Generation of Isogenic Human Pluripotent Stem Cell-Derived
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University of Connecticut OpenCommons@UConn Doctoral Dissertations University of Connecticut Graduate School 11-9-2018 Generation of Isogenic Human Pluripotent Stem Cell-Derived Neurons to Establish a Molecular Angelman Syndrome Phenotype and to Study the UBE3A Protein Isoforms Carissa Sirois University of Connecticut - Storrs, [email protected] Follow this and additional works at: https://opencommons.uconn.edu/dissertations Recommended Citation Sirois, Carissa, "Generation of Isogenic Human Pluripotent Stem Cell-Derived Neurons to Establish a Molecular Angelman Syndrome Phenotype and to Study the UBE3A Protein Isoforms" (2018). Doctoral Dissertations. 1988. https://opencommons.uconn.edu/dissertations/1988 Generation of Isogenic Human Pluripotent Stem Cell-Derived Neurons to Establish a Molecular Angelman Syndrome Phenotype and to Study the UBE3A Protein Isoforms Carissa L. Sirois, Ph.D. University of Connecticut, 2018 Abstract Angelman Syndrome (AS) is a neurodevelopment disorder for which there is currently no cure that is characterized by severe seizures, intellectual disability, absent speech, ataxia, and happy affect. Loss of expression from the maternally inherited copy of UBE3A, a gene regulated by genomic imprinting, causes AS. Currently there are multiple promising therapeutic approaches being explored and developed for AS, some of which involve targeting or expression of the human genetic sequence. Subsequently, it is necessary to establish robust cellular models for AS that can be used to test these, as well as future, potential AS therapies. Toward this aim, here we have used the CRISPR/Cas9 genome editing system to generate several isogenic human pluripotent stem cell lines two achieve two primary goals. First, we aimed to establish a robust quantitative molecular phenotype for cultured human AS neurons using the transcriptome. We identified and validated a list of genes that are consistent differentially expressed in AS neurons when compared to isogenic controls that can be assayed following drug treatments. Second, we aimed to study the abundance and localization of the three human UBE3A protein isoforms. We found that isoform 1 is the predominant protein isoform, and that UBE3A, regardless of isoform, appears to localize mostly to the cytoplasm, with low levels of expression in the nucleus and other organelles. The work in this thesis demonstrates that differentially expressed genes can be used as a phenotype for AS neurons to measure the effects of potential therapies, and provides important and previously unknown information as to the abundance and localization of the human UBE3A protein isoforms in human neurons. Generation of Isogenic Human Pluripotent Stem Cell-Derived Neurons to Establish a Molecular Angelman Syndrome Phenotype and to Study the UBE3A Protein Isoforms Carissa L. Sirois B.S., Eastern Connecticut State University, 2010 M.S., University of Hartford, 2013 A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy at the University of Connecticut 2018 i Copyright by Carissa L. Sirois 2018 ii APPROVAL PAGE Doctor of Philosophy Dissertation Generation of Isogenic Human Pluripotent Stem Cell-Derived Neurons to Establish a Molecular Angelman Syndrome Phenotype and to Study the UBE3A Protein Isoforms Presented by Carissa L. Sirois, M.S. Major Advisor ________________________________________________________________ Stormy J. Chamberlain Associate Advisor _____________________________________________________________ Eric. S. Levine Associate Advisor _____________________________________________________________ Stephen J. Crocker Associate Advisor _____________________________________________________________ James Li University of Connecticut 2018 iii Acknowledgements I would first and foremost like to thank my advisor and mentor, Dr. Stormy Chamberlain. I am truly lucky to have had the opportunity to have Stormy has my mentor throughout my PhD because she was exactly the type of mentor that I needed. Stormy was always available to talk to about anything, whether it was my project, answering questions about various aspects of the PhD, or offering advice for after graduation. She always encouraged not only my work in the lab, but pursuing other endeavors such as applying for fellowships, giving talks, or attending conferences. She has greatly shaped the type of scientist that I am and that I strive to be. Secondly, I would like to thank my thesis committee members: Dr. Eric Levine, Dr. Stephen Crocker, and Dr. James Li. My committee always supported the work that I did and provided great advice about my project throughout my PhD. Dr. Levine was very willing to collaborate with our lab on various aspects of my project, which was greatly appreciated. Dr. Crocker always had suggestions for follow up experiments at each of my committee meetings, as well as other helpful advice. Dr. Li rounded out my committee by providing great feedback on the genetic and developmental aspects of my project. I would also like to acknowledge Dr. Xue- jun Li, who served on my committee for part of my PhD before she left the university, and Dr. Marc Lalande, who I always considered to be my unofficial co-PI before he also left the university. I have been incredibly lucky to work with a great group of people in the Chamberlain lab and, by close association, the Lalande lab and UConn Stem Cell Core. There are not enough pages in this thesis to fully describe here how much help and support I have received from Drs. Noelle Germain, Ivy Chen, and Jack Hsiao. These three individuals were the original members of the Chamberlain lab when I joined and they all taught me so much. I am grateful for all of their help and their continued friendship throughout my time here (and beyond!). I would also like to thank Chris Stoddard for always being willing to teach myself and other members of the lab, not only about genome editing, but also other various aspects of molecular biology. Thank iv you also to the previous members of the Lalande lab, who were always such a joy to work with and who also taught me so much: Dr. Maeva Langouet, Dr. Souour Mansour, Dr. Elodie Mathieux, Erin Banda, Heather Glatt-Deely, and Michael Chung, who is now a member of the Chamberlain lab. I would also like to thank Dea Gorka, who worked with me as an undergraduate for two summers before joining our lab as a PhD student. Dea is a delight to work with and I hope that her and Michael continue the tradition of amazing students produced by our lab. I would like to thank the faculty and students of both the Department of Neuroscience and Department of Genetics and Genome Sciences for any assistance and feedback that they provided during my time at UConn. A huge thank you to Dr. Justin Cotney, the members of the Cotney lab (Andrea Wilderman, Tara Yankee, and Jen Venoudenhove), and to Scott Adamson for all of their help with bioinformatics. Everyone was extremely patient with the many questions I had and was always willing to provide advice. Thank you to all of the friends that I made during my time in the program: Judy Bloom, John Wizeman, Cory Brennick, Cory Willis, Alex Nicaise, Sarah Benjamin, Mitali Adlahka. This entire process was made so much better by sharing it with such an amazing group of people. Thank you also to all of my friends and family who have always been so supportive of my decision to be a perpetual student. A special thanks to my parents and sister, who have always encouraged me along the way and instilled in me the values of hard work, motivation, and dedication. Finally, I would like to thank my husband Sean, who has patiently waited ten years for me to no longer be a student. He has been the biggest source of support and I am so lucky to have him in my life. v Table of Contents Chapter 1 – Angelman Syndrome, a genomic imprinting disorder of chromosome 15q11-q13.1 ................................................................................................................... 1 General introduction .................................................................................................... 2 Genomic imprinting at chromosome 15q11-q13.1 ........................................................ 2 Disorders arising from mutations in 15q11-q13.1 ......................................................... 5 UBE3A ......................................................................................................................... 7 The prospect of molecular therapies for AS ................................................................. 9 Chapter 2 – Generation of isogenic stem cell lines using CRISPR/Cas9-mediated genome editing .......................................................................................................................... 17 Background & Rationale ............................................................................................ 18 Materials & Methods .................................................................................................. 20 Results ...................................................................................................................... 24 Correction of UBE3A point mutation in AS iPSCs ................................................... 24 Mutate translational start sites for UBE3A protein isoforms in H9 hESCs ............... 26 Deletion of UBE3A in H9 hESCs ............................................................................ 27 Knockout of UBE3A in normal iPSCs .....................................................................