Content Consultant: Larry H Bernstein, MD, FCAP
Genomics Orientations for Personalized Medicine
Volume One
http://www.amazon.com/dp/B018DHBUO6
![genomicsebook3[1]](https://i0.wp.com/pharmaceuticalintelligence.com/wp-content/uploads/2013/02/genomicsebook31.jpg)
Image Collage by SJ Williams, PhD, Google Images in Assembly
Larry H Bernstein, MD, FCAP, Senior Editor
Triplex Medical Science, Trumbull, CT
Stephen J. Williams, PhD, Editor
Leaders in Pharmaceutical Business Intelligence, Philadelphia
and
Aviva Lev-Ari, PhD, RN, Editor
Editor-in-Chief BioMed E-Book Series
Leaders in Pharmaceutical Business Intelligence, Boston
avivalev-ari@alum.berkeley.edu
Volume Two:
Genomics Methodologies: NGS, BioInformatics & Simulations and the Genome Ontology
Volume Three:
Five Leading Genomics Research Centers in the US
![DNAMicro copy[1]](https://i0.wp.com/pharmaceuticalintelligence.com/wp-content/uploads/2013/02/dnamicro-copy1.jpg)
Image of DNA by Sondra Barrett, PhD [DNAMicro.jpg with permission]
Other e-Books in the BioMedicine e-Series
Series A: e-Books on Cardiovascular Diseases
Content Consultant: Justin D Pearlman, MD, PhD, FACC
Volume One: Perspectives on Nitric Oxide
Sr. Editor: Larry Bernstein, MD, FCAP, Editor: Aviral Vatsa, PhD and Content Consultant: Stephen J Williams, PhD
available on Kindle Store @ Amazon.com
http://www.amazon.com/dp/B00DINFFYC
Volume Two: Cardiovascular Original Research: Cases in Methodology Design for Content Co-Curation
Curators: Justin D Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP, Aviva Lev-Ari, PhD, RN
- Causes
- Risks and Biomarkers
- Therapeutic Implications
Volume Three: Etiologies of CVD: Epigenetics, Genetics & Genomics
Curators: Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
- Causes
- Risks and Biomarkers
- Therapeutic Implications
Volume Four: Therapeutic Promise: CVD, Regenerative & Translational Medicine
Curators: Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
- Causes
- Risks and Biomarkers
- Therapeutic Implications
Volume Five: Pharmaco-Therapies for CVD
Volume Curators: Justin D Pearlman, MD, PhD, FACC and Aviva Lev-Ari, PhD, RN
- Causes
- Risks and Biomarkers
- Therapeutic Implications
Volume Six: Interventional Cardiology and Cardiac Surgery for Disease Diagnosis and Guidance of Treatment
Volume Curators: Justin D Pearlman, MD, PhD, FACC and Aviva Lev-Ari, PhD, RN
- Causes
- Risks and Biomarkers
- Therapeutic Implications
In addition to the Seven Volumes of SERIES A: Cardiovascular Diseases, Not included in SERIES A is a Three Volume Series by Dr. Pearlman, Editor, on Cardiovascular Diseases, positioned as Academic Textbooks for Training Residents in Cardiology and Texts for CEU Courses in Cardiology [Hardcover, Softcover, e-Books].
- CVD 1: Causes of Cardiovascular Diseases
- CVD 2: Risk Assessment of Cardiovascular Diseases
- CVD 3: Management of Cardiovascular Diseases
- CVD 4: Volume Seven: Cardiac Imaging
Series B: e-Books on Genomics & Medicine
Content Consultant: Larry H Bernstein, MD, FCAP
Volume 1: Genomics and Individualized Medicine
Sr. Editor: Stephen J Williams, PhD
Editors: Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
Volume 2: Latest in Genomics Methodologies for Therapeutics: Gene Editing, NGS & BioInformatics, Simulations and the Genome Ontology
Editors: Stephen J Williams, PhD and TBA
Volume 3: Institutional Leadership in Genomics
Editors: Aviva Lev-Ari, PhD, RN and TBA
Series C: e-Books on Cancer & Oncology
Content Consultant: Larry H Bernstein, MD, FCAP
Volume 1: Cancer and Genomics
Sr. Editor: Stephen J Williams, PhD
Editors: Ritu Saxena, PhD, Tilda Barliya, PhD
Volume 2: Cancer Therapies: Metabolic, Genomics, Interventional, Immunotherapy and Nanotechnology in Therapy Delivery
Author, Curator and Editor: Larry H Bernstein, MD, FCAP
Guest Authors: Stephen J Williams, PhD, Dror Nir, PhD and Tilda Barliya, PhD, Demet Sag, PhD
Volume 3: Cancer Patients’ Resources on Therapies
Sr. Editor: TBA
Series D: e-Books on BioMedicine
Content Consultant: Larry H Bernstein, MD, FCAP
Volume 1: Metabolic Genomics & Pharmaceutics
Author, Curator and Editor: Larry H Bernstein, MD, FCAP
Volume 2: Infectious Diseases
Editor: TBA
Volume 3: Immunology and Therapeutics
Author, Curator and Editor: Larry H Bernstein, MD, FCAP
Series E: Patient-centered Medicine
Content Consultant: Larry H Bernstein, MD, FCAP
Volume 1: The VOICES of Patients, HealthCare Providers, Care Givers and Families: Personal Experience with Critical Care and Invasive Medical Procedures
Author, Curator and Editor: Larry H Bernstein, MD, FCAP
Volume 2: Medical Scientific Discoveries for the 21st Century & Interviews with Scientific Leaders
Author, Curator and Editor: Larry H Bernstein, MD, FCAP
Volume 3: Milestones in Physiology & Discoveries in Medicine and Genomics
Author, Curator and Editor: Larry H Bernstein, MD, FCAP
Volume 4: Medical 3D BioPrinting – The Revolution in Medicine
Our DOMAINS in Scientific Media
I. Pharmaceutical: Biologics, Small Molecules, Diagnostics
II. Life Sciences: Genomics and Cancer Biology
III. Patient-centered Medicine: Focus on #1: Cardiovascular, #2: Cancer, #3: Physiology Metabolomics, Immunology
IV. Biomedicine, BioTech, and MedTech (Medical Devices)
V. HealthCare: Patient-centered Medicine and Personalized/Precision Medicine
This e-Book is a comprehensive review of recent Original Research on Genomics and Individualized Medicine
and related opportunities for Targeted Therapy written by Experts, Authors, Writers. The results of Original Research are gaining value added for the e-Reader by the Methodology of Curation. The e-Book’s articles have been published on the Open Access Online Scientific Journal, since April 2012. All new articles on this subject, will continue to be incorporated, as published with periodical updates.
Open Access Online Journal
http://www.pharmaceuticalIntelligence.com
is a scientific, medical and business, multi-expert authoring environment for information syndication in several domains of Life Sciences, Medicine, Pharmaceutical and Healthcare Industries, BioMedicine, Medical Technologies & Devices. Scientific critical interpretations and original articles are written by PhDs, MDs, MD/PhDs, PharmDs, Technical MBAs as Experts, Authors, Writers (EAWs) on an Equity Sharing basis.
List of Contributors to Volume One
Larry Bernstein, MD, FCAP, Senior Editor
Introduction 1.1, 1.2, 1.4, 1.5, 2.2, 2.6, 3.1, 3.2, 3.3, 3.6, 4.6, 4.8, 5.8, 5.9, 5.10, 6.1, 6.2, 6.3, 6.5, 6.7, 6.8, 6.9, 6.10, 6.11, 6.12, 6.13, 6.14, 6.16, 6.17, 8.5, 8.6, 9.6, 10.4, 10.5, 10.6, 10.7, 11.1, 11.7, 11.10, 11.11, 12.2, 12.3, 12.4, 12.6, 12.8, 13.8, 13.9, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, 15.5, 15.8, 15.9, 15.9.4, 15.11, 16.1, 16.2, 16.3, 16.4, 16.5, 17.2, 18.1, 18.2, 18.5, 18.6, 20.2, 20.3, 20.4, 20.5, 20.6, Introduction-21, Summary-21, Volume Summary, Epilogue
Stephen J. Williams, PhD, Editor
2.3, 2.7, 6.15, 7.6, 8.8, 11.8, 12.5, 12.7, 15.3, 20.7, Introduction-21
Aviva Lev-Ari, PhD, RN, Editor-in-Chief, BioMed e-Books Series
1.6, 2.1, 2.5, 3.4, 3.5, 3.7, 3.8, 4.1, 4.4, 4.5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 6.18, 7.1, 7.2, 7.3, 7.4, 7.5, 8.1, 8.2, 8.3, 8.7, 8.9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.8, 10.1, 10.2, 10.3, 10.8, 11.2, 11.3, 11.5, 11.9, 12.1, 13.5 13.7, 15.1, 15.2, 15.4, 15.6, 15.7, 15.9.1, 15.9.2, 15.9.3, 15.9.5, 15.10, 17.1, 18.3, 18.4, 19.4, 19.5, 20.1, 20.8, 21.1.1, 21.1.2, 21.1.3, 21.1.4, 21.2.1, 21.2.2, 21.2.3, 21.2.4, 21.3.1, 21.3.2, 21.4.2
Sudipta Saha, PhD
1.3, 6.6, 11.6, 13.2, 13.3, 13.4, 19.1, 19.2, 19.6, 19.7, 19.8, 19.9, 19.10
Ritu Saxena, PhD
4.2, 6.4, 9.7, 13.6, 14.1, 17.3, 17.4, 17.5, 19.3
Tilda Barlyia, PhD
8.4, 13.1, 14.2
Marcus W Feldman, PhD, Professor of Computational Biology, Stanford University, Department of Biology
2.4
4.7, 4.9, 4.10
11.4
electronic Table of Contents
Chapter 1
1.2 CRACKING THE CODE OF HUMAN LIFE: Milestones along the Way – Part IIA
1.3 DNA – The Next-Generation Storage Media for Digital Information
1.4 CRACKING THE CODE OF HUMAN LIFE: Recent Advances in Genomic Analysis and Disease – Part IIC
1.5 Advances in Separations Technology for the “OMICs” and Clarification of Therapeutic Targets
1.6 Genomic Analysis: FLUIDIGM Technology in the Life Science and Agricultural Biotechnology
Chapter 2
2.2 DNA structure and Oligonucleotides
2.3 Genome-Wide Detection of Single-Nucleotide and Copy-Number Variation of a Single Human Cell
2.6 The Binding of Oligonucleotides in DNA and 3-D Lattice Structures
2.7 Finding the Genetic Links in Common Disease: Caveats of Whole Genome Sequencing Studies
Chapter 3
3.1 Big Data in Genomic Medicine
3.2 CRACKING THE CODE OF HUMAN LIFE: The Birth of Bioinformatics & Computational Genomics – Part IIB
3.3 Expanding the Genetic Alphabet and linking the Genome to the Metabolome
3.5 MIT Scientists on Proteomics: All the Proteins in the Mitochondrial Matrix identified
3.6 Identification of Biomarkers that are Related to the Actin Cytoskeleton
Chapter 4
4.1 ENCODE Findings as Consortium
4.2 ENCODE: The Key to Unlocking the Secrets of Complex Genetic Diseases
4.5 Human Genome Project – 10th Anniversary: Interview with Kevin Davies, PhD – The $1000 Genome
4.6 Quantum Biology And Computational Medicine
4.7 The Underappreciated EpiGenome
4.8 Unraveling Retrograde Signaling Pathways
4.9 “The SILENCE of the Lambs” Introducing The Power of Uncoded RNA
4.10 DNA: One man’s trash is another man’s treasure, but there is no JUNK after all
Chapter 5
5.1 Paradigm Shift in Human Genomics – Predictive Biomarkers and Personalized Medicine – Part 1
5.2 Computational Genomics Center: New Unification of Computational Technologies at Stanford
5.3 Personalized Medicine: An Institute Profile – Coriell Institute for Medical Research: Part 3
5.4 Cancer Genomics – Leading the Way by Cancer Genomics Program at UC Santa Cruz
5.5 Genome and Genetics: Resources @Stanford, @MIT, @NIH’s NCBCS
5.6 NGS Market: Trends and Development for Genotype-Phenotype Associations Research
5.9 Transphosphorylation of E-coli Proteins and Kinase Specificity
5.10 Genomics of Bacterial and Archaeal Viruses
Chapter 6
6.1 Directions for Genomics in Personalized Medicine
6.3 Mitochondrial Damage and Repair under Oxidative Stress
6.4 Mitochondria: More than just the “Powerhouse of the Cell”
6.5 Mechanism of Variegation in Immutans
6.7 Cardiac Ca2+ Signaling: Transcriptional Control
6.8 Unraveling Retrograde Signaling Pathways
6.12 Zebrafish—Susceptible to Cancer
6.13 RNA Virus Genome as Bacterial Chromosome
6.14 Cloning the Vaccinia Virus Genome as a Bacterial Artificial Chromosome
6.15 Telling NO to Cardiac Risk- DDAH Says NO to ADMA(1); The DDAH/ADMA/NOS Pathway(2)
6.16 Transphosphorylation of E-coli proteins and kinase specificity
6.17 Genomics of Bacterial and Archaeal Viruses
Chapter 7
7.2 Consumer Market for Personal DNA Sequencing: Part 4
7.6 Personalized Medicine: Clinical Aspiration of Microarrays
Chapter 8
8.1 Personalized Medicine as Key Area for Future Pharmaceutical Growth
8.2 Inaugural Genomics in Medicine – The Conference Program, 2/11-12/2013, San Francisco, CA
8.4 Nanotechnology, Personalized Medicine and DNA Sequencing
8.6 Transcript Dynamics of Proinflammatory Genes
8.8 Intratumor Heterogeneity and Branched Evolution Revealed by Multiregion Sequencing[1]
Chapter 9
9.1 Personal Tale of JL’s Whole Genome Sequencing
9.6 The Initiation and Growth of Molecular Biology and Genomics – Part I
9.7 Personalized Medicine-based Cure for Cancer Might Not Be Far Away
9.8 Personalized Medicine: Cancer Cell Biology and Minimally Invasive Surgery (MIS)
Chapter 10
10.2 Imatinib (Gleevec) May Help Treat Aggressive Lymphoma: Chronic Lymphocytic Leukemia (CLL)
10.4 Treatment for Metastatic HER2 Breast Cancer
10.5 Personalized Medicine in NSCLC
10.6 Gene Sequencing – to the Bedside
10.7 DNA Sequencing Technology
10.8 Nobel Laureate Jack Szostak Previews his Plenary Keynote for Drug Discovery Chemistry
Chapter 11
11.1 mRNA Interference with Cancer Expression
11.2 Angiogenic Disease Research Utilizing microRNA Technology: UCSD and Regulus Therapeutics
11.4 A microRNA Prognostic Marker Identified in Acute Leukemia
11.5 MIT Team: Microfluidic-based approach – A Vectorless delivery of Functional siRNAs into Cells.
11.7 When Clinical Application of miRNAs?
11.8 How mobile elements in “Junk” DNA promote cancer. Part 1: Transposon-mediated tumorigenesis,
11.9 Potential Drug Target: Glycolysis Regulation – Oxidative Stress-responsive microRNA-320
11.10 MicroRNA Molecule May Serve as Biomarker
11.11 What about Circular RNAs?
Chapter 12
12.1 The “Cancer Establishments” Examined by James Watson, Co-discoverer of DNA w/Crick, 4/1953
12.2 Otto Warburg, A Giant of Modern Cellular Biology
12.3 Is the Warburg Effect the Cause or the Effect of Cancer: A 21st Century View?
12.4 Hypothesis – Following on James Watson
12.5 AMPK Is a Negative Regulator of the Warburg Effect and Suppresses Tumor Growth In Vivo
12.6 AKT signaling variable effects
12.8 Phosphatidyl-5-Inositol signaling by Pin1
Chapter 13
13.1 Nanotech Therapy for Breast Cancer
13.5 Prostate Cancer: Androgen-driven “Pathomechanism” in Early onset Forms of the Disease
13.6 In focus: Melanoma Genetics
13.8 Breast Cancer and Mitochondrial Mutations
13.9 Long noncoding RNA network regulates PTEN transcription
Chapter 14
14.1 HBV and HCV-associated Liver Cancer: Important Insights from the Genome
14.2 Nanotechnology and HIV/AIDS treatment
14.3 IRF-1 Deficiency Skews the Differentiation of Dendritic Cells
14.5 Five Malaria Genomes Sequenced
14.6 Rheumatoid Arthritis Risk
14.7 Approach to Controlling Pathogenic Inflammation in Arthritis
14.8 RNA Virus Genome as Bacterial Chromosome
14.9 Cloning the Vaccinia Virus Genome as a Bacterial Artificial Chromosome
Chapter 15
15.1 Personalized Cardiovascular Genetic Medicine at Partners HealthCare and Harvard Medical School
15.3 DDAH Says NO to ADMA(1); The DDAH/ADMA/NOS Pathway(2)
15.6 Gene Therapy Into Healthy Heart Muscle: Reprogramming Scar Tissue In Damaged Hearts
15.8 Ca2+ signaling: transcriptional control
15.9 Lp(a) Gene Variant Association
15.9.5 Gene, Meis1, Regulates the Heart’s Ability to Regenerate after Injuries.
15.11 How Might Sleep Apnea Lead to Serious Health Concerns like Cardiac and Cancers?
Chapter 16
16.1 Can Resolvins Suppress Acute Lung Injury?
16.2 Lipoxin A4 Regulates Natural Killer Cell in Asthma
16.3 Biological Therapeutics for Asthma
16.4 Genomics of Bronchial Epithelial Dysplasia
16.5 Progression in Bronchial Dysplasia
Chapter 17
17.1 Breakthrough Digestive Disorders Research: Conditions Affecting the Gastrointestinal Tract.
17.2 Liver Endoplasmic Reticulum Stress and Hepatosteatosis
17.3 Biomarkers-identified-for-recurrence-in-hbv-related-hcc-patients-post-surgery
17.4 Usp9x: Promising Therapeutic Target for Pancreatic Cancer
17.5 Battle of Steve Jobs and Ralph Steinman with Pancreatic cancer: How We Lost
Chapter 18
18.1 Ubiquitin Pathway Involved in Neurodegenerative Disease
18.3 Neuroprotective Therapies: Pharmacogenomics vs Psychotropic Drugs and Cholinesterase Inhibitors
18.5 Cell Transplantation in Brain Repair
18.6 Alzheimer’s Disease Conundrum – Are We Near the End of the Puzzle?
Chapter 19
19.1 Genetics and Male Endocrinology
19.2 Genomic Endocrinology and its Future
19.4 Therapeutic Targets for Diabetes and Related Metabolic Disorders
19.6 Personal Recombination Map from Individual’s Sperm Cell and its Importance
19.7 Gene Trap Mutagenesis in Reproductive Research
19.8 Pregnancy with a Leptin-Receptor Mutation
19.10 Reproductive Genetic Testing
Chapter 20
20.1 Genomics & Ethics: DNA Fragments are Products of Nature or Patentable Genes?
20.2 Understanding the Role of Personalized Medicine
20.3 Attitudes of Patients about Personalized Medicine
20.4 Genome Sequencing of the Healthy
20.5 Genomics in Medicine – Tomorrow’s Promise
20.6 The Promise of Personalized Medicine
20.8 Genomic Liberty of Ownership, Genome Medicine and Patenting the Human Genome
Chapter 21
Recent Advances in Gene Editing Technology Adds New Therapeutic Potential for the Genomic Era: Medical Interpretation of the Genomics Frontier – CRISPR – Cas9
Introduction
21.1 Introducing CRISPR/Cas9 Gene Editing Technology – Works by Jennifer A. Doudna
21.1.1 Ribozymes and RNA Machines – Work of Jennifer A. Doudna
21.2 CRISPR in Other Labs
21.2.1 CRISPR @MIT – Genome Surgery
21.2.2 The CRISPR-Cas9 System: A Powerful Tool for Genome Engineering and Regulation
Yongmin Yan and Department of Gastroenterology, Hepatology & Nutrition, University of Texas M.D. Anderson Cancer, Houston, USADaoyan Wei*
21.2.5 CRISPR & MAGE @ George Church’s Lab @ Harvard
21.3 Patents Awarded and Pending for CRISPR
21.3.1 Litigation on the Way: Broad Institute Gets Patent on Revolutionary Gene-Editing Method
21.3.2 The Patents for CRISPR, the DNA editing technology as the Biggest Biotech Discovery of the Century
21.4 CRISPR/Cas9 Applications
21.4.2 CRISPR: Applications for Autoimmune Diseases @UCSF
21.4.3 In vivo validated mRNAs
21.4.6 Level of Comfort with Making Changes to the DNA of an Organism
21.4.8 CRISPR/Cas9 Finds Its Way As an Important Tool For Drug Discovery & Development
Summary
List of Videos by Chapter
Introduction
VIEW VIDEO – Courtesy of National Human Genome Research Institute on GenomeTV
The Human Genome and Individualized Medicine – David Valle
http://www.youtube.com/watch?v=rnMW3fdCV5g
Part I
VIEW VIDEO – Courtesy TRINITYCOLLEGEDUBLIN
‘What is Life? A 21st Century Perspective’ by Dr Craig Venter
http://www.youtube.com/watch?v=qi2MhsUSu0U
Chapter 1
VIEW VIDEO – Courtesy of National Human Genome Research Institute on GenomeTV
Genome-Wide Association Studies – Karen Mohlke (2012)
http://www.youtube.com/watch?v=HHvdupHgeFg
Chapter 2
VIEW VIDEO – Courtesy of National Human Genome Research Institute on GenomeTV
Human Genome Structural Variation, Disease, and Evolution – Evan Eichler
http://www.youtube.com/watch?v=KVJV_pg5zaM
Chapter 3
VIEW VIDEO – Courtesy of National Human Genome Research Institute on GenomeTV
Conceptualization of the Human Genome Project & Development of Data
http://www.youtube.com/watch?feature=player_detailpage&v=NF2Ew1E1kZE
Chapter 4
VIEW VIDEO – Courtesy of National Human Genome Research Institute on GenomeTV
The Genomic Landscape circa 2012 – Eric Green
http://www.youtube.com/watch?v=GLwCs370IGI&playnext=1&list=PL7BF28B5835280CFC&feature=results_video
Chapter 5
VIEW VIDEO – Courtesy of GoogleTechTalks
Human Genetics and Genomics: The Science for the 21st Century
http://www.youtube.com/watch?feature=player_embedded&v=9SzwiZMSBeQ
Part III
VIEW VIDEO Courtesy UCBerkeleyEvents
Published on Jan 30, 2013, Regents’ Lecture, 1/24/13
Genomic Medicine Challenge: Translating Basic Research
http://www.youtube.com/watch?v=7Soz7uOMAcA
Chapter 6
VIEW VIDEO – Courtesy of GeneNetwork
Chapter 7
VIEW VIDEO – Courtesy of National Human Genome Research Institute on GenomeTV
Molecular Pathology will move to NGS, College of American Pathologists – Debra Leonard
published on Feb 8, 2013
http://www.youtube.com/watch?feature=player_detailpage&v=52IYHGfEoNo
Chapter 8
VIEW VIDEO – Courtesy of Stanford University
Genomics and Personalized Medicine
http://www.youtube.com/watch?v=pgHAXCMMcro
Chapter 9
VIEW VIDEO – Courtesy of Programmingoflife
Programming of Life
http://www.youtube.com/watch?v=00vBqYDBW5s
Chapter 10
VIEW VIDEO – Courtesy of Vanderbilt University
Dr. Dan Roden: “Personalized Medicine: Your genome and the future of medicine”
http://www.youtube.com/watch?v=wfLMZQCZYu4
Part IV
VIEW VIDEO – Courtesy of Vanderbilt University
Your Genome and the Future of Medicine: Laying the Groundwork
http://www.youtube.com/watch?v=I2En61Pz5So
Chapter 11
VIEW VIDEO – Courtesy of JHUAAP
Cancers Genomes and their Implications for Curing Cancer by Bert Vogelstein
http://www.youtube.com/watch?v=KE8TY0gT–g
Chapter 12
VIEW VIDEO – Courtesy of UCtelevision
On the Same Page: Personalized Medicine
http://www.youtube.com/watch?v=I1qdpWZR1_c
Chapter 13
VIEW VIDEO – Courtesy of National Human Genome Research Institute on GenomeTV
Pharmacogenomics – Howard McLeod (2012)
http://www.youtube.com/watch?v=A4IV7MC_x08
Chapter 14
VIEW VIDEO – Courtesy of UCtelevision
Genomics and Infectious Diseases
http://www.youtube.com/watch?v=cgSTP84qDp0
Chapter 15
VIEW VIDEO – Courtesy of National Human Genome Research Institute on GenomeTV
The heart of the matter: genomics and cardiovascular disease – Leslie Biesecker
http://www.youtube.com/watch?v=Kg82C4di5Ck
Chapter 19
VIEW VIDEO – Courtesy of 23andMe·
23andMe and PPH Partner for DNA Testing
http://www.youtube.com/watch?v=MJQ3FBitlJ0&playnext=1&list=PL1F27A9171C88CF77&feature=results_main
Chapter 20
AUDIOS
Barriers and Solutions to the Implementation of Personalized Medicine
Genomic and Personalized Medicine Forum Duke Institute for Genome Sciences & Policy. Geoffery Ginsburg, MD, PhD – Executive Director, Center for Personalized Medicine; Director, Genomic Medicine; Institute for Genome Sciences & Policy
Modeling the Morbid Human Genome
Nicholas Katsanis, PhD – Director, Center for Human Disease Modeling, Professor – Departments of Cell Biology and Pediatrics
Chapter 21
https://www.physicsforums.com/threads/breakthrough-prize-genome-editing-with-crispr-cas9.798959/
https://www.quantamagazine.org/20150206-crispr-dna-editor-bacteria/
VIDEOS
VIEW VIDEO – Courtesy of Indiana University School of Medicine via youtube.com
Ethical Issues in Personalized Medicine
http://www.youtube.com/watch?feature=player_detailpage&v=1LDZPdZlt0c
VIEW VIDEO – Courtesy of UBCInterdisciplinary via youtube.com
Who Should Determine the Nature of Genome Testing in ‘Genomic Medicine in 2015’
http://www.youtube.com/watch?feature=player_detailpage&v=0QKs0b8qJys
Introduction
Larry H Bernstein, MD, FCAP
This volume of articles addresses the current dilemma of our time. This is because we have had a succession of wars in a post WWII that on the one hand, spurred technology innovation to a level of accomplishment in the 20th century surpassing the previous three hundred years, and which has accelerated in the first ten years of the 21st century. This has been equally good for the very young, and it remains to be seen, for the post-baby boomers. We are also faced with the costs of a long term debt from a prolonged engagement in hegemonic foreign policy and a 15 year period of risky investments, dominated by a housing market crash. Despite these problems the basic research on the anatomy, physiology, and pathophysiology has reaped benefits that are leading to a new emerging framework for a more consolidated pharmaceutical industry required to set higher standards, to identify drug targets, to diminish toxicity risks and identify problems in the earliest phase of Clinical Trials, and to take on the greatest challenges that seemed insurmountable before.
This result is leading to a not nearly mature, but enthusiastic embrace of personalized medicine. This is already resulting in a different engagement between the patient and physician, a redefinition of how clinical trials are to be carried out, and a better underpinning of the systems biology approach to medical discovery – by cooperative arrangements between government and universities, and with industry. The advances in the basic science of the chromosome and cell proliferation, of the genome regulatory function, and the discovery of a functional role in the “dark matter”, euphemistically called “Junk DNA”, subcellular “cross talk”, and cell signaling pathways has opened a “Pandora’s Box”. The future continues to be just around the corner!
Even more impressive, as the reader takes this journey in reading, there will be an “emergence” and discovery that our thinking about the genome and genetics has fundamentally changed with a convergence of biophysics, chemistry, biology, medicine, and biotechnology ad bioengineering with a compression of the “OMICS” as a result of a realization of the evolutionary consistency of retained functions in cell substructure over long stretches of time and across species, and the major function of chromatin structure takes on a regulatory role, far exceeding the simple Watson-Crick and early translational models (DNA to RNA to protein; mRNA and ER; mitochondrion; lysosome). This becomes more clear when we examine how disturbances arise by small mutational changes in non-coding DNA as well as coding DNA that result in what we have always considered “disease”.
We find that disease manifests in different ways in some organ systems, and to some extent, reflected in the ontogeny (which recapitulates phylogeny). The best example is the observation that carcinoma of the lung, liver, gall bladder, oral cavity, esophagus, and colon, have genomic imprints in common that are not strongly featured in other types of cancer. So we have on the one hand, cancer cells that are less differentiated than their parent, and on the other hand, more like cancer cells from the same post-embryonic cell line than those from other cell lines. A challenge that came out of this is to determine at what time in the stage of disease progression, intervention is most effective. As a result of this coming into a “systems biology” approach, we have discovered signaling pathways, “driver mutations”, and ligand-binding interactions that are crucial for cellular metabolism. We can no longer think that it’s all about the “CODE”, although the code is the link to cellular metabolism, both ordered and dysfunctional.
VIEW VIDEO – Courtesy of National Human Genome Research Institute on GenomeTV
The Human Genome and Individualized Medicine – David Valle
http://www.youtube.com/watch?v=rnMW3fdCV5g
Table of Contents
Select Ordered Keywords
GpA replication, transcription, covalent bond, nucleoside, nucleotide, nucleotide sequences, DNA, Double Helix, RNA, protein, chromosome, nucleus, mitochondrion
GpB metabolic control, histone, nucleosome, telomere, telomerase, phosphorylation, growth and morphogenesis, polymeric structure, cellular proliferation, intercellular adhesion
GpC ENCODE, expanded genetic code, fractal gene, helical folding models,
GpD noncovalent bond, mutagenesis, DNA repair, apoptosis and mitophagy , response to oxidative stress, mitochondrial dysfunction,
GpE cell organelle interactions, ubiquitination, ribosomes and ribophagy, methylation, synthetic nucleotides, hydrophobic repulsion, phosphofructokinase (PFK), allostericity, cytochromes and electron transport
GpF isomers, nitric oxide, nitric oxide synthases, signaling pathways, cytokine, DNA-histone interaction, intron
GpG cellular transformation, cell membrane plasticity, cellular movement, cell type and shape, lipid structures
GpH cardiovascular disease and cancer, neurodegenerative and muscular diseases, aging, inducible progenitor stem cell (iPSC), drug target
Part I
The Classical Model of the Gene
The story begins after WWII with Niels Bohr directing his student to upgrade biology to the established principles of Physics. This was not long after Feynman lost his wife to cancer, and he concluded without reservations that the doctors at the Mayo Clinic had nothing to work with.
Von Neumann constructed the first computer, and Turing had broken the “code”, then constructed the Turing “machine”, the basis for a “thinking machine”. Claude Shannon took from “statistical thermodynamics” to create information theory, which became information-induction in the hands of Solomon Kullback (logarithmic measures of information and their application to testing statistical hypotheses; Amazon). You think, “What does this happen to do with biology and medicine?” Read on, because it was a terrific enabler. Biology was leaving the “descriptive stage”, and would be able to explain the diversity seen in the era of Darwin and Humboldt. Physics was shaken by the rigidity of the 19th century “determinism”, but in Part I we do have a simplistic model for genetics.
Issues to consider:
Prior to the discovery of the DNA base pairs, what scientific work precedes the marvelous story?
[1] Linus Pauling’s “Nature of the Chemical Bond”
[2] Radioisotope labels and Lawrence Livermore Laboratory under EO Lawrence
[3] Otto Warburg’s work on oxidative metabolism, and the as yet unexplained “Pasteur Effect” in an oxygen environment (Warburg hypothesis)
[4] Discovery and isolation of adenine nucleotides, purines, pyrimidines, pyridine nucleotides, FMN
[5] Krebs cycle intermediates and the tie in with electron transport chain
[6] Fritz Lipmann’s discovery of Coenzyme A, The Lynen cycle (fatty acid synthesis), Cori cycle
[7] The terms mitochondria, ribosome, lysosome are added to the nucleus to describe a cell
What is the big question that remains:
[1] The mechanism of cell division
[2] The transmission of information related to observed traits
[3] An organo-mechanical explanation for Darwin’s evolution from Gregor Mendel, one-gene one-enzyme, Thomas Hunt Morgan’s work on fruit flies, and Ronald Fisher’s discovery of the discriminant function and treatment of leaf and petal length and width.
Keywords: replication, transcription, covalent bond, nucleoside, nucleotide, nucleotide sequences, DNA, Double Helix, RNA, protein, chromosome, nucleus, ribosome, nucleotide base-pairs
VIEW VIDEO – Courtesy TRINITYCOLLEGEDUBLIN
‘What is Life? A 21st Century Perspective’ by Dr Craig Venter
http://www.youtube.com/watch?v=qi2MhsUSu0U
Chapter 1
Basic Science Foundation for the Genome in Cell Proliferation and Cell Death
The emergence of molecular biology inserts itself at Cold Spring Harbor Laboratories with Max Delbruck’s annual lectures attended by young scientists flocking to participate in the most relevant studies in biology. The future Nobel laureate James Watson attends with his mentor, Nobel laureate Salvador Luria. He will present at the 1953 CSHL meetings, the year he share the Nobel Prize for the “Watson-Crick” model of DNA based on the crystallography of the deceased Rosalind Franklin. The new model for discovery in the field is bacteria, cells grown in culture, sea urchin, plant seeds, eukaryotes,..not humans, maybe cells of aflatoxin (www.ansci.cornell.edu) – induced hepatic carcinoma in rats.
aflatoxin 
The regulatory function of the genome is waiting to be discovered. The focus is on DNA replication, cell division, uncontrolled proliferation, an explanation for the balance between synthetic and catabolic processes, and the effects of oxidative stress that leads to signaling pathways and the allosteric behavior of phosphofructokinase (PFK). The model is being built by a “deconstructionist” work effort.
Keywords: replication, transcription, covalent bond, nucleus, nucleoside, nucleotide, nucleotide sequences, DNA, Double Helix, RNA, chromosome, telomere, telomerase
VIEW VIDEO – Courtesy of National Human Genome Research Institute on GenomeTV
Genome-Wide Association Studies – Karen Mohlke (2012)
http://www.youtube.com/watch?v=HHvdupHgeFg
Larry H Bernstein, MD, FCAP
1.2 CRACKING THE CODE OF HUMAN LIFE: Milestones along the Way – Part IIA
Larry H Bernstein, MD, FCAP
1.3 DNA – The Next-Generation Storage Media for Digital Information
Sudipta Saha, PhD
1.4 CRACKING THE CODE OF HUMAN LIFE: Recent Advances in Genomic Analysis and Disease – Part IIC
1.5 Advances in Separations Technology for the “OMICs” and Clarification of Therapeutic Targets
Larry H Bernstein, MD, FCAP
1.6 Genomic Analysis: FLUIDIGM Technology in the Life Science and Agricultural Biotechnology
Aviva Lev-Ari, PhD, RN
Chapter 2
Going Beyond the Classical Model
This work is now proceeding in several prestigious academic centers, and leads from the gene to the deconstruction of the genome. The discovery of nucleotide base pairs that code for the entire genome is underway. There are changes in nucleotide sequences that become a basis for mutation analysis. At this time, there is an emerging revolution in computational and applied mathematical support, just as the 21st millenium arrives. Now we find variation in copy-number, short sequence repeats, single-nucleotide polymorphisms. What do these findings mean? In addition, some portions of the genome are carried over from ancient ancestral roots.
“Interdisciplinary Science” September 2010 issue, J.C. Perez published a peer-reviewed paper proving that the whole human genome codon populations are managed by a “DRAGON fractal paper folding curve” fine-tuned around the “Golden ratio”. Particularly, this main paper entitled “Codon populations in single-stranded whole human genome DNA Are fractal and fine-tuned by the Golden Ratio 1.618.” shows that the Universal Genetic Code Table not only maps codons to amino acids, but serves as a global checksum matrix at the whole genome macro-structural scale.
Keywords: metabolic control, histone, nucleus, nucleosome, phosphorylation, polymeric structure, cellular proliferation, Single-nucleotide (SNP), copy-number variation, oligonucleotide, alleles
An image on Jean-Clode Perez’s first discovery (1990) NUMBER OF Fibonacci in the DNA coding for genes …. The discovering consists of the fact that DNA consists of the set of “resonances” of the considered kind, that is, as a rule, sections of the genetic code of the length, equal Fibonacci number Fn are divided by the golden section into the set of the T-bases.
In “Interdisciplinary Science” September 2010 issue, J.C. Perez published a peer-reviewed paper proving that the whole human genome codon populations are managed by a “DRAGON fractal paper folding curve” fine-tuned around the “Golden ratio”. Particularly, this main paper entitled “Codon populations in single-stranded whole human genome DNA Are fractal and fine-tuned by the Golden Ratio 1.618.” shows that the Universal Genetic Code Table not only maps codons to amino acids, but serves as a global checksum matrix at the whole genome macro-structural scale.
The surprising discovery by Jean-Clode Perez allows an interesting conclusion regarding an analogy between music, poetry, market processes (“Elliott Waves”) and genetic code. (Fibonacci’s “resonance’s “, underlying the SUPRA-code). Fibonacci numbers (1, 2, 3, 5, 8, 13, 21, 34, 55, 89, 144, …). It is the same proportion, which controls by morphology of natural organisms such, as a pinecone, cactus, pineapple, etc.
http://www.goldenmuseum.com/1611001.jpg
Biperiodic Table of Petoukov (2010)
http://www.goldenmuseum.com/1611003.gif
An Artistic Image of Dynamic Molecular-Genetic Structure
Zenkin’s WEB site http://www.docstoc.com/docs/127713303/Intellectual-Aesthetics-Of-Scientific-Discoveries
JC Perez’s analysis of sensitivity of DNA to noise
http://creationwiki.org/pool/images/thumb/4/4f/Golden_ratio.jpg/180px-Golden_ratio.jpg
Golden Ratio emerges from Fractal Chaos
2.1 2013 Genomics: The Era Beyond the Sequencing of the Human Genome: Francis Collins, Craig Venter, Eric Lander, et al.
2.2 DNA structure and Oligonucleotides
2.3 Genome-Wide Detection of Single-Nucleotide and Copy-Number Variation of a Single Human Cell
Stephen J. Williams, PhD
Marcus W Feldman, PhD
Aviva Lev-Ari, PhD, RN
2.6 The Binding of Oligonucleotides in DNA and 3-D Lattice Structures
Larry H Bernstein, MD, FCAP
2.7 Finding the Genetic Links in Common Disease: Caveats of Whole Genome Sequencing Studies
Stephen J. Williams, Ph.D.
VIEW VIDEO – Courtesy of National Human Genome Research Institute on GenomeTV
Human Genome Structural Variation, Disease, and Evolution – Evan Eichler
http://www.youtube.com/watch?v=KVJV_pg5zaM
Chapter 3
Big Data and Relating the Code to Metabolic Signatures
The support of computational power and price decreases in the cost of storage leads to Big Data. It is this factor that gives life to bioinformatics and computational biology. This enables the linking of the genome, or polynucleotide sequences to cellular metabolic activity. What will emerge is referred to the “OMICs” revolution. The rapid evolution of instruments in GC, GC/MS, NMR, and such enables the discovery of small molecules, opening up the proteome and the metabolome, which is set to become “translational medicine”.
Keywords: genome, proteome, metabolome, transcriptome, computational models, big data, spectrometry, cytoskeleton, mitochondrion, mDNA, cell membrane plasticity, cellular movement
VIEW VIDEO – Courtesy of National Human Genome Research Institute on GenomeTV
Conceptualization of the Human Genome Project & Development of Data
http://www.youtube.com/watch?feature=player_detailpage&v=NF2Ew1E1kZE
3.1 Big Data in Genomic Medicine
Larry H. Bernstein, MD, FCAP
3.2 CRACKING THE CODE OF HUMAN LIFE: The Birth of Bioinformatics & Computational Genomics – Part IIB
Larry H. Bernstein, MD, FCAP
3.3 Expanding the Genetic Alphabet and linking the Genome to the Metabolome
Larry H. Bernstein, MD, FCAP
3.5 MIT Scientists on Proteomics: All the Proteins in the Mitochondrial Matrix identified
Aviva Lev-Ari, PhD, RN
3.6 Identification of Biomarkers that are Related to the Actin Cytoskeleton
Larry H. Bernstein, MD, FCAP
Aviva Lev-Ari, PhD, RN
Aviva Lev-Ari, PhD, RN and Larry Bernstein, MD, FCAP
Chapter 4
The Expansion of the Genetic Code
Indexing encyclopedic catalog of sequence variants and Open Access Resources on DNA Research
This chapter deals entirely with the cataloguing of the genetic code, and unlocking polynucleotide sequences that have association with identified diseases. This will lead to specific genomic targets for therapeutic intervention.
Keywords: ENCODE, expanded genetic code, fractal gene, helical folding models
VIEW VIDEO – Courtesy of National Human Genome Research Institute on GenomeTV
The Genomic Landscape circa 2012 – Eric Green
http://www.youtube.com/watch?v=GLwCs370IGI&playnext=1&list=PL7BF28B5835280CFC&feature=results_video
4.1 ENCODE Findings as Consortium
Aviva Lev-Ari, PhD, RN
4.2 ENCODE: The Key to Unlocking the Secrets of Complex Genetic Diseases
Ritu Saxena, PhD
Anamika Sarkar, Ph.D
Aviva Lev-Ari, PhD, RN
4.5 Human Genome Project – 10th Anniversary: Interview with Kevin Davies, PhD – The $1000 Genome
Aviva Lev-Ari, PhD, RN
4.6 Quantum Biology And Computational Medicine
Larry H. Bernstein, MD, FCAP
4.7 The Underappreciated EpiGenome
Demet Sag, PhD
4.8 Unraveling Retrograde Signaling Pathways
Larry H. Bernstein, MD, FCAP
4.9 “The SILENCE of the Lambs” Introducing The Power of Uncoded RNA
Demet Sag, PhD
4.10 DNA: One man’s trash is another man’s treasure, but there is no JUNK after all
Demet Sag, PhD
Part II
Emergence of the Genomic Network
This concept of DNA as a static entity eventually gave rise to a more dynamic biomolecule as studies discovered how gene regulation, alternative splice sites, single nucleotide polymorphisms, transposable DNA elements, microRMAs, and epigenetics could alter the cellular phenotype and function in physiology and disease. As a result, researchers started to shift from studying the effects of single genes to a more global genetic view that alterations of genetic networks were important in disease manifestations. These studies became possible with the advent of high throughput technologies and increased computing power. As a result, a paradigm shift from studying one gene at a time to studying thousands of genes at one time, resulted in the research presented in this Chapter.
Chapter 5
Quest for the Key to Life – Promise of the Paradigm Shift
There are a number of very active genomic research centers investigating genomic sequence changes on a large scale and linking the genetic data to disease.
Keywords: Next-generation sequencing, whole-genome sequencing, personalized medicine, signaling pathways, cytokine, DNA-histone interaction
VIEW VIDEO – Courtesy of GoogleTechTalks
Human Genetics and Genomics: The Science for the 21st Century
http://www.youtube.com/watch?feature=player_embedded&v=9SzwiZMSBeQ
5.1 Paradigm Shift in Human Genomics – Predictive Biomarkers and Personalized Medicine – Part 1
Aviva Lev-Ari, PhD, RN
5.2 Computational Genomics Center: New Unification of Computational Technologies at Stanford
Aviva Lev-Ari, PhD, RN
5.3 Personalized Medicine: An Institute Profile – Coriell Institute for Medical Research: Part 3
Aviva Lev-Ari, PhD, RN
5.4 Cancer Genomics – Leading the Way by Cancer Genomics Program at UC Santa Cruz
Aviva Lev-Ari, PhD, RN
5.5 Genome and Genetics: Resources @Stanford, @MIT, @NIH’s NCBCS
Aviva Lev-Ari, PhD, RN
5.6 NGS Market: Trends and Development for Genotype-Phenotype Associations Research
Aviva Lev-Ari, PhD, RN
Aviva Lev-Ari, PhD, RN
Larry H Bernstein, MD, FCAP
5.9 Transphosphorylation of E-coli Proteins and Kinase Specificity
Larry H Bernstein, MD, FCAP
5.10 Genomics of Bacterial and Archaeal Viruses
Larry H Bernstein, MD, FCAP
Chapter 6
Gene Regulatory Role and Cellular Disturbances
The discussion now moves further into the previously unexplored regulatory functions of the gene. There is a profound interaction between the mitochondrion and the nucleus, the cell membrane, and the ribosome. There is much that has come to light on the “dark matter” of the gene, previously considered “junk DNA”. This is the link between our understanding an important function in the genome that is essential to get to an understanding of how to set therapeutic targets.
Keywords: noncovalent bond, mutagenesis, DNA repair, apoptosis and mitophagy , response to oxidative stress, mitochondrial dysfunction, cell organelle interactions, ubiquitination, ribosomes and ribophagy, methylation, synthetic nucleotides, hydrophobic repulsion, phosphofructokinase (PFK), allostericity, cytochromes and electron transport
6.1 Directions for Genomics in Personalized Medicine
Larry H Bernstein, MD, FCAP
6.3 Mitochondrial Damage and Repair under Oxidative Stress
Larry H. Bernstein, MD, FCAP
6.4 Mitochondria: More than just the “Powerhouse of the Cell”
Ritu Saxena, PhD
6.5 Mechanism of Variegation in Immutans
Larry H. Bernstein, MD, FCAP
Sudipta Saha, Ph.D.
VIEW VIDEO
6.7 Cardiac Ca2+ Signaling: Transcriptional Control
Larry H Bernstein, MD, FCAP
6.8 Unraveling Retrograde Signaling Pathways
Larry H. Bernstein, MD, FCAP
Larry H Bernstein, MD, FCAP
Larry H Bernstein, MD, FCAP
6.12 Zebrafish—Susceptible to Cancer
Larry H Bernstein, MD, FCAP
6.13 RNA Virus Genome as Bacterial Chromosome
Larry H Bernstein, MD, FCAP
6.14 Cloning the Vaccinia Virus Genome as a Bacterial Artificial Chromosome
Larry H Bernstein, MD, FCAP
6.15 Telling NO to Cardiac Risk- DDAH Says NO to ADMA(1); The DDAH/ADMA/NOS Pathway(2)
Stephen J. Williams, PhD
6.16 Transphosphorylation of E-coli proteins and kinase specificity
Larry H. Bernstein, MD, FCAP
6.17 Genomics of Bacterial and Archaeal Viruses
Larry H. Bernstein, MD, FCAP
Aviva Lev-Ari, PhD, RN
Part III
Genomics Enters the Clinic
Advances in technologies such as microarray, CGH analysis, proteomics, methylation arrays, and tissue arrays allowed us to 1) investigate the global changing landscape within the cell and tissues, and 2) apply these analyses on a massive scale. Medical research began to realize the utility and application of these technologies to clinical problems, both to disease etiology and later to tailoring therapeutic strategies based on molecular signatures unique to a patient. Genomic strategies are now mainstream for the detection, diagnosis, and treatment of multiple diseases. The following chapters describe some of the recent advances using Genomics in Medicine.
VIEW VIDEO Courtesy UCBerkeleyEvents
Published on Jan 30, 2013, Regents’ Lecture, 1/24/13
Genomic Medicine Challenge: Translating Basic Research
http://www.youtube.com/watch?v=7Soz7uOMAcA
Chapter 7
Personalized Medicine and Genomics Directions
Personalized medicine is the individualized treatment of patients based on a historical knowledge, and more exactly, a bioinformational adjustment of the treatment to the patient based on 1) known pharmacologic toxicity because of genetically determined kinetically-based modification of the expected response to a drug, 2) application of a drug matched to the patient based on drug targeting for the illness, 3) targeted therapy to give the maximal response and to minimize toxicity. The medications used for a significant number of diseases were based on targeting the physiochemical dysfunction without the ability to attack the root cause of illness. This has brought repeated reapplication of old drugs for new uses, development of drug resistance, and limited long term success for chronic diseases. Genomics-based personalized medicine is the promise to realize a greater benefit to the patient, and at reduced long term costs.
Keywords: personalized medicine, individualized treatment, genomic-compatible treatment, biopharmaceutical, targeted therapy, toxicities, dose-response curve, liver metabolism, bioelimination, side-effects
VIEW VIDEO – Courtesy of National Human Genome Research Institute on GenomeTV
Molecular Pathology will move to NGS, College of American Pathologists – Debra Leonard
published on Feb 8, 2013
http://www.youtube.com/watch?feature=player_detailpage&v=52IYHGfEoNo
Aviva Lev-Ari, PhD, RN
7.2 Consumer Market for Personal DNA Sequencing: Part 4
Aviva Lev-Ari, PhD, RN
Aviva Lev-Ari, PhD, RN
Aviva Lev-Ari, PhD, RN
Aviva Lev-Ari, PhD, RN
7.6 Personalized Medicine: Clinical Aspiration of Microarrays
Stephen J. Williams, Ph.D.
Chapter 8
The Implicit Promise of Genome-based Therapeutic Targeting
Genomic therapy is under rapid development in biopharmaceutic therapeutics. It requires the blocking of regulatory sites on the genome, or the insertion of a “negating” code-sequence.
Keywords: personalized medicine, genome therapeutics, code switches, nanotechnology, downregulation, signaling pathways, epigenome, translational medicine
VIEW VIDEO – Courtesy of Stanford University
Genomics and Personalized Medicine
http://www.youtube.com/watch?v=pgHAXCMMcro
8.1 Personalized Medicine as Key Area for Future Pharmaceutical Growth
Aviva Lev-Ari, PhD, RN
8.2 Inaugural Genomics in Medicine – The Conference Program, 2/11-12/2013, San Francisco, CA
Aviva Lev-Ari, PhD, RN
Aviva Lev-Ari, PhD, RN
8.4 Nanotechnology, Personalized Medicine and DNA Sequencing
8.6 Transcript Dynamics of Proinflammatory Genes
Larry H Bernstein, MD, FCAP
Aviva Lev-Ari, PhD, RN
8.8 Intratumor Heterogeneity and Branched Evolution Revealed by Multiregion Sequencing[1]
Stephen J. Williams, Ph.D.
Aviva Lev-Ari, PhD, RN
Chapter 9
Personalized Medicine to the Clinics
Personalized medicine has been introduced with respect to the patient and the promise. It won’t be realized in any substantial way until the barriers to introduction are removed. This doesn’t mean a lower standard of surveillance. The long time required to bring a drug to market is related to the Clinical Trials prior to introduction. FDA is making progress in meeting the surge in biopharmaceutical, although some drug classes have had multiple failures. A part of the picture that is overlooked is the development of biomarkers, either proteomic, or peptide, or polynucleotide assays that can be used to follow the treatment. In the future, drugs will not be approved without the introduction of such a biomarker. In addition, the analytical tools are being developed for use near the patient, and nanotechnology with multispecimen automation is not far away. This progress will directly affect physician decision-making.
Keywords: WGS, NGS, SNPs, applying genome sequencing to disease, mutagenesis, DNA repair, apoptosis and mitophagy, response to oxidative stress, mitochondrial dysfunction, cell organelle interactions, ubiquitination, methylation, synthetic nucleotides
VIEW VIDEO – Programmingoflife
Programming of Life
http://www.youtube.com/watch?v=00vBqYDBW5s
9.1 Personal Tale of JL’s Whole Genome Sequencing
Aviva Lev-Ari, PhD, RN
Aviva Lev-Ari, PhD, RN
Aviva Lev-Ari, PhD, RN
Aviva Lev-Ari, PhD, RN
Aviva Lev-Ari, PhD, RN
9.6 The Initiation and Growth of Molecular Biology and Genomics – Part I
Larry H Bernstein, MD, FCAP
9.7 Personalized Medicine-based Cure for Cancer Might Not Be Far Away
Ritu Saxena, PhD
9.8 Personalized Medicine: Cancer Cell Biology and Minimally Invasive Surgery (MIS)
Aviva Lev-Ari, PhD, RN
Chapter 10
PharmacoGenomics Achievements and New Drug Indications
Genomics is shaping the future of the pharmaceutical industry with a shift from organic medicinal chemistry aimed at targeting neuroendocrine receptors and drugs that interact with accessible circulating proteins or cell receptors with the knowledge that the approach may have limited efficacy, to a targeting of key genomic functions that have established links to specific diseases, which moves into bioinformatics, biotechnology, biopharmaceuticals individual tailored treatment goals.
Keywords: medicinal chemistry, biopharmaceutical genomics, personalized medicine, patient-specific therapy, treatment targets, genomic medicine
VIEW VIDEO – Courtesy of Vanderbilt University
Dr. Dan Roden: “Personalized Medicine: Your genome and the future of medicine”
http://www.youtube.com/watch?v=wfLMZQCZYu4
Aviva Lev-Ari, PhD, RN
10.2 Imatinib (Gleevec) May Help Treat Aggressive Lymphoma: Chronic Lymphocytic Leukemia (CLL)
Aviva Lev-Ari, PhD, RN
Aviva Lev-Ari, PhD, RN
10.4 Treatment for Metastatic HER2 Breast Cancer
Larry H Bernstein, MD, FCAP
10.5 Personalized Medicine in NSCLC
10.6 Gene Sequencing – to the Bedside
Larry H Bernstein, MD, FCAP
10.7 DNA Sequencing Technology
Larry H Bernstein, MD, FCAP
10.8 Nobel Laureate Jack Szostak Previews his Plenary Keynote for Drug Discovery Chemistry
Aviva Lev-Ari, PhD, RN
Part IV
Targeting Cancer
Cancer treatment has historical been elusive and palliative because of the known tissue and/or organ type behaviors, delay in diagnosis, differences in behaviors of leukemias vs solid tumors, and other factors not elucidated until recently. The discovery of leukemia was by Rudolph Virchow, who was the father of modern pathology, but he also visited the wards on a daily basis. He observed the leukemic cells under the microscope, and he identified the proliferation of myeloid and lymphocytic cell lines that later led to classifications based on nuclear to cytoplasmic ratio, abnormal nuclear changes, and later, changes identified with histologic stains that have not shifted to genomic biomarkers. Next was the closely related, solid tumor of the lymphoid-immune system, which could be irradiated, and which had variable prognosis depending on the mix of small and large lymphocytes and the distortion of the structure. Virchow had concerns about his students becoming over-reliant on the morphology. Hodgkin’s lymphoma was describes in 1832 by the individual of that name. It has eosinophils sprinkled in the lymphocytic tumor.
Dorothy Hodgkin’s name is also attached to the lymphoma (an anatomist at University of Minnesota). Hodgkin’s lymphoma has a relatively benign course. The early pioneers were Lawrence Berman (Detroit), Rappaport (Chicago), and the early classification was developed in 1966 before lymphoid cells were divided into B-cells and T-cells. Then came the Lukes Collins modifications in 1974. The current 2008 WHO classification system, developed in 2008 has been adopted by most hematologists/oncologists.
Beginning shortly after the initial experiments of Billingham, Brent and Medawar, Robert A. Good and colleagues made, extensive studies of the bases of immunologic tolerance and strategies for producing immunologic tolerance experimentally (1955-1991). Good discovered that plasma cells are the major antibody elements in the mammalian system, independent of, but parallel to the critical contributions of Fagraeus of Sweden (1947-1951). Bisection of the lymphoid cell universe into antibody producing plasma cells and lymphocytes responsible for cell mediated immunity in X-linked agammaglobulinemia in 1954 was described as ‘Good’s Syndrome’. Bruce Glick and later by Max Cooper and Robert Good, demonstrated that the bursa of Fabricius is necessary for B (antibody producing) cell development in birds by removal of the bursa in newly born chicks.
- Ribatti D, Crivellato E, Vacca A (2006). “The contribution of Bruce Glick to the definition of the role played by the bursa of Fabricius in the development of the B cell lineage”. Clin. Exp. Immunol. 145 (1): 1–4. doi:10.1111/j.1365-2249.2006.03131.x. PMC 1942006. PMID 16792666.^ http://www.nlm.nih.gov/cgi/mesh/2009/MB_cgi?mode=&term=Bursa+of+Fabricius
- Sternberg SS, “Bottoms Up to a Nobel-Worthy Chicken’s Bottom” American Journal of Surgical Pathology Volume 27(11), November 2003, pp 1471-1472
- Cooper, M. D., R. D. A. Peterson, M. A. South, R. A. Good. (1966). The functions of the thymus system and the bursa system in the chicken. J. Exp. Med. 123:75.
The bursa is an epithelial and lymphoid organ that is found only in birds, and its equivalent in mammals is the lymphatic system derived from bone marrow and spleen, distinguished from thymic derived lymphocytes, or T-cells. The role of the thymus in the immune response was also identified shortly after the discovery of bursa’s role in antibody responses. In thymectomized animals, the ability to reject allografts, and to mount delayed hypersensitivity responses, was drastically reduced. By the mid-1960s, immunologists were convinced that there were indeed two separate arms of the immune system: one dealing exclusively with the production of circulating antibodies (humoral immunity), and another that is involved in the delayed hypersensitivity-type reactions and graft rejections (cell-mediated immunity). Good also demonstrated that bone marrow transplantation in mice may be regularly used to completely correct immunodeficiency, caused by fatal irradiation, without producing graft vs. host disease, if the bone marrow is first purged of all post-thymic committed cells. Both immunocompetent T cells and immunoincompetent T-cell precursors must first be removed.
In collaboration with Reisner, Kapoor and O’Reilly, Good successfully applied this principle to correct severe combined immunodeficiency disease of humans using marrow from mismatched haploidentical parental donors. Along with others around the world, Good has shown that marrow transplantation using his matched sibling donor methodology can be used to treat successfully and provide normal life for patients suffering from some 60 genetically determined, or acquired, lethal diseases. From investigations with his German and Japanese students, Wustow, Onoe, Ikehara and Jyonouchi, and along with Kincade and Fernandes, Good showed that bone marrow transplantation both within and across major histocompatibility barriers can be used as a means of introducing resistance genes against leukemia, and as the means to correct completely genetically based immunologic abnormalities in autoimmune-prone NZB mice.
Neuroblastoma (N.B.) is the most common extracranial solid cancer in childhood and the most common cancer in infancy. This form of cancer is a malignant growth of neural tissue in or around the adrenal medulla. Neuroblastoma most commonly affects children age 5 or younger, and some recede without treatment. Melanoma is not a cancer of childhood. It is only expressed after puberty.
The birth of chemotherapy is credited with Sidney Farber at Harvard Medical School. The next in a series of successes were other early childhood cancers. The discovery of oophorectomy for breast cancer in Chicago, and the use of minimally invasive breast surgery in Cleveland were landmarks in the war on cancer. There was a stigma associated with breast cancer, and we no longer see the fungating, necrotic breast cancers that were not uncommon 40 years ago. But successes were largely limited and palliative, and 5 year survivals was the measure for comparison of all tumors resected.
Keywords: malignant neoplasm, benign tumor, cancer, cancer progression, cancer resection, cancer radiation, time to recurrence, cancer immunity, RNA, protein, chromosome, nucleus, mitochondrial dysfunction, DNA repair, apoptosis and mitophagy , response to oxidative stress
VIEW VIDEO – Courtesy of Vanderbilt University
Your Genome and the Future of Medicine: Laying the Groundwork
http://www.youtube.com/watch?v=I2En61Pz5So
Chapter 11
RNA Manipulation and Disease Management
RNA has shown promise as the intermediate in transcription of the genetic code because it is a small polynucleotide that ties the genome to protein synthesis and organelle function. However, while it can be engineered for therapy, it is unclear whether a change in the sequence that gave rise to the cancer would be associated with resistance.
Keywords: mRNA, mtRNA, siRNA, RNA polymerase, gene-deletion, gene-insertion
11.1 mRNA Interference with Cancer Expression
Larry H. Bernstein, MD, FCAP
11.2 Angiogenic Disease Research Utilizing microRNA Technology: UCSD and Regulus Therapeutics
Aviva Lev-Ari, PhD, RN
Aviva Lev-Ari, PhD, RN
11.4 A microRNA Prognostic Marker Identified in Acute Leukemia
Prabodh Kandala, PhD
11.5 MIT Team: Microfluidic-based approach – A Vectorless delivery of Functional siRNAs into Cells.
Aviva Lev-Ari, PhD, RN
Sudipta Saha, Ph.D
11.7 When Clinical Application of miRNAs?
Larry H Bernstein, MD, FCAP
11.8 How mobile elements in “Junk” DNA promote cancer. Part 1: Transposon-mediated tumorigenesis,
Stephen J Williams, PhD
11.9 Potential Drug Target: Glycolysis Regulation – Oxidative Stress-responsive microRNA-320
Aviva Lev-Ari, PhD, RN
11.10 MicroRNA Molecule May Serve as Biomarker
Larry H Bernstein, MD, FCAP
11.11 What about Circular RNAs?
Larry H Bernstein, MD, FCAP
VIEW VIDEO – Courtesy of JHUAAP
Cancers Genomes and their Implications for Curing Cancer by Bert Vogelstein
http://www.youtube.com/watch?v=KE8TY0gT–g
Chapter 12
Genomics and Cancer
This chapter discusses a fundamental problem in dealing with cancer genomics. We have a good knowledge of some key mechanisms that have to be attacked, such as, the ubiquitination/ apoptosis and the methylation processes, as well as oxidative stress. James Watson gives a critical viewpoint on the benefit of antioxidant therapy. There is also a detailed look at the problem of the metabolic changes that are inherent in all cancers that were described by Otto Warburg in the 1920s as a conversion of aerobic metabolizing cells to mitochondrial-defective cells that rely on anaerobic metabolism, as bacteria (Pasteur Effect). This is actually adaptive for the cell, blocking the mitochondrial activity in TCA metabolism and using it for synthesis of new cells at the expense of the organism (which would be cancer cachexia).
Keywords: aerobic glycolysis, TCA cycle, facultative anaerobe, gluconeogenesis, phosphofructokinase, allostericity, fumarate, entry into mitochondrial pathways, cancer mutagenesis, apoptosis, mitophagy and ribophagy, cancer progression, cell proliferation, control of cellular functions, intercellular adhesion, metastasis.
VIEW VIDEO – Courtesy of UCTelevision
On the Same Page: Personalized Medicine
http://www.youtube.com/watch?v=I1qdpWZR1_c
12.1 The “Cancer Establishments” Examined by James Watson, Co-discoverer of DNA w/Crick, 4/1953
Aviva Lev-Ari, PhD, RN
12.2 Otto Warburg, A Giant of Modern Cellular Biology
Larry H. Bernstein, MD, FCAP
12.3 Is the Warburg Effect the Cause or the Effect of Cancer: A 21st Century View?
Larry H. Bernstein, MD, FCAP
12.4 Hypothesis – Following on James Watson
Larry H. Bernstein, MD, FCAP
12.5 AMPK Is a Negative Regulator of the Warburg Effect and Suppresses Tumor Growth In Vivo
Stephen J Williams, PhD
12.6 AKT signaling variable effects
Larry H. Bernstein, MD, FCAP
Stephen J Williams, PhD
12.8 Phosphatidyl-5-Inositol signaling by Pin1
Larry H Bernstein, MD, FCAP
Chapter 13
Genomic Identification and Potential Treatments of Specific Cancers
This chapter reviews the genomic identification and treatments of some specific cancers. Of some interest is that the lens of the eye doesn’t undergo carcinogenesis because the lens cells lose their nuclei. While the mature red cell is also without a nucleus, the active bone marrow precursors generate leukemia, but erythroleukemia is rare. The lens of the eye and the mature red cell have 85 percent of their metabolism tied up in glycolysis, and much of the remainder in the pentose phosphate shunt (which is a driver of purine metabolism).
Keywords: nanotechnology, therapeutic targets, leukemias and lymphomas, cancers of solid organs, biomarkers, premalignant, cellular proliferation, cell membrane plasticity, cellular transformation, cellular movement, metastasis, regression, apoptosis, tumor suppressor, pathway activation, aerobic glycolysis, exome sequencing, transcription, ubiquitination, DNA repair, chromatin remodeling, somatic mutations, endocrine-driven
VIEW VIDEO – Courtesy of National Human Genome Research Institute on GenomeTV
Pharmacogenomics – Howard McLeod (2012)
http://www.youtube.com/watch?v=A4IV7MC_x08
13.1 Nanotech Therapy for Breast Cancer
Tilda Barlyia, PhD
Sudipta Saha, Ph.D
Sudipta Saha, Ph.D
13.5 Prostate Cancer: Androgen-driven “Pathomechanism” in Early onset Forms of the Disease
Aviva Lev-Ari, PhD, RN
13.6 In focus: Melanoma Genetics
Ritu Saxena, PhD
Aviva Lev-Ari, PhD, RN
13.8 Breast Cancer and Mitochondrial Mutations
Larry H Bernstein, MD, FCAP
13.9 Long noncoding RNA network regulates PTEN transcription
Larry H Bernstein, MD, FCAP
Part V
Systemic and Organ System Genomics
This is an ordered series of presentations based on distinctive organ systems, and the metabolic environment engendered by each organ. The metabolism and the causes of disease were not known when Rokitanski in Vienna devised the systematic examination of organs by the changes in gross pathology viewed in the autopsy organ by organ. We look at the heart, lung, GI tract, kidney and lower urinary tract, adrenals, thyroid, and the brain, pituitary, and spinal cord. In time, there was a classification of diseases as inflammatory, immunological, neoplastic, circulatory, genetic, metabolic.
The student of Rokitansky, Ignatz Semelweis, observed that women could give birth on the street or with a midwife more safely than in the delivery room and instituted rigorous hand-washing for physicians delivering (nurses were not transmitters). It was also the anatomist and surgeon, John Hunter, who observed that outcomes from war inflicted wounds (Seven Year War) as he pulled the wounded out of the mud. Hunter’s favorite student was the country physician, Edward Jenner. In 1796, Dr. Jenner observed that milkmaids developed cowpox and were immune to developing smallpox and discovered a way to protect people from getting smallpox with the development of the first smallpox vaccine. Before widespread vaccination, mortality rates in individuals with smallpox were high—up to 35% in some cases. The disease was known as Pox or Red Pox in England.
The disease had reoccurred for centuries going back to ancient Egypt. The first clear reference to smallpox inoculation was made by the Chinese author Wan Quan (1499–1582) in his Douzhen xinfa (痘疹心法) published in 1549. Inoculation for smallpox does not appear to have been widespread in China until the reign era of the Longqing Emperor (r. 1567–1572) during the Ming Dynasty. In China, powdered smallpox scabs were blown up the noses of the healthy. The patients would then develop a mild case of the disease and from then on were immune to it. The technique did have a 0.5-2.0% mortality rate, but that was considerably less than the 20-30% mortality rate of the disease itself. Variolation was also practiced throughout the latter half of the 17th century by physicians in Turkey, Persia, and Africa. In 1714 and 1716, two reports of the Ottoman Empire Turkish method of inoculation were made to the Royal Society in England, by Emmanuel Timoni, a doctor affiliated with the British Embassy in Constantinople, and Giacomo Pylarini.
Lady Mary Wortley Montagu, wife of the British ambassador to Ottoman Constantinople, is widely credited with introducing the process to Great Britain in 1721. Source material tells us on Montagu; “When Lady Mary was in the Ottoman Empire, she discovered the local practice of inoculation against smallpox called variolation.” The procedure had been performed on her son and daughter, aged five and four, respectively. They both recovered quickly. In 1721, an epidemic of smallpox hit London and left the British Royal Family in fear. Reading of Lady Wortley Montagu’s efforts, they wanted to use inoculation on themselves. Doctors told them it was a dangerous procedure, so they decided to try it on other people first. The test subjects they used were condemned prisoners.
The doctors inoculated the prisoners and all of them recovered in a few weeks. So assured, the British royal family inoculated themselves and reassured the English people that it was safe. Stimulated by a severe epidemic, variolation was first employed in North America in 1721. The practice had been known in Boston since 1706, when Cotton Mather (of Salem witch trial fame) discovered his slave, Onesimus had been inoculated while still in Africa, and many slaves imported to Boston had also received inoculations. The practice was, at first, widely criticized. However, a limited trial showed six deaths occurred out of 244 who were vaccinated (2.5%), while 844 out of 5980 died of natural disease, and the process was widely adopted throughout the colonies.
It was considerably later that microbiology came to be a science based on the work of Louis Pasteur (1822-1895) and Robert Koch (1843-1910). It was Pasteur who applied vaccination to rabies in 1886. Koch developed what is known as Koch’s Postulates, and he is credited with discovery of tuberculosis bacterium. In 1903 it was suggested for the first time that transduction by viruses might cause cancer.
In 1911 Peyton Rous reported the transmission of chicken sarcoma, a solid tumor, with a virus, and thus Rous became “father of tumor virology”. Several years later, the cause of the devastating Spanish flu pandemic of 1918 was unclear until French scientists showed that a “filter-passing virus” could transmit the disease to people and animals, fulfilling Koch’s postulates. In 1935, Wendell Stanley crystallized the tobacco mosaic virus for electron microscopy and showed that it remains active even after crystallization. Concurrently, Max Delbrück described the basic “life cycle” of a virus in 1937: rather than “growing”, a virus particle is assembled from its constituent pieces in one step; eventually it leaves the host cell to infect other cells. Clear X-ray diffraction pictures of crystallized TMV were obtained by Bernal and Fankuchen in 1941. Then, in 1949 John F. Enders, Thomas Weller and Frederick Robbins reported growth of poliovirus in cultured human embryonal cells, the first significant example of an animal virus grown outside of animals or chicken eggs. This work aided Jonas Salk in deriving a polio vaccine from deactivated polio viruses; this vaccine was shown to be effective in 1955. The Hershey-Chase experiment in 1952 was important in that it showed that only DNA and not protein enters a bacterial cell upon infection with bacteriophage T2.
Crystallography had arrived with the tobacco mosaic virus (TMV) crystalized and its structure elucidated in detail. Based on such pictures, Rosalind Franklin proposed the full structure of the tobacco mosaic virus. She also elucidated the structure of DNA. In 1955, Heinz Fraenkel-Conrat and Robley Williams showed that purified TMV RNA and its capsid (coat) protein can self-assemble into functional virions, suggesting that this assembly mechanism is also used within the host cell, as Delbrück had proposed earlier.
Keywords: embryogenesis, proliferation, pluripotent stem cell, cell function, stability, nuclear regulation, signaling pathways, organ systems, endocrine, inflammatory, carcinoma and sarcoma, epithelial cell, endothelial cell, circulatory collapse, infarction, regeneration, microbiome, parasite, viriome, signaling pathways, oxidative stress, mitochondrial damage and repair, endoplasmic reticulum, intron, exome, apoptosis and mitophagy, methylation, phosphorylation, aging, intercellular adhesion, metastasis
Chapter 14
Genomics in Infectious and Inflammatory Disease, Immunity
The science of infectious disease is relatively young compared to the long, recurring history of plagues, most notably in the crusade years, syphilis, tuberculosis, leprosy, tick-borne disease, and war related diseases preceding the seven-year war and in medieval Europe, Asia, and the middle east. The frequency and severity of infectious disease increased with the movement of the farm population into crowded cities. The most interesting part of the story is that bacteria and fungi have lived with man for centuries, and they have adapted to both man and the viruses that infect them. The more chronic inflammatory diseases tie in with the stimulation of the cellular immune system. These are mainly in the thymic derived lymphocytes, and other supporting cells. The secondary reactive system is in the B-cells, which are antibody producing. Both cells have “memory” and cell surface recognition. The immunology fills textbooks, but the genomics of the immunology is still emerging. Why are the B-cells most populated in the gastrointestinal tract? Because that is where the bacteria reside!
Keywords: Inflammatory reaction, T- and B-lymphocytes, mast cells, monocytes, granulacytes, antibody-mediated immunity, adaptive immunity, NFkB, cytokines, cell signaling, IL-1.
VIEW VIDEO – Courtesy of UCtelevision
Genomics and Infectious Diseases
http://www.youtube.com/watch?v=cgSTP84qDp0
14.1 HBV and HCV-associated Liver Cancer: Important Insights from the Genome
Ritu Saxena, PhD
14.2 Nanotechnology and HIV/AIDS treatment
Tilde Barliya, PhD
14.3 IRF-1 Deficiency Skews the Differentiation of Dendritic Cells
Larry H Bernstein, MD, FCAP
Larry H Bernstein, MD, FCAP
14.5 Five Malaria Genomes Sequenced
Larry H Bernstein, MD, FCAP
14.6 Rheumatoid Arthritis Risk
Larry H Bernstein, MD, FCAP
14.7 Approach to Controlling Pathogenic Inflammation in Arthritis
Larry H Bernstein, MD, FCAP
14.8 RNA Virus Genome as Bacterial Chromosome
Larry H Bernstein, MD, FCAP
14.9 Cloning the Vaccinia Virus Genome as a Bacterial Artificial Chromosome
Larry H Bernstein, MD, FCAP
Chapter 15
Cardiovascular and Angiogenesis: Genomics in Cardiac Disease
Cardiovascular disease is as old as man. It gained more attention in the 19th century with population growth and the industrialization, and more so as Walter Reed and Ronald Ross had success leading to decline of some infectious diseases with the improvement in swamp drainage, and later introduction of penicillin, and other drugs. The crowding into cities with industrialization also affected cardiovascular disease. A study in England showed that the stressed workers had more heart disease than the “bosses”. The interest in diet also preceded Atkins. A very large man in England reduced his weight by eating lean meat, which Dr. Atkin’s seized upon. Of course the picture gets far more interesting with comparing heart attack rates in Scotland and China!
Then we had the lipidemia classifications in the 1950s, and the study of lipogenesis by the liver coming from UT Southwestern SOM, and Burton Sobel’s work on infarct size, and the “save muscle” program that has brought up both surgical and medical interventions. It was about 60 years ago that patients were “chewing” leaf for digitalization. The introduction of the stethoscope and the EKG were seminal events. So here we are today with the most advanced cardiovascular medicine on the planet, and surgeons and cardiologist needing a piece of the pie.
Keywords: Acute myocardial infarct, nitric oxide, oxidative stress, congestive heart failure, myocardial metabolism, coronary circulation, shock, myocardiocyte, cardiac biomarker, mitochondria, actomyosin, troponins, natriuretic peptides, digoxin, beta-blocker, arteriole, large artery, capillary, intima, media, plaque, plaque rupture, generation of plaque, hypercoagulable state, anti-coagulant medications, omega-3/omega-6 ratio, glycation, diabetes, cardiomegaly, cardiogenic shock, stroke, iNOS, eNOS, circulatory intravascular flow and resistance, platelet aggregation, lipoproteins, hyperlipidemia, triglycerides, LDL cholesterol, total cholesterol, cardio-pharmaceuticals, ventricular and atrial dysrhythmias.
VIEW VIDEO – Courtesy of National Human Genome Research Institute on GenomeTV
The heart of the matter: genomics and cardiovascular disease – Leslie Biesecker
http://www.youtube.com/watch?v=Kg82C4di5Ck
15.1 Personalized Cardiovascular Genetic Medicine at Partners HealthCare and Harvard Medical School
Aviva Lev-Ari, PhD, RN
15.3 DDAH Says NO to ADMA(1); The DDAH/ADMA/NOS Pathway(2)
Larry H Bernstein, MD, FCAP
15.6 Gene Therapy Into Healthy Heart Muscle: Reprogramming Scar Tissue In Damaged Hearts
Aviva Lev-Ari, PhD, RN
Aviva Lev-Ari, PhD, RN
15.8 Ca2+ signaling: transcriptional control
Larry H Bernstein, MD, FCAP
15.9 Lp(a) Gene Variant Association
Larry H Bernstein, MD, FCAP
Aviva Lev-Ari, PhD, RN
Aviva Lev-Ari, PhD, RN and Larry H. Bernstein, MD, FCAP
Curator: Aviva Lev-Ari, PhD, RN
Larry H Bernstein, MD, FCAP
15.9.5 Gene, Meis1, Regulates the Heart’s Ability to Regenerate after Injuries.
Aviva Lev-Ari, PhD, RN
Aviva Lev-Ari, PhD, RN
15.11 How Might Sleep Apnea Lead to Serious Health Concerns like Cardiac and Cancers?
Larry H. Bernstein, MD, FCAP
Chapter 16
Pulmonary & Genomics
The lung and the heart go together making up a systemic circulation and a pulmonary circulation by way of the right pulmonary artery leaving the right ventricle and the right pulmonary vein returning oxygenated blood to the left atrium. The lung is constructed as a branching main bronchi from the trachii, the branches and terminal bronchi, leading into air sacs with only two layers of epithelium with endothelium beneath. It is in the space that air containing oxygen, carbon dioxide, and a constant portion of nitrogen traverse exchanging O2 and CO2 between the circulating hemoglobin containing RBC, allowing for delivery of O2 to peripheral tissues. It’s quite unremarkable in health.
However, it is subject to congestion from backflow of blood from the heart, interstitial fibrosis, inflammatory and infectious disease, and can seed bacteria into the circulation. Asthma and chronic obstructive pulmonary disease are among the common diseases affecting the lung, and they are highly influenced by the environment and smoking. Cancer of the lung has been the most common cancer, but it is common in women as well. Genomics might not come to mind when you think about lung, but this is where biopharmaceuticals is going, and not jus for carcinoma.
Keywords: carcinoma, asthma, inflammatory disease, interstitial fibrosis, cytokines, mutagenesis, precancerous bronchopulmonary dysplasia, epithelial proliferation, progression, metastasis, drug targets.
16.1 Can Resolvins Suppress Acute Lung Injury?
Larry H Bernstein, MD, FCAP
16.2 Lipoxin A4 Regulates Natural Killer Cell in Asthma
Larry H Bernstein, MD, FCAP
16.3 Biological Therapeutics for Asthma
Larry H Bernstein, MD, FCAP
16.4 Genomics of Bronchial Epithelial Dysplasia
Larry H Bernstein, MD, FCAP
16.5 Progression in Bronchial Dysplasia
Larry H Bernstein, MD, FCAP
Chapter 17
GI and Liver
The gastrointestinal tract is vital for connecting the gut to the liver, with its own portal circulation. We receive nutrients through the first segment of small intestine, and we excrete waste delivered from the descending colon to the rectum. The active microbiome has a vital role in interacting with the GI immune system. The structure of the small intestinal villi are affected by disease that flattens the villi, and causes malabsorption. Most interesting is that the lung is an “outpouching” of the GI tract in embryologic origin, which is consistent with findings reported on cancer genomics. The liver is the largest organ and is very critical in protein synthesis, lipogenesis, production of circulating proteins and lipoproteins, and is sensitive to starvation. The circulation is divided between the portal system and the hepatic artery. So it is very difficult to infarct the liver, referred to as a Zahn infarct. Hepatocellular carcinoma is not as common as gastric cancer, which has been increasing. Hepatocellular carcinoma will increase with a rise in HCV virus, and an increase in fatty liver with fibrosis.
Keywords: steatosis, fibrosis, hepatocellular carcinoma, viral hepatitis, amebiasis, malaria, tropical diseases, malabsorption, glycolysis, glycogenesis, glycogenolysis, cancer cachexia, metastasis, lipogenesis, albumin, globulin family, transthyretin, transferrin, starvation, Crohn’s disease, diarrhea, vomiting, gastric cancer, esophageal cancer, pancreatic cancer, rectal cancer, oropharyngeal cancer
17.1 Breakthrough Digestive Disorders Research: Conditions Affecting the Gastrointestinal Tract.
Aviva Lev-Ari, PhD, RN
17.2 Liver Endoplasmic Reticulum Stress and Hepatosteatosis
Larry H Bernstein, MD, FCAP
17.3 Biomarkers-identified-for-recurrence-in-hbv-related-hcc-patients-post-surgery
Ritu Saxena, PhD
17.4 Usp9x: Promising Therapeutic Target for Pancreatic Cancer
17.5 Battle of Steve Jobs and Ralph Steinman with Pancreatic cancer: How We Lost
Ritu Saxena, PhD
Chapter 18
Neuromuscular and Brain
Muscle, peripheral motor and sensory nerves, the cranial nerves, skeletal muscle, smooth muscle, retina, and inner ear, and the brain and brain stem all are a system of “motor and sensory” functions. The muscle has a contractile apparatus the is sensitive to Ca++ mediated signals from the nervous system. The brain is a complex network that records experience, and is at a high level of cognition, and is also the center of creativity in intellectual pursuits, music, science, the arts, architecture, mathematics, literature, and communication. It is also subject to traumatic damage, to the effects of social experience or lack thereof, and changes of late or early aging degenerative changes.
Keywords: Brain, neuronal connections, brain stem, cerebral circulation, blood-brain barrier, learning, perception, sensory loss, aging, Huntington’s chorea, Alzheimer’s disease, Parkinson’s disease, gene links, neuropharmacology, neuro-imaging, mutations, epigenomics, stroke
18.1 Ubiquitin Pathway Involved in Neurodegenerative Disease
Larry H. Bernstein, MD, FCAP
Larry H. Bernstein, MD, FCAP
18.3 Neuroprotective Therapies: Pharmacogenomics vs Psychotropic Drugs and Cholinesterase Inhibitors
Aviva Lev-Ari, PhD, RN
Aviva Lev-Ari, PhD, RN
18.5 Cell Transplantation in Brain Repair
Larry H Bernstein, MD, FCAP
18.6 Alzheimer’s Disease Conundrum – Are We Near the End of the Puzzle?
Larry H Bernstein, MD, FCAP
Chapter 19
Genomic Endocrinology and Reproductive Biology Genomics
This portion includes thyroid, pituitary, adrenals, and the male and female sex organs, as well as the breast. This is because the synthesis of the pancreatic islets, adrenal corticosteroids is an end-point in the synthesis of steroids by that organ. However, the important role of the ovaries and the testes in the synthesis of sex hormones is as vital as the reproductive function as well, and is tied in with genetic disorders of sex determination, and is also related to hormonal effects in cancer. One might also consider the kidneys, heart, and gastrointestinal tract have hormonal production that is important in the scheme of functioning, but will used wherever the fit is best. An example is the natriuretic peptides, which go with heart failure.
We now move into the emerging reality of genomics closer to the bedside. There is testing in tertiary care medical center laboratories, and molecular testing is a strategic plan for medicine and pathology.
Keywords: cellular transformation, cell type and shape, lipid structures, cancer targets
VIEW VIDEO – Courtesy of 23andMe·
23andMe and PPH Partner for DNA Testing
http://www.youtube.com/watch?v=MJQ3FBitlJ0&playnext=1&list=PL1F27A9171C88CF77&feature=results_main
19.1 Genetics and Male Endocrinology
19.2 Genomic Endocrinology and its Future
Ritu Saxena, PhD
19.4 Therapeutic Targets for Diabetes and Related Metabolic Disorders
Aviva Lev-Ari, PhD, RN
Aviva Lev-Ari, PhD, RN
19.6 Personal Recombination Map from Individual’s Sperm Cell and its Importance
Sudipta Saha, Ph.D
19.7 Gene Trap Mutagenesis in Reproductive Research
Sudipta Saha, Ph.D
19.8 Pregnancy with a Leptin-Receptor Mutation
Sudipta Saha, Ph.D
Sudipta Saha, Ph.D.
19.10 Reproductive Genetic Testing
Sudipta Saha, Ph.D.
Chapter 20
Genomics & Ethics
Introduction
Larry H Bernstein, MD, FCAP
This chapter deals specifically with the issues that have arisen within the last decade, particularly since 2005, with the rapid advances in Genome-Wide Association Sequencing (GWAS). While much testing has been done with microarrays and there has been intensive work in advancing our knowledge about breast cancer, ovarian cancer, and the leukemias, much more is yet to be done. The most widely used methods have not had the power of GWAS, and they have had an advantage of favorability as a result of cost, familiarity, and ease of use. But the situation is changing rapidly, and deployment of more sophisticated, rapid throuput methods are always around the corner. In addition, miniaturization and consolidation of steps in the process will make high volume of use more attractive. This development has opened up new possibilities for genomic testing in clinical practice, but at the same time has opened up clear dangers posed by the changing structure of institutional medicine, and by the conflicting premises that are posed by physician-directed vesus consumer-directed testing. Much will be covered in the following six sections.
In advance of the reader going there, we’ll begin with a few points to keep in mind:
- The distinction between predictability and complicated indeterminate risk
- Privacy of patient information
- Informed consent
- Physician obligation to do no harm
- Patenting of genetic sequences
- Evidence-based decision-making
- Patient populations
Keywords : personalized medicine, targeted therapy, evidence-based medicine, consumer-directed marketing, predictive testing, biomarkers, molecular-directed treatment, cofactors in risk-analysis, physician-patient relationship, patient education, information generated anxiety, Mendelian genetics, non-mendelian traits, multifactorial disease elements, nature and nurture
VIEW VIDEO – Courtesy of Indiana University School of Medicine via youtube.com
Ethical Issues in Personalized Medicine
http://www.youtube.com/watch?feature=player_detailpage&v=1LDZPdZlt0c
AUDIO – Curtesy 0f Duke University
Modeling the Morbid Human Genome
Nicholas Katsanis, PhD – Director, Center for Human Disease Modeling, Professor – Departments of Cell Biology and Pediatrics
An Opinion by Patrick Taylor of Harvard Medical School
Does personalized genomics pit privacy against ethics? | Ars Technica
Nov 6, 2008 … As genomic technology becomes available to the public and the cost of …
Patrick Taylor of Harvard Medical School largely agrees with one aspect of the other essay, namely that genomic information will only really make sense when integrated into a data framework that includes electronic medical records. The problem is, however, that this forces an uncomfortable balance between privacy and ethical concerns, nicely summed up by the essay’s title, “When consent gets in the way.”
Taylor says that, so far, we’ve tended to equate privacy with data ownership and control; if people dictate the access to their medical data, the reasoning goes, then they retain effective ownership of the data, and can safeguard their privacy. Because of both medical ethics and past abuses, the medical research community has extremely strict guidelines about how it obtains consent from people enrolled in medical studies. In the current age, that informed consent has often involved an agreement to provide access to medical records.
But that model breaks down when it comes to large collections of digitized genomic data. For one thing, it’s relatively easy to inform someone about the potential of receiving a placebo during a drug trial; it’s much harder to get them to the point where they can provide informed consent about datamining medical records that include genomic data. The flipside is that datamining expeditions will be harder to run through the ethical approval process, since, by their nature, they tend to be rather open-ended.
What gets lost in this focus on privacy and private ethics, Taylor argues, is a focus on the big picture of public ethics. All of society benefits from medical research, and often the members that will benefit the most (he cites the elderly and minorities) are the least likely to give informed consent. The solution, in his view, is to develop a framework in which the privacy of genomics data can be protected without invoking the consent of the genome’s owner for every access. “If we protect privacy effectively,” Taylor writes, “we will not reduce ethics to autonomy, and autonomy to data ownership.”
SOURCE:http://arstechnica.com/tech-policy/2008/11/does-personalized-genomics-pit-privacy-against-ethics/
20.1 Genomics & Ethics: DNA Fragments are Products of Nature or Patentable Genes?
Aviva Lev-Ari, PhD, RN
20.2 Understanding the Role of Personalized Medicine
20.3 Attitudes of Patients about Personalized Medicine
20.4 Genome Sequencing of the Healthy
Larry H. Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
20.5 Genomics in Medicine – Tomorrow’s Promise
20.6 The Promise of Personalized Medicine
Stephen J. Williams, Ph.D
20.8 Genomic Liberty of Ownership, Genome Medicine and Patenting the Human Genome
Aviva Lev-Ari, PhD, RN
AUDIO
Barriers and Solutions to the Implementation of Personalized Medicine
Genomic and Personalized Medicine Forum Duke Institute for Genome Sciences & Policy. Geoffery Ginsburg, MD, PhD – Executive Director, Center for Personalized Medicine; Director, Genomic Medicine; Institute for Genome Sciences & Policy
VIEW VIDEO – Courtesy of UBCInterdisciplinary via youtube.com
Who Should Determine the Nature of Genome Testing in ‘Genomic Medicine in 2015’
http://www.youtube.com/watch?feature=player_detailpage&v=0QKs0b8qJys
Summary to Chapter 20
Larry H Bernstein, MD, FCAP
You, the reader, have gone through a series of presentations dealing with ethical and social issues that naturally arise within the years since the human genome project (HGP) and ENCODE, and more recently, the 2004 International HapMap project, and 2005 GWAS. We now have a convergence of different issues relating to the practice of medicine, the education of physicians, the communication between physician and patients. I initiated these presentations with some items to keep in mind. They are the problems that have to be resolved.
The physician has to be committed to the value – the patient comes first. The corollary to that is “do no harm”.
1. The physician has to make decision as they have always been done, by a total view of the patient, based on
- patient history
- family history
- EKG, imaging, lab results, as related to the present condition
Does this change the way that medicine is to be practiced? Not really. This has been and will continue to be. What does change is that the physician will be more cognizant of the patient’s life style, and there will be more active guidance, even by using professional dietitians and physiotherapists to enable the patient to change their lifestyle. This is not genomic. But only 5-10% of genomic risk can be identified to account for the patient’s condition (non-mendelian), with the exception of familial breast and ovarian cancer (80%), at this time. Nevertheless, there will be the emergence of therapies that have to be treated according to genomic findings, and the treatment will have to be followed with accurate biomarkers that reflect the improvement of failure of treatment. This is genomic. This is also within the precepts of ethical medical practice.
This is the most clearly identifiable task imposed on the physician. There are others that are not as hard and fast.
- The role of genomic testing in prenatal diagnosis
- The conflicting desire of a patient to want to have testing, either because of family history (legitimate), or because of ability to pay for a consumer-driven, for-profit, genomic find a needle in a haystack, risk assessment program.
- There will be litigation issues arising out of unqualified consumer-driven practices.
- There is the problem of explaining to the uneducated patient what the meaning of the risk estimate is.
2. There is an unresolved issue of patenting of exomes, or gene targets for suppression or for upregulation
3. There is a real positive value in all of this that needs mention. The use of biomarkers to follow treatment, and improved treatments will be rolled out. We are already seeing prospects for identifying a potential disease “alarm” perhaps a decade before disease presents. This means that it would be possible to decrease the cost of screening and improve the benefit by targeting for what it is best to follow at regular intervals over time.
4. There is an undiscussed benefit that is related to the change in lifestyle of the patient. Oxidative stress is a term used prolifically, but the systemic damage due to OS is remediable. This is the case for our most common ailments. Therefore, the use of diet, exercise, and stress reduction methods will be used to reduce the effects of OS substantially, and to provide for a better quality of life for the patient.
5. A social issue is related to the characteristics of a “person”, and questionable conclusions that are drawn based upon studies purported to be able to assess risk between “black” and “white” that have no validity today.
6. Results obtained from whole genome testing –
- Required findings
- incidental findings
- physicians responsibility to control nature and extent of testing
7. It follows that testing is required to obtain a meaningful answer, thereby, minimize false positives, and the necessity to reduce the potential for misunderstanding.
8. Informed consent
- Geneva Conventions
- beneficence, autonomy
- Nuremberg Code (articulated in 1947 by U.S. judges): “ The voluntary consent of the human subject is absolutely essential . . . [and includes] legal capacity . . . free power
of choice . . . sufficient knowledge and comprehension of the [nature, duration, and purpose of the experiment] . . . to make an understanding and enlightened decision.” - GJ Annas. Globalized Clinical Trials and Informed Consent NEJM 2009; 360:2050-53. http://dx.doi.org/10.1056/NEJMp0901474
- International Covenant on Civil and Political Rights (international treaty became effective in 1976): “ No one shall be subjected to torture or to cruel, inhuman or degrading treatment or punishment. In particular, no one shall be subjected
without his free consent to medical or scientific experimentation.” - Declaration of Helsinki of the World Medical Association (promulgated in 1964 and revised eight times since): “ The physician should obtain the subject’s freely-given informed consent, preferably in writing. . . . [But in clinical research] if the physician considers it essential not to obtain informed consent, the specific reasons for this proposal should be stated in the experimental protocol for transmission to [an] independent committee.”
- International Ethical Guidelines for Biomedical Research Involving Human Subjects (published in 1993, and since revised, by the Council for International Organizations of Medical Science): “ The investigator must obtain the voluntary, informed consent of the prospective subject [or legally authorized representative]. . . . Waiver of informed consent is to be regarded as uncommon and exceptional, and must in all cases be approved by an ethical review committee.”
- January 2009 opinion of the U.S. Court of Appeals for the Second Circuit: whether researchers who experiment on humans without their informed consent violate a substantially similar international human rights law. Furthermore, the Court ruled the informed consent requirement is sufficiently “(i) universal and obligatory, (ii) specific and definable, and (iii) of mutual concern,” to be considered a “customary international law norm” that can support a claim under the Alien Tort Statute.
- the first precept is the requirement for voluntary, competent, informed, and understanding consent of the research subject. In the Second Circuit court’s words, “The American tribunal’s conclusion that action that contravened the Code’s first principle constituted a crime against humanity is a lucid indication of the international legal significance of the prohibition on nonconsensual medical experimentation.”5 Moreover, the requirement of informed consent in research has been widely adopted in international treaties (including the International Covenant on Civil and Political Rights and the Geneva Conventions), domestic law, and nonbinding international codes of ethics such as the Declaration of Helsinki (see Codes Relied on by the Second Circuit).
- The Second Circuit’s persuasive opinion that the doctrine of informed consent has attained the status of an international human rights norm that can be enforced in the world’s courts should help persuade international corporations and researchers alike to take informed consent — and perhaps the other principles of the Nuremberg Code — much more seriously.
- Informed consent applies to necessity for procedures outside of prospective clinical trials, and was a critical issue in the provision of transfusion at the outset of the HIV epidemic – the person concerned is told why it was collected, and the recipient is told the risks and benefits of transfusion – his/her informed consent secured.
Ethical Physician Incentives
An important note on ethical physician incentives appeared in the March 2013 NEJM {Ethical Physician Incentives — From Carrots and Sticks to Shared Purpose. by N Biller-Andorno, and TH Lee. N Engl J Med 2013; 368:980-982. http://dx.doi.org/10.1056/NEJMp1300373
The authors state, “We believe that shared-purpose orientations are not only a precondition for an ethical use of incentives but also essential for organizational effectiveness. When teams feel ownership of the shared goal, they can display creativity and flexibility that go beyond what’s possible with incentives based on tradition, self-interest, or affective responses alone, while maintaining health professionals’ sense of moral agency and responsibility. Practically speaking, however, a shared-purpose orientation alone is frequently not sufficient.” They envision a role for genomic screening for improved public health. This is put forth in reference to a soon to be published commentary in the May 2013 issue of Genetics in Medicine, the peer-reviewed journal of the American College of Medical Genetics and Genomics (ACMG), that now is the time to explore genetic testing to identify people at high risk for carefully selected, preventable disease, as it appears likely that in ten years time, routine preventive health care for adults may include genetic testing alongside the now familiar tests for cholesterol levels, mammography and colonoscopy.
As has been noted, the technology is available, and the price is coming down so rapidly that it will soon be possible and practical to offer a carefully selected panel of genetic tests that could avert disastrous health consequences in people at high risk for serious life-threatening diseases. Thus, it is sensible to try to identify those people early who carry a strong predisposition to developing a preventable condition with respect to specific cancers and catastrophic vascular events that they can seek preventive care. The colonoscopy example was cited earlier, directed to 1:400 of the population.
Chapter 21
Advances in Gene Editing Technology: New Gene Therapy Options in Personalized Medicine – Medical Interpretation of the Genomics Frontier – CRISPR – Cas9
Chapter Curators: Larry H Bernstein, MD, FCAP, Stephen J Williams, PhD and Aviva Lev-Ari, PhD, RN
This Chapter will appear with modifications as Part One in
Content Consultant: Larry H Bernstein, MD, FCAP
Volume Two:
Genomics Methodologies: NGS, BioInformatics & Simulations and the Genome Ontology
Stephen J. Williams, PhD, Senior Editor
Co-Editor(s) – TBA
Recent Advances in Gene Editing Technology Adds New Therapeutic Potential for the Genomic Era
Author and Curator: Stephen J Williams, PhD
The fundamental shift presently occurring within the medical field as well as our understanding of underlying biology has been brought on by revolutionary advances in the disciplines referred to as ‘OMICS’ (genomics, metabolomics, transcriptomics, proteomics). This paradigm shift has brought a new, more “personalized” mindset in investigating, treating, detecting, and policy-decision making disease as well as the physician-patient relationship. This Volume One of Genomics explains this paradigm shift as our classical understanding of the gene has evolved with rapid development of molecular technologies and high-end computational methods to a vision beyond the classic model. This new model involves big data to focus of the “code of OMIC signature”, moving from our investigational focus of “one gene at a time” to analysis of the changes in the networks of protein and gene expression occurring during disease progression.
Moving toward this promise of genome-based therapeutics has required the concomitant development of methodologies unavailable to the researcher and drug developer for most of the 20th century. These new technologies have allowed for the sequencing of the whole genome (advanced and inexpensive pyrosequencing), analyze the proteome for changes in post-translational modifications (new mass spectroscopy techniques combined with automated high-throughput gel electrophoresis on robotic platforms), ability to track all the changes happening to a patient’s metabolic profile (LC-MS in combination with an array of biocurated database functions), and develop new therapeutics based on discrete disease-specific changes in protein, enzyme, and DNA/RNA (mutational analysis, and advanced molecular techniques to allow for manipulation of DNA/RNA such as gene editing and therapeutic vectors) all advancements being dependent on the massive advancements in computing power and software development.
Although this final chapter on a specific technology (Cas9-mediated gene editing) might seem out of place to the reader for the subject of this Genomics volume, as discussed above, the development of these omics-related technologies have spurred the advent of personalized therapies. For example, in the 1990’s (as highlighted in the earlier chapters of this book) Dr. Craig Venter founded Celera Genomics with the goals of 1) sequencing the human genome in a cost effective manner (using new DNA sequencing technology and workflow he and colleagues had developed, and 2) use the information from whole genome sequencing to develop a new line of genomic-based therapeutics. Other companies such asHuman Genome Sciences, Myriad Genetics, Seattle Genetics and recently new ventures from 23andMe and Google Ventures were also founded based on the promise that high-end sequencing information could directly lead to this new era of genome-based therapeutics. And although many in the medical field have felt that the primary goal of these companies, in particular using genomic analysis to enhance drug development has been a bit disappointing, AS IN ALL SCIENTIFIC AND MEDICAL DISCOVERY, which involves both SERENDIPITY and INDIRECT HAPPENSTANCE, three important breakthroughs, directly related to the development of a post-genomics era personalized medicine approach, resulted from the aforementioned efforts. These were:
- The detection of disease-specific mutations in exomes resulting in “druggable” protein targets and ability to define the respective drug-responsive patient cohorts
Chronic myelogenous (or myeloid or myelocytic) leukemia (CML) was one of the first cancers attributed to a specific chromosomal aberration, namely the translocation event resulting in a fusion protein between part of theBCR(“breakpoint cluster region”) gene from chromosome 22 with the ABL gene on chromosome 9. Early drug development efforts were directed against the tyrosine kinase activity of the aberrant BCR/ABL protein. The first of this new class of drugs was imatinib mesylate (Gleevec™) showed early success but was later noticed that a subset of patients had significantly greater response rates. This led to more detailed investigation of Gleevec’s mechanism of action and was determined that Gleevec’s therapeutic action depended on the drug’s ability to bind to an ATP binding pocket within the BCR/ABL. Patients with a specific mutation in this ATP pocket (C944T and T1052C) were found resistant to Gleevec. This finding, that pateint’s DNA could be sequenced to stratify them in responder versus nonresponder groups became a cornerstone for tyrosine kinase inhibitor (TKI) development for various cancers. One example is the development of crizotanib, a TKI directed against a mutant version of the anaplasticlymphomakinase (ALK) enzyme, namely in patients carrying the ALK-EML4 fusion gene. As with Gleevec, certain mutations in the ATP binding pocket confer resistance to the inhibitory effects of crizotanib. Therefore, the Whole Exome Sequencing (WES) has shown its utility not only in drug development against cancer-specific mutant targets but stratifies patient cohorts into eligible versus non-eligible for a specific personalized therapy.
- Ability to define at-risk populations based on genomic data and development of corresponding genetic risk assessment for disease
Tremendous advances have been made in the area of risk-assessment for a plethora of diseases, including various malignancies, heart disease, and metabolic diseases. These risk factors have been identified given our advances in whole genome sequencing and proteomic and metabolomics. And, although the aforementioned companies had not developed therapeutic agents using these technologies, their major contribution has been the development of the diagnostic tests which identify at-risk patients and susceptible populations for a given disease. For example, the development of tests for carriers of the BRCA1/BRAC2 breast/ovarian cancer susceptibility mutation or APC (for colon cancer) has led to the appearance of Family Risk Assessment Programs and radically changed the discourse between patient and physician. And although determining risk factors to a disease such as cardiac disease in a large population can be fraught with complexities, the advanced research tools together with gene-directed technologies discussed in this Volume and current chapter may give better clarity in this regard. In essence, the technology had been developed well before its use in the clinic had been identified.
- Supplying and verifying linkages of specific genetic alterations to heritable diseases and offering a framework for future advances in gene-replacement and mutation-correction therapy
Our abilities to phenotypically correct inheritable diseases thru a gene-therapy (either by gene replacement or correction of mutated genes) have been hampered by three main areas. First identifying the specific mutations for a given inheritable disease used to be an arduous time-consuming process (linkage analysis), especially in small affected populations. However as whole exome sequencing rapidly evolved this had no longer become a rate-limiting step toward the development of a gene-directed therapy. Second and more troubling was determining a process which could deliver therapeutic genes in a safe, reliable and persistent manner. The first attempts at gene-therapy, relying on DNA virus and retroviral based delivery met with disaster and set back the field of gene therapy for decades (this story is too long for an introduction but for reference see thelink.) Recently there have been improvements in therapeutic gene-therapy delivery systems such as the use of conditionally replicative adenovirus (cRADs) and novel serotype AAV (Recombinant adeno-associated virus, a nonpathogenic single stranded DNA human parvovirus) which have greatly improved safety and therapeutic profiles). The third issue, directly related to this chapter on Cas9-mediatied DNA editing) is the ability to integrate therapeutic DNA into the genome in a safe manner or correct mutations in their proper place. It is well established that the random integration of pieces of DNA has spurious effects on gene expression or contribute to transformation by an insertional mutagenesis mechanism.
This chapter will discuss how CRISPR/Cas9-mediated gene editing is being used in ex vivo strategies, namely to insert T-cell specific genes, in definable and safe loci, for the development of the new CAR-T cancer immuno-based therapies. In addition CRISPR/Cas9-mediated gene editing has much hope and promise for correcting specific mutations related to inheritable diseases, although investigations are at an infantile yet rapidly expanding area. As discussed above, new technologies have preceded their clinical use, mostly in a serendipitous and advantageous manner. Therefore it is a natural progression, using the concepts and curations in previous chapters, to investigate how a new technology, such as CRISPR/Cas9 medicated gene editing will fit into the ‘OMICS era of medicine.
Introduction to Chapter 21
Larry H Bernstein, MD, FCAP
The recent development of advanced methods for genome engineering has superceded methods already in used in recent years of the 21st century. Genome editing technologies enable the deletion, insertion or correction of DNA at specific targeted sites within an organism’s genome. The power of the technology lies in its ability to specifically target any site in the genome and to alter the DNA sequence at that site. Researchers working in the biomedical field use these techniques to address diseases that are known to have a genetic origin. Early genome-editing research focused on the use of zinc finger nucleases and transcription activator-like effector nucleases (TALENs), which laid important foundations in establishing genome engineering as a potential approach for treating human diseases. These were superceded by the recent discovery of CRISPR-Cas9, followed by work demonstrating its advantages over traditional approaches. CRISPR-Cas9 has truly democratized genome editing and transformed the potential for treatment of genetic disorders. Genetic disorders may or may not be heritable, i.e., passed down from the parents’ genes. In non-heritable genetic disorders, defects may be caused by new mutations or changes to the DNA. In such cases, the defect will only be heritable if it occurs in the germ line.
Genes causing rare heritable childhood diseases are being discovered at an accelerating pace driven by the decreasing cost and increasing accessibility of next-generation DNA sequencing combined with the maturation of strategies for successful gene identification. The findings are shedding light on the biological mechanisms of childhood disease and broadening the phenotypic spectrum of many clinical syndromes. Most are due to a defect in an enzyme or transport protein, which results in a block in a metabolic pathway. Nearly every metabolic disease has several forms that vary in age of onset, clinical severity, and, often, mode of inheritance. Newborn screening tests can identify some of these disorders.
This chapter introduces some giants in the 20th century study of genetic medicine, such as, Victor McKusick, widely known as the “father of medical genetics” and Elizabeth F. Neufeld, among others.
RNA has a role in suppressing translation, as do proteins by allosteric effects. In addition, the most common diseases involved in age related change are strongly responsive to extracellular matrix effects, ionic fluxes, effects on the cellular matrix, and involve multicentric genome expression. This mode of expression leads one to think hard about the therapeutic target, or targets. The effect of RNA or of protein interacting with the genome is not an element of the classic construct. The phenotypic presentations may have genomic associations, and there may also be population variants.
The use of genomic profiling has rapidly emerged in the laboratory armamentarium. We consider Pompe’s disease, polymorphisms in the long non-coding RNA, and the role of Lipoprotein Lipase in Atherosclerosis, altered Stress Hormones that affect the ability to bounce back from trauma, and genome engineering with CRISPR-Cas9 (Jennifer A. Doudna and Emmanuelle Charpentier) in this review as examples of important work in recent years.
This document is a review and of the brilliant accomplishment of the Doudna Laboratory at University of California, Berkeley. It also traces the developments leading up to this groundbreaking work. The principle investigator is a young woman of significant accomplishments with the astounding publication of 4 papers at this time in 2015 and 20 in 2014. She is a member of the National Academy of Sciences, and recipient of the Breakthrough Prize and the Lurie Prize in Biomedical Sciences, R. B. Woodward Visiting Professor, Harvard University (2000-2001). She achieved the Henry Ford II Professor of Molecular Biophysics and Biochemistry, Center for Structural Biology, Department of Molecular Biophysics and Biochemistry, Yale University (1994-2002) nine years after completion of her B.A. at Pomona College, and her Ph.D. under Jack Stozak at Harvard in 1989, became a Searle Scholar in 1996, and a Howard Hughes Investigator in 1997.
Her work has encompassed the editing of genes using the CRISPR-Cas9 system, and her team replaced a gene in a human cell which was convincing replicated in the Broad Laboratory at Harvard. The laboratory is currently working on the just reported immunological implications for CRISPR-Cas9 with respect to editing prokaryotic CRISPR-Cas genomic loci that encode RNA-mediated adaptive immune systems that bear some functional similarities with eukaryotic RNA interference. This is because acquired and heritable immunity against bacteriophage and plasmids begins with integration of ∼30 base pair foreign DNA sequences into the host genome.
Of special note are the following applications:
21.4.2 CRISPR: Applications for Autoimmune Diseases @UCSF
Reporter: Aviva Lev-Ari, PhD, RN
21.4.3 In vivo validated mRNAs
Doudna’s Interview from the National Academy of Science in 2004
Doudna discusses her current work with signal recognition particles, a type of RNA that is found in virtually all cell types and is responsible for directing specific proteins to specific membranes. She also discusses how advances in genomic sequencing may help catalog the complete range of functional RNA molecules. (9 minutes)
SOURCE
http://www.nasonline.org/news-and-multimedia/podcasts/interviews/jennifer-doudna.html
The Doudna lab pursues mechanistic understanding of fundamental biological processes involving RNA molecules. Research in the lab is currently focused on three major areas:
- bacterial immunity via the CRISPR system,
- RNA interference in eukaryotes, and
- translational control logic.
http://rna.berkeley.edu/crispr.html
http://rna.berkeley.edu/rnai.html
Different subunits are colored with invader RNAs in the background. Art by Gerard W.M. Staals
http://rna.berkeley.edu/translation.html
Alu-element regulated miRNA interactions
The Voice of Aviva Lev-Ari, PhD, RN
Big Pharma, CRISPR and Cancer
In January, the pharmaceutical giant Novartis announced that it would be using Doudna’s CRISPR technology for its research into cancer treatments. It plans to edit the genes of immune cells so that they will attack tumors.
https://www.quantamagazine.org/20150206-crispr-dna-editor-bacteria/#ixzz3UaJdJh5t
- The biggest biotech discovery of the century is about to change medicine forever
https://www.quantamagazine.org/20150206-crispr-dna-editor-bacteria/#ixzz3UaIwAbQU
Evolution of the Discovery
- In 1987, Yoshizumi Ishino and colleagues at Osaka University in Japan published the sequence of a gene called iap belonging to the gut microbe E. coli.
https://www.quantamagazine.org/20150206-crispr-dna-editor-bacteria/#ixzz3UaL6md3C
- In 2007, Blake Wiedenheft joined Doudna’s lab as a postdoctoral researcher, eager to study the structure of Cas enzymes to understand how they worked.
https://www.quantamagazine.org/20150206-crispr-dna-editor-bacteria/#ixzz3UaNXI2R4 - In a new paper in Nature Reviews Genetics, Koonin and Mart Krupovic of the Pasteur Institute in Paris argue that the CRISPR-Cas system got its start when mutations transformed casposons from enemies into friends.
https://www.quantamagazine.org/20150206-crispr-dna-editor-bacteria/#ixzz3UaN6Ucnk
- In January 2013, the scientists went one step further: They cut out a particular piece of DNA in human cells and replaced it with another one.
- In the same month, separate teams of scientists at Harvard University and the Broad Institute reported similar success with the gene-editing tool.
https://www.quantamagazine.org/20150206-crispr-dna-editor-bacteria/#ixzz3UaOF9xi4
- At Editas, a company based in Cambridge, Massachusetts, scientists have been investigating the Cas9 enzyme made by another species of bacteria, Staphylococcus aureus and Streptococcus pyogenes
https://www.quantamagazine.org/20150206-crispr-dna-editor-bacteria/#ixzz3UaMPy9Yw
- Writing last year in the journal Reproductive Biology and Endocrinology, Motoko Araki and Tetsuya Ishii of Hokkaido University in Japan predicted that doctors will be able to use CRISPR to alter the genes of human embryos “in the immediate future.”
https://www.quantamagazine.org/20150206-crispr-dna-editor-bacteria/#ixzz3UaOg9jyD
International regulatory landscape and integration of corrective genome editing into in vitro fertilization, Motoko Araki and Tetsuya Ishii*
http://www.rbej.com/content/12/1/108
The Human Germ Line
Don’t edit the human germ line
Heritable human genetic modifications pose serious risks, and the therapeutic benefits are tenuous, warn Edward Lanphier, Fyodor Urnov and colleagues.
The CRISPR technique has dramatically expanded research on genome editing. But we cannot imagine a situation in which its use in human embryos would offer a therapeutic benefit over existing and developing methods. It would be difficult to control exactly how many cells are modified. Increasing the dose of nuclease used would increase the likelihood that the mutated gene will be corrected, but also raise the risk of cuts being made elsewhere in the genome.
http://www.nature.com/news/don-t-edit-the-human-germ-line-1.17111
Engineering the Perfect Baby
Industry Body Calls for Gene-Editing Moratorium
Doudna’s Interview from the National Academy of Science in 2004
Track 6: Future Directions (requires RealPlayer)
Doudna discusses her current work with signal recognition particles, a type of RNA that is found in virtually all cell types and is responsible for directing specific proteins to specific membranes. She also discusses how advances in genomic sequencing may help catalog the complete range of functional RNA molecules. (9 minutes)
SOURCE
http://www.nasonline.org/news-and-multimedia/podcasts/interviews/jennifer-doudna.html
VIEW VIDEOS on Gene Editing
https://www.physicsforums.com/threads/breakthrough-prize-genome-editing-with-crispr-cas9.798959/
https://www.quantamagazine.org/20150206-crispr-dna-editor-bacteria/
The Structural Biology of CRISPR-Cas Systems
Curr Opin Struct Biol. 2015 Feb 24;30C:100-111
Authors: Jiang F, Doudna JA
Abstract
Prokaryotic CRISPR-Cas genomic loci encode RNA-mediated adaptive immune systems that bear some functional similarities with eukaryotic RNA interference. Acquired and heritable immunity against bacteriophage and plasmids begins with integration of ∼30 base pair foreign DNA sequences into the host genome. CRISPR-derived transcripts assemble with CRISPR-associated (Cas) proteins to target complementary nucleic acids for degradation. Here we review recent advances in the structural biology of these targeting complexes, with a focus on structural studies of the multisubunit Type I CRISPR RNA-guided surveillance and the Cas9 DNA endonuclease found in Type II CRISPR-Cas systems. These complexes have distinct structures that are each capable of site-specific double-stranded DNA binding and local helix unwinding.
PMID: 25723899 [PubMed – as supplied by publisher]
SOURCE
About the Significance of the CRISPR Discovery
“This technology will revolutionize biology in the same way PCR did,” Rudolf Jaenisch introducing Jennifer Doudna, 6/13/2014 @KI Symposium @MIT.
Koch Institute for Integrative Cancer Research @MIT – Summer Symposium 2014: RNA Biology, Cancer and Therapeutic Implications, June 13, 2014 8:30AM – 4:30PM, Kresge Auditorium @MIT
http://pharmaceuticalintelligence.com/2014/06/16/lecture-contents-delivered-at-koch-institute-for-integrative-cancer-research-summer-symposium-2014-rna-biology-cancer-and-therapeutic-implications-june-13-2014-mit/
Top CRISPR Related Publications
http://blog.appliedstemcell.com/top-crispr-related-publications/
What is CRISPR? Why are Cas9-CRISPR services so popular?
http://blog.appliedstemcell.com/what-is-crispr-why-are-cas9-crispr-services-so-popular/
Custom Rat Model Generation Service Using CRISPR/Cas9
http://www.appliedstemcell.com/services/animal-models/
Jennifer A. Doudna
Dr. Jennifer Doudna is a member of the departments of Molecular and Cell Biology and Chemistry atUC Berkeley, the Howard Hughes Medical Institute, and Lawrence Berkeley National Lab, along with the National Academy of Sciences, and the American Academy of Arts and Sciences.
http://rna.berkeley.edu/people.html
AWARDS for the Discovery
Jennifer Doudna, cosmology teams named 2015 Breakthrough Prize winners
Jennifer Doudna, The winner of the 2014 Lurie Prize in the Biomedical Sciences
Doudna was a Searle Scholar and received a 1996 Beckman Young Investigators Award, the 1999 NAS Award for Initiatives in Research and the 2000 Alan T. Waterman Award. She was elected to the National Academy of Sciences in 2002 and to the Institute of Medicine in 2010. In 2014, Doudna was awarded the Lurie Prize in Biomedical Sciences from the Foundation for the National Institutes of Health as well as the Dr. Paul Janssen Award for Biomedical Researchand Breakthrough Prize in Life Sciences, both shared with Emanuelle Charpentier.
SOURCE
http://en.wikipedia.org/wiki/Jennifer_Doudna
21.1 Introducing CRISPR/Cas9 Gene Editing Technology – Works by Jennifer A. Doudna
21.1.1 Ribozymes and RNA Machines – Work of Jennifer A. Doudna
Reporter: Aviva Lev-Ari Ph.D. RN
Reporter: Aviva Lev-Ari Ph.D. RN
Reporter: Aviva Lev-Ari Ph.D. RN
Curator: Aviva Lev-Ari, PhD, RN
21.2 CRISPR in Other Labs
21.2.1 CRISPR @MIT – Genome Surgery
21.2.2 The CRISPR-Cas9 System: A Powerful Tool for Genome Engineering and Regulation
Yongmin Yan and Department of Gastroenterology, Hepatology & Nutrition, University of Texas M.D. Anderson Cancer, Houston, USADaoyan Wei*
Reporter: Aviva Lev-Ari Ph.D. RN
Reporter: Aviva Lev-Ari Ph.D. RN
21.2.5 CRISPR & MAGE @ George Church’s Lab @ Harvard
Genome Engineering: CRISPR & MAGE
Multiplex Automated Genome Engineering (MAGE), is an intentionally broad term. In practice, it has come to be associated with a very efficient oligonucleotide allele-replacment (lambda red beta), so far restricted mainly to E.coli. CRISPR, in contrast, works in nearly every organism tested.
Relevant companies: EnEvolv, Egenesis, Editas.
News:
Editas (NextBigFuture, 28-Nov-2013, Brian Wang)
A Call to Fight Malaria One Mosquito at a Time by Altering DNA (NY Times, 17-Jul-2014, Carl Zimmer)
Resources:
* Vectors: Addgene
* Computational: Center for Causal Consequences of Variation (CCV)
Relevant Lab Publications:
2013 Probing the limits of genetic recoding in essential genes. Science.
2013 Genomically Recoded Organisms Impart New Biological Functions. Science.
2013 CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nature Biotech.
2009 Programming cells by multiplex genome engineering and accelerated evolution. Nature.
SOURCE
http://arep.med.harvard.edu/gmc/B2.html
21.3 Patents Awarded and Pending for CRISPR
21.3.1 Litigation on the Way: Broad Institute Gets Patent on Revolutionary Gene-Editing Method
Reporter: Aviva Lev-Ari, PhD, RN
21.3.2 The Patents for CRISPR, the DNA editing technology as the Biggest Biotech Discovery of the Century
Reporter: Aviva Lev-Ari, PhD, RN
2.4 CRISPR/Cas9 Applications
Kennedy EM1, Kornepati AV1, Goldstein M2, Bogerd HP1, Poling BC1, Whisnant AW1, Kastan MB2, Cullen BR3.
21.4.2 CRISPR: Applications for Autoimmune Diseases @UCSF
Reporter: Aviva Lev-Ari, PhD, RN
21.4.3 In vivo validated mRNAs
Author & Curator: Larry H. Bernstein, MD, FCAP
21.4.5 Where is the most promising avenue to success in Pharmaceuticals with CRISPR-Cas9?
Author: Larry H. Bernstein, MD, FCAP
21.4.6 Level of Comfort with Making Changes to the DNA of an Organism
Curator: Aviva Lev-Ari, PhD, RN
Reporter: Aviva Lev-Ari, PhD, RN
21.4.8 CRISPR/Cas9 Finds Its Way As an Important Tool For Drug Discovery & Development
Author: Stephen J. Williams, Ph.D.
Summary
Larry H Bernstein, MD, FCAP
The field of biology is now experiencing a transformative phase with the advent of facile genome engineering in animals and plants using RNA-programmable CRISPR-Cas9. The CRISPR-Cas9 technology originates from type II CRISPR-Cas systems, which provide bacteria with adaptive immunity to viruses and plasmids. The CRISPR associated protein Cas9 is an endonuclease uses a guide sequence within an RNA duplex, tracrRNA:crRNA, to form base pairs with DNA target sequences, enabling Cas9 to introduce a site-specific double-strand break in the DNA. This Review illustrates the power of the technology to systematically analyze gene functions in mammalian cells, study genomic rearrangements and the progression of cancers or other diseases, and potentially correct genetic mutations responsible for inherited disorders. The development of specific methods for efficient and safe delivery of Cas9 and its guide RNAs to cells and tissues will also be critical for applications of the technology in human gene therapy.
Targeted gene knockdown by RNA interference (RNAi) has provided researchers with a rapid, inexpensive and high-throughput alternative to homologous recombination. However, knockdown by RNAi is incomplete, varies between experiments and laboratories, has unpredictable off-target effects, and provides only temporary inhibition of gene function. These restrictions impede researchers’ ability to directly link phenotype to genotype and limit the practical application of RNAi technology.
CRISPR/Cas9 allows the targeted genome editing for efficient and reliable ways to make precise, targeted changes to the genome of living cells. Recently, a new tool based on a bacterial CRISPR-associated protein-9 nuclease (Cas9) from Streptococcus pyogenes has generated considerable excitement. This follows several attempts over the years to manipulate gene function, including homologous recombination and RNA interference (RNAi).
In the acquisition phase, foreign DNA is incorporated into the bacterial genome at the CRISPR loci. CRISPR loci is then transcribed and processed into crRNA during crRNA biogenesis. During interference, Cas9 endonuclease complexed with a crRNA and separate tracrRNA cleaves foreign DNA containing a 20-nucleotide crRNA complementary sequence adjacent to the PAM sequence.
There are several CRISPR biopharmaceutical companies using CRISPR-Cas9 technologies to develop transformative medicines for serious human diseases.CRISPR Therapeutics is focused on translating CRISPR-Cas9 gene-editing technology into transformative medicines for serious human diseases. There are also Cambridge-based Editas Medicine and Caribou Biosciences, among the biotech startups working to advance a much-watched new technology for precise gene editing. Doudna and her collaborator, Emmanuelle Charpentier of the Helmholtz Center for Infection Research in Braunschweig, Germany, and Umeå University in Sweden, figured out how to transform a bacterial defense against viral infection into a tool to edit out abnormal sections of genes, such as those that cause hereditary diseases.
In addition, researchers from North Carolina State University (NC State) and the University of North Carolina at Chapel Hill (UNC-CH) have created and utilized a nanoscale vehicle composed of DNA to deliver the CRISPR-Cas9 gene editing complex into cells both in vitro and in vivo.
Three Areas of Importance of CRISPR/Cas9 as a TOOL in Preclinical Drug Discovery Include:
- Gene-Function Studies: CRISPR/CAS9 ability to DEFINE GENETIC LESION and INSERTION SITE
- CRISPR/CAS9 Use in Developing Models of Disease
- Using CRISPR/Cas9 in PRECLINICAL TOXICOLOGY STUDIES
The advent of the first tools for manipulating genetic material (cloning, PCR, transgenic technology, and before microarray and other ‘omic methods) allowed scientists to probe novel, individual gene functions as well as their variants and mutants in a “one-gene-at-a time” process. In essence, a gene (or mutant gene) was sequenced, cloned into expression vectors and transfected into recipient cells where function was evaluated.
The current methods of producing the CRISPR–Cas9 components provide great flexibility in terms of expression and delivery, and biologists can exploit these options to control when and where DSBs are generated in an organism. To introduce DSBs and generate modifications early in development, the CRISPR–Cas9 components can be injected as DNA, RNA, or protein into most developing organisms. This approach, which has been widely used, generates mosaic organisms for analysis. To gain control over which tissues are affected, a plasmid expressing Cas9 under the control of tissue-specific enhancers can be used. Since each cell has a choice of whether to repair a breakthrough NHEJ or HDR, a variety of different repair events will be present in the injected organism (and in individual cells).
The relative ease of generating mutant animals will yield many additional animal models of disease and supply a means of testing whether specific polymorphisms are the proximal cause of disease in vivo. Additionally, the CRISPR–Cas9 system is amenable to application in organisms not widely used for genetic studies. Organisms that may be better suited to mimic human disease can now be more easily used to generate disease models. For example, mouse models of the bleeding disorder von Willebrand disease fail to fully recapitulate the human disease.
Apart from point mutations and gene deletions, large chromosomal rearrangements can drive specific cancers. By simultaneously introducing gRNAs targeting two different chromosomes or two widely separated regions of the same chromosome, RGNs have been used to introduce targeted inversions and translocations into otherwise wild-type human cells. These engineered cells will ultimately allow for studies of the causative role of these gene fusions in cancer progression. The first RGN based genetic screens were recently carried out in cultured mammalian cells. The screens identified targets affecting the DNA mismatch repair pathway, resistance to bacterial and chemical toxins, and cell survival and proliferation. The Zheng group also compared the results of their screen for genes involved in resistance to a drug that inhibits B-Raf with a prior RNAi screen that used the same cell line and drug, which revealed that gRNAs identified targets that could be validated more consistently and efficiently than shRNAs, pointing to the potential advantages of using gRNAs to knock out, rather than knock down, gene function in genetic screens.
Recent advances in genome editing have greatly accelerated and expanded the ability to generate animal models. These tools allow generating mouse models in condensed timeline compared to that of conventional gene-targeting knock-out/knock-in strategies. Moreover, the genome editing methods have expanded the ability to generate animal models beyond mice.
A critical component of producing transgenic animals is the ability of each successive generations to pass on the transgene. In her post on this site, A NEW ERA OF GENETIC MANIPULATION Dr. Demet Sag discusses the molecular biology of Cas9 systems and their efficiency to cause point mutations which can be passed on to subsequent generations
The conclusions from a recent meeting held in Napa have been summarized as follows.
The summary statement is as follows: “A framework for open discourse on the use of CRISPR-Cas9 technology to manipulate the human genome is urgently needed.”
They make 4 more specific recommendations.
- Strongly discourage clinical application of this technology at this time.
- Create forums for education and discussion
- Encourage open research to evaluate the utility of CRISPR-Cas9 technology for both human and nonhuman model systems.
- Hold an international meeting to consider these issues and possibly make policy recommendation.
The need for the meeting resulted from the following work in Japan:
- Concerns surface on Chinese paper on genetic modification of human embryos
- http://www.ipscell.com/2015/04/doudna/
CRISPR-Cas is a prokaryotic defense system against invading genetic elements. In a collaboration with John van der Oost’s laboratory, we are studying the structure and function of the effector complex of the Type III-A CRISPR-Cas system of Thermus thermophilus: the Csm complex (TtCsm). Recently, we showed that multiple Cas proteins and a crRNA guide assemble to recognize and cleave invader RNAs at multiple sites . Our negative stain EM structure of the TtCsm complex exhibits the characteristic architecture of Type I and Type III CRISPR-associated ribonucleoprotein complexes, suggesting a model for cleavage of the target RNA at periodic intervals (in collaboration with Eva Nogales, UC Berkeley, HHMI).
Double-stranded RNA induces potent and specific gene silencing in a broad range of eukaryotic organisms through a pathway known as RNA interference (RNAi). RNAi begins with the processing of endogenous or introduced precursor RNA into micro-RNAs (miRNAs) and small interfering RNAs (siRNAs) 21-25 nucleotides in length by the enzyme Dicer. We previously determined the crystal structure of an intact Dicer enzyme, revealing how Dicer functions as a molecular ruler to measure and cleave duplex RNAs of a specific length. Current work focuses on the mechanism of a complex of proteins known as the RISC loading complex (RLC) which load miRNA into the endonuclease Argonaute. The RLC contains the enzyme Dicer as well as TRBP, an RNA-binding protein hypothesized to interact with miRNA and Dicer during RISC loading. We seek to determine the molecular underpinnings of these interactions, along with the role of TRBP in RISC loading.
MicroRNAs (miRNAs) regulate endogenous eukaryotic genes by repressing gene expression through direct base-pairing interactions with their target messenger RNAs (mRNAs). To date, the rules used to predict miRNA-mRNA interactions have been based on one-dimensional sequence analysis. A more complete picture of miRNA-mRNA interactions should take into account the ability of RNA to form two- and three-dimensional structures. We are investigating the role of mRNA structure in the efficiency and specificity of targeting by miRNAs. Specifically, we are investigating the structure of Alu elements found within the 3′ untranslated regions (UTRs) of many human mRNAs and whether these structured domains serve as targets of a subset of human miRNAs. We are using in vitro biochemical methods and cell-based assays to probe the relationship between miRNA binding and mRNA structure.
The 5’ UTR of mRNA is also the site of multiple regulatory mechanisms, including upstream open reading frames (uORFs), internal ribosome entry sites (IRESs), protein binding sites, and stable secondary structures. Genes that profoundly influence cellular state often are controlled by multiple of these regulatory mechanisms. We are attempting to further understand regulatory elements in the 5′ UTR of mammalian mRNA using a combination of in vitro, cell-based and high-throughput techniques.
What does this mean for the development of therapeutics in the near future?
New methods for programming cell phenotype have broadly enabled drug screening, disease modeling, and regenerative medicine. Current research explores genome engineering tools, such as CRISPR/Cas9-based gene regulation and epigenome editing, to more precisely reprogram gene networks and control cellular decision making.
Donald Zack, M.D., Ph.D., Associate Professor of Ophthalmology and Neuroscience, Johns Hopkins University School of Medicine, is using CRISPR/Cas9 technology to generate retinal cell type-specific reporter ES and iPS lines and to introduce retinal degeneration-associated mutations. These reporter lines can be used to follow retinal neuronal specification during differentiation, they allow the purification of specific cell types by sorting and immunopanning, and they also are useful for the development of drug screening assays.
Jacquin C. Niles, M.D., Ph.D., Associate Professor of Biological Engineering, Massachusetts Institute of Technology is using CRISPR-Cas9 technology to study functional genetics in the human malaria parasite, Plasmodium falciparum. The team has established strategies for achieving controllable gene expression, and has integrated these into an experimental framework that facilitates efficient interrogation of virtually any target parasite gene using CRISPR/Cas9 editing.
Sidi Chen, Ph.D., Postdoctoral Fellow, Laboratory of Dr. Feng Zhang, Broad Institute and the Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology is observing that cancer genomics has revealed hundreds to thousands of mutations associated with human cancer. To test the roles of these mutations, we applied CRISPR/Cas9-mediated genome editing platform to engineer specific mutations in oncogenes and tumor suppressor genes. This results in tumorigenesis in several internal organs in mice. Our method expedites modeling of multigenic cancer with virtually any combination of mutations.
Samuel Hasson, Ph.D., Principal Investigator, Neuroscience, Pfizer, Inc., observes that while RNAi-based functional genomics is a staple of gene pathway and drug target exploration, there is a need for tools to provide rapid orthogonal validation of gene candidates that emerge from RNAi campaigns. CRISPR, CRISPRi, and CRISPRa are not only developing into primary screening platforms, they are a promising method to compliment RNAi and enhance the quality of functional genomic datasets.
These are some of the developments that will be discussed in detail at an upcoming meeting in Boston, MA in June titled ‘Gene Editing for Drug Discovery’.
SOURCE
http://rna.berkeley.edu/crispr.html
http://rna.berkeley.edu/rnai.html
http://rna.berkeley.edu/translation.html
Doudna Lab Publications
http://rna.berkeley.edu/publications.html
The structural biology of CRISPR-Cas systems.
Jiang F, Doudna JA
Curr Opin Struct Biol 2015 Feb 24;30C:100-111
Rational design of a split-Cas9 enzyme complex.
Wright AV, Sternberg SH, Taylor DW, Staahl BT, Bardales JA, Kornfeld JE, Doudna JA
Proc Natl Acad Sci U S A 2015 Feb 23
Genomic Engineering and the Future of Medicine.
Doudna JA
JAMA 2015 Feb 24;313(8):791-792
Integrase-mediated spacer acquisition during CRISPR-Cas adaptive immunity.
Nuñez JK, Lee AS, Engelman A, Doudna JA
Nature 2015 Feb 18
Dicer-TRBP Complex Formation Ensures Accurate Mammalian MicroRNA Biogenesis.
Wilson RC, Tambe A, Kidwell MA, Noland CL, Schneider CP, Doudna JA
Mol Cell 2014 Dec 30
Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery.
Lin S, Staahl B, Alla RK, Doudna JA
Elife 2014 Dec 15;3
Cutting it close: CRISPR-associated endoribonuclease structure and function.
Hochstrasser ML, Doudna JA
Trends Biochem Sci 2014 Nov 18
RNA Targeting by the Type III-A CRISPR-Cas Csm Complex of Thermus thermophilus.
Staals RH, Zhu Y, Taylor DW, Kornfeld JE, Sharma K, Barendregt A, Koehorst JJ, Vlot M, Neupane N, Varossieau K, Sakamoto K, Suzuki T, Dohmae N, Yokoyama S, Schaap PJ, Urlaub H, Heck AJ, Nogales E, Doudna JA, Shinkai A, van der Oost J
Mol Cell 2014 Nov 20;56(4):518-530
Genome editing. The new frontier of genome engineering with CRISPR-Cas9. (Free Full Text)
Doudna JA, Charpentier E
Science 2014 Nov 28;346(6213):1258096
Doudna JA, Sontheimer EJ
Methods Enzymol 2014;546C:xix-xx
New tools provide a second look at HDV ribozyme structure, dynamics and cleavage.
Kapral GJ, Jain S, Noeske J, Doudna JA, Richardson DC, Richardson JS
Nucleic Acids Res 2014 Oct 17
Programmable RNA recognition and cleavage by CRISPR/Cas9.
O’Connell MR, Oakes BL, Sternberg SH, East-Seletsky A, Kaplan M, Doudna JA
Nature 2014 Sep 28
RNA-guided assembly of Rev-RRE nuclear export complexes.
Bai Y, Tambe A, Zhou K, Doudna JA
Elife 2014;3:e03656
Evolutionarily Conserved Roles of the Dicer Helicase Domain in Regulating RNAi Processing.
Kidwell MA, Chan JM, Doudna JA
J Biol Chem 2014 Aug 18
Structure-Guided Reprogramming of Human cGAS Dinucleotide Linkage Specificity.
Kranzusch PJ, Lee AS, Wilson SC, Solovykh MS, Vance RE, Berger JM, Doudna JA
Cell 2014 Aug 12
Insights into RNA structure and function from genome-wide studies.
Mortimer SA, Kidwell MA, Doudna JA
Nat Rev Genet 2014 May 13
Cas1-Cas2 complex formation mediates spacer acquisition during CRISPR-Cas adaptive immunity.
Nuñez JK, Kranzusch PJ, Noeske J, Wright AV, Davies CW, Doudna JA
Nat Struct Mol Biol 2014 May 4
CasA mediates Cas3-catalyzed target degradation during CRISPR RNA-guided interference.
Hochstrasser ML, Taylor DW, Bhat P, Guegler CK, Sternberg SH, Nogales E, Doudna JA
Proc Natl Acad Sci U S A 2014 Apr 18
Structures of Cas9 Endonucleases Reveal RNA-Mediated Conformational Activation.
Jinek M, Jiang F, Taylor DW, Sternberg SH, Kaya E, Ma E, Anders C, Hauer M, Zhou K, Lin S, Kaplan M, Iavarone AT, Charpentier E, Nogales E, Doudna JA
Science 2014 Feb 6
DNA interrogation by the CRISPR RNA-guided endonuclease Cas9.
Sternberg SH, Redding S, Jinek M, Greene EC, Doudna JA
Nature 2014 Jan 29
http://rna.berkeley.edu/fun/D-lab%202013/SMALL/crisprconf.jpg
http://www.nasonline.org/news-and-multimedia/podcasts/interviews/jennifer-doudna.html
The Doudna lab pursues mechanistic understanding of fundamental biological processes involving RNA molecules. Research in the lab is currently focused on three major areas:
- bacterial immunity via the CRISPR system,
- RNA interference in eukaryotes, and
- translational control logic.
http://rna.berkeley.edu/crispr.html
http://rna.berkeley.edu/rnai.html
http://rna.berkeley.edu/translation.html
Other related curation on Gene Editing published as
Recent Advances in Gene Editing Technology Adds New Therapeutic Potential for the Genomic Era
Author and Curator: Stephen J Williams, PhD
Volume Summary
Larry H Bernstein, MD, FCAP
We have journeyed through about a century of scientific development that has changed the face of both physics and biology, and that has changed the face of medicine, before which there was no standard for the education of physician practitioners. In 1910 Abraham Flexner (1866-1959) published a report entitled “Medical Education in the United States and Canada.” It is the most important event in the history of American and Canadian medical education, and the Johns Hopkins Medical School was started based on the Flexnerian Model. (He founded the Institute for Advanced Study in Physics in 1930).
Before the Second World War the progress in medical science was divorced from advances in chemistry and physics, but the advances in medicine, initially related to public health and immunology stood out, and progressed to vitamins and physiology as follows:
A. Infectious Agents and Insecticides
Ross (1902) role of insects as vectors in the infectious cycle (malaria)
Koch (1905) identification of the tubercle bacillus and other work on tuberculosis
Laveran (1907) role of protozoa in causing disease (malaria)
Nicolle (1928) role of clothes lice in the transmission of typhus
B. Immunology
Behring (1901) serum therapy and its application against diphtheria
Ehrlich (1908) immunity
Mechnikov (1908) phagocytosis
Richet (1913) anaphylaxis
Bordet (1919) antigens and antibodies in immune reactions
Landsteiner (1930) blood groups and blood typing
C. Chemotherapy/Drug Development
C Domagk (1939) prontosil (sulphonamides)
Fleming, Chain & Florey (1945) penicillin
D. Classical Genetics
J Hunt Morgan (1933) role of chromosomes in heredity
Muller (1946) production of mutations by X-ray irradiation
C. Molecular and Cell Biology/metabolism
Kossel (1910) work on protein including the nucleic substance
AV Hill (1922) heat production in muscle
Otto Meyerhof (1922) oxygen consumption and the metabolism of lactic acid in muscle
Otto Warburg (1931) nature and mode of action of the respiratory enzyme
Spemann (1935) organiser effect in embryonic development
D. Hormones and Vitamins
Kocher (1909) physiology, pathology and surgery of the thyroid gland
Banting & Macleod (1923) insulin
Eijkman (1929) antineuritic vitamin
Hopkins (1929) growth-stimulating vitamin
Whipple, Minot & Murphy (1934) liver therapy in cases of anaemia
von Szent-Györgyi (1937) vitamin C and the catalysis of fumaric acid
Dam (1943) vitamin K
Doisy (1943) chemical nature of vitamin K
E. Physiology
Pavlov (1904) physiology of digestion (conditioned reflexes)
Golgi & Ramón y Cajal (1906) structure of the nervous system
Alexis Carrel (1912) Carrel-Dakin method of treating war wounds (1914-1919), end-to-end anastomosis of blood vessels (1902), method for supplying sterile respiratory system to organs removed from body (1908)
Krogh (1920) capillary motor regulating mechanism
Einthoven (1924) electrocardiography
Sir Charles Scott Sherrington and Edgar Douglas (1932) functions of neurons
Heymans (1938) role of sinus and aortic mechanisms in the regulation of respiration
Of note in the field of Chemistry in the same period were:
Friedrich Wilhelm Ostwald received the Nobel Prize in Chemistry in 1909 for his work on catalysis, chemical equilibria and reaction velocities. Ostwald, van ‘t Hoff, and Arrhenius are usually credited with being the modern founders of the field of physical chemistry. The Ostwald process (patent 1902) for production of nitric acid and Haber and Bosch’s work on nitrogen fixation led to large-scale production of fertilizers and explosives. http://upload.wikimedia.org/wikipedia/commons/thumb/3/35/Ostwald_vant_Hoff.jpg/300px-Ostwald_vant_Hoff.jpg
Walther Hermann Nernst – a German physical chemist and physicist who is known for his theories behind the calculation of chemical affinity as embodied in the third law of thermodynamics, for which he won the 1920 Nobel Prize in chemistry. Nernst helped establish the modern field of physical
Notable work in the early medical chemistry at that time is with Wieland, Windaus, and Hans (not Emil) Fischer for work with bile acids, haemin and chlorophyll, and sterol synthesis and Vit D (1927, 28, 30).
Irving Langmuir published the 1919 article “The Arrangement of Electrons in Atoms and Molecules” in which, building on Gilbert N. Lewis’s (both students of Ostwald) cubical atom theory and Walther Kossel’s chemical bonding theory, he outlined his “concentric theory of atomic structure”. He was awarded the 1932 Nobel Prize in Chemistry for his work in surface chemistry.
There is no clue to the post WWII developments in medicine, genetics, and biology until the developments after the Manhattan project, as developed in this volume, though following a strong foundation from a European tradition in Physics and Physical Chemistry. Many of the greatestest scientists had emigrated from Germany with the rise of Hitler and the Nazi Party.
We have seen the emergence of biochemistry, organic chemistry, medicinal chemistry, a dynamic pharmaceutical industry, NIH support of expanding research and postdoctoral training in cardiology, endocrinology, cancer, and pathology since the establishment of a scientifically based medical academy, which led to full collaboration of the research described between strong academic institutions across the US, and between US and both UK and continental Europe, with the developments described here.
After the completion of the HGP in 2003, the work was ripe for accelerated discovery, and we have seen new issues in the years since the human genome project (HGP) and ENCODE, and more recently, the 2004 International HapMap project, and 2005 GWAS. This is because there is a now a confluence of circumstances relating to the practice of medicine, the education of physicians, the communication between physician and patients changing from what is referred to “god handing down an edict” to evidence-based medicine. This is also complicated at a time that we have a national state-by-state implementation of a remodeled Medicare and Medicaid plan based on the program already successful in Massachusetts.
In the reorganization, there will be more regional hospital, academic and clinic consolidations, and even possible statewide organizations, movement of patients from inpatient beds sooner with a high skill level of outpatient support, greater concentration of physician staffs aligned to PHO type arrangements, and a need to fill PCP gaps with qualified Advanced Nurse Providers.
All of this is happening now. This is a realignment to meet the needs of the Payor (Fed, HMO, Big Insurance), with tighter margins per stay and critical decisions about capital needs and depreciation, at the same time, required to meet a higher risk of performance standard. Eric topol refers to the need to education of this generation of physicians in Personalized Medicine. But that has never been so easy for those advanced in their careers, and even bright new entries into the profession are faced with productivity guidelines. It is an assignment that will be a new challenge for the Pathology profession, just as the student lab was long ago replaced by the laboratory, with microbiology, blood bank, hematology, chemistry and immunology, to which was added molecular testing. It will be a very challenging undertaking compared to past experience, and it will be a very big adjunct to microscopy, while imaging technology, in the hands of radiology, is undergoing a parallel transformation.
We derive the following major points from what has been presented in this work:
Genomics will become a key component integrated into patient-care, preventive-medicine, and what is going to become a standard of practice for personalized medicine, or individualized-care of a patient defined by individuality, culture, and personal goals for treatment outcomes. A personal goal may be a likely or unlikely point of view in the eye of the observer:
- Let me live with my illness, but relieve my pain
- Give me a realistic time to prepare for dying so I can tie up loose ends
- A cure would be a gift if there adverse effects are minimal
The expanded view of this expectation resides in a more accepting view of what lies ahead and of what is behind. The choice before us lacked clarity in the past. The view was limited, and might still be for some with an unfulfilled life, whether imposed or chosen.
The medical requirement that supercedes all others is:
- Clinical medicine context … clinically guided
- physician/patient relationship … not a consumer relationship
- First do no harm… directly related to priority for care
- must know significance … disease recessive traits ..
- can we offer anything?
common complex diseases…
- both genetic & environmental factors
- not inherited in predictable ways
- gene-gene interactions
- variants usually account for a small amount of risk
examples where both clinical assessment and genomic personalized medicine are expected to realize potential real concordance are:
- macular degeneration
- alzheimer’s disease
- colon cancer
The increased benefit to the pathology-diagnostic imaging -surgical-oncology team is seen as
- tailoring treatment though genomic guidance:
- microscopic doesn’t dictate treatment
- determine choice of treatment
- drug reactions may be avoided
Medical Gutenberg
Eric Topol refers to the “Medical Gutenberg” in a recent lecture in the Medscape series “Creative Destruction of Medicine”. He says ” If we go back to the 1400s and the printing press invented by Johannes Gutenberg, you know how transformative that invention was. The high priests were no longer the only ones who could read; the ability to read books was unleashed to the public. Many years, many centuries have passed since those times, but here in the 21st century we’re getting consumers — the public — to read medical stuff.” He goes on that “now we’re moving from information asymmetry to information parity. This really sets up a unique experience, but it won’t [happen] for all consumers because they’re not all going to want to learn to read and get into this [medical information]. But who has the most vested interest in one’s health if it isn’t that individual, that patient?”
That’s Medical Gutenberg. That’s the opportunity that lies ahead with digital medicine — shifting that information and data to the patient requiring the guidance, knowledge, and experience from physicians.
A Tale of Two Nominal Super-Drugs.
A Success Story? Perhaps too early to know. New York Times reported on March 19 , 2013 that Amgen, had met the primary goal of a Phase 3 clinical trial in patients with advanced melanoma, with 16 percent of the patients in the trial who had the treatment, called talimogene laherparepvec, or TVEC, experienced a significant shrinkage of their tumors that lasted at least six months compared with only 2 percent of the patients in a control group. TVEC is a herpes simplex virus modified in such a way that it replicates in fast-growing cancer cells but not healthy ones, and it also contains an implanted gene for GM-CSF (colony stimulating factor), a protein that stimulates the immune system. When the the replicating viruses cause the cell to burst, freeing the virus and the GM-CSF in the presence of tumor components, it elicits a systemic immune response that can kill cancer cells throughout the body. Recall that this is a late-stage response, and a long term disease free survival is not determined.
A Failure. {Marker for NSCLC Chemo Response Doesn’t Hold Up. by Crystal Phend, MedPage Today, March 20, 2013} A DNA repair biomarker thought to predict benefit from platinum-based chemotherapy in non-small cell lung cancer (NSCLC) doesn’t actually do that good a job. The problem is both technical and due to the inability of the assay to distinguish the key form of the protein for DNA repair. The ERCC1 protein expression level didn’t predict a boost in overall survival (OS) from adjuvant cisplatin (Platinol)-based chemotherapy compared with observation alone in two clinical trials (P=0.23 for interaction). There was no effect seen in the ERCC1-negative group, which was the basis for proposing the protein as a predictive biomarker.
Why is that significant surprise? The inability of the assay to distinguish the key form of the protein for DNA repair, the group reported in the March 21 issue of the NEJM. The antibodies do not have adequate discrimination for therapeutic decision making regarding cisplatin-containing treatment in patients with NSCLC, which requires the specific detection of the unique functional isoform of ERCC1 — ERCC1-202. There are three other isoforms of ERCC1 (excision repair cross-complementation group 1) protein that aren’t critical in fighting the cytotoxic effect of platinum chemotherapy.
So here we have it. It’s not yet, far from the worst of times, but equity barriers remain for a time. The science is critical important, and the implementation of good science can reap huge benefits in time.
New Recommendations for Genetic Reporting
GENNewsHighlights Mar 22, 2013
Finally, there is now emerging a standard of care for providing and reporting of genetic information. The American College of Medical Genetics and Genomics (ACMG) released landmark recommendations on the handling of incidental findings in clinical genome and exome sequencing.This was published within days of completion of this work. It is only the beginning of a process expected to go through many revisions.
http://www.genengnews.com/gen-news-highlights/new-recommendations-for-genetic-reporting/81248136/
A minimum list of genetic conditions, genes, and variants that laboratories performing clinical sequencing should seek and report to the physicians that ordered the testing—regardless of the original reasons for which the test was ordered.
In assembling this list, the Working Group prioritized the disclosure of disorders where:
- Preventative measures and treatments exist
- Patients might not experience symptoms for a long period of time
- The genetic mutations are well recognized and known to have a strong link of causation
Examples of diseases recommended for disclosure include rare hereditary cancers and rare heart diseases that could result in sudden cardiac death.
According to Robert C. Green, MD, medical genecist at Brigham and Women’s Hospital, Harvard, laboratories are looking for guidance on how and what should be communicated to clinicians when results are analyzed. These recommendations will allow a small percentage of families to learn unexpected but potentially life-saving information about an illness they may have never suspected they were at risk for.” The Working Group did not recommend giving patients the choice of whether or not their physician would receive results from the list of recommended incidental findings. This makes sense in the realization that the actual strength of the finding is uncertain. The Working group also recommended that adult-onset conditions on the list be reported, perhaps with the expectation of life-style modification for prevention.
Epilogue
What is the Future for Genomics in Clinical Medicine?
Larry H Bernstein, MD, FCAP
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