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TELOMERE EXTENSION USING MODIFIED TERT Mrna TO TELOMERE EXTENSION USING MODIFIED TERT mRNA TO LENGTHEN HEALTHSPAN A DISSERTATION SUBMITTED TO THE NEUROSCIENCES PROGRAM AND THE COMMITTEE ON GRADUATE STUDIES OF STANFORD UNIVERSITY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY John Ramunas May 2014 © 2014 by John Ramunas. All Rights Reserved. Re-distributed by Stanford University under license with the author. This work is licensed under a Creative Commons Attribution- Noncommercial 3.0 United States License. http://creativecommons.org/licenses/by-nc/3.0/us/ This dissertation is online at: http://purl.stanford.edu/vb798wq6556 ii I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. Helen Blau, Primary Adviser I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. Michael Longaker I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. Juan Santiago I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. Robert Sapolsky Approved for the Stanford University Committee on Graduate Studies. Patricia J. Gumport, Vice Provost for Graduate Education This signature page was generated electronically upon submission of this dissertation in electronic format. An original signed hard copy of the signature page is on file in University Archives. iii ABSTRACT One of our long-term goals with this work is to extend the human health span, the period of life when humans are relatively free from age-related disease. Molecular mechanisms that limit the human health span include epigenetic drift, accumulation of cellular waste products, DNA damage, and telomere shortening, and here we demonstrate a method to address telomere shortening. Telomeres comprise DNA sequences that protect the ends of chromosomes but shorten over time due to oxidative damage and incomplete DNA replication during S phase of the cell cycle. Short telomeres can lead to activation of p53, cell cycle arrest or apoptosis, chromosome- chromosome fusions, or malignancy. Short telomeres are implicated not only in age-related diseases including cancer and heart disease, but in diseases in which cells are under high replicative demand, such as muscular dystrophy. The nucleoprotein telomerase comprising the protein component TERT and RNA component TERC extends telomeres, and several efforts at extending telomeres have focused on increasing the amount of telomerase in cells, which conveys proliferative capacity to cells in culture and can reverse phenotypes of aging in animal models. Currently small molecule activators of telomerase are in use by humans, but have no detectable effects on telomere length in many subjects. Adeno-associated viral delivery of TERT is another promising approach, one that extends rodent lifespan without increasing the incidence of cancer possibly because it is usually episomal and diluted out in fast-dividing cells, but risks genomic integration and resulting constitutive TERT expression which may not be acceptable in longer-lived species. Here we present an alternative method for telomere extension made possible by the recent discovery that delivery of mRNA comprising modified nucleotides such as pseudouridine iv modulates the Toll-like receptor mediated innate immune response that is activated in most cells in response to unmodified mRNA. Nucleoside modifications occur naturally in mammalian RNA and provide a means of distinguishing endogenous from exogenous RNA such as bacterial RNA which have fewer or no such modifications. We find that delivery of modified mRNA encoding TERT to fibroblasts and myoblasts results in transient elevation of telomerase activity, telomere extension, and increased proliferative capacity. All cells treated to date have eventually senesced and expressed markers of senescence to the same degree as untreated cells, important for the safety of our approach. Repeated treatment increases proliferative capacity further, suggesting that the approach may be useful over a prolonged period. We have taken initial steps at delivering mRNA in vivo and this work continues. v ACKNOWLEDGEMENTS I would like to thank my thesis advisor and mentor Helen Blau for her unwavering and strong support; creativity; highly energetic connecting of ideas, technologies, and people; excellent judgment about what matters and what does not in science and in life; for an exceptionally well-run and well-stocked lab; and for never shying from raising the bar higher. I am also deeply grateful to my co-advisor Juan Santiago for being a role model in applying engineering thinking to biological problems, for his outstanding intuition about how anything works, his excellent advice, strong support, and wisdom over the years. I would also like to thank my thesis committee members Robert Sapolsky and Michael Longaker for their excellent biological judgment and advice that has steered us well. Robert went to great lengths at the start of my graduate program to help guide my rejuvenation dreams in the right direction, and his advice was spot-on then and continues to be in each of our meetings. I would like to thank our key collaborator John Cooke for his perspective, insights, and positive energy, and for recruiting my scientific brother Eduard Yakubov. I would like to thank Eduard for his friendship, creativity, molecular biology expertise and transcribing many thousands of micrograms of mRNA, wisdom, and constancy. I am grateful to Jen Brady for expertly answering every one of my endless questions about molecular biology. I thank my father Tony Ramunas and mother Susan Fletcher for their love that has shaped my perspective and default mental state well, for focusing my attention on health on a daily basis, for teaching me to aspire to change the human condition, and for their infinite support as we all follow that path together. I would like to thank my step-father John Freeman for his support and many insights, and my brother Alan and his wife Jen and their kids for their love. I would like to thank Colin Holbrook for his passion for perfection and deep interest in science, Moritz Brandt for inspiring me to know more, and Viktor vi Shkolnikov for his friendship, deep knowledge of Russian and other science, technical skill, dedication, and the many successes and failures we have enjoyed and learned from together. I would also like to thank Robin Holbrook, Peggy Kraft, and Kassie Koleckar for your immense skills and your essential help that have, on a daily basis, helped immensely to make this project happen. I would like to thank the other Blau lab members for their valuable advice and insight, and for their good nature and humor that never ends: Glenn Markov, Russ Haynes, David Burns, Faye Mourkioti, Matt Decker, Andrew Ho, Penney Gilbert, Karen Havenstrite, Paul Cook, Stephane Corbel, Ben Cosgrove, Ermelinda Porpiglia, Erika Cornell, Nora Yucel, Srihari Sampath, Srinath Sampath, and Eva Moreno. I would like to thank the members of the Santiago lab and the AntiHero team for your engineering skill, help, and camaraderie: Karl Stahl, Angus Pacala, Mary Reynolds, Curran Kaushik, Francisco de la Paz, Ken Lopez, Michele Dragoescu, Giancarlo Garcia, Anita Rogacs, and David Fenning. I would like to thank Dr. Zane Cohen for a surgery very well done. I would like to thank Ross Colvin for his expert help at every turn in my degree. Finally I would like to thank John Huguenard for being open to pursuing rejuvenation many years ago and for supporting me through the years. I am very happy to be able to continue to work on this project with the same wonderful, passionate, and skillful team. May we see the products of our work in use. vii TABLE OF CONTENTS ABSTRACT ................................................................................................................................... iv ACKNOWLEDGEMENTS ........................................................................................................... vi List of Figures ................................................................................................................................. x List of Tables ................................................................................................................................ xii Chapter 1 Introduction to telomere shortening and extension ...................................................... 1 Telomeres .................................................................................................................................... 2 Mechanisms of telomere shortening ........................................................................................... 3 Telomerase .................................................................................................................................. 5 Telomere shortening and disease ................................................................................................ 7 Genetic mutations in telomere maintenance genes ................................................................. 8 Muscular dystrophy ................................................................................................................ 8 Cognitive decline ...................................................................................................................
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