/jimmunol.178.3.1523 http://www.jimmunol.org/content/178/3/1523 Downloaded from References This article cites 40 articles, 12 of which you can access for free at: http://www.jimmunol.org/content/178/3/1523.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 28, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology BTNL2, a Butyrophilin/B7-Like Molecule, Is a Negative Costimulatory Molecule Modulated in Intestinal Inflammation Heather A. Arnett,1* Sabine S.
    [Show full text]
  • A Computational Approach for Defining a Signature of Β-Cell Golgi Stress in Diabetes Mellitus
    Page 1 of 781 Diabetes A Computational Approach for Defining a Signature of β-Cell Golgi Stress in Diabetes Mellitus Robert N. Bone1,6,7, Olufunmilola Oyebamiji2, Sayali Talware2, Sharmila Selvaraj2, Preethi Krishnan3,6, Farooq Syed1,6,7, Huanmei Wu2, Carmella Evans-Molina 1,3,4,5,6,7,8* Departments of 1Pediatrics, 3Medicine, 4Anatomy, Cell Biology & Physiology, 5Biochemistry & Molecular Biology, the 6Center for Diabetes & Metabolic Diseases, and the 7Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202; 2Department of BioHealth Informatics, Indiana University-Purdue University Indianapolis, Indianapolis, IN, 46202; 8Roudebush VA Medical Center, Indianapolis, IN 46202. *Corresponding Author(s): Carmella Evans-Molina, MD, PhD ([email protected]) Indiana University School of Medicine, 635 Barnhill Drive, MS 2031A, Indianapolis, IN 46202, Telephone: (317) 274-4145, Fax (317) 274-4107 Running Title: Golgi Stress Response in Diabetes Word Count: 4358 Number of Figures: 6 Keywords: Golgi apparatus stress, Islets, β cell, Type 1 diabetes, Type 2 diabetes 1 Diabetes Publish Ahead of Print, published online August 20, 2020 Diabetes Page 2 of 781 ABSTRACT The Golgi apparatus (GA) is an important site of insulin processing and granule maturation, but whether GA organelle dysfunction and GA stress are present in the diabetic β-cell has not been tested. We utilized an informatics-based approach to develop a transcriptional signature of β-cell GA stress using existing RNA sequencing and microarray datasets generated using human islets from donors with diabetes and islets where type 1(T1D) and type 2 diabetes (T2D) had been modeled ex vivo. To narrow our results to GA-specific genes, we applied a filter set of 1,030 genes accepted as GA associated.
    [Show full text]
  • Location Analysis of Estrogen Receptor Target Promoters Reveals That
    Location analysis of estrogen receptor ␣ target promoters reveals that FOXA1 defines a domain of the estrogen response Jose´ e Laganie` re*†, Genevie` ve Deblois*, Ce´ line Lefebvre*, Alain R. Bataille‡, Franc¸ois Robert‡, and Vincent Gigue` re*†§ *Molecular Oncology Group, Departments of Medicine and Oncology, McGill University Health Centre, Montreal, QC, Canada H3A 1A1; †Department of Biochemistry, McGill University, Montreal, QC, Canada H3G 1Y6; and ‡Laboratory of Chromatin and Genomic Expression, Institut de Recherches Cliniques de Montre´al, Montreal, QC, Canada H2W 1R7 Communicated by Ronald M. Evans, The Salk Institute for Biological Studies, La Jolla, CA, July 1, 2005 (received for review June 3, 2005) Nuclear receptors can activate diverse biological pathways within general absence of large scale functional data linking these putative a target cell in response to their cognate ligands, but how this binding sites with gene expression in specific cell types. compartmentalization is achieved at the level of gene regulation is Recently, chromatin immunoprecipitation (ChIP) has been used poorly understood. We used a genome-wide analysis of promoter in combination with promoter or genomic DNA microarrays to occupancy by the estrogen receptor ␣ (ER␣) in MCF-7 cells to identify loci recognized by transcription factors in a genome-wide investigate the molecular mechanisms underlying the action of manner in mammalian cells (20–24). This technology, termed 17␤-estradiol (E2) in controlling the growth of breast cancer cells. ChIP-on-chip or location analysis, can therefore be used to deter- We identified 153 promoters bound by ER␣ in the presence of E2. mine the global gene expression program that characterize the Motif-finding algorithms demonstrated that the estrogen re- action of a nuclear receptor in response to its natural ligand.
    [Show full text]
  • Pediatric and Perinatal Pathology (1842-1868)
    VOLUME 33 | SUPPLEMENT 2 | MARCH 2020 MODERN PATHOLOGY ABSTRACTS PEDIATRIC AND PERINATAL PATHOLOGY (1842-1868) LOS ANGELES CONVENTION CENTER FEBRUARY 29-MARCH 5, 2020 LOS ANGELES, CALIFORNIA 2020 ABSTRACTS | PLATFORM & POSTER PRESENTATIONS EDUCATION COMMITTEE Jason L. Hornick, Chair William C. Faquin Rhonda K. Yantiss, Chair, Abstract Review Board Yuri Fedoriw and Assignment Committee Karen Fritchie Laura W. Lamps, Chair, CME Subcommittee Lakshmi Priya Kunju Anna Marie Mulligan Steven D. Billings, Interactive Microscopy Subcommittee Rish K. Pai Raja R. Seethala, Short Course Coordinator David Papke, Pathologist-in-Training Ilan Weinreb, Subcommittee for Unique Live Course Offerings Vinita Parkash David B. Kaminsky (Ex-Officio) Carlos Parra-Herran Anil V. Parwani Zubair Baloch Rajiv M. Patel Daniel Brat Deepa T. Patil Ashley M. Cimino-Mathews Lynette M. Sholl James R. Cook Nicholas A. Zoumberos, Pathologist-in-Training Sarah Dry ABSTRACT REVIEW BOARD Benjamin Adam Billie Fyfe-Kirschner Michael Lee Natasha Rekhtman Narasimhan Agaram Giovanna Giannico Cheng-Han Lee Jordan Reynolds Rouba Ali-Fehmi Anthony Gill Madelyn Lew Michael Rivera Ghassan Allo Paula Ginter Zaibo Li Andres Roma Isabel Alvarado-Cabrero Tamara Giorgadze Faqian Li Avi Rosenberg Catalina Amador Purva Gopal Ying Li Esther Rossi Roberto Barrios Anuradha Gopalan Haiyan Liu Peter Sadow Rohit Bhargava Abha Goyal Xiuli Liu Steven Salvatore Jennifer Boland Rondell Graham Yen-Chun Liu Souzan Sanati Alain Borczuk Alejandro Gru Lesley Lomo Anjali Saqi Elena Brachtel Nilesh Gupta Tamara
    [Show full text]
  • A Private 16Q24.2Q24.3 Microduplication in a Boy with Intellectual Disability, Speech Delay and Mild Dysmorphic Features
    G C A T T A C G G C A T genes Article A Private 16q24.2q24.3 Microduplication in a Boy with Intellectual Disability, Speech Delay and Mild Dysmorphic Features Orazio Palumbo * , Pietro Palumbo , Ester Di Muro, Luigia Cinque, Antonio Petracca, Massimo Carella and Marco Castori Division of Medical Genetics, Fondazione IRCCS-Casa Sollievo della Sofferenza, San Giovanni Rotondo, 71013 Foggia, Italy; [email protected] (P.P.); [email protected] (E.D.M.); [email protected] (L.C.); [email protected] (A.P.); [email protected] (M.C.); [email protected] (M.C.) * Correspondence: [email protected]; Tel.: +39-088-241-6350 Received: 5 June 2020; Accepted: 24 June 2020; Published: 26 June 2020 Abstract: No data on interstitial microduplications of the 16q24.2q24.3 chromosome region are available in the medical literature and remain extraordinarily rare in public databases. Here, we describe a boy with a de novo 16q24.2q24.3 microduplication at the Single Nucleotide Polymorphism (SNP)-array analysis spanning ~2.2 Mb and encompassing 38 genes. The patient showed mild-to-moderate intellectual disability, speech delay and mild dysmorphic features. In DECIPHER, we found six individuals carrying a “pure” overlapping microduplication. Although available data are very limited, genomic and phenotype comparison of our and previously annotated patients suggested a potential clinical relevance for 16q24.2q24.3 microduplication with a variable and not (yet) recognizable phenotype predominantly affecting cognition. Comparing the cytogenomic data of available individuals allowed us to delineate the smallest region of overlap involving 14 genes. Accordingly, we propose ANKRD11, CDH15, and CTU2 as candidate genes for explaining the related neurodevelopmental manifestations shared by these patients.
    [Show full text]
  • Fine Mapping Studies of Quantitative Trait Loci for Baseline Platelet Count in Mice and Humans
    Fine mapping studies of quantitative trait loci for baseline platelet count in mice and humans A thesis submitted in fulfillment of the requirements for the degree of Doctor of Philosophy Melody C Caramins December 2010 University of New South Wales ORIGINALITY STATEMENT ‘I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project's design and conception or in style, presentation and linguistic expression is acknowledged.’ Signed …………………………………………….............. Date …………………………………………….............. This thesis is dedicated to my father. Dad, thanks for the genes – and the environment! ACKNOWLEDGEMENTS “Nothing can come out of nothing, any more than a thing can go back to nothing.” - Marcus Aurelius Antoninus A PhD thesis is never the work of one person in isolation from the world at large. I would like to thank the following people, without whom this work would not have existed. Thank you firstly, to all my teachers, of which there have been many. Undoubtedly, the greatest debt is owed to my supervisor, Dr Michael Buckley.
    [Show full text]
  • Essential Genes and Their Role in Autism Spectrum Disorder
    University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2017 Essential Genes And Their Role In Autism Spectrum Disorder Xiao Ji University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Bioinformatics Commons, and the Genetics Commons Recommended Citation Ji, Xiao, "Essential Genes And Their Role In Autism Spectrum Disorder" (2017). Publicly Accessible Penn Dissertations. 2369. https://repository.upenn.edu/edissertations/2369 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/2369 For more information, please contact [email protected]. Essential Genes And Their Role In Autism Spectrum Disorder Abstract Essential genes (EGs) play central roles in fundamental cellular processes and are required for the survival of an organism. EGs are enriched for human disease genes and are under strong purifying selection. This intolerance to deleterious mutations, commonly observed haploinsufficiency and the importance of EGs in pre- and postnatal development suggests a possible cumulative effect of deleterious variants in EGs on complex neurodevelopmental disorders. Autism spectrum disorder (ASD) is a heterogeneous, highly heritable neurodevelopmental syndrome characterized by impaired social interaction, communication and repetitive behavior. More and more genetic evidence points to a polygenic model of ASD and it is estimated that hundreds of genes contribute to ASD. The central question addressed in this dissertation is whether genes with a strong effect on survival and fitness (i.e. EGs) play a specific oler in ASD risk. I compiled a comprehensive catalog of 3,915 mammalian EGs by combining human orthologs of lethal genes in knockout mice and genes responsible for cell-based essentiality.
    [Show full text]
  • Anti-Inflammatory Role of Curcumin in LPS Treated A549 Cells at Global Proteome Level and on Mycobacterial Infection
    Anti-inflammatory Role of Curcumin in LPS Treated A549 cells at Global Proteome level and on Mycobacterial infection. Suchita Singh1,+, Rakesh Arya2,3,+, Rhishikesh R Bargaje1, Mrinal Kumar Das2,4, Subia Akram2, Hossain Md. Faruquee2,5, Rajendra Kumar Behera3, Ranjan Kumar Nanda2,*, Anurag Agrawal1 1Center of Excellence for Translational Research in Asthma and Lung Disease, CSIR- Institute of Genomics and Integrative Biology, New Delhi, 110025, India. 2Translational Health Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, 110067, India. 3School of Life Sciences, Sambalpur University, Jyoti Vihar, Sambalpur, Orissa, 768019, India. 4Department of Respiratory Sciences, #211, Maurice Shock Building, University of Leicester, LE1 9HN 5Department of Biotechnology and Genetic Engineering, Islamic University, Kushtia- 7003, Bangladesh. +Contributed equally for this work. S-1 70 G1 S 60 G2/M 50 40 30 % of cells 20 10 0 CURI LPSI LPSCUR Figure S1: Effect of curcumin and/or LPS treatment on A549 cell viability A549 cells were treated with curcumin (10 µM) and/or LPS or 1 µg/ml for the indicated times and after fixation were stained with propidium iodide and Annexin V-FITC. The DNA contents were determined by flow cytometry to calculate percentage of cells present in each phase of the cell cycle (G1, S and G2/M) using Flowing analysis software. S-2 Figure S2: Total proteins identified in all the three experiments and their distribution betwee curcumin and/or LPS treated conditions. The proteins showing differential expressions (log2 fold change≥2) in these experiments were presented in the venn diagram and certain number of proteins are common in all three experiments.
    [Show full text]
  • Structural Capacitance in Protein Evolution and Human Diseases
    Article Structural Capacitance in Protein Evolution and Human Diseases Chen Li 1,2, Liah V.T. Clark 1, Rory Zhang 1, Benjamin T. Porebski 1,3, Julia M. McCoey 1, Natalie A. Borg 1, Geoffrey I. Webb 4, Itamar Kass 1,5, Malcolm Buckle 6, Jiangning Song 1, Adrian Woolfson 7 and Ashley M. Buckle 1 1 - Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, Victoria 3800, Australia 2 - Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, Zurich 8093, Switzerland 3 - Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH, UK 4 - Faculty of Information Technology, Monash University, Clayton, Victoria 3800, Australia 5 - Amai Proteins, Prof. A. D. Bergman 2B, Suite 212, Rehovot 7670504, Israel 6 - LBPA, ENS Cachan, CNRS, Université Paris-Saclay, F-94235 Cachan, France 7 - Nouscom, Baumleingasse, CH-4051 Basel, Switzerland Correspondence to Adrian Woolfson and Ashley M. Buckle: [email protected]; [email protected] https://doi.org/10.1016/j.jmb.2018.06.051 Edited by Dan Tawfik Abstract Canonical mechanisms of protein evolution include the duplication and diversification of pre-existing folds through genetic alterations that include point mutations, insertions, deletions, and copy number amplifications, as well as post-translational modifications that modify processes such as folding efficiency and cellular localization. Following a survey of the human mutation database, we have identified an additional mechanism that we term “structural capacitance,” which results in the de novo generation of microstructure in previously disordered regions. We suggest that the potential for structural capacitance confers select proteins with the capacity to evolve over rapid timescales, facilitating saltatory evolution as opposed to gradualistic canonical Darwinian mechanisms.
    [Show full text]
  • Supplementary Table S1. Correlation Between the Mutant P53-Interacting Partners and PTTG3P, PTTG1 and PTTG2, Based on Data from Starbase V3.0 Database
    Supplementary Table S1. Correlation between the mutant p53-interacting partners and PTTG3P, PTTG1 and PTTG2, based on data from StarBase v3.0 database. PTTG3P PTTG1 PTTG2 Gene ID Coefficient-R p-value Coefficient-R p-value Coefficient-R p-value NF-YA ENSG00000001167 −0.077 8.59e-2 −0.210 2.09e-6 −0.122 6.23e-3 NF-YB ENSG00000120837 0.176 7.12e-5 0.227 2.82e-7 0.094 3.59e-2 NF-YC ENSG00000066136 0.124 5.45e-3 0.124 5.40e-3 0.051 2.51e-1 Sp1 ENSG00000185591 −0.014 7.50e-1 −0.201 5.82e-6 −0.072 1.07e-1 Ets-1 ENSG00000134954 −0.096 3.14e-2 −0.257 4.83e-9 0.034 4.46e-1 VDR ENSG00000111424 −0.091 4.10e-2 −0.216 1.03e-6 0.014 7.48e-1 SREBP-2 ENSG00000198911 −0.064 1.53e-1 −0.147 9.27e-4 −0.073 1.01e-1 TopBP1 ENSG00000163781 0.067 1.36e-1 0.051 2.57e-1 −0.020 6.57e-1 Pin1 ENSG00000127445 0.250 1.40e-8 0.571 9.56e-45 0.187 2.52e-5 MRE11 ENSG00000020922 0.063 1.56e-1 −0.007 8.81e-1 −0.024 5.93e-1 PML ENSG00000140464 0.072 1.05e-1 0.217 9.36e-7 0.166 1.85e-4 p63 ENSG00000073282 −0.120 7.04e-3 −0.283 1.08e-10 −0.198 7.71e-6 p73 ENSG00000078900 0.104 2.03e-2 0.258 4.67e-9 0.097 3.02e-2 Supplementary Table S2.
    [Show full text]
  • Supplemental Methods
    Supplemental Methods: Sample Collection Duplicate surface samples were collected from the Amazon River plume aboard the R/V Knorr in June 2010 (4 52.71’N, 51 21.59’W) during a period of high river discharge. The collection site (Station 10, 4° 52.71’N, 51° 21.59’W; S = 21.0; T = 29.6°C), located ~ 500 Km to the north of the Amazon River mouth, was characterized by the presence of coastal diatoms in the top 8 m of the water column. Sampling was conducted between 0700 and 0900 local time by gently impeller pumping (modified Rule 1800 submersible sump pump) surface water through 10 m of tygon tubing (3 cm) to the ship's deck where it then flowed through a 156 µm mesh into 20 L carboys. In the lab, cells were partitioned into two size fractions by sequential filtration (using a Masterflex peristaltic pump) of the pre-filtered seawater through a 2.0 µm pore-size, 142 mm diameter polycarbonate (PCTE) membrane filter (Sterlitech Corporation, Kent, CWA) and a 0.22 µm pore-size, 142 mm diameter Supor membrane filter (Pall, Port Washington, NY). Metagenomic and non-selective metatranscriptomic analyses were conducted on both pore-size filters; poly(A)-selected (eukaryote-dominated) metatranscriptomic analyses were conducted only on the larger pore-size filter (2.0 µm pore-size). All filters were immediately submerged in RNAlater (Applied Biosystems, Austin, TX) in sterile 50 mL conical tubes, incubated at room temperature overnight and then stored at -80oC until extraction. Filtration and stabilization of each sample was completed within 30 min of water collection.
    [Show full text]