Southwestern Medical Perspectives 2019: The CRISPR Revolution by Southwestern Medical Foundation

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CRISPR REVOLUTION

The gene-editing tool behind an unprecedented launch of hopes and dreams for humanity A T C G

ON THE COVER The sky lantern â&#x20AC;&#x201C; a small hot-air balloon made of wire and paper â&#x20AC;&#x201C; is used in celebrations around the world as a symbol of hope and the wish for a brighter future.

Possibilities and Responsibilities

Kathleen M. Gibson P r e s i d e n t & C EO

Southwestern Medical Foundation and its founders have played a leading role in defining the medical standards for our community. This legacy and the capital it attracted has broadly and deeply inspired progress in medicine. Few breakthroughs in the history of science have had the kind of sweeping and immediate impact of CRISPR (pronounced “crisper”) gene editing in bringing rapid and monumental change to the way scientists approach key questions of life. If you haven’t heard of CRISPR beyond this mention, you most certainly will soon. Every month, hundreds of advances in medical and clinical research are published related to the technology. This game-changing tool has the potential to alter not just the world of health care but every living thing. We believe that the better society understands the issues, the better we’ll be able to engage in meaningful discourse on the future of medical care and public policy. The power to alter the code of life itself brings with it profound moral and ethical questions. To that end, we invited thought leaders from across the country and around the world to contribute their perspectives on this subject. Together, they bring a diverse set of thinking on what CRISPR means to the future of medical and genetic research in key areas of biotechnology, regulation, and human somatic and germline editing. We hope to facilitate an understanding that turns care and concern into cures and positive change for humankind. Along with our feature on CRISPR technology, we take a moment to celebrate a few of the many people on whom, and the programs on which, the success of the Foundation rests. We believe philanthropy begins with getting acquainted. It is individualized and personal – yet it is by connecting with one another that we accomplish great things. We hope this issue of Perspectives inspires.

Southwestern Medical Foundation Officers, Trustees, and Honorary Trustees OFFICERS James R. Huffines, Chairman of the Board Kathleen M. Gibson, President and CEO

Kay Schlankey, Senior Vice President and CFO Emily Davis, Corporate Secretary

BOARD OF TRUSTEES John L. Adams Charles Anderson Charlotte Jones Anderson Ralph W. Babb Jr. Alice Worsham Bass Jill C. Bee Randy Best Robert W. Best Lucy Billingsley Jan Hart Black* Randy Bowman David O. Brown J. Robert Brown Leland R. Burk W. Plack Carr Jr. Nita P. Clark Mary McDermott Cook* David R. Corrigan* Timothy P. Costello Harlan R. Crow Linda Pitts Custard Robert H. Dedman Jr.* Joseph M. DePinto Jennifer Eagle Timothy Eller

Matrice Ellis-Kirk Sandra Street Estess Robert A. Estrada Gloria Eulich Linda Perryman Evans Hill A. Feinberg Andersen C. Fisher Richard W. Fisher Stuart Fitts Cate Ford Holland P. Gary Judy Gibbs Kathleen M. Gibson Mark D. Gibson Satish Gupta Rolf R. Haberecht, Ph.D. Ronald W. Haddock Nancy S. Halbreich David C. Haley* Kathryn W. Hall LaQuita C. Hall Paul W. Harris Julie K. Hersh J. Hale Hoak Richard E. Hoffman, M.D.

David B. Holl James R. Huffines* Rex V. Jobe* Eric Johnson Amb. Robert Jordan (ret.) Robert L. Kaminski Gary C. Kelly James Keyes Caren Kline Harlan Korenvaes Peter A. Kraus Joyce Lacerte Mark Langdale Samuel D. Loughlin Bobby B. Lyle* S. Todd Maclin Nancy Cain Marcus, Ph.D. Charles W. Matthews William S. McIntyre IV Pauline Medrano Howard M. Meyers David B. Miller* Sarah K. Miller Kit Tennison Moncrief Carter Montgomery

Kay Y. Moran Charles E. Nearburg J. Ray Nixon Jr. Alfreda B. Norman Lydia H. Novakov James C. Oberwetter Connie O’Neill Carlos G. Peña Guillermo Perales Jeanne L. Phillips Daniel K. Podolsky, M.D.* Richard R. Pollock Matthew S. Ramsey Kelly E. Roach Katie H. Robbins Linda Harbin Robuck Catherine M. Rose* Matthew K. Rose William E. “Billy” Rosenthal Daniel G. Routman Robert B. Rowling* Steven S. Schiff Robert J. Schlegel Debbie Scripps David T. Seaton

Robert T. Enloe III Roy Gene Evans Jerry Farrington Robert I. Fernandez Lee Fikes Edwin S. Flores, Ph.D. Terry J. Flowers, Ed.D. Gerald J. Ford Kay Carter Fortson Alan D. Friedman Gerald W. Fronterhouse Printice L. Gary William R. Goff Joseph M. “Jody” Grant Joe M. Haggar III Howard Hallam Charles M. Hansen Jr. Linda W. Hart Joe V. Hawn Jr. Frederick B. Hegi Thomas O. Hicks Lyda Hill Laurence E. Hirsch

James M. Hoak Sally S. Hoglund T. Curtis Holmes Jr. Keith W. Hughes Walter J. Humann Hunter L. Hunt Ray L. Hunt Kay Bailey Hutchison Judith K. Johnson Philip R. Jonsson Dale V. Kesler Gary Kusin David M. Laney Laurence H. Lebowitz Thomas C. Leppert John I. Levy Wendy A. Lopez Sarah Losinger Tom Luce Ann E. Margolin John D. McStay Harvey R. Mitchell W. A. “Tex” Moncrief Jr.

Susan Byrne Montgomery Jennifer T. Mosle J. Fulton Murray Jr. Mike A. Myers Joseph B. Neuhoff Teresa Haggerty Parravano Rena M. Pederson Jack Pew Jr. J. Blake Pogue Caren H. Prothro Mary Stewart Ramsey Carolyn Perot Rathjen Michael S. Rawlings Leonard M. Riggs Jr., M.D. Jean W. Roach Lizzie Horchow Routman Stephen H. Sands Pete Schenkel John Field Scovell George E. Seay III Paul R. Seegers Carl Sewell Jr. George A. Shafer

Florence Shapiro Ted C. Skokos Nicole G. Small Bonnie B. Smith Jerry V. Smith William S. Spears, Ph.D. Marvin J. Stone, M.D. Sam L. Susser Catherine B. Taylor Richard K. Templeton Jere W. Thompson Jr.* McHenry T. Tichenor Jr. Kip Tindell John C. Tolleson Lisa Troutt Margaret B. Vonder Hoya W. Kelvin Walker Kelcy L. Warren George W. Wharton, M.D. Kern Wildenthal, M.D., Ph.D. Martha S. Williams Todd Williams Mark Zale * Executive Committee

HONORARY TRUSTEES Sara Melnick Albert Rafael M. Anchia Gilbert Aranza Marilyn H. Augur Doris L. Bass Peter Beck Gil J. Besing David W. Biegler Albert C. Black Jr. Cecilia G. Boone Daniel H. Branch Diane M. Brierley Jean Ann Brock Robert W. Brown, M.D. Stuart M. Bumpas Stephen Butt Edward H. Cary III Jeffrey A. Chapman Berry R. Cox Joe D. Denton Robert J. DiNicola Thomas M. Dunning Thomas J. Engibous

Karen L. Shuford Lisa K. Simmons Emmitt J. Smith William T. Solomon Roger T. Staubach Joanne H. Stroud, Ph.D. Ellen C. Terry Michelle R. Thomas Gifford O. Touchstone Jim L. Turner Jack C. Vaughn Jr. John J. Veatch Jr. Kent Waldrep J. Thomas Walter Jr. Carol R. West Jimmy Westcott Laura L. Wheat Evelyn Whitman-Dunn Terry M. Wilson Kneeland C. Youngblood, M.D. Donald Zale

contents

Cover Story

The CRISPR Revolution

CRISPR, which stands for Clustered Regularly Interspaced Short Palindromic Repeats, is a leap forward in the field of biotechnology that exploits a primitive, adaptable immune system found in bacteria and is being repurposed to edit genes in any organism — from microbes to plants to humans. When added to our vast and rapidly expanding knowledge base of genes, gene function, and gene regulation, CRISPR might become the most extraordinary revolution in the history of civilization.

A T C G EDITORIAL DIRECTOR J. Kim Brayton the BraytonGroup EDITORIAL TEAM Lisa Dennison, Ph.D. Renee English Kathleen Gibson Megan Jenkins Brittany Lebling Ronnie Rittenberry Stephanie Vidikan WRITERS J. Kim Brayton Brittany Lebling Various authors and sources* D E S I G N D I RE C TOR J. Kim Brayton PHOTOGRAPHERS Lara Bierner Steve Foxall David Gresham Grant Miller Stock and archival resources*

Introduction CHAPTER 1.0 The CRISPR Revolution CHAPTER 2.0 A Brief Refresher CHAPTER 3.0 CRISPR Applications 3.1 Research 3.2 Gene Drives 3.3 Microbes, Plants, and Animals 3.4 Diagnostics and Drug Discovery 3.5 Human Somatic Cells 3.5.1 Duchenne Muscular Dystrophy 3.6 Human Germline Cells CHAPTER 4.0 The History of Genetic Modification CHAPTER 5.0 The Business of CRISPR CHAPTER 6.0 Bioethics and Regulation CHAPTER 7.0 The Future

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Contributors David Baltimore, Ph.D., California Institute of Technology, President Emeritus Rodolphe Barrangou, Ph.D., North Carolina State University George M. Church, Ph.D., Harvard University, Harvard Medical School, and Massachusetts Institute of Technology Francis S. Collins, M.D., Ph.D., National Institutes of Health, Director Marcy Darnovsky, Ph.D., Center for Genetics and Society, Berkeley Jennifer A. Doudna, Ph.D., University of California, Berkeley Christi Dupree, TracyLocke, Dallas Kathleen M. Gibson, Southwestern Medical Foundation, President Christopher Gyngell, Ph.D., The University of Melbourne William Hurlbut, M.D., Stanford Medical School Jeffrey Leiden, M.D., Ph.D., Vertex Pharmaceuticals, Chairman, President, and CEO Eric Olson, Ph.D., University of Texas Southwestern Medical Center Daniel K. Podolsky, M.D., University of Texas Southwestern Medical Center, President Marsha Saxton, Ph.D., University of California, Berkeley Larry Schlesinger, M.D., Texas Biomedical Research Institute, President Feng Zhang, Ph.D., Massachusetts Institute of Technology Articles, Features, and Thank Yous

* see pages 90-91 Editorial comments and contributions are welcome. Send correspondence to:

64 Breakthrough Prize

Southwestern Medical Foundation Parkland Hall at Old Parkland 3889 Maple Avenue, Suite 100 Dallas, Texas 75219

Zhijian “James” Chen, Ph.D., was one of five researchers awarded a 2019 Breakthrough Prize in Life Sciences.

info @ swmedical.org p 214-351- 6143 f 214-352-9874

Every Issue

66 We Connect

By connecting with one another, we further medical innovation, medical research, and the health of the public.

90 Acknowledgments

This issue was made possible through the contributions of many people. To them, we offer our deepest thanks.

1 President’s Letter 92 A Moment in Time On December 12, 2018, the Texas Historical Commission recognized the founding of Southwestern Medical College – known today as UT Southwestern – as a significant event of Texas history by dedicating an official Texas Historical Marker.

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What if we could develop sustainable, cost-effective biofuels to power our cars, our homes, our world?

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What if we could develop an abundance of transplant organs ?

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What if we could create droughttolerant and disease-resistant crops, reducing our dependence on irrigation and pesticides?

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What if we could repair genes and prevent â&#x20AC;&#x201C; or even cure â&#x20AC;&#x201C; debilitating and life-threatening genetic disease?

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CRISPR.

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The unprecedented launch of hopes and dreams for humanity has begun.

HUMANKIND HAS ALWAYS had an innate desire

to improve itself and its environment. We have thrived in large part because we’ve been able to modify our surroundings, passing this knowledge on to the next generation to be built upon, refined, and improved. Along the way, we’ve accomplished a great many things. But the speed at which fundamental discoveries are being transformed into real-world applications – from innovations like artificial intelligence to virtual reality to blockchain technology – has never been greater than it is right now. In the world of medicine, a key driver of that acceleration is the fact that we now have a far deeper understanding of the molecular basis of disease. Add to this the arrival of CRISPR technology – which gives scientists the ability to precisely edit the DNA molecules in any living thing – and the potential scope and impact of what is possible boggles the mind. CRISPR has taken once-distant boundaries and blurred them, bringing the world to a crucial decision point: How far can or should we go in the engineering of life? Though the answers will vary not only among different people but also different cultures, one thing seems clear: medicine is undergoing a transformation. We are moving from the management of disease to its prevention and correction at the molecular level. To what extent CRISPR researchers will be able to achieve all that we can imagine remains to be seen. But as you will soon discover, CRISPR has given us much to think about.

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The CRISPR Revolution

I n 1968, The Beatles released the song Revolution. You say you want a revolution Well, you know We all want to change the world. At the time it was recorded, beyond a cultural revolution, another transformative revolution was about to begin. No one then imagined that computers would so thoroughly change the world. Computers were refrigerator-size mainframes weighing thousands of pounds and costing $250,000 – more than $1.8 million in today’s dollars. Who could have anticipated the scope of the digital revolution? That computers would shrink to the size of a wristwatch, become thousands of times more powerful than a mainframe, and cost only a few hundred dollars? That a device you hold in your hand could be a window to the world? That billions of people around the globe could connect with each other, instantly, to share ideas or search for answers on nearly any topic? That an online store would become the world’s biggest retailer (Amazon)? Or that you’d be able to watch more than 2 million cat videos on something called YouTube? (To date, cat videos have been viewed more than 25 billion times. Cat videos.) To begin with, in the ’60s such an enormous leap in technology would have seemed impossible. What’s more, it would have been cause for concern — because it’s a natural human reaction to be cautious of great leaps forward in technology. But the digital revolution took the world by storm, changing nearly every aspect of how we live. What once seemed like science fiction has become our everyday reality. Today, we find ourselves just a few years down another, even more powerful, transformative path: that of genome editing. “Most of the public,” said Dr. Jennifer Doudna, a co-discoverer of CRISPR, “does not appreciate what is coming.” The revolution has begun. And it’s about to change the world.

PERSPECTIVE

Dr. Jennifer A.

Dr. Doudna is an Investigator with the Howard Hughes Medical Institute, the Innovative Genomics Institute, and the Gladstone Institute and a Professor of Chemistry and Molecular and Cell Biology at the University of California, Berkeley. Drs. Doudna and Emmanuelle Charpentier were the first to propose that CRISPR-Cas9 could be used for programmable editing of genomes.

DOUDNA THE CRISPR REVOLUTION

The elegant two-stranded helical structure of DNA, discovered in the 1950s, immediately explained how genetic information is copied and inherited, and also suggested the potential for its alteration. Over the past several decades, precise and predictable gene editing has been achieved by inducing double-stranded DNA breaks in chromosomes, triggering cells to alter the DNA sequence during doublestrand break repair. Although powerful in principle, the practice of such gene-editing methods was strictly for specialists with extensive technical and financial resources until the advent of CRISPR-Cas genome-editing tools. Based on the programmable DNA cutting activity of the The CRISPR revolution has not only accelerated CRISPR-associated (Cas) protein Cas9, it is now possible to and advanced biomedical research, it is now induce site-specific alterations to genomes in cells, tissues, and whole organisms. This transformative technology provides on the cusp of providing clinical treatments or important scientific opportunities for curing genetic diseases even cures for genetic diseases. and engineering desirable genetic traits, as well as new approaches to live-cell imaging and point-of-care diagnostics. The basis for the CRISPR revolution goes beyond inherent programmability, building upon the naturally evolved diversity of bacterial immune systems that extend CRISPR-based technology beyond precision gene editing. The DNA-targeting protein Cas9 (the first and most widely used Cas protein to be harnessed for genome engineering) has several properties that ensure precise and efficient editing. Cas9 assembles with specific RNA molecules that “guide” the recognition of matching DNA sequences, a process that can be controlled by scientists and hence used to edit desired genes in cells. In addition, the Cas9 protein has proven remarkably accurate when used in limited amounts and in primary (as opposed to cancerous) cells. Furthermore, Cas9 is amenable to a wide range of manipulations to enhance accuracy and control the way DNA repair takes place, ensuring desired editing outcomes. The resulting CRISPR revolution has not only accelerated and advanced biomedical research, it is now on the cusp of providing clinical treatments or even cures for genetic diseases, including sickle cell anemia and muscular dystrophy. Clinical trials that are expected to get underway over the next few years could ultimately lead to “one-and-done” treatments that fix disease-causing mutations, offering patients truly personalized therapies with lifesaving benefits. Scientists and clinicians are working to realize its exciting potential and also to ensure responsible use of this powerful technology. In the years ahead, CRISPR tools will continue to drive fundamental research, enabling discoveries about the genes and pathways that underlie health, disease, and therapeutic outcomes. The CRISPR revolution will also undoubtedly build on other technologies, including machine learning to accelerate the pace of new discoveries and, hopefully, the clinical advances to come from them.

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THE WORD “REVOLUTIONARY” is often overused by the media to describe new scientific discoveries. But CRISPR’s impact on the world might be so far-reaching that even a thesaurus full of superlatives would fall short. CRISPR-Cas technology has sparked the imagination of scientists across a wide range of disciplines. It has made gene editing inexpensive, easy, and accessible – igniting optimism but, at the same time, also giving rise to ethical concerns over how to use such a powerful tool. The rising adoption of CRISPR technology has quickly taken hold: ■ New companies have been founded on the initial CRISPR intellectual property rights granted to various individuals and institutions. A few examples of what has been done using CRISPR-Cas technology over the past few years: In the United States, a treatment for the blood disorder beta thalassemia is the closest CRISPR therapy to market. Chinese scientists have made wheat resistant to a common fungal disease, dogs more muscular, and pigs with leaner meat. UC Berkeley and Keck Graduate Institute created a CRISPR “chip” to diagnose genetic diseases in minutes and to evaluate the performance of a gene therapy on a patient. By combining CRISPR with CAR T, scientists can edit cancerous T-cells and send them back into the body effectively. Without editing, an affected CAR T-cell would have difficulty distinguishing between itself and a cancer cell and destroy both in the process. At the University of California, Riverside, a team is working to reprogram a yeast strain to convert sugars into biofuels, adhesives, and fragrances. A plant pathologist at Penn State University has created a mushroom that doesn’t brown. Researchers at Temple University and the University of Nebraska Medical Center have eliminated the virus responsible for AIDS from the genomes of living animals – a critical step toward the development of a possible cure for human HIV infection. At UT Southwestern Medical Center, a team lead by Dr. Eric Olson has corrected Duchenne muscular dystrophy in mice and dogs, and in human cells outside the body. Using CRISPR-Cas9, researchers have edited out the faulty region of the gene that causes Huntington’s disease in cells derived from patient samples. Researchers at the University of California, San Francisco have shown CRISPR, without cutting DNA, can ramp up the activity of certain genes and prevent severe obesity in mice with genetic mutations that predispose them to extreme weight gain. At Harvard University, Dr. George Church is exploring the possibility of saving the Asian elephant by enabling it to thrive in an entirely new habitat: the tundra of Siberia. How? By using genes from an extinct woolly mammoth.

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Strategic alliances with heavyweight companies and start-ups have formed. ■ Licenses to use CRISPR-Cas9 technology have been granted to pharmaceutical, material manufacturing, and agricultural companies, supercharging their R&D labs. ■ The United States and China, in particular, are now locked in an intellectual property arms race – or perhaps more accurately, a “gold rush.” ■ CRISPR has inspired new rules for research. International conferences have issued statements and governments have provided guidelines, and yet there can be no guarantees rules won’t be broken. As if to punctuate the concern, in November 2018, a Chinese biophysics researcher announced the birth of twin girls whose genetic makeup had been altered using CRISPR-Cas9 technology. This revelation led many CRISPR experts to propose a five-year global moratorium on heritable genome editing in humans. Without question, CRISPR makes it easier for people with ill-intent to do great harm – a concern that led former Director of National Intelligence James Clapper to include genetic editing technologies on a list of threats posed by “weapons of mass destruction and proliferation.” And yet CRISPR has given humanity the chance to enter a new era in medicine with the potential to fulfill the promise that started with the sequencing of the human genome. For the first time, we have the ability to precisely dictate changes at the molecular level and cure genetic disease. Beyond treating patients, CRISPR’s most important application might lie in the discovery of new drugs and inexpensive diagnostic tools. It will likely usher in a new era in agriculture to feed a growing world population and could be used to control malaria-carrying mosquitoes, saving millions of lives worldwide. Together we must decide by what scientific and moral guidelines we should steer the world’s genetic destiny. We have reached a turning point in human history because CRISPR-Cas represents a freedom the likes of which mankind has never experienced. The story of CRISPR is laid out before you. It’s a lot to digest, but we believe it is well worth an investment of your time. The future isn’t just coming. It’s here.

PERSPECTIVE

Dr. Feng

ZHANG STAYING CURIOUS, STAYING OPTIMISTIC

Dr. Zhang is a core member of the Broad Institute of MIT and Harvard. He is also a Howard Hughes Medical Institute Investigator, McGovern Investigator, and Professor in MIT’s Departments of Brain and Cognitive Sciences and of Biological Engineering. Dr. Zhang was a key pioneer in the development of CRISPR-Cas9 and its use in eukaryotic cells. He continues to discover and develop CRISPR tools with the potential to diagnose and treat disease.

Many medical treatments have humble origins: Compounds from tree bark, for example, have been used for antimalarial properties; bacteria that live in the dirt also have been found to contain potent antibiotics. One of the most promising new therapeutics, CRISPR, leverages bacterial defense systems that provide microbes with adaptive immunity against invading foreign genetic material, some of which have been engineered to allow us to precisely edit genes in human cells to understand and treat genetic diseases. Through the combined efforts of many outstanding scientists studying and applying these systems, CRISPR is As we continue to explore nature and its now being widely applied in biological research and developed as a new kind of medicine. diversity, we are certain to discover new The first CRISPR system that was harnessed for genome processes that may be engineered to advance editing, CRISPR-Cas9, is based on one of thousands of human health and improve sustainable living. naturally occurring CRISPR systems. Beyond Cas9, there also exist numerous novel CRISPR systems that have different properties that are now being engineered for a wide range of applications for improving human health. For example, going forward, CRISPR-based tools may be used to correct disease-causing mutations, fine-tune the levels of gene expression products to bring diseased cells into balance, engineer cells to help the body fight off cancer and other diseases, promote wound healing, and aid in the detection and monitoring of infectious diseases. As we continue to explore nature and its diversity, we are certain to discover new processes that may be engineered to advance human health and improve sustainable living. The development of CRISPR-based technologies reflects this, beginning with an obscure observation of an unusual pattern of sequences and continuing with the engineering and optimization of CRISPR-associated enzymes for genome engineering and beyond. Now, with broad knowledge of gene function, increasingly affordable sequencing technology, and a burgeoning database of microbial sequences, it is possible to conduct direct, targeted searches to identify natural systems with properties suitable for development of pre-specified technologies. There are still a number of critical challenges to achieving the full therapeutic potential of CRISPR technologies, and we are hopeful that partial solutions to these challenges can be found in the natural world. The work on CRISPR systems has been filled with exciting discoveries, but, above all, it has inspired scientists to look beyond the horizon to find the next thing, to explore natural diversity, and seek inspirations that can guide the development of new solutions for improving human health.

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A Brief Refresher

S ome of our readers have their Ph.D. in molecular biology, but for most of us it’s

been awhile since we cracked open our molecular biology textbook. Or was it eighth-grade biology? Because CRISPR has the potential to change the world, we offer a brief refresher in order to better understand the science behind the revolution. Metaphors can often help explain complex ideas. CRISPR has been referred to as a molecular scalpel and compared to the findand-replace feature on a word processor. But such comparisons ignore many aspects of the technology’s nuanced capabilities. A deeper understanding of CRISPR-Cas technology requires only a passing knowledge of genetics and molecular biology. Over the next few pages, we cover some of the basics. Things like DNA and RNA, genes and proteins, mutations and DNA repair. And how certain processes link them together. We promise to keep it all as easily digestible as possible. And when you’re done, you’ll not only impress your friends with your knowledge but maybe even amaze yourself. That said, if your molecular biology education is on solid ground, feel free to skip ahead to page 20. For everyone else, we ask that you close your eyes. Imagine yourself at a picnic, on a mountain hilltop. Wait, is that Julie Andrews, singing? Let’s start at the very beginning. A very good place to start. When you read you begin with A-B-C... When you code for life, you begin with A and T ... C and G. DNA. Deoxyribonucleic acid. You remember. The molecule in which all living things, from microorganisms to insects, plants, and animals – including humans – store genetic information by means of an embedded code.

THE FUNDAMENTALS

DNA

The structure of the DNA molecule consists of two strands of sugar and phosphate molecules that wrap around each other like a twisted ladder. Each rung of the ladder consists of a pair of chemicals called nitrogenous bases, or bases for short. It is the order of these bases that creates a code. Every organism uses the same four bases: adenine (A), thymine (T), guanine (G), and cytosine (C). And because A pairs with T, and C with G, when the DNA strands separate or “unzip,” each serves as a template to complete the other strand. A T C G

IF ALL OF THE DNA molecules from every cell in your body were lined up end to end, they would form a strand 6 billion miles long.

DNA replication is an amazing biological phenomenon. Every time a human cell divides, it must copy and transmit the exact same sequence of base pairs to its two “daughter” cells. Do mistakes happen? They do. During human cell division, mistakes occur at a rate of about 1 per every 100,000 nucleotides (one base and the “side of the ladder” where it’s attached). This might sound minimal, but with 6 billion base pairs per diploid cell – cells with two sets of chromosomes – 60,000 mistakes would be made in

DNA and Odile Speed Crick One of the most important events in modern biology occurred in 1953 when James Watson and Francis Crick “elucidated” the doublehelix structure of the DNA molecule, seen at left as drawn by his artist wife, Odile. (Contrary to what you may remember, Watson and Crick were not DNA discoverers but rather the first scientists to determine an accurate description of its double-helical design, thus elucidated.)

each one of the two daughter cells. Sometimes the wrong nucleotide is inserted – an A instead of a G, for example. Sometimes part of the strand is given an extra nucleotide. Or one too few. Or 10,000 either way. Fortunately, our cells are equipped with highly sophisticated DNA repair machinery that can fix the majority of these mistakes. Most of them are corrected through a process, aptly named proofreading, that fixes about 99% of the errors. As good as that sounds, it’s not enough to ensure normal cell functioning. So, another process, known as mismatch repair, reduces the error rate even further – to less than one mistake per 100 million base pairs. Any errors that remain at this point become permanent mutations after the next cell division because the cell no longer recognizes them as errors.

According to Watson, Crick immediately recognized the significance of their conceptual breakthrough, declaring to an assembly of lunch patrons that they had “found the secret of life.” Crick said he had no memory of making such an announcement but recalled telling his wife that evening, “We seem to have made a big discovery.” “Years later she told me that she hadn’t believed a word of it,” Crick recalled. “You were always coming home and saying things like that,” she told him, “so naturally I thought nothing of it.”

It is worth noting that successful organisms have evolved the means to repair their DNA efficiently but not too efficiently. They leave just enough genetic variability to strike a balance between ensuring a stable population and allowing for the evolution of the species. A T C G

DNA REPAIR IS A CONSTANT, extremely active, and natural part of every organism’s daily life.

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IN HUMAN CELLS, if DNA damage cannot be fixed, the cell will undergo programmed cell death (apoptosis) to avoid passing on faulty DNA.

A Spark of Inspiration In 1962, the Nobel Prize in Physiology or Medicine was awarded jointly to Francis Crick, James Watson, and Maurice Wilkins “for their discoveries concerning the molecular structure of nucleic acids and its significance for information transfer in living material.” Of note: Rosalind Franklin, a British biophysicist, provided data that was critical to Crick and Watson’s work, but Franklin was not eligible for Nobel Prize consideration because she passed away four years before it was awarded. The Nobel Prize is not awarded posthumously. The double-helix structure of DNA is arguably the most recognizable icon in biology, yet Crick and Wilkins were trained as physicists. All three men acknowledged that their interest in DNA was sparked by a series of lectures delivered by Erwin Schrödinger at Trinity College in Dublin in February 1943. Schrödinger, an Austrian physicist, had become famous in the 1920s for his work on quantum theory (fans of physics will remember his famous cat). Key among his achievements was his wave equation, for

which he was awarded the Nobel Prize in Physics in 1933. At his Trinity lectures, Schrödinger argued that life could be thought of in terms of storing and passing along biological information, and he proposed a mindexpanding metaphor: Because so much information had to be packed into every cell, it must be stored in what Schrödinger called a “hereditary code-script” embedded in the molecular fabric of chromosomes. Crack the code, and we would understand life. Schrödinger’s metaphor – a decipherable Erwin Schrödinger genetic code book – might seem obvious now, but at the time it was a bold new concept. “The notion that life might be perpetuated by means of an instruction book inscribed in a secret code appealed to me,” Watson would later say.

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THE FUNDAMENTALS

Organisms and Cells

Every organism has at least one cell and is classified as either a prokaryote (pronounced pro-CARRY-oat) or eukaryote (you-CARRY-oat). Prokaryotes are single-celled microorganisms and include bacteria and archaea. Their DNA floats freely about inside the cell because they have no nucleus. Eukaryotes can be single-celled or multicelled. Each cell contains its DNA in a nucleus. Eukaryotes include amoebas, yeast, fungi, insects, plants, animals – and humans. A T C G

CALCULATING THE TOTAL number of cells in the human body is tricky, but it’s safe to say there are trillions. Most recently, researchers have placed the number at 37.2 trillion. To put the number in perspective, a million seconds is 12 days. A billion seconds is 31.7 years. A trillion seconds is 31,709 years. Now imagine 31,709 years times 37.2, with every second representing a different cell in your body.

Chromosomes and Genes

A chromosome is a single, long molecule of DNA. Each chromosome is tightly coiled around proteins called histones that support its structure. In eukaryotes, the DNA is divided into paired sets of different chromosomes. Humans have 23 pairs of chromosomes. A fruit fly has four. The family dog? 39 pairs. As we all remember, one set of chromosomes comes from the mother, the other from the father, and when brought together they produce offspring that carry a new set of genetic instructions – which get reshuffled in each subsequent generation. Genes are smaller sections of chromosome that store the information necessary to make a specific protein. More precisely, a gene provides instructions to build a chain of amino acids in a certain order – as proteins are long chains of amino acids. Protein-coding genes account for less than 3% of our total DNA. The 16

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IN THE 1960S, researchers thought the human genome might contain as many as 2 million genes, the assumption being that human beings are extremely complex. Since then, the number has been steadily declining. THE MOST RECENT ESTIMATE is that the human genome contains between 19,000 and 20,000 genes – a range that seems profoundly counterintuitive when compared to a water flea, which has 31,000 genes, the most found in any animal.

function of the remaining 97% is not clear, although scientists think much of it has to do with gene regulation – turning genes on and off. Many genes respond to environment and behavioral factors, such as changes in diet, level of exercise, exposure to toxins, stress, or certain medications.

or ATA. These three-letter sequences, called codons, correspond to specific amino acids. There are also “start” and “stop” codons that mark the beginning and end of a gene. An RNA called transfer RNA, or tRNA, delivers the correct amino acid to the ribosome, where a third RNA (ribosomal RNA, or rRNA) builds the amino acid chain one at a time. An error in the original DNA code, or during the transcription and translation process, might have no effect, partially cripple, or completely disable the protein’s intended function. The degree to which the error affects the organism depends on what function that protein serves. How proteins are made DNA

Nucleus Gene

RNA and Protein Making

DNA is famous. RNA? Not so much. RNA, however, is just as deserving of the spotlight. One of the first things taught in Molecular Biology 101 is: DNA codes for RNA, and RNA codes for proteins. And while a simplification, it’s generally true. All proteins are unique chains made up of as many as 21 different amino acids. The cells in our body construct hundreds of trillions of proteins every second of our lives. It’s what keeps us alive. The sequence of the amino acids in a protein determines its unique, threedimensional structure, and shape is what gives each protein a specific function. Protein making consists of two major steps: transcription and translation. During the transcription process, the information stored in a gene’s DNA is transferred into a molecule called messenger RNA (mRNA), which leaves the nucleus to deliver the information to a ribosome – a complex molecular machine found in the cytoplasm of all living cells. Translation, the second step, begins when ribosomes “read” the mRNA code in groups of three letters, such as CCG

Growing amino acid chain

mRNA

tRNA

2 Ribosome

Cytoplasm

Free amino acids

A DNA segment is copied into mRNA.

It leaves the nucleus for a ribosome.

tRNA and rRNA assemble an amino acid chain per mRNA instructions.

Enzymes

Most enzymes are proteins (amino acid chains) folded into complex shapes. At any given moment, enzymes are performing all of the work inside a cell. Enzymes break (or cut) molecules apart and put molecules together. There is a specific enzyme for every chemical reaction needed to make the cell work properly. Enzymes speed up chemical reactions in living systems millions of times over. Without them, chemical reactions would happen too slowly to sustain life.

Viruses, Bacteria, and CRISPR

The battle between viruses and bacteria has raged for millennia, with each grappling for temporary superiority. Viruses that attack prokaryotes are called bacteriophages – phages for short. All phages attack with one purpose: to inject their own genetic code into a bacterial cell and use it as a factory to produce more phages.

Color-enhanced transmission electron micrograph showing phages (in red) attacking a prokaryote cell (E. coli).

If a bacterium survives a phage attack, it adds a segment of the phage’s DNA into its own genetic code for later use. CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats and refers to the unique region of a bacterium’s DNA where these snippets of phage DNA are added. This process and its purpose were discovered only decades ago. Researchers found that the snippets and a section of phage DNA were a perfect match. It soon became clear that CRISPR was a primitive bacterial immune system and CRISPR was a chronological record of all the viruses the cell and its ancestors had encountered and defeated. Near CRISPR regions are genes called CRISPR-associated (Cas) genes, which code for building Cas proteins. Cas proteins are typically made up of two kinds of enzymes, those that unwind DNA and those that cut DNA. To defend itself, the bacteria copies the genetic material of each phage DNA segment into an RNA molecule. The RNA molecules pair up with the Cas proteins, and together they drift through the cell looking for intruders. If they encounter a genetic match, the RNA latches onto the attacker and the Cas protein chops the phage DNA in two, effectively killing the intruder.

CRISPR-Cas Gene Editing

While CRISPR-Cas is amazing enough just as a primitive, adaptive immune system, the field of genome engineering changed once scientists discovered that CRISPR could be easily and inexpensively programmed. By swapping the stored phage DNA “spacers” with a synthesized, custom RNA, researchers realized they could latch on to any specific genetic segment they wanted to address. The synthesized RNA is called guide RNA (gRNA), which shepherds Cas9 to the precise spot on DNA where a cut is called for. Cas9 locks onto the double-strand section of DNA and “unzips” it, which allows the gRNA to pair up with the region of the targeted DNA.

Cas9 then snips the DNA, creating a break across both strands of the DNA molecule. The cell, sensing a problem, attempts to repair the break. Cells usually repair a break in their DNA by “gluing” the loose ends back together. This process most often results in a mistake that disables or “knocks out” the gene – which can be a desired result. Or, the repair can correct the mistake or even insert new genetic material. Below is a simplified explanation that illustrates gene silencing and gene insertion using CRISPR-Cas9. CRISPR-Cas systems built with other Cas enzymes offer researchers an array of ways to silence, edit, skip, suppress, interfere with, or activate genes in any organism.

How CRISPR-Cas9 works in gene silencing and gene correction CRISPR-Cas9 can be programmed to recognize a particular DNA sequence by creating a guide RNA (gRNA), which guides Cas9 to make a double-strand cut at the targeted site. Cells have the ability to quickly detect broken DNA and attempt to repair it. The CRISPRCas9 system takes advantage of the cell’s natural repair mechanisms. Cas9 requires a simple and common sequence of base pairs called a PAM sequence in order to bind to its target. One of Cas9’s limitations is that it cannot affect areas of the genome that do not have a PAM sequence nearby. 1

CRISPR-Cas9 contains a Cas9 protein and a guide RNA (gRNA) that matches a sequence in the targeted gene.

Cas9 gRNA

MATCHING SEQUENCE

Cas9 locks on to the double strand of DNA, unwinds it, and then cuts across both strands of DNA.

PAM SEQUENCE

BREAK

3a The cell’s attempt to repair the break can either silence or repair the gene using a

natural repair mechanism called non-homologous end joining (NHEJ).

REPLACEMENT DNA SEGMENT

3b Alternatively, a faulty gene can be corrected using a replacement segment of DNA

whose end regions match the target sequence. This natural cell repair mechanism is known as homologous directed repair (HDR).

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QUESTIONS & ANSWERS

What is gene therapy?

The realization of the genetic basis of hereditary disease led to an early concept of gene therapy in which “good” DNA could be added to the cells of those who suffer from genetic defects. It was first tested on humans in 1990. Gene therapy is done using something called a vector to insert tiny fragments of DNA into a cell’s nucleus. A vector is simply a shuttle used to carry DNA into a cell and leave it behind for the cell to use. Generally, the vectors used in gene therapy are viruses. A vector can be injected into a person’s body directly or done in cells outside the body and then reinjected. Once DNA is delivered to the target cells, the hope is that The Story of Jesse Gelsinger 18-year-old Jesse Gelsinger suffered from a genetic disease called ornithine transcarbamylase (OTC) deficiency, a metabolic disorder that affects 1 in 40,000 newborns. OTC prevents the body from breaking down ammonia. In OTC patients, the excessive build-up of ammonia in the body often causes death soon after birth unless the patient’s diet is immediately adjusted and monitored throughout his or her entire life. Jesse was able to live on a strict non-protein diet. Because a single-gene defect is responsible for OTC, researchers considered it a prime candidate for Jesse Gelsinger gene therapy. In 1999, Jesse volunteered for a gene therapy experiment at the University of Pennsylvania designed to test possible treatments for OTC, believing he could help OTC-afflicted newborns. A vector carrying a normal OTC gene was injected into his liver. The vector was an adenovirus, a modified version of the virus that causes the common cold. Jesse had been told that other test patients had received adenovirus without serious complications. But Jesse’s body had an unexpected, violent reaction. On September 17, 1999, four days after the injection, Jesse died from multiple organ failure. In January 2000, the FDA halted all of the University of Pennsylvania’s gene therapy human trials and began investigating 69 other gene therapy trials underway in the U.S. Eventually, 28 trials were reviewed, with 13 requiring remedial action. Months later, the FDA and the National Institutes of Health worked together to enhance patient protection through two new programs: the Gene Therapy Clinical Trial Monitoring Plan and the Gene Transfer Safety Symposia. Initially, it was believed that Jesse’s deadly reaction was random and unforeseeable. However, further investigation revealed that past research subjects as well as experimental animals had become sick from the vector. This revelation raised ethical concerns because these results were known and not fully communicated to Jesse, his family, or to the other volunteers. One of the lessons of this tragic event is that researchers must be focused on effective ways to ensure that their research subjects – who courageously give of themselves – do so with a complete and unbiased understanding of the risks and benefits involved in their participation.

the cells will absorb the DNA – a healthy, functional gene – and integrate it into the cells’ own genetic makeup. However, more than 40 years of research has shown this simple process is much more challenging and technically complex to do safely and effectively than initially believed. Gene therapy has more recently become a loosely defined umbrella term for any technique that uses genes to treat or prevent disease. What is a viral vector?

Viruses cause infections by injecting their own genetic material through cell membranes. Since the 1970s, scientists have exploited this capability, stripping viruses of their infectious features and using the resulting “viral vectors” to transport DNA into targeted cells. Creating viral vectors is a painstaking, expensive process, and a shortage of clinical-grade vectors has led to a manufacturing bottleneck for various therapies. What is the difference between genetic engineering and gene editing ?

Genetic engineering is a process that introduces new DNA (called recombinant DNA, or rDNA) into an organism to alter its genome, generally with limited control of where it will be added. The new DNA may even come from a different species. The resulting organism is called a genetically modified organism or GMO. Gene editing is a much more precise method of manipulating the genome of a plant, animal, or other living thing than previous methods. It allows for the intentional addition, substitution, or deletion of specific nucleotides in an organism’s genome. What is gene regulation?

The human body makes different kinds of cells – heart, muscle, bone, brain – using the same set of genes. It can do this because in any cell at any time, certain genes are switched on (or expressed) and others are switched off (or silenced). A bone cell makes the proteins needed for a bone cell; a brain cell, the proteins for a brain cell; and so on. Each cell uses only a tiny fraction of the total genetic information available. The process of turning genes on and off is known as gene regulation. The ongoing regulation of protein-coding genes is a mind-boggling system of control that is far from fully understood. Scientists have learned that genes are switched on and off by proteins called transcription factors. And thousands of “transcripts” are produced every second in every cell. A CRISPR system called CRISPR Interference or CRISPRi can modify gene expression using a “dead” Cas

protein that binds to a location but is incapable of making a cut. This interference is reversible. What is a genetic mutation?

A genetic mutation is a permanent alteration in the DNA sequence that makes up a gene, such that the sequence differs from what is found in most people. Mutations range in size, affecting a single base pair to a large segment of a chromosome that includes multiple genes. The impact of a particular mutation depends on how it alters a protein’s function, as well as how vital that protein is to survival. Gene mutations are classified in two major ways: Hereditary (or germline) mutations are inherited and remain present throughout a person’s life in virtually every cell in the body – including egg and sperm cells. In humans, acquired (or somatic) mutations occur at some point during a person’s life and are present only in certain cells. Such mutations can be caused by environmental factors such as ultraviolet radiation from the sun or lifestyle factors such as smoking or diet, or they can occur during cell division. Acquired mutations cannot be passed to the next generation. Genetic alterations that occur in more than 1% of the population are called polymorphisms and are considered a normal DNA variation. Polymorphisms are responsible for many of the differences between people, such as eye and hair color and blood type. What is genetic disease?

A genetic disease is any disease that is caused by an abnormality in an individual’s DNA. There are three main types of nuclear genetic disorders: single-gene inheritance, multifactorial or complex inheritance, and chromosomal abnormalities. Single-gene disorders are mutations that affect only one gene; these are the most common of genetic diseases.

A T C G

SICKLE CELL ANEMIA is a single-gene disorder that happens when single base – one “letter” among hundreds in the gene that codes for the protein beta hemoglobin – is incorrect. This simple but life-changing error changes the three-letter code (codon) for the sixth amino acid (in what is a 146-long amino acid protein chain) from glutamic acid to valine.

Researchers have estimated there are more than 6,500 single-gene disorders, which occur in about 1 of every 100 births. Examples include cystic fibrosis, Huntington’s disease, hemochromatosis, and sickle cell anemia. Multifactorial or complex disorders are the result of many determinants. One of biology’s challenging problems is to understand how a set of genes contributes to diseases with complex patterns of inheritance – such as diabetes, heart disease, asthma, cancer, and mental illness. In these cases, no one gene has the power to determine whether a person will develop the disease or not. Chromosomal abnormalities typically occur as a result of a problem with cell division. For example, Down syndrome (or trisomy 21) occurs when a person has three copies of chromosome 21. Where does CRISPR-Cas fit in the gene-editing spectrum?

CRISPR is the latest and, by far, most powerful of modern gene-editing technologies. Previous technologies – including ZFN and TALEN – rely on time-consuming and expensive protein engineering approaches. CRISPR-Cas is the first viable tool that can explore, interrogate, and provide us with the ability to repair the genome in diseases where limited treatment options are available. CRISPR puts an advanced set of powerful scientific tools, usually reserved for the well-funded few, into the hands of many. As a result, the fields of biology and genetics have exploded.

Congratulations

Feeling a little smarter? You should. And it was certainly faster than wading

through the 1,300-plus pages of Molecular Biology of the Cell, 6th edition. Now that you know a few basics about genetics and molecular biology, you can better understand why CRISPR has pushed the field of gene editing to a new level and how the world is about to change forever. S O U T H W E S T E R N M E D I C A L P E R S P E C T I V E S . 2 019

A T C G

C H A P T E R 3.1 ———————————————————————————————————————————————————————————

Applications: Research

C RISPR has lowered the cost and increased the output of genomic research into

the gene function of every living organism and become the go-to genetic research platform in hundreds of labs around the world. The first and most widely researched CRISPR – CRISPR-Cas9 – continues to be further refined and now has thousands of variants. But over the past few years, scientists have searched for and found alternatives to the Cas9 enzyme. To understand CRISPR is to first understand that CRISPR is not one thing. “It’s like ‘fruit’ – it describes a whole category,” says Dr. Feng Zhang of the Broad Institute. Some of the new Cas enzymes cut DNA in different ways that make certain edits more likely to work – making a staggered cut instead of Cas9’s neater, blunt cut. Still others are smaller, which allows scientists to more easily insert them into cells. CRISPR-Cas12a was the first system after CRISPR-Cas9 to be used for gene editing in the lab. (Just as there are a variety of apples, there are a variety of Cas12 enzymes.) The hunt for Cas9 alternatives has scientists scouring the planet for unique bacteria. CasX and CasY were found in the DNA of bacteria that inhabit underground aquifers. And one of the earliest known forms of Cas12b was discovered in the clean room where NASA’s Viking spacecraft was assembled. These discoveries, along with dozens of others, are being used to switch genes on, turn genes off, insert DNA, delete it, or even make genes glow to reveal their location. The second key to understanding CRISPR is that it does only part of the work. When a Cas enzyme cuts DNA at a specific location, the cut triggers the cell’s alarm bells and its natural repair mechanisms quickly go to work. The repairs, however, can be inefficient, error-prone, or made in the wrong place, which has led to further research challenges. New insights continue to be made into why and under what circumstance CRISPR gene editing works, though not with equal success in all cells. For the layperson, keeping up with all that is going on is not possible, but if you’re thinking something like, “Whoa, this is amazing” – you’re spot on.

Dr. George M.

PERSPECTIVE

Dr. Church, Professor at Harvard & MIT, has co-authored more than 500 papers, 143 patent publications, and the book Regenesis. He co-initiated the BRAIN Initiative (2011) and Genome Projects (1984, 2005) to provide and interpret the world’s only open-access personal precision medicine datasets. And, along with Dr. Feng Zhang, he was the first to successfully adapt CRISPR-Cas9 for genome editing in eukaryotic cells.

CHURCH CRISPR AS ICON OF THE NEW NORMAL

”I don’t know what the language of the year 2000 will look like, but I know it will be called Fortran.”

Tony Hoare, 1980 Turing Award winner about the 1957 invention

Riffing on the quote above, CRISPR gets credit for broad swaths of the biotech revolution that are not limited to, and often not even related to, CRISPR. CRISPR is merely a subset of “gene editing,” which itself is a tiny subset of genetic engineering and gene therapy. The latter can be categorized into additive, subtractive, and precise changes. Nuclease editors (HEG, ZFN, TALEN, CRISPR) are mainly subtractive and have Engineered microbial, plant, and animal non-ideal reproducibility and toxicity profiles. The holy grail applications to green manufacturing, food, is “precise editing,” which leverages custom deaminases, integrases, recombinases, and homology directed repair and carbon sequestration are improving (HDR) – all of which predate CRISPR. exponentially. Furthermore, CRISPR is shaping up to be the most expensive system of therapies in history – close to $1 million per dose. In contrast, powerful and cost-effective alternatives don’t involve any of the above “writing” technologies but instead focus on “reading.” We have reduced the cost of reading our genomes from $3 billion for a low-quality (haploid) genome in 2004 to $1,000 in 2015 for a high-quality, clinically interpreted genome to as little as $0 to the patient today. This enables avoidance of serious Mendelian diseases via pre-conception, in vitro fertilization (IVF), or noninvasive prenatal testing (NIPT). So, with CRISPR as the emoji icon for biotech, what then might the “new normal” become? Engineered microbial, plant, and animal applications to green manufacturing, food, carbon sequestration, etc., are improving exponentially. DNA now extends beyond conventional biological uses — to build 3D nanorobots and to store digital information, for example, trillions of bits of DNA barcodes actively record the full developmental lineage of mammals from zygote to death. We now engineer a handful of changes in “universal donor” human chimeric antigen receptor T-cells to achieve precise elimination of cancer cells in patients. Many dozens of changes in the pig genome make the animal suitable for organ transplant donation. Thousands of mutations can result in resistance to all viruses, even those that we haven’t met yet. We are getting rapidly better at making many human tissues and organs in vitro and editing their genomes to help discover genetic disease causes, cures, and preventions. Human germline enhancement seems to get attention wildly out of proportion to both risks and benefits, yet arguably the top achievement of the new normal, aging reversal, will most likely be via adult (not embryo) gene therapy because feedback on efficacy requires a shorter wait and adults outnumber newborns 75 to 1. Resistance to cognitive decline may be achieved via enhancements — with far faster feedback if tested in adults (vs. embryos – weeks vs. decades).

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CRISPR HAS BECOME both biological buzzword

and research cornerstone in universities and academic medical centers and across a wide spectrum of industry. New tools, regularly added to the CRISPR toolkit, make it even more powerful. While progress in agriculture, energy, and material manufacturing, among others, has proven remarkable – it is CRISPR’s therapeutic potential that could hold the most promise. Yet since its discovery, key obstacles stand in the way of fulfilling that promise. At the top of the list is accuracy. Without high levels of accuracy, any proposed CRISPR gene therapy risks unintended consequences. Extensive efforts have been made to understand how guide RNAs bind to a particular site and why mismatches occur. Software tools that can predict offtarget sites are improving but further development is needed. And ways to better manage or circumvent the cell’s natural repair machinery continue. Another key challenge is delivery. Researchers can edit cells either ex vivo (outside the body) or in vivo (inside the body). With ex vivo, cells are removed from

the body, edits are made – which allows for some kind of risk assessment – and the cells are returned to the patient. With in vivo, CRISPR-Cas components are packaged in a delivery vehicle and delivered systemically (to the whole body) or to a target organ. Viral, nonviral, and physical means are being tested, but challenges remain. A third issue relates to the long-term effects of CRISPR’s component pieces. Where do the various parts of CRISPR end up in the body? How long do they stay there? And is there any risk associated – e.g., cancer – with any individual component? The fourth issue concerns the body’s innate and adaptive immune defense responses. During in vivo delivery, components need to be sheltered from the immune system until they reach their target cells. Enabling CRISPR to induce epigenetic changes (modifying gene expression without making permanent changes to the DNA) might equire CRISPR elements to remain in the body for weeks, or even months, to be effective. This is why effectively dealing with the body’s immune response to Cas systems becomes an even greater consideration. Prime editing CRISPR technology brings new promise

Jumping genes: a giant leap for CRISPR? Something called transposons – or jumping genes – might transform CRISPR-Cas technology into something even more powerful. Transposons are pieces of DNA that sit within a genome but for reasons that are largely unknown have the ability to cut themselves out of their original site and jump to another. In June 2019, two teams announced variants of a new CRISPR method based on “jumping genes” that might make it much easier to add sections of DNA to cells. “I think we will see a flurry of excitement around this,” says Columbia University’s Dr. Samuel Sternberg, who leads one of the teams. Dr. Sternberg studied under Dr. Jennifer Doudna, and, with her, co-authored A Crack in Creation: Gene Editing and the Unthinkable Power to Control Evolution. The second team is lead by Dr. Feng Zhang, co-

Transposons were discovered in corn, where they cause varied expression of color. discoverer of CRISPR-Cas9. Dr. Zhang and his colleagues describe turning a jumping gene into a task-oriented machine. With an assist from CRISPR enzymes, it zips to the part of the genome whose address it is given and delivers a package of DNA, pronto. Dr. Zhang calls his system “CRISPR-associated transposase,” or CAST. Proof of principle was shown in bacteria in both studies. So the question becomes: Can the idea be translated to mammalian cells? Both teams think that it can.

According to Dr. Sternberg, while researchers will need to figure out various aspects involved with moving from bacterial to eukaryotic systems, given the progress made in CRISPR research, he believes any problems are easily solvable. “We are excited to be discovering as many new things as we can and add them to the CRISPR toolbox to give the community all the components we might want to start tinkering around and making these systems even more effective,” Dr. Sternberg says. “CRISPR-based tools are often DNA-cutting tools, and they’re very efficient at disrupting genes. In contrast, CAST is naturally set up to integrate genes,” says Jonathan Strecker, co-author of the Zhang study. “This just underscores how diverse nature can be and how many unexpected features we have yet to find,” adds Dr. Zhang.

A new version of CRISPR, called “prime editing,” could make it possible to insert or delete specific sequences at genome targets with less off-target effects according to a new study appearing online in Nature in October 2019. “Prime editors offer more targeting flexibility and greater editing precision,” says Dr. David Liu, a chemist at the Broad Institute who led the study. Dr. Liu, his postdoc Andrew Anzalone, and co-workers tested variations of their prime editors doing more than 175 different edits on human and mouse cells. They created and then corrected mutations that cause sickle cell anemia and Tay-Sachs disease. In its paper, the team claims the technology “in principle can correct about 89% of known pathogenic human genetic variants.” Prime editing modifies both the Cas9 protein and the guide RNA. When it

locates its target DNA, the Cas9 “nicks” one of the DNA strands (instead of cutting both strands), and the CRISPR system then adds the corrected DNA sequence. “It’s a huge step in the right direction,” said Dr. George Church. Prime editing “well may become the way that diseasecausing mutations are repaired,” says Dr. Fyodor Urnov at the Innovative Genomics Institute but adds that it’s too soon to be certain. A research explosion

2011

2019

In 2011, there were fewer than 100 published papers on CRISPR. In late 2019, more than 15,000 were published.

PERSPECTIVE

Dr. Daniel K.

Dr. Podolsky is President of UT Southwestern Medical Center and holds the Philip O’Bryan Montgomery, Jr., M.D. Distinguished Presidential Chair in Academic Administration. He is also a Professor of Internal Medicine and holds the Doris and Bryan Wildenthal Distinguished Chair in Medical Science.

PODOLSKY RETHINKING WHAT IS POSSIBLE

Advances in science and technology are regularly pushing past present boundaries of medicine. But there are times when a major discovery truly alters the course of medical progress, presenting both unexpected promise and unseen challenges. Such is the case with CRISPR, the gene-editing tool that is inspiring new approaches to many diseases. The mapping of the human genome and subsequent expansion of genetics research has opened the door to a world of precision medicine that offers new hope for solutions to previously untreatable disorders. Still in its infancy, CRISPR already promises possible treatments for a number of diseases including Duchenne muscular dystrophy, sickle cell anemia, and a rare genetic disorder that causes blindness. At UT Southwestern, we are taking a This life-changing technology is also raising important comprehensive approach to gene therapy to take ethical concerns, as evidenced by last year’s disclosure that genetically modified twins had been born in China. How full advantage of the CRISPR revolution. and when should we modulate the very basis of human life? Will changing a person’s genetic structure have unintended consequences? Is it ethical to use these approaches to improve human performance capability unrelated to any malady? Can our society devise ways to pay for costly gene therapies so that they are available to all who need them? Academic medical centers, by their nature, are designed to operate at the forefront of medical innovation and engage in such issues. Through active involvement in the laboratory, the hospital, and the classroom, physician-scientists regularly contemplate ethical and regulatory issues as they strive to bring new treatments and therapies from the bench to the bedside through clinical trials. At UT Southwestern, we are taking a comprehensive approach to gene therapy to take full advantage of the CRISPR revolution. In addition to incorporating this technology into ongoing research, we are creating a gene therapy center with its own viral vector facility that will produce viral constructs that meet standards for clinical use to deliver potentially lifesaving therapies to patients. In our multidisciplinary Peter O’Donnell Jr. Brain Institute, a team of researchers and physicians is building a program to develop gene therapies to treat rare neurological diseases that afflict children. Just as other technologies have opened up frontiers in health care, CRISPR provides anew the ability to rethink what is possible in medicine. As the pace of innovation continues to accelerate, our challenge is to embrace the new possibilities while maintaining our academic and clinical standards. Let us strive to apply creativity, collaboration, and integrity to make the best use of this revolutionary tool and expand our understanding of human disease.

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A T C G

C H A P T E R 3.2 ———————————————————————————————————————————————————————————

Applications: Gene Drives

Why do mosquitoes exist? What purpose do they serve?

Go ahead. We’ll wait. Bat food? And therein lies the concern. If we made some mosquito species extinct, would bat populations suffer? What then? Would something else suddenly tumble out of balance? Can scientists confidently predict what would happen? Ecosystems are complex. Even experts disagree on outcomes. On the other hand, the mosquito is, by far, the most dangerous animal on earth. An estimated 435,000 people died from malaria in 2017 – mostly children under age 5 – more than 90% of them in Africa. While there are roughly 3,000 species of mosquitoes, only about 200 of them bite humans, and only a handful of those are responsible for deadly diseases such as malaria, dengue, yellow fever, Zika, and West Nile. And while it’s true that mosquitoes are a key food source for some birds, lizards, frogs, and, yes, bats – no species relies solely on them. Gene drives exist in nature, but they’re rare and random. Yet with CRISPR, researchers can design them. Normally, offspring have a 50% chance of inheriting any given gene from a parent. But a gene drive allows scientists to push a particular gene – one that makes mosquitoes sterile or unable to carry the malaria parasite, for example – through a given population, quickly. The potential to adapt CRISPR for use in gene drives was first presented by Dr. Kevin Esvelt at Massachusetts Institute of Technology. “There is an overwhelming moral imperative to do something about malaria,” Dr. Esvelt said. While options are being actively researched, questions and concerns mount. For example, should any country, group, or individual have the right to change even a small element of nature in ways that could affect everyone? And yet, should one country, that is not affected, be able to tell another country, whose children are dying, what to do? 24

MALARIA IS

caused by a one-celled parasite. Female Anopheles mosquitoes pick up the parasite from infected people when getting blood needed to nurture their eggs. Two main schools of thought are being explored to target the three species of mosquito most responsible for malaria’s transmission – Anopheles gambiae, Anopheles coluzzii, and Anopheles arabiensis. At Imperial College, London, the focus is on reducing the number of malaria-transmitting mosquitoes. That means spreading genes that create more males, causing a population crash or genes that lead to female infertility. Gene drives that do this are called “suppression drives” because they suppress the population. In September 2018, a paper was published revealing that Imperial’s suppression drive reached 100% prevalence among mosquitoes in the lab after 7 to 11 generations. At UC San Diego, UC Irvine, and CalTech, groups of scientists are focused on a different approach called “alleviation drives.” The idea is to spread traits to mosquitoes that will make them unable to carry the malaria infection in the first place. Both groups are working toward the same goal: to save hundreds of thousands of lives every year. Importantly, even if these approaches were 100% successful, only a tiny fraction of the world’s mosquito population would be affected. The Bill and Melinda Gates Foundation vs. the mosquito

Malaria occurs in nearly 100 countries, taking a toll on human health and imposing a heavy social and economic burden in developing countries, particularly in sub-Saharan Africa and South Asia. In recent years, malaria funding has increased dramatically and major gains have been made through a combination of interventions. This includes diagnosis and treatment using reliable tests and effective drugs, indoor spraying with insecticides, and the use of bed nets treated with long-lasting

insecticide to protect people at night. “In 2016, for the first time in years, the number of malaria cases in the world went up. This is not a blip. It is not noise. It is a signal,” said Bill Gates. “What it signals is this: We have reached the point of diminishing returns from our current strategy.” Current tools and treatments are insufficient to achieve elimination in many countries, and the cost of maintaining the interventions has reached several billion dollars a year. Further, the malaria parasite

RODENTS MIGHT also be a target for gene drives.

One of the greatest threats to island plant and animal species are invasive rats and mice. Invasive rodents can be found on more than 80% of islands worldwide. Brought by ships over hundreds of years, they have taken over, causing and threatening to cause the further extinction of native seabirds and other wildlife. The significant environmental threat posed by rats and mice makes their removal a critical conservation goal, but the species have proven difficult to eradicate. Gene drives could change that. The Genetic Biocontrol of Invasive Rodents (GBIRd) program – a partnership of experts from seven universities, government, and not-for-profit organizations managed by the non-profit group Island Conservation – is investigating the feasibility and suitability of a gene-drive initiative with the goal of being able to answer key questions: Could we create a self-limiting, gene-drive modified mouse that biases future generations to be male (or female) only, thereby achieving eradication by attrition? If so, should we do it? And under what conditions or restrictions? Although such a technology is far from ready for release, GBIRd is working with risk assessors, conservation geneticists, bioethicists, and ecologists to identify an island for a potential field trial.

and three African countries – Mali, Burkina Faso, and has begun to develop Uganda – where secure resistance to available mosquito facilities are insecticides and drugs. currently being outfitted. The Gates Foundation Officials with the Gates recently announced plans Foundation have stated to increase its support of Target Malaria – a project led they now believe that gene drives are necessary to by Austin Burt at Imperial end malaria. According to College, London – bringing a business plan developed the foundation’s total commitment to $75 million. for the foundation, the gene drive mosquitoes could be The investment is being released in 2029. used to fund a CRISPR gene The plan is to release drive that causes female edited Anopheles gambiae mosquitoes to become mosquitoes across substerile within 11 generations Saharan Africa, with the – or about a year. hope the drive will spread In recent years, Target across a large area, causing Malaria has expanded mosquitoes to disappear, to involve 16 institutions saving lives. and includes teams in Italy No organization has fought devastating effects of malaria with more tenacity and resources than the Bill and Melinda Gates Foundation. To date, it has committed more than $2.9 billion in grants to combat the disease worldwide.

DARPA funds Safe Genes program In a 2016 assessment report, James Clapper, then Director of National Intelligence, added gene editing to the list of threats posed by “weapons of mass destruction and proliferation.” In July 2017, the U.S. Defense Advanced Research Projects Agency (DARPA) created the Safe Genes program, awarding $65 million in four-year contracts to seven scientific teams led by The Broad Institute of MIT and Harvard; Harvard Medical School; Massachusetts General Hospital; MIT; North Carolina State University; UC Berkeley; and UC Riverside. The goal is to find ways to halt or even reverse the effects of a CRISPR gene drive by removing engineered genes from the environment, allowing the biosphere to normalize.

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A p p l i c a t i o n s : Microb e s, Plants, an d A nimals

CRISPR can unlock the genomic potential of microbes to create all manner of

biobased chemicals – from fuels to flavors, as well as to modify food bacteria, optimize fermentation, reduce spoilage, or create the next generation of probiotics for healthier intestinal microbiomes. Unlike GMO, CRISPR doesn’t require transgenics (inserting genes from one species to another). If there are dangers to GMO-created foods, CRISPR can avoid them by using DNA sequences from similar species. The United States Department of Agriculture (USDA) has given all CRISPR-edited crops the green light as long as they could also have been developed through traditional breeding methods. By contrast, the European Court of Justice ruled that CRISPRedited crops will be strictly regulated, the same as it does GMO crops. The USDA regulation gives American seed companies a significant competitive advantage. What took 10 years can now take place in a single crop cycle, saving millions of dollars. CRISPR can be used on all manner of crops, fruits, and vegetables to induce higher yield, climate resilience, and disease resistance. It can also introduce a myriad of new traits that directly benefit consumers – from improving taste and nutritional value to creating a coffee variety that is naturally decaffeinated. That said, the issue of food labeling disclosure awaits, and the degree of social and consumer acceptance remains to be seen. When it comes to food animals, the Food and Drug Administration (FDA) has held that CRISPR-edited animals will be regulated in the same way as veterinary drugs. The designation strikes many animal breeders as shortsighted because the ruling includes animals that could otherwise be developed through traditional breeding techniques. For researchers and companies wanting to use CRISPR to solve animal disease and welfare problems, at this time there does not appear to be a viable path forward. Beyond animals raised for food, CRISPR has far-reaching implications – from replenishing wildlife to creating compatible pig organs for transplant, not to mention populating Siberia with a hybrid version of the extinct woolly mammoth. 26

PERSPECTIVE

Dr. Rodolphe

BARRANGOU

Dr. Barrangou is a Distinguished Professor of Food Science at North Carolina State University engaged in probiotics research on the evolution and functions of CRISPR-Cas systems and applications in bacteria used in food manufacturing. His work on CRISPR began years before it was understood. He co-founded Locus Biosciences and Intellia Therapeutics and serves as the editor-in-chief of The CRISPR Journal.

WHY CRISPR WILL LEAD THE NEXT FOOD REVOLUTION The advent of CRISPR-based technologies has revolutionized genetics and democratized genome editing, enabling applications in medicine, biotechnology, and agriculture. Now, scientists across the globe are exploiting CRISPR-based molecular machines to breed next-generation crops and livestock and screen for enhanced bacteria used throughout the food supply chain. In food crops, seed developers can mine the genomes of plants such as corn, soy, wheat, and rice, as well as those of fruits and vegetables such as bananas, berries, and tomatoes, to improve water usage, increase yield, develop pest resistance and prevent agriculture disease, enabling the genesis of healthier plant and a more sustainable agricultural system. Higher-yield corn, healthier rice, tastier tomatoes, and drought-resistant Overall, this disruptive technology and wheat are being bred by scientists across the globe in publictransformative field are enabling the genesis of private partnerships that develop a better agriculture. a healthier food supply, a more sustainable plant Likewise, applications in livestock encompassing poultry, swine, and cattle enable breeding next-generation animals agriculture, and a more humane livestock. with increased resistance to infectious disease, notably bacterial pathogens and viruses. Cows resistant to mastitis bacterial infections, chickens resistant to avian viruses, and pigs with reduced environmental release of phosphorous and nitrogen are being selected across the globe. Besides crops and livestock, a diversity of CRISPR-based technologies have been used in food bacteria to alter the composition and functions of microbial populations from farm to fork, to optimize food fermentation, prevent spoilage, and enhance the functional attributes of bacterial cultures impacting food taste, quality, and safety. The food microbiome is being optimized to prevent bacterial spoilage, eliminate pathogens, and enhance fermentation starter cultures and health-promoting probiotics. Ironically, CRISPR-Cas systems, which were originally characterized in dairy starter cultures, have been used to enhance resistance against bacteriophage in milk fermentation, across the globe, for better pizza, cheeseburgers, nachos, cheese, and yogurt. Food bacteria have been screened to improve food texture and flavor during the fermentation process, and next-generation probiotics promise better gut health and healthier intestinal microbiomes. Overall, this disruptive technology and transformative field are enabling the genesis of a healthier food supply, a more sustainable plant agriculture, and a more humane livestock. Yet, there is a need to support science and technology and ensure that regulatory agencies enable the use of disruptive and transformative technologies with science-based, informed decisions. Science stewardship is perhaps even more important when it comes to public engagement and consumer opinions, with the need to engage a broad audience on the benefits of next-generation crop and livestock breeding and highlight the critical impacts of CRISPR on sustainability and the environment.

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SEED DEVELOPMENT GIANTS such DowDuPont,

BASF, Bayer Crop Science, and Syngenta AG (stateowned by the Chinese government) have dominated genetically modified crop technology since the 1990s. But CRISPR-Cas helps to level the playing field by bringing down the cost of creating a new seed line, enabling smaller competitors like Inari, Plantedit, Pairwise, and Benson Hill Biosystems, as well as universities, to participate. As a result, the seed market will become far more diverse. A new corn variety is projected to be the first commercialized CRISPR-Cas9 gene-edited crop. DuPont plant scientists partially knocked out a gene that results in kernels with a higher starch content, making the corn variety a superior choice for use in paper adhesives and food thickeners. Other researchers are investigating the cotton genome to find genes that control the shape, structure, length, and strength of cotton fibers. “When you push cotton quality up, you can make stronger, finer yarns so garments require less total mass and are more durable,” explains Kater D. Hake with Cotton Inc.

White button mushrooms that resist browning when cut

Mmmm, chocolate

A world without chocolate might not be a world many of us would want to live in. Cacao plants occupy a precarious position on the planet, growing in rainforest conditions roughly 20 degrees north and south of the equator, where temperature, rain, and humidity stay relatively constant throughout the year. More than half of the world’s chocolate comes from just two countries: Côte d’Ivoire and Ghana. But some scientists have predicted that cacao plants could disappear by as early as 2050 as a result of warmer temperatures and drier weather conditions. In addition, cocoa plants have long battled the swollen shoot virus, which

Some 800 billion tomatoes are produced globally each year – bred to stay firm enough to be shipped long distances. But over the years flavor and nutrition have been sacrificed. Researchers are now working with heirloom and wild varieties, taking the fruit’s “tastier” and more nutritious genes and incorporating them back into the genomes of commercially grown tomatoes. There is no shortage of ideas. Soybean oils that can withstand high cooking heat without producing trans fats; apples and mushrooms that don’t brown when sliced; sweeter peas; a plan to turn the ground cherry (aka Cape gooseberry) into a major berry crop; and gluten-free and high-fiber wheat varieties – all appear to be just a few short years from making their way onto grocery store shelves. If that’s not enough, a small CRISPR startup, Kultevat, has identified a Russian dandelion that can make rubber exactly like rubber from a tree. It’s easier to grow, more sustainable, and less expensive. Other researchers are working on a corn variety that enhances the photosynthesis process, taking more carbon out of the air while growing more quickly.

can kill a cacao tree within three years. The disease has forced Ghana to cut down 200 million trees since 1946. The Innovative Genomics Institute (IGI), a research lab overseen by Dr. Jennifer Doudna, is working with Mars Inc. as part of the candy company’s $1 billion commitment to reduce its carbon footprint. Mars previously funded research to map the cacao genome. Myeong-Je Cho, director of plant genomics at IGI, has used CRISPR to alter the genes of the cacao plant. If all goes as planned, the edits will lead to the development of cacao plants that don’t wilt or rot at their current elevations. Chocoholics, rejoice.

Once sliced, the white button mushroom doesn’t stay white for long. The browning that occurs is caused by the pigment melanin. In 2015, Dr. Yinong Yang, an associate professor at Penn State University, traced the melanin production to a polyphenol oxidase gene in the mushroom and used CRISPR-Cas9 to turn it off.

The result? Non-browning mushrooms. In October 2015, Dr. Yang wrote a letter to the

USDA to confirm that his mushroom, which did not contain any foreign DNA, would not be regulated. In April 2016, the white button mushroom became the first CRISPR-edited food to get approval from the USDA – a milestone in that it opened the door for countless other CRISPR-edited crops to be developed.

CRISPR research will allow targeted cannabis-based products With CRISPR, instead of spending years of trial and error to crossbreed new cannabis strains, researchers will be able to determine which genes are responsible for different functions. As of July 2019, marijuana is legal in 11 states and Washington, D.C., with 33 states supporting medical In January 2018, a team of marijuana programs. scientists at Sunrise Genetics “People either demonize presented the first completed cannabis or make it sound like map of the cannabis genome. the most amazing thing,” Now breeders and says Dr. Ryan Vandrey, at the researchers have access to a Johns Hopkins School of comprehensive view of the Medicine. “The ideal path 10 pairs of chromosomes in forward is where we figure cannabis, which include both out which components hemp and marijuana. of the cannabis plant help

with specific symptoms or health conditions, and we develop refined and targeted medicines.” As interest grows in pharmaceutical uses for CBD – which was approved last year to treat a form of epilepsy – experts anticipate growing interest in high-CBD varieties and securing plant patents. Dr. Todd Michael, Director of Informatics at the J. Craig Venter Institute, is excited about the future of high-CBD products as a substitute for opioids. “We’ve got a huge opioid epidemic,” Dr. Michael says, “and it’s been shown that CBD, and THC, can play a role in managing pain.”

An Asian elephant- woolly mammoth hybrid

Woolly mammoths were last seen some 3,600 years ago. But a Harvard-based team, led by Dr. George Church, is using CRISPR to copyand-paste DNA from the mammoth genome into living elephant cell cultures. So far, a number of genes have been successfully rewritten into Asian elephant cell lines, resulting in increasingly mammothlike cells with each edit. Key mutations – for mammoth hemoglobin, extra hair growth, fat production, and nuanced climate adaptations

– have already been engineered. Once a first generation of modern mammoths are born, they will be in the care of Asian elephant family groups in zoos, providing them with the social imprinting they will need to form herds of their own. When the population becomes large enough, herds can be established at suitable sites throughout the Arctic, the prime location under consideration being Russia’s Pleistocene Park in northeastern Siberia.

Pig organ transplants According to the U.S. Health Resources and Services Administration, there are more than 100,000 people in the U.S. on the waiting list for organ transplants. Organs from pigs, which are close in size and work similarly to human organs, could be the solution. The problem has been that pig organs are quickly rejected by the human body, causing severe immune reactions. There are also viruses in pig DNA known as porcine endogenous retroviruses, or PERVs, that can be passed down during transplantation and infect human cells. And

Anti-aging research One avenue to developing a new human medical therapy is to begin with veterinary use. In May 2019, Rejuvenate Bio announced it will launch a gene-therapy trial in dogs in late 2019 to combat

ANIMAL HUSBANDRY presents far more challenges

than plant cultivation. One genetically modified plant can generate enough seeds to grow commercially viable crops, but creating a sustainable animal herd is much more involved. Dr. Alison Van Eenennaam at UC Davis has used CRISPR to introduce a gene that triggers all male characteristics in cows. The resulting animals could enable more economical beef production. Other scientists have used CRISPR to target the genes encoding common allergens in eggs in an effort to produce hypoallergenic eggs. That said, given current FDA regulations, any innovation in the U.S. – as well as across Europe – will remain in the lab unless there is a policy change. For now, China has given its researchers far more flexibility, having not yet decided whether or how it will regulate CRISPR-modified food. Chinese researchers have used CRISPR to knock out two genes that suppress hair growth and muscles in goats with the goal of increasing cashmere and meat production. Other teams have created “low-fat” pigs that would be less expensive to raise and suffer less in cold weather, made pigs resistant to classical swine fever, and created cows that are better protected from tuberculosis, which can spread to humans.

if these virsues are spread to certain tissues, they can cause cancer. eGenesis is working to make porcine tissue PERVfree and as human-like as possible to enable lifesaving medical interventions. The company has four key areas under development: cellular genome engineering

mitral valve disease (MVD), a condition commonly encountered in the Cavalier King Charles Spaniel breed and a direct result of the aging processes. The gene therapy adds a new piece of DNA into the cells of the dogs in order to halt the

to assess both PERV and immunology, organ production, preclinical testing, and studies to enable clinical development. The company is currently testing organs taken from CRISPR-edited pigs in monkeys at Massachusetts General Hospital in Boston. The experiments are being led by the hospital’s chief of transplant surgery, Dr. James Markmann. eGenesis is also working closely with the FDA to explore the technology, its ramifications, and regulatory considerations while the clinical side is developed.

buildup of fibrotic scar tissue in the heart. If Rejuvenate Bio can show that processes of aging have been reversed in dogs, it will lend added justification for human trials, where regulatory approval would otherwise be far more challenging.

BY RE-ENGINEERING MICROBES, CRISPR can

both modify and create new chemical products – from industrial cleaning products to new fragrances. Take the example of producing biofuel from algae. For years, the challenge for researchers has been to keep algae “fat” enough to produce oil but nimble enough to grow quickly. Synthetic Genomics, founded by Dr. J. Craig Venter, has been working with ExxonMobil to develop algae-based biofuels since 2009. Their research team successfully used CRISPR to modify the algae Nannochloropsis gaditana, increasing the algae’s oil content from 20% to more than 40% without a significant reduction in the algae’s growth rate. “We’re still at the research phase in this program,” explains Dr. Vijay Swarup, a VP at ExxonMobil. “There’s a long way to go in making an algae that can produce even more fat, live comfortably in saltwater pools outside, and be processed into fuel for cars, planes, and trains.”

A T C G

DR. J. CRAIG VENTER is a biochemist, geneticist, and businessman and is acknowledged, along with Francis S. Collins, as being a primary force behind the Human Genome Project. Dr. Venter, using private funding, independently mapped and sequenced human DNA. He is currently CEO of the J. Craig Venter Institute, a nonprofit that conducts research in synthetic biology. SOUTHWESTERN MEDICAL PERSPECTIVES . 2019

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A p p l i c a t i o n s : Diagnostic s an d D rug Disc ove r y

The gene-editing ability of CRISPR has been compared to the find-and-replace

feature on a word processor. The diagnostic ability of CRISPR can be understood by thinking of DNA as the internet and CRISPR as a search engine. Consider the dengue fever virus. Scientists begin by identifying a unique segment of its genome, and then a guide RNA is designed to match up with that sequence. If a match is found, a paper strip test will confirm its reading. Instead of using Cas9 to make a single cut, two newly discovered Cas enzymes – Cas12 and Cas13 – are deployed because of their unique abilities. Once the guide RNA finds a match, they aggressively cut any single strands of DNA in the vicinity. To know whether or not a match has been found, single strand “recorder molecules” are added to the component mix so that, when cut, they send a signal that can be read – such as fluorescence. For most infectious diseases, diagnosis requires specialized expertise, sophisticated equipment, and electricity, all of which can be in short supply in many places where illnesses such as dengue fever occur. CRISPR-based diagnostic tools could become as simple as at-home pregnancy tests and work even with smartphone apps. They can bring results faster, cost far less, and require fewer trained professionals to administer than current methods – bringing unprecedented accessibility to people around the world. CRISPR’s role in drug discovery could prove to be as important as its therapeutic use, as it has redefined what is possible with R&D. The ability of CRISPR technology to activate or inhibit individual genes allows researchers to determine which genes (and the proteins they code for) cause or prevent disease, which in turn identifies the key targets for potential drugs to affect. CRISPR-Cas also makes it easier to create cellular and whole-animal models that precisely mimic human diseases, and to do so quickly and cheaply. This helps scientists more accurately verify the safety and efficacy of potential drug therapies and make them better predictors of what will happen in clinical trials. 30

DIAGNOSTIC SYSTEMS

In June 2017, Dr. Jennifer Doudna joined a team of researchers to launch Mammoth Biosciences. In February 2018, Drs. Doudna and Feng Zhang simultaneously published research in Science detailing the use of the Cas12 enzyme as diagnostic detection tool, naming it DETECTR and SHERLOCK, respectively. DETECTR was able to identify two types of human papillomavirus (HPV), the most common sexually transmitted infection, in human samples. Compared with currently available molecular diagnostics, DETECTR is faster, less expensive and easier to use and requires no specialized equipment. Sherlock Biosciences, cofounded by Dr. Zhang in March 2019, switched enzymes – to Cas13a – to pick out multiple viruses, such as Zika and dengue, in one sample simultaneously. The system uses disposable paper strips and costs only a few dollars to make. CRISPR’s role as a diagnostics platform has the potential to extend well beyond health care into agriculture to determine what’s making animals sick or to identify what microbes are in the soil – or to the oil and gas industry to detect corrosive microbes in pipelines. It has potential applications in non-invasive prenatal testing, food security, environmental

monitoring and detection, and facilitating rapid response by identifying harmful biological agents.

DRUG DEVELOPMENT

The drug discovery process involves compounds being carefully screened and evaluated for therapeutic use. However, the process typically spans more than a decade and exceeds a billion dollars. And only a small percentage of candidates ever make it to market. Due to its ease and versatility, CRISPR can overcome many technical challenges of drug discovery – making gene editing more controllable and precise and identifying target drugs more rapidly. Collaborations between pharmaceutical companies and academia are transpiring – most recently between the University of California and GlaxoSmithKline, which in June 2019 invested $67 million over five years to harness CRISPR-Cas9 to find new medicines. CRISPR can also generate animal disease models that are more realistic to test the efficacy and safety of candidates. For example, mouse models with multiple mutations can now be generated in a single step. CRISPR holds promise not only for developing therapies faster and at lower cost but for facilitating the advancement of personalized medicine. Soon, the tailoring of therapies to individual patients might no longer be just an idea but a distinct reality.

The serious threat of drug-resistant infection

Clostridium difficile can cause fatal infections. One of the most alarming facts concerning public health is that as bacteria resistance evolves, we are running low on effective antibiotics. In recent years, antibiotic resistance has risen to dangerous levels. According to the Centers for Disease

Control and Prevention (CDC), at least 2 million Americans become infected with germs resistant to antibiotics each year and more than 23,000 die from these infections. Current antibiotics don’t specifically target the harmful bacteria attacking

our bodies when we’re sick. Instead, they attack both good and bad bacteria. In addition, it is both difficult and costly to develop fresh antibiotics to combat deadly infections. But CRISPR systems could, in theory, be used to kill a single species of germ while leaving good bacteria untouched. Consider the bacterium Clostridium difficile, which causes fatal infections in hospitals and nursing homes. C. difficile has been ranked by the CDC as a top drugresistant threat responsible for about 15,000 deaths each year. Dr. Jan Peter van Pijkeren, a food scientist from the University of WisconsinMadison, is creating a probiotic cocktail of bacteria that patients can swallow as a liquid or pill that carries a customized CRISPR message to C. difficile, causing it to make lethal cuts to its

own DNA. “The downside of antibiotics is they are a sledgehammer that depletes and destroys the gut microbial community,” Dr. van Pijkeren explains. “You want to instead use a scalpel in order to specifically eradicate the microbe of interest.” While a solution remains in the future, if proven successful CRISPR could become not just the world’s most effective gene-editing tool but also the best bacteria-killing technology. And, perhaps, just in time because according to The Review on Antimicrobial Resistance (a 2014 UKcommissioned report ), drug-resistant infections and their resulting diseases or injury are expected to rise dramatically, killing more than 10 million people each year by 2050 if left unchecked. For comparison, by 2050, cancer is projected to kill 8.2 million.

Researchers find gene that provides resistance to West Nile and Zika UT Southwestern researchers used CRISPR to identify a gene active in resistance to flavivirus infection, a class of pathogens that includes West Nile virus, dengue fever, and Zika virus.

A natural defense is found.

The study was led by Dr. John Schoggins, Associate Professor of Microbiology, who identified the IFI6 gene as a likely candidate. From there, researchers used traditional cell culture studies to confirm the gene’s protective role. “In the CRISPR screen, we used human liver cells and knocked out every gene in the genome – about 19,000 genes – one at a time,” Dr. Schoggins explained.

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Applications: Human Somatic Cells

There are two kinds of cells in the human body: somatic cells and germline cells.

Germline cells are either cells that make sperm and eggs (known as germ cells) or the cells in an early embryo that later differentiate into different functions. Somatic cells are everything else – things such as skin, liver, and heart cells – the cells in particular organs or tissues that perform a specific function. Somatic cell editing is the ground zero of the CRISPR revolution. It’s the potential game changer. Researchers everywhere, working with a growing number of CRISPRCas systems, are laser-focused on curing genetic diseases. How soon will it happen? “We tend to overestimate the effect of a technology in the short run and underestimate its effect in the long run,” was an observation made more than 50 years ago by Dr. Roy Amara, a scientist and President of the Institute for the Future. It became known as Amara’s Law. The computer, invented at the end of World War II, was first thought to have unlimited potential but came to be seen as a clunky, chunky beast lurking in the basement of giant corporations. The feeling the digital revolution had been overhyped was summarized in 1977 when Ken Olsen, CEO of Digital Equipment, famously said, “There is no reason anyone would want a computer in their home.” In 1998, Nobel prize-winning economist Paul Krugman wrote, “By 2005 or so, it will become clear that the internet’s impact on the economy has been no greater than the fax machine’s.” In the world of genetics, Amara’s Law has played out with the mapping of the Human Genome Project. Grand visions of a new era of sweeping change in medicine led to disappointment and a lack of tangible results. Will CRISPR live up to expectations? Or will obstacles and fatigue over hyperbolic headlines lead to impatience and naysayers? CRISPR is still early in its development. It might simply be a matter of time. For now, the first U.S. human clinical trial to cure a genetic disease using CRISPR is underway, and the results could change the future of medicine forever. 32

PERSPECTIVE

Dr. Francis S.

Dr. Collins, a physician-geneticist noted for his landmark discoveries of disease genes and his leadership of the international Human Genome Project, is the Director of the National Institutes of Health (NIH). In that role, he oversees the work of the world’s largest supporter of biomedical research.

COLLINS TEAMING BOLDNESS WITH RESPONSIBILITY

Scientists have identified the molecular causes of nearly 6,500 human diseases, yet treatments currently exist for only about 500. Within the next 10 years, biomedical researchers, disease advocates, and clinical experts will seek to realize the promise of new technologies to treat or even cure many conditions that once seemed out of reach. Particularly exciting in this arena is the potential of CRISPR genome-editing systems. Yet, as research moves boldly forward in this fast-paced field, it is imperative that our pursuit of cures also proceeds responsibly. The promise of gene editing is great. But at the National Among NIH’s highest priorities is research aimed Institutes of Health (NIH), the world’s leading public supporter of biomedical research, we are among the many who think at using somatic cell genome editing to treat or that, for the time being, the only CRISPR therapeutic strategies cure life-threatening inherited disorders. that should be pursued in humans involve somatic cell genome editing. In this approach, genetic material is edited only in relevant tissues without the risk of passing those changes on to future offspring. Among NIH’s highest priorities is research aimed at using somatic cell genome editing to treat or cure life-threatening inherited disorders, such as sickle cell disease and muscular dystrophy. Germline genome editing is quite a different story: NIH strongly opposes the use of genome editing in heritable cells in humans for the foreseeable future. We contend that society is not ready – and potentially may never be ready – for this approach, which will irreversibly alter the human DNA instruction book in ways that will have consequences for future generations of humankind. Based on this concern, the NIH supports recent calls for an international moratorium on the clinical use of human germline genome editing. A moratorium period of five years or more could give scientific, economic, and thought leaders around the globe an opportunity to engage in serious discussions about the safety, ethical, philosophical, and theological concerns raised by this application of CRISPR technology. It is a debate that, while difficult, we simply cannot afford to postpone. The consequences of failing to provide an international moratorium are highlighted by the recent CRISPR editing experiments in human embryos that took place in China. Should such epic scientific misadventures continue to proceed, a technology with enormous potential for the prevention and treatment of disease could become overshadowed by justifiable public outrage, fear, and disgust. As with many other emerging technologies, it will take much thoughtful deliberation by all sectors of society to weigh CRISPR’s exceedingly bright promise for cure of cancer and genetic diseases against its potentially devastating pitfalls when applied to the germline. Yet, we are confident that the biomedical research community will rise to the challenge by standing up for what is both bold and ethical on behalf of the hundreds of millions of people all around the world who are still awaiting cures.

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SOMATIC CELL RESEARCH using CRISPR-Cas

technology is moving out of the lab and into the clinic. Proven therapeutic effects include the inhibition of viral infection, reversal of deafness and muscular

dystrophy (pages 38-43), and the elimination of tumors in cancer models. As of this writing, clinical trials in the U.S. are underway in the areas of cancer, HIV, and sickle cell disease.

The first attempt to use CRISPR to treat a genetic disorder in the U.S. Sickle cell disease affects about 100,000 people in the U.S., most of whom are African American. A genetic defect causes bone marrow to produce a defective protein that makes blood cells that are sickleshaped, hard, and sticky. The deformed cells get stuck inside blood vessels and don’t carry oxygen normally, causing debilitating and, often, eventually lifeshortening complications. The defective blood cells also increase the risk for infections and damage to vital organs such as the heart and can cause lifethreatening strokes. Many people with sickle cell disease don’t live past their 40s. The only curative treatment is transplantation of donor blood stem cells, but there aren’t enough donors, and treatment isn’t without

Painful episodes can occur when sickled red blood cells, which are stiff and inflexible, get stuck in small blood vessels. These episodes deprive tissues and organs of oxygen-rich blood and can lead to organ damage, especially in the lungs, kidneys, spleen, and brain. potential complications. Modifying the patient’s own hematopoietic stem and progenitor cells is a better strategy – an option that is, in theory, available to every patient because the risk of rejection wouldn’t exist. This clinical trial is considered one of most anticipated medical

experiments in decades: the first attempt to use the gene-editing technique CRISPR to treat a genetic disorder in the U.S. For the study, doctors are using cells taken from patients’ own bone marrow that will be genetically modified with CRISPR to make them produce a

CRISPR cures deafness in “Beethoven” mice

In July 2019, scientists at Harvard Medical School and Boston Children’s Hospital used a novel geneediting approach to salvage the hearing of mice with genetic hearing loss, and they have succeeded in doing so without any apparent offtarget effects resulting from the treatment. The animals – known as Beethoven mice – were treated for the same genetic mutation that causes progressive hearing loss in 34

humans, culminating in profound deafness by the mid-20s. The new approach, described online in Nature Medicine, involves an optimized version of the classic CRISPR-Cas9 geneediting system that is better at recognizing the diseasecausing mutation seen in Beethoven mice. The refined tool allowed scientists to selectively disable the defective copy of a hearing gene called Tmc1

protein, fetal hemoglobin, which is the main oxygen transport protein in the human fetus during the last seven months of development and continues in the newborn until roughly the baby is roughly 2 to 4 months old. By restoring the body’s production of fetal hemoglobin, the hope is it will compensate for the defective protein that causes sickle cell disease and enable patients to live normally for the rest of their lives. “It’s exciting to see that we might be on the cusp of a highly effective therapy for patients with sickle cell,” says Dr. David Altshuler, chief scientific officer at Vertex Pharmaceuticals. Vertex is co-sponsoring the study with the CRISPR Therapeutics lab in Cambridge, Massachusetts.

Eight sites have recruited patients for the research in the United States, Canada, and Europe. Up to 45 patients, ages 18 to 35, will eventually be enrolled. “People with sickle cell disease have been waiting a long time for therapies that just let them live a normal life,” Dr. Altshuler says. “It’s exciting to see that we might be on the cusp of a highly effective therapy for patients with sickle cell.” The scientists expect it will take months before they see signs that the CRISPRmodified cells are producing hemoglobin at clinically useful levels and determine the degree of improved health. And it will take years, with careful monitoring, to note any complications; establish health and safety risks; and determine how long the benefits will last.

Fast Track for CRISPR treatment of beta thalassemia while sparing the healthy copy. The system was able to recognize a single incorrect DNA letter in the defective copy among 3 billion letters in the mouse genome. “Our results demonstrate that this more-refined, better-targeted version of the now-classic CRISPRCas9 editing tool achieves an unprecedented level of identification and accuracy,” said co-senior investigator David Corey. The team said the results set the stage for using the same approach to treat other dominantly inherited genetic diseases that arise from a single defective copy of a gene. While more work remains to be done before the system can be used in humans, the work represents a milestone because it greatly improves the efficacy and safety of standard geneediting techniques.

In December 2017, CRISPR Therapeutics and Vertex Pharmaceuticals announced a co-development and cocommercialization program centering on a CRISPR cell therapy called CTX001 for patients suffering from severe hemoglobinopathies such as beta thalassemia a fairly common blood disorder. In February 2019, it was reported that the first patient received the treatment, which marked the first CRISPR clinical trial done outside of China.

While the estimated completion date of the study – which is being held in hospitals in Canada, Germany, and the United Kingdom – is September 2022, in April 2019 the FDA granted Fast Track Designation to the two companies for CTX001 to treat transfusion-dependent beta thalassemia (TDT). The Fast Track program is designed to facilitate the development and expedite the review of drugs that treat serious conditions and fill unmet medical needs.

CRISPR-Cas used to edit cystic fibrosis mutations In August 2019, a research team from the University of Trento, Italy, used a CRISPRCas system to correct two of the mutations that cause cystic fibrosis. The technique, called SpliceFix, fixed the gene and restored its protein production mechanism at the same time.

“We demonstrated that our repair strategy works on patient-derived organoids [ 3D models made from stem cells] and with a high level of precision: It targets only the mutated sequences, leaving non-mutated DNA untouched,” said Giulia Maule, author of the study.

Brain cancer’s “immortality switch” turned off with CRISPR

Immortality is one of the key traits of cancer cells. In contrast to healthy cells, which are limited in the number of times they are able to divide, cancer cells can go on dividing and multiplying forever. A normal cell’s lifespan is determined by telomeres, which protect the ends of chromosomes. With each division, the telomeres get a little shorter until they can no longer protect the chromosomes, at which point cell division stops.

“immortality switch.” Researchers focused on an aggressive form of brain cancer, called glioblastoma (Senator John McCain and Beau Biden both died from glioblastomas) and found that cancer cells used a part of the GABP protein called GABPbeta1L to activate the switch. In lab mice, researchers removed the GABPbeta1L section of the gene using CRISPR. The edited GABP protein had a detrimental effect on the cancer cells but no effect on other cells. Dr. Costello’s team and other collaborators are now pursuing two approaches: the creation of a smallmolecule drug that targets GABPbeta1L and the development of a CRISPRbased therapy that can alter human genes so they will not produce GABPbeta1L.

Tumor cells in most cancers are able to “steal” immortality from our stem cells, which can also divide indefinitely thanks to a telomere-extending enzyme called telomerase. Cancer drugs designed to block telomerase also affect a patient’s stem cells, which limit their ability to produce new blood cells and other vital cells. Dr. Joseph Costello and his team at UC San Francisco have discovered a way to limit cancer’s access to this

CRISPR used to treat U.S. cancer patients for the first time The first clinical trial in the U.S. to use CRISPR in a treatment got underway in September 2018. Led by oncologist Dr. Edward Stadtmauer at the University of Pennsylvania, it consists of genetically modifying patients’ own T-cells to make them more efficient at fighting certain kinds of cancer cells. Each of the 18 patients slected for the trial has a type of relapsed cancer that tends to overproduce an antigen called NY-ESO-1. Pharmaceutical drugs known as immunotherapies have been dramatically effective in treating some cancers. The idea is to install the same capacity directly into the DNA of T-cells. Researchers added a gene to make the T-cells attack cancer, but they also used CRISPR to delete a gene called PD-1, which can prevent the Tcells from killing cancer cells. The treatment involves taking immune system T-cells that the body uses to fight cancer, modify their genes in a lab, and reintroduce the newly edited T-cells into the patient. The goal is to circumvent the kinds of tricks

CRISPR tested in possible “bubble boy” therapy About 1 in 50,000 boys are born with no immune cells – no way to molecularly protect themselves. The disease, called X-linked severe combined immunodeficiency, or SCID-X1, is commonly known as the “bubble boy” disease because if babies born with SCID-X1 are not secluded in a hygienic “bubble,” they could contract an infection and die. In people with SCID-X1 these cells are robbed of their immune cell-generating abilities due to a mutation in a single gene called ILR2 gamma. Stanford scientist Dr. Matthew Porteus is using a CRISPR-Cas9 treatment

that harnesses the particular class of stem cells that gives rise to immune and blood cells. In preclinical trials, Stanford scientists and their collaborators were able to replace the mutated gene behind the devastating immune disease. Now, scientists at the School of Medicine and their collaborators have used CRISPR-Cas9 to devise a new treatment to replenish immune cells in mouse models of SCID-X1. The results are promising, the scientists said, because they believe the treatment could potentially work in humans as well.

Neuroscientists develop a new model for autism Using CRISPR, researchers at MIT and in China have engineered macaque monkeys to express a gene mutation that is linked to autism and other neurodevelopmental disorders in humans. The monkeys show some behavioral traits and brain connectivity patterns similar to those seen in humans with these conditions. Mouse studies of autism have yielded drug candidates that have been tested in clinical trials, but

none have succeeded. Many pharmaceutical companies have given up on testing such drugs because of the poor track record so far. The new model could lead to better treatment options for some neurodevelopmental disorders, says Dr. Guoping Feng of MIT’s McGovern Institute for Brain Research. “Our goal is to generate a model to ... discover treatment options that will be much more translatable to humans,” he says.

CRISPR in clinical trial to cure childhood blindness

T-cells can be engineered to not only attack but delete a gene. that cancer cells use to evade or otherwise thwart their destruction by the immune system. The first two patients, one with sarcoma and one with multiple myeloma, have been treated. It’s not certain how effective the treatment has been, and, according to university officials, we won’t know until the trial has treated a total of 18 patients. Also likely, we won’t hear more about it until there’s been a presentation or a peer-reviewed paper, the university said.

The Penn study is funded by the Parker Institute for Cancer Immunotherapy, an organization started by Napster co-founder and early Facebook investor Sean Parker, as well as by a startup firm, Tmunity. The trial is scheduled to conclude in 2033. The assessment will include both safety (whether the edited T-cell treatment leads to any negative side effects) as well as efficacy (measured by outcomes such as whether the cancer disappears, the length of remission, and overall patient survival).

Two companies have trials scheduled to treat Leber congenital amaurosis, which is the most common cause of inherited childhood blindness, affecting about 2 to 3 of every 100,000 births. The condition is caused when people are born without the gene that converts light into signals the brain can interpret. Using CRISPR technology, scientists hope to add that gene to a person’s makeup, permanently curing this type of blindness. The two companies, Editas Medicine and Allergan, will test the new technique in up to 18 people – both adults and children age 3 or older – in the United States starting in late 2019.

Previous studies proved that the disease can be treated on a genetic level. The upcoming trial marks the maturation of the CRISPR-Cas9 toolset into a clinical-grade technology. An earlier gene therapy called Luxturna, which was the first in vivo gene therapy approved by the FDA, has shown it can cure the ailment by injecting a replacement gene into cells in the retina.

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A p p l i c a t i o n s : D u c h e n n e M u s c u l a r D y s tr o p h y by J. Kim Brayton, Editor of Perspectives

D uchenne muscular dystrophy is the most common fatal genetic disorder to affect

children – almost exclusively boys – around the world. For those afflicted, every skeletal muscle in the body slowly deteriorates. And while I cannot imagine having such a disease, watching it slowly take its toll comes with a profound feeling of helplessness. I know. My younger brother was born with Duchenne. I was 7 years old when David was born, more than 40 years before the human genome was sequenced. As a baby, David was diagnosed with severe mental disability. No one knew he also had Duchenne until he started to fall five or six years later when he ran through the house or tried to climb stairs. Fate had dealt my brother a miserable hand. When I was young, there was a period of hope when I believed it might be possible to cut a deal with God. “If you fix my brother, I promise I will….” I can remember watching the Jerry Lewis Muscular Dystrophy Telethon on Labor Day weekends, listening to researchers report their findings and seeing my mother’s silent tears when Jerry Lewis sang “You’ll Never Walk Alone.” And I remember David deftly maneuvering the fancy, motorized wheelchair our grandparents bought him, equipped with a joystick he could operate with his chin. David’s disease progressed, misshaping, twisting, and weakening his body until he died, in his late 20s. There was, of course, nothing that could be done. He maintained a sense of humor all of his life – keeping a hockey stick under his bed, just in case – a bittersweet image that never failed to make me laugh. David never complained about his condition. Not once. That there is a potential cure in the making for this horrific disease is beyond remarkable. It is my sincerest wish that a safe and effective treatment for this and other severe neuromuscular diseases will soon be a reality. What a day that will be, when hope turns into help.

PERSPECTIVE

Dr. Eric

Dr. Olson is Director of UT Southwestern’s Hamon Center for Regenerative Science and Medicine and Professor and Chair of Molecular Biology. He co-founded Exonics Therapeutics to advance his research on DMD and other neuromuscular diseases.

OLSON ALL MY LIFE HAS LED TO THIS MOMENT

There are moments in life when experience meets opportunity. I am in the midst of such a moment with the convergence of muscle biology and the advent of gene editing. I have dedicated my career to deciphering the mechanisms that govern development and disease of muscles. Every activity of life, from movements of the body, to the beating of the heart, to flow of blood in the circulatory system and the functions of tissues and organs, depends on different types of muscles. Given the centrality of muscle to every facet of life, and the many genes that control the structure and function of muscle, it is not surprising that there are hundreds of genetic disorders of muscle. Many of these are among mankind’s most devastating disorders, and there is not a single cure for any. Duchenne muscular dystrophy (DMD) is the holy grail of muscle diseases. Caused by mutations in the dystrophin gene, which encodes a massive membrane protein required to To see muscle cells from a boy whose body maintain the integrity of muscle membranes, the absence of has never made dystrophin in its life produce dystrophin in DMD leads to degeneration of skeletal muscles normal levels of dystrophin is something that, and the heart. This disease typically afflicts boys, leading honestly, takes your breath away. to loss of ambulation by the second decade of life, followed by respiratory and cardiac failure and premature death. Approximately 300,000 boys are afflicted worldwide. With the advent of CRISPR gene editing, we devised a strategy to correct a majority of the thousands of dystrophin mutations that cause DMD. This new method has enabled the restoration of dystrophin expression in cells from DMD patients. By engineering a harmless virus to carry CRISPR gene-editing components to muscle and the heart, we have also successfully corrected DMD mutations in mice and large mammals with the most common human dystrophin mutations and restored muscle function. It’s no longer a question of whether this gene editing method can work – it can work! The key issues now are to confirm safety, which looks good so far, and to optimize virus production. We feel we are now poised to advance this gene-editing strategy toward eventual human clinical trials. In contrast to all other therapies, which treat the symptoms of DMD, CRISPR has the potential to correct the underlying genetic errors that cause DMD and thereby prevent disease progression. I feel my entire career has led me to this moment, and I am committed to developing a safe and effective therapy for the many DMD patients in desperate need. To see muscle cells from a boy whose body has never made dystrophin in its life produce normal levels of dystrophin is something that, honestly, takes your breath away. I also believe DMD will be an initial test case for CRISPR-based gene-editing therapies that can eventually be applied to other diseases of muscle for which there are no available therapies.

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Ben Dupree on growing up with Duchenne Ben Dupree was diagnosed with Duchenne muscular dystrophy (DMD) at age 9. He’s spent much of his life dealing with the realities of the disease. As a young boy, Ben remembers struggling to keep up with other children during soccer games or school exercises. “Other kids would be running around, and I would be sitting with my hands on my knees trying to catch my breath,” he said. “It was hard to go through that.” Gradually it became difficult to climb stairs, and even standing became a strenuous effort. By the time he reached middle school, Ben was using a wheelchair full time. His teenage years were an especially frustrating period as the disease complicated many facets of his life. While playing in the high school

Dr. Eric Olson shows Ben Dupree that dystrophin is being produced in gene-edited heart muscle cells taken from Dupree’s blood.

band, he often had to sit away from the group because parts of the stadium weren’t wheelchair accessible. He gave it up, as he did other activities that reminded him of his physical limitations. “I was very depressed,” Ben said. “I think that at age 15, 16, kids want to try to be independent. They really

struggle with the idea of relying on other people. I had to come to terms with that.” Things began to change while Ben was in college at SMU (he graduated in 2015 with a degree in biochemistry), after deciding to join his mother on trips to speak with other families across the country dealing

SingleCut CRISPR corrects DMD in dogs

In July 2018, scientists, for the first time, used a CRISPR-Cas9 technology, called SingleCut, to halt the progression of DMD in dogs sharing this mutation, raising hopes that a CRISPR therapy could be used to treat this crippling and deadly disease in people. Dr. Olson’s team collaborated with the Royal Veterinary College (RVC). The RVC’s dog colony program was supported by grants from the Wellcome Trust, Muscular Dystrophy UK, and Duchenne Ireland. “Children with DMD often die, either because 38

their heart loses the strength to pump or their diaphragm becomes too weak to breathe,” said Dr. Eric Olson, who led the study. “This encouraging level of dystrophin expression would hopefully prevent that from happening.” To reach the animals’ billions of muscle cells, two dogs were injected with an adeno-associated virus (AAV) carrying the new CRISPR components that target skeletal muscle and heart tissues. Six weeks later, muscle cells were making dystrophin.

with DMD. “I was able to come out of that dark place through the community of support,” he said. “The people I came to know fostered a feeling that we’re all in this together, and that brought me back to where I am today.” Coincidentally, Dr. Eric Olson – one of the world’s leading experts on muscle disease – and the Duprees were neighbors. “From the first time I met Ben, I felt we had a real connection,” Dr. Olson said. In 2015, Dr. Olson invited Ben to UT Southwestern to provide a blood sample to test whether a new CRISPR method would work on human cells. “Ben has played an instrumental role in our research. Early on, he was our patient zero,” Dr. Olson said. “One of the most inspiring aspects of the work

was the moment when Ben came to the lab and together we looked through the microscope at his own cells that came from his blood sample and they were beating in a dish, and then we showed him that they were producing the dystrophin protein that his body cannot make.” “Being able to see that gave me a lot of hope for the future – kind of seeing that this is a concrete thing that can happen. It could be done,” Ben said. “I’m hoping to use social work training to more directly help people get through what I’ve gone through. “I’m not necessarily hoping for a cure but for anything that could slow the progression of the disease,” he added. “Even if I’m not here to see the lifesaving treatment, my hope is that people being born now with the disease can still benefit.”

Vertex Pharmaceuticals acquires Exonics Therapeutics The researchers then administered the vectors into the bloodstream of two more dogs. The dog receiving the higher dose produced dystrophin at up to 70% of normal levels in skeletal muscle after eight weeks and 92% in heart muscle. The positive findings “brought tears to the eyes and were jaw-dropping,” Dr. Olson said. “We’re very mindful of ethical concerns and have done our best to keep our use of dogs to an absolute minimum,” he added. “I can’t help but feel tremendously excited,” said Dr. Jennifer Doudna. “This is really an indication of where the field is heading, to deliver gene-edited molecules to the tissues that need them and have a therapeutic benefit.” Beyond the scientific confirmation, Dr. Olson remarked that the dogs “showed obvious signs of behavioral improvement – running, jumping – it was quite dramatic.”

In July 2019, Exonics Therapeutics, co-founded by Dr. Eric Olson, became a wholly owned subsidiary of Vertex Pharmaceuticals. “Vertex has a proven track record of developing important therapies for serious diseases, and we are excited to combine our efforts to potentially develop a safe and efficacious onetime treatment for severe neuromuscular diseases,” Dr. Olson said. Along with the approximately $1 billion acquisition, Vertex will expand its licensing agreement with CRISPR Therapeutics, potentially a $2.5 billion collaboration. “These transactions are highly aligned with our strategy of investing in scientific innovation to create transformative medicines for people with serious diseases,” Vertex Chairman, President and CEO Jeffrey Leiden said. “The Duchenne community needs novel approaches to treat and cure this devastating disease,

and Exonics’ technology has the potential to dramatically improve the lives of Duchenne patients,” stated Debra Miller, CEO and founder of CureDuchenne, which provided initial seed funding to co-found Exonics. Dr. Olson will continue as Exonics’ chief science advisor and provide oversight and guidance on research and development. “We are excited about the possibility of developing potentially curative therapies for DMD and DM1 together with Vertex,” said CRISPR Therapeutics CEO Dr. Samarth Kulkarni. Vertex has proven gene therapy treatments for cystic fibrosis (CF). The expanded agreement with CRISPR Therapeutics allows it use of Exonics’ SingleCut technology to discover and develop potential new treatments for CF as well as for sickle cell disease.

PERSPECTIVE

Christi

Christi Dupree graduated with a double major in Journalism and Psychology from the University of Missouri in 2018. She works as an Art Director at TracyLocke advertising in Dallas, Texas.

DUPREE MY BROTHER, BEN

While my classmates were learning multiplication tables, I was learning about gene mutations. More specifically, about my brother’s mutation. It’s not an easy thing to explain to a 9-year-old, but my mother had the patience of a saint and a knack for coming up with creative metaphors. Excited to be in the loop, I would then try to explain genetics to my friends at school, but most of the metaphors got lost in translation. And even back then, my brother would interject whenever my analogies didn’t add up. Ben is nothing if not persistent in his loyalty to the factual basis of all things. That’s who he is. CRISPR has given Ben new possibilities, and As we grew, Ben and I pursued our own interests, Ben choosing the scientific path of biochemistry while I for all of us, a hope that now fills a space in our explored my creative side through journalism. Ironically, we minds once riddled with anxiety. found ourselves dabbling in each other’s worlds, all thanks to CRISPR. My ears perked up the day my professor mentioned CRISPR in my intro biology class. I hadn’t expected to hear about a familiar topic, yet here I was, learning about it in detail through Ben’s preferred medium: a classroom. Meanwhile, Ben had become the face of CRISPR at UT Southwestern and was navigating the art of storytelling for the first time (usually my stomping grounds). Now, we both have a fuller understanding of the technology itself, but we also have a deeper understanding of each other and the way our brains work. We’ve learned the value of his persistent loyalty to facts and my unfocused metaphorical curiosity. He’s learned to explore the creative parts of his brain and I’ve developed an analytical side. And we’ve both come to understand that no family is the same. Even though families within the Duchenne Muscular Dystrophy community may share similar experiences and struggles, at the end of the day each piece of a family unit is a human being with an incredibly diverse set of experiences, emotions, and quirks. I’m thankful for CRISPR because of the part it has played in strengthening our love for and understanding of each other. It hasn’t been easy. We’ve endured loss and we’ve struggled at times to find happiness. But even in that, we’ve grown closer than ever before. CRISPR has given Ben new possibilities, and for all of us, a hope that now fills a space in our minds once riddled with anxiety. Thanks to that hope, which now can exist for all families affected by Duchenne, we’ve been able to live in the present and lean into our love for each other. We’ve laughed more, we’ve been more spontaneous, and we’ve allowed ourselves to be vulnerable with each other. Our family’s perspective on CRISPR is complicated and talking about it brings up a jumble of joy and fear. But as we move forward, I can honestly say that I’ve never been more excited than I am right now about what the future holds.

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DUCHENNE AND THE SEARCH FOR A CURE

OLSON

ON EXON SKIPPING, SINGLECUT CRISPR, AND THE HUMAN EXPERIENCE AT 2.6 MILLION base pairs in size,

DMD is the largest gene in the human body, which, among other things, means there is tremendous opportunity for errors to occur. DMD comprises 79 separate coding regions, called exons, which fit neatly together, end to end, like a puzzle. The gene holds the genetic code needed to produce the protein dystrophin. Dystrophin is essential for muscles to function properly. Duchenne muscular dystrophy is caused by mutations in the DMD gene, which prevent muscle cells from making dystrophin. As a result, the cells become damaged and, over time, are replaced with scar tissue and fat in a process called fibrosis. Duchenne muscular dystrophy affects 1 in every 5,000 boys. Currently, there are about 300,000 boys in the world suffering from the disease. There are more than 3,000 different mutations that occur in the DMD gene. Some suggest there might be as many as 7,000. Suffice it to say there are thousands. Despite heroic efforts supported by hundreds of millions of dollars over many decades, scientists have been unable to produce a cure. Among those working to change that are Dr. Eric Olson and members of the Olson lab. In July 2019, Perspectives had a chance to sit down with Dr. Olson to better understand why, for the first time, a cure could be within grasp.

How does one even begin to solve Duchenne muscular dystrophy? I’ve been thinking about muscle for a long time. Thirty years. Or more. Studying the biology of muscle — how it forms, how it functions. The students and postdoctoral fellows in my lab have discovered many of the key gene regulatory proteins, molecules, and mechanisms that form muscle in our body. There is no activity by an animal or human being since the beginning of time that has occurred without muscle. All movement – all art, all music, all athleticism, everything – requires sophisticated functions of muscle. The dystrophin protein is like a shock absorber. You need the ends to be intact, but there’s a long, coiled section in the middle. One of the key insights is that not all 79 exons that code for dystrophin are needed. If a mutation occurs somewhere in the coiled area, chances are good that the exon can be skipped, the section removed, and the gene put back together. It just makes it a little shorter. A few less coils. Although, in some cases, the mutation is of a kind that by simply bypassing the problem area the gene is perfectly restored. But there are thousands of possible mutations. Right. So you can’t make a genetic therapy that’s customized for every person. It’s too expensive, and you can’t test it. If you’re making a genetic therapy, you have to be able to show that it’s safe. You can’t do that as a one-off on every person. So what we’ve done is cluster mutations that occur in certain areas. For example, the most common group of DMD mutations – around 12% of all cases – occurs in exons 45 to 50, a region that is especially fragile and susceptible to disruption or mutation. When mutations occur in this area, exon 51 is thrown out of frame because of its unique shape. It’s called a hotspot. And for boys with mutations in this particular hotspot we can consolidate all of their mutations by skipping exon 51. How many hotspots have you found? We’ve identified 10 hotspots that we believe we can address. We can cluster hundreds of different

mutations within one hotspot and can use a specific guide RNA to correct them all with a one-time genetic therapy. We’ve tested the idea in patient-derived cells and showed it could work. Taken together, they account for 60% to 80% of all Duchenne cases. How do you skip exons? After CRISPR-Cas technology was discovered, we started looking into what it could do. With CRISPR you can devise a system that makes two cuts in the DNA, rejoin the ends, and eliminate the mutated code in the middle. But the problem is that solution, at least at present, is unreliable. It’s complicated. The two cuts have to be made at the same time. Too many things can go wrong. It’s very inefficient.

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The simplest possible way that you could ever correct a genetic mutation is with SingleCut CRISPR.

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So we developed a technology called SingleCut CRISPR. We make a single cut in such a way that the ends will reliably rejoin. One cut and it nibbles on a little piece of the DNA and the gene comes right back together. It’s relatively straightforward. In fact, the simplest possible way that you could ever correct a genetic mutation is with SingleCut CRISPR. It’s important to keep in mind this technology is confined to patients with the disease. Nothing we are doing will affect the gene pool. And to that point, Duchenne can never be eliminated from the gene pool. While two-thirds of all cases are inherited from an X-chromosome from the mother, one-third are a spontaneous mutation because the gene is so massive and fragile. How is CRISPR delivered to the muscle cells? That’s the challenge, not only for CRISPR but for any therapeutic drug. It’s always about effective delivery. Delivery is what matters.

Fortuitously, there’s an AAV — an adeno-associated virus — specific to muscle and heart tissue. So when you inject it, it saturates the body but collects in muscle tissue. Also, in the liver, but we added some DNA to prevent the virus from expressing there. So we’ve got a reliable delivery system to target the exact cells in the body that we want to address. Do all muscle cells need to be corrected? This is another aspect of the biology of muscle that lent itself to this technology. You don’t have to correct every nucleus in every muscle cell to be effective. We’ve found that dystrophin is overproduced by the body, meaning far more is made than is needed. The percentage of muscle cells that need to be making dystrophin is around 20%. We’ve surpassed that number. What has stood out in this journey? It’s not that common for a basic scientist to actually see how his or her work directly affects someone. Up until this point, I had never really known a person with any of the diseases we were working on. But with Duchenne, I got wrapped into the whole thing. I got to know Ben and the Dupree family. I went to patient advocacy groups and became immersed in the whole human experience. Duchenne really connects you to that. It affects boys in their most sensitive periods of life. Their social growth and all that. It affects their siblings. The whole family. Every aspect. It’s a humbling disease. Exonics was recently sold. How does that feel? And what does it mean for you going forward? Exonics was acquired by Vertex Pharmaceuticals, a terrific company. The merger of Exonics with Vertex will enable the work to move faster than it ever could have moved in Exonics alone. This was the best outcome for the technology and the patients. Some of the people from my lab who did the pioneering work will continue with Vertex. I’ll serve on its scientific advisory board and be heavily involved. This work is the most exciting thing I’ve ever done. The idea has all the resources it needs. Amazing people are working on it. It’s not a slam dunk, but I’m really optimistic. S O U T H W E S T E R N M E D I C A L P E R S P E C T I V E S . 2 019

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C H A P T E R 3.6 ———————————————————————————————————————————————————————————

Applications: Human Germline Cells

Perhaps no scientific discovery in history has held more promise or sounded

more alarms. Making genetic modifications to human embryos and reproductive cells such as sperm and eggs is known as germline editing. Through germline editing, any edits would pass to that person’s children, and their children, in perpetuity. The full implications of those changes are difficult, if not impossible, to foresee. In a startling announcement in November 2018, it was revealed that a Chinese biophysicist had created the first CRISPR-edited babies with the goal of imbuing the twin girls with HIV resistance. The announcement – met with immediate and harsh rebuke – propelled humanity to the threshold of uncharted territory. The key question for researchers and bioethicists is not whether CRISPR-Cas technology should be used on human embryos now to make babies – the consensus is that the technology is nowhere near ready, ethically or scientifically – but whether it should be permitted in the future, and if so, under what conditions. Proponents suggest there is a moral imperative to move toward reproductive use of germline modification. Done safely, openly, and responsibly, it could potentially lead to the decrease, or even elimination, of the incidence of many single-gene diseases and reduce human suffering in profound and meaningful ways not otherwise possible. Opponents make the case that human germline modification would be fraught with unknown risk; that there are alternative ways to prevent inherited diseases; that it could negatively impact society in fundamental ways; and that it forces genetic changes onto future generations who have no say in accepting that risk. Members of the disabled community argue that their quality of life and level of happiness is no less than that of others. Should children with their “condition” be “fixed” before birth? Before they have the option to decide for themselves? And how would society view disabled parents who chose to retain their child’s disability? One thing is certain: As CRISPR technology continues to evolve, it will require considerable, ongoing, and thoughtful evaluation. 42

PERSPECTIVE

Dr. William

Dr. Hurlbut is a physician, Adjunct Professor and Senior Research Scholar in Neurobiology at the Stanford Medical School. His primary areas of interest involve the ethical issues associated with advancing biomedical technology, the biological basis of moral awareness, and studies in the integration of theology with the philosophy of biology.

HURLBUT A NEW PARADIGM

The visionary investor and philanthropist Sir John Templeton spoke of the present age as “the blossoming time ... reaping the fruits of generations of scientific thought.” Yet, he understood that such powers were “a tremendous, awe-inspiring responsibility,” and spoke of the need for the deepest humility and wisdom. Nowhere is this more evident than in our recent advances in gene editing – and their potential application in human germline genetic engineering. The difficult ethical dilemmas of gene editing are already apparent at the most fundamental levels of laboratory research. During the earliest stages of embryogenesis, individual genes can be selectively altered to study their role in healthy development and disease. This yields valuable scientific knowledge, but, at the same time, reopens unresolved controversies over the moral standing of human embryos and their instrumental use in biomedical research. Indeed, extensive studies of genetically edited embryos would be an unavoidable step in establishing the safety of any future applications of germline editing. In moving from the lab to the clinic, additional ethical issues arise. What level of disease or disability justifies such an intervention and the associated risks? Are albinism, deafness, or dyslexia proper targets of germline At their foundations, the guidance and intervention? At what point is such action a disrespect of governance of genetic technologies are “species” diversity and a dishonor to natural human variation? As germline genetic engineering moves “beyond therapy” issues, matters for the whole human family. to elective interventions such as hair color or height, what role does parental preference and presumed reproductive rights play in such decisions? Even with the best of intentions such practices might promote a subtle shift in the character of procreation toward laboratory production, provoking a competitive social attitude incompatible with unconditional love. Moreover, there are proposals for projects of de-extinction of human ancestral species, a “cure” for aging, and guided evolution of the human future. The questions raised by such projects go beyond issues of individual rights and social responsibilities to considerations of the very source and significance of the natural world, its integrated and interdependent processes, and the way these provide the foundational frame for the physical, psychological, and spiritual meaning of human life. As both practical and theoretical matters, these issues involve values, beliefs, and goals that are beyond the scope of empirical science and require the full breadth of human experience and wisdom. At their foundations, the guidance and governance of genetic technologies are “species” issues, matters for the whole human family. Within both the scientific community and the general public, there is wide recognition that our new gene editing tools are a “threshold technology,” an opportunity to reenvision and reorder our place and purpose within the natural world. The wise governance of these technologies is a key practical and moral challenge of our age, and our response to them could significantly alter the very future of life on Earth.

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GERMLINE EDITING — A BRIEF RECAP

In April 2015, a Chinese team first reported using CRISPR-Cas9 on human embryos. Experiments were conducted on 86 discarded embryos obtained from a fertility clinic. Researchers encountered serious obstacles. In December 2015, Congress passed legislation specifying no public funding could be used for research “in which a human embryo is intentionally created or modified to include a heritable genetic modification.” In February 2016, a team led by developmental biologist Dr. Kathy Niakan of the Francis Crick Institute in London used CRISPR-Cas9 to disrupt the production of a protein called OCT4 in human embryos in order to better understand embryonic development. In 2017, Dr. Shoukhrat Mitalipov, Director of the Center for Embryonic Cell and Gene Therapy at Oregon Health and Science University (OHSU),

secured private funding and edited human embryos to repair a mutation in a gene called MYBPC3, which causes a deadly heart disease – hypertrophic cardiomyopathy (HCM), a thickening of the heart muscle. In July 2018, the United Kingdom’s Nuffield Council on Bioethics determined that heritable genome editing could be “ethically acceptable in some circumstances.” In August 2018, Chinese researchers announced that CRISPR base editing had been used to correct a single DNA letter – a “C” to a“T” in the gene FBN1 that causes Marfan syndrome, an incurable connective tissue disorder that affects about 1 in 5,000 people. Success was achieved in 16 out of 18 embryos, a significant improvement over Dr. Mitalipov’s results (42 out of 58). But no event has impacted the discussion over germline editing as much as the announcement made in Hong Kong in November 2018 (below left).

Chinese biophysicist announces birth of the first CRISPR-edited babies In November 2018, on the eve of the Second International Summit on Human Genome Editing in Hong Kong, Dr. He Jiankui, a Chinese biophysics researcher and Associate Professor in the Department of Biology at the Southern University of Science and Technology (SUSTech) in Shenzhen, announced the birth of twin girls (Lulu and Nana) from embryos whose genomes had been edited using CRISPR. Initially, Dr. He did not present any solid proof that this experiment had, in fact, taken place, which cast doubt on his motives and triggered international concerns from scientists. The world scientific community reacted with shock, outrage, and nearunanimous condemnation. Summit Chairman and Nobel Laureate Dr. David Baltimore called Dr. He’s experiment “irresponsible” and criticized his lack of transparency. Dr. Feng Zhang called for a moratorium on gene-edited babies. Dr. Jennifer Doudna said in a statement that “this work reinforces the urgent need to confine the use of gene editing in human embryos to settings where

Dr. He Jiankui addresses the Second International Summit on Human Genome Editing in Hong Kong. a clear unmet medical need exists, and where no other medical approach is a viable option, as recommended by the National Academy of Sciences.” And NIH Director Dr. Francis Collins declared in a statement that “this work represents a deeply disturbing willingness by Dr. He and his team to flout international ethical norms.” Concern was also expressed over the fact that several Western scientists knew of Dr. He’s research, and it was felt they had not done enough to stop him. In January 2019, it was confirmed that Dr. He’s experiments had, in fact, taken place – and the twin girls were under medical observation. An additional

pregnancy was also confirmed. That same month, Chinese investigators released their initial findings, promising stiff penalties, and Dr. He’s university fired him. Tests confirmed that one of the twins will be resistant to HIV because the gene edits silenced both copies of her CCR5 gene. The other twin might still be susceptible to infection because one copy of her CCR5 gene was left intact. In March, Chinese officials drafted stricter rules for human gene editing. According to a May 2019 Wall Street Journal article, Dr. He had originally been investigating editing a gene that can offer protection from familial hypercholesterolemia, a rare cholesterol-related disease that can cause broken bones in children. He changed his mind after visiting a village where he saw HIV-positive families facing discrimination. In China, children born to infected individuals are not able to attend regular schools. Dr. He saw the trial as a way to use science to fight that injustice and, at the same time, bring glory to China, the Journal reported.

Russian biologist renews controversy with two plans to create more CRISPR-edited babies Dr. Denis Rebrikov, who heads a genome-editing laboratory at Russia’s largest fertility clinic, the Kulakov National Medical Research Center for Obstetrics, Gynecology and Perinatology in Moscow, told the journal Nature he plans to use CRISPR to edit the CCR5 gene in embryos – the same gene Dr. He Jiankui targeted – to create HIV-resistant babies. Dr. Rebrikov explained he hopes to take a different approach than Dr. He, who conducted his experiments using sperm from HIVpositive fathers. Dr. Rebrikov plans to implant edited embryos into only a subset of HIV-positive mothers who do not respond to anti-HIV drugs as the risk of the mother transmitting the infection to the child is far greater. In response, Nature has tried to rally opinion to stop him. “Time is of the essence,” Nature warned. Most scientists remain adamant that there is no justification for editing the CCR5 gene in embryos because the risks don’t outweigh the benefits. In addition, New Scientist reported that Dr. Rebrikov

Dr. Denis Rebrikov

also intends to use CRISPR to give deaf couples babies who can hear. He has found five pairs of deaf parents who have agreed to let him use CRISPR technology to ensure that children born from their edited embryos would not inherit a deafnesscausing mutation in the GJB2 gene. Dr. Rebrikov told Nature he won’t actually create any CRISPR babies unless the Ministry of Health of the Russian Federation gives him the green light. In 2018, Vladimir Putin allocated roughly $2 billion for genetic research and named Dr. Maria Vorontsova, an endocrinologist who some say is Putin’s oldest daughter, to the 30-person panel overseeing the work – an area of study Putin has said will “determine the future of the whole world.”

PERSPECTIVE

Dr. Marcy

Dr. Darnovsky is Executive Director of the Center for Genetics and Society, a public interest organization working to encourage responsible uses and effective governance of human genetic and assisted reproductive technologies. She speaks and writes widely on these topics.

DARNOVSKY DO NOT OPEN THE DOOR

Should CRISPR be used to alter the genes and traits of future children and generations? Dozens of nations have considered this prospect and decided that manipulating the human germline should be legally off limits. The U.S. and China have not. Some gene-editing enthusiasts now want to reopen the question. The answer will affect us all and shape the future. Yet despite the stakes being so high, many discussions of heritable genome editing are distorted by dubious assumptions. The claim that editing human embryos is needed to save babies from inherited disease and could “reduce human suffering in profound and meaningful ways,” for example, is misleading for several reasons. First is the substantial risk of introducing rather than Affluent parents could soon find themselves preventing harm. Editing an embryo’s genes can go wrong contemplating fertility clinic ad campaigns for in multiple ways, including off-target edits, on-target but inaccurate edits, and a condition called mosaicism that genetically upgraded embryos. produces a mix of altered and unaltered cells in an embryo – and in the resulting child. No one knows what the health effects of these unintended changes might be, for that child or for future generations. Second: Safe options for preventing transmission of genetic disease already exist. 100% of at-risk parents can accomplish this by using donated sperm or eggs. Alternatively, the overwhelming majority can use a well-established embryo screening technique to ensure an unaffected child who is also genetically related to both biological parents. Embryo screening poses ethical dilemmas; it raises challenging questions about what kinds of people we welcome into the world. But it is far less ethically fraught than manipulating the genes of future children. Discussions of heritable genome editing tend to focus on these issues of safety and efficacy. We hear much less about the dangerous societal consequences that could ensue if “CRISPR babies” were to become a widespread phenomenon. And let’s face it: Opening the door to heritable genome editing just a crack is highly unlikely. However well intentioned, efforts to allow it for “therapy” but not “enhancement” couldn’t be expected to hold in the face of commercial pressures. Affluent parents could soon find themselves contemplating fertility clinic ad campaigns for genetically upgraded embryos. To be sure, traits like intelligence are too complex to be effectively controlled. But CRISPR babies could nonetheless find a market based on the allure of perceived superiority. Perceptions matter: We know from the sordid history of racism that social disparities and discrimination can be powerfully fueled by false beliefs that some humans are biologically “fitter” than others. There is no compelling reason to develop heritable genome editing. Unless we want to build a world divided into genetic “haves” and “have nots,” let’s keep the door shut.

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PERSPECTIVE

Dr. Christopher

Dr. Gyngell is a Research Fellow in Biomedical Ethics at the Murdoch Children’s Research Institute and University of Melbourne in Melbourne, Australia. His research interests lie primarily in the ethical and legal implications of biotechnologies and the philosophy of health and disease.

GYNGELL THE MORAL IMPERATIVE

When discussing the immense potential of genome editing, it has become common to quote a phrase made famous in Spider-Man: “With great power comes great responsibility.” What does “great responsibility” mean in the context of genome editing? Using technology responsibly clearly entails using it safely. Lack of responsibility in this sense is what troubled so many people about the recent announcement that the first-ever gene-edited babies had been born. There is still tremendous uncertainty about the effects of genome editing in human cells, not to mention germline cells. A basic tenet of research ethics is that any risk to research participants must be balanced by appropriate benefits. We are still some way off this barrier being cleared for any reproductive application of CRISPR. But responsibility doesn’t just mean avoiding unsafe uses – it also entails positive obligations. When you have powerful capacities and can therefore achieve great good, then morality compels you to do so. It is widely accepted that we have a moral imperative to Responsibility doesn’t just mean avoiding unsafe uses – it also entails positive obligations. benefit future generations. If we can reduce the impact of climate change for those who come after us, then we ought When you have powerful capacities and to do it. If we can reduce the burden of disease in future can therefore achieve great good, then morality generations, then we are morally compelled to do so. compels you to do so. This moral imperative to benefit future generations has implications for the ongoing development of technologies. The imperative to reduce the impact of climate change implies an obligation to develop renewable energy technologies. The imperative to reduce the disease burden on future generations implies an obligation to undertake medical research. Genome editing has enormous potential to reduce rates of disease and promote human flourishing. Most immediately, genome editing could reduce the incidence of lethal single-gene disorders – like Tay-Sachs disease and spinal muscular atrophy – in cases where genetic selection is not effective. In the long term, it may be possible to use genome editing to reduce or delay polygenic disorders – like cancer, heart disease, and diabetes – that cause huge numbers of premature deaths and place great financial stress on our health systems. In the very long term, genome editing could potentially help us respond to threats caused by antibiotic resistance, climate change, or other future hazards. To use CRISPR responsibly, we need to develop it as a tool that can help us fulfill our obligations to future generations.

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PERSPECTIVE

Dr. Marsha

Dr. Saxton is an advocate for and member of the disability community. She works as Director of Research at the World Institute on Disability in Berkeley, and Lecturer in Disability Studies at the University of California, Berkeley.

SAXTON VOICES THAT MUST BE HEARD

Leaders and participants in science and public interest argue against taking the risks of CRISPR-Cas 9. I agree with these concerns, so I will focus solely on the disability community perspective. Many who declare themselves spokespeople or experts on CRISPR are family members or medical providers. They speak of ending the suffering of disabled people. People with disabilities must be the ones to speak about our experiences, our lives. Our community slogan is “nothing about us without us.” Do disabled people want to be “edited” via genetics? The disability rights movement has made significant gains – access to education, employment, transportation, participation in the community. Yet, we still Central to our objections is the manipulative use face discrimination, and lack enforcement of disability laws of suffering from disability as a justification for in the U.S. and around the world. An irony is that disability CRISPR. This promotes stereotypes, reinforcing discrimination is slowly but successfully being challenged in the same era as stereotypes of “suffering and burden” are discriminatory policies and practices, limiting being exploited in marketing of genetic technologies to progress in our inclusion. eliminate disability. Of course we welcome helpful treatment, but “the quest for cure” is complex. The general public, and the medical and genetic research communities continue to hold stereotyped views about us. Hollywood often employs disability as a metaphor for suffering ( Me Before You, Million Dollar Baby) reinforcing messaging that our lives are not worth living. Yes, there are disabled people who may suffer. Disability can be difficult, cause pain, end lives early. Yet, much of the “suffering” results from discriminatory attitudes – marginalization, exclusion. Discrimination is what is largely disabling about disability. Seeking cures “at all cost” is not our goal. Some segments of the disability community wish to retain their bodies and lives unedited ‑ for example, many in the Deaf community, and those with dwarfism. If you judge this as wrong, perhaps it is because you don’t know us – how hard we’ve fought for inclusion, even for our very existence. Don’t base your assumptions of the lives and needs of disabled people on your family member, or a patient in your practice. You need to meet the disability community and find out our lives are good. Central to our objections is the manipulative use of suffering from disability as a justification for CRISPR. This promotes stereotypes, reinforcing discriminatory policies and practices, limiting progress in our inclusion. Instead, actively engage us in discussion. And please stop distorting our lives in order to override the many valid objections to this unpredictable, dangerous, and unnecessary approach which could negatively affect human beings for generations ahead.

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The History of Genetic Modification

G enetic modification isn’t new.

For thousands of years, humans have been altering the genomes of plants and animals through selective breeding – long before anyone knew what genes were, let alone how they worked. While we humans are a highly adaptive species, the story of humanity can also be seen as the story of how we have chosen to genetically modify much of the world around us to better suit our health, well-being, and happiness. Two monumental events united genetics with biotechnology. The first was the 1953 discovery of the double helical structure of DNA. The second was the discovery of a recombinant DNA technique in 1973. For the first time, molecular biologists gained the ability to manipulate DNA molecules, making it possible to study genes and attempt to harness them. Recombinant DNA has proven important to the production of vaccines and protein therapies such as human insulin, interferon, and human growth hormone. The milestones of genetic modification prior to CRISPR are vast. The following pages highlight a few key achievements – from selective breeding to irradiation to the first modern gene-editing techniques. The discovery of CRISPR is the story of how an adaptable, prokaryotic viral defense system – first co-opted from a single species of bacteria, Streptococcus pyogenes (which gives us strep throat), and followed by Staphylococcus aureus (which gives us a staph infection) – became the start of the world’s most powerful and versatile genome-editing platform. As the possibilities and challenges for CRISPR-Cas systems accelerate, we take a moment to appreciate how curiosity into basic science has brought us to a point where molecular biology is widely regarded as the science of the future and biotechnology is set to become one of its leading industries. Oh, and while some of you may remember that in 1997 scientists cloned a sheep named Dolly, you might not know that Barbra Streisand recently cloned her dog. Twice. 48

BEFORE CRISPR

Our ancestors were able to influence the living things around them through a process called “selective breeding” – a term coined by Charles Darwin – in which humans actively chose which traits should be passed on to offspring. The dog is thought to be the first organism selectively bred. Around 32,000 years ago, while our ancestors were busy hunting and gathering, they bred dogs to help them hunt, herd, and stand guard. Since the onset of the agricultural age some 12,000 years ago, countless fruits and vegetables have also undergone radical transformation. One of the more dramatic changes in plant genetics has been corn, or maize, which began as a wild grass called teosinte. It had tiny ears and few kernels, but thousands of years of selectively breeding have given us the varieties of modern corn we feed to livestock and butter for ourselves. Over the past century, our ability to modify genes became more direct. In 1927, Dr. Hermann J. Muller –

an American geneticist who taught at the University of Texas in Austin – demonstrated that radiation from X-rays would change the genetic makeup of fruit flies and that the mutations would be passed to subsequent generations. By the 1930s, scientists were bombarding seeds and insect eggs with X-rays, and during the 1950s and 1960s, radiation breeding produced a sizable percentage of the world’s crops – everything from varieties of rice, wheat, and barley to pears, peas, and peppermint. These mutations have improved yield, taste, size, and resistance to disease and have helped plants adapt to more diverse climates. In 1973, a monumental breakthrough occurred when Dr. Herbert Boyer at Stanford University and Dr. Stanley Cohen at UC San Francisco developed a method by which they could cut out a gene from one organism and paste it into another. They transferred a gene that codes for antibiotic resistance. The technology became known as recombinant DNA Stem cell restrictions and breakthroughs

Dr. Norman Borlaug and the Green Revolution Dr. Borlaug is most famous for having launched what came to be known as the Green Revolution – an agricultural phenomenon responsible for an exponential increase in the amount of food produced on Earth. During his 20 years in Mexico, Dr. Borlaug and his colleagues perfected a dwarf wheat variety that could produce large amounts of grain, resist diseases, and resist lodging – the bending and breaking of the stalk that often occurs in highyielding grains. It’s estimated that the genetically modified wheat developed by Dr. Borlaug and his team has saved some 2 billion people in the underdeveloped world from death by starvation. He was awarded the Nobel Peace Prize in 1970. In 1984, Dr. Borlaug began teaching and conducting research at Texas A&M University. Two years later, he created The World Food Prize to recognize the

achievements of individuals who have improved the quality, quantity, or availability of food. The prize was first awarded to Dr. Borlaug’s former colleague, Dr. M.S. Swaminathan, for his leadership and success in introducing and further developing high-yielding varieties of wheat in India. More than a decade before the advent of CRISPR, Dr. Borlaug stated, “...[T]he world has the technology – either available or well advanced in the research pipeline – to feed

On August 9, 2001, after months of lobbying from both sides, President George W. Bush gave an 11-minute speech from his ranch in Crawford, Texas, on the ethics and fate of U.S. federal funding for stem cell research. President Bush presented arguments in favor of and opposing embryonic stem cell research and explained his decision to limit but

on a sustainable basis a population of 10 billion people. The more pertinent question today is whether farmers and ranchers will be permitted to use this new technology.” Dr. Borlaug died at the age of 95 on September 12, 2009. His legacy lives on in the work being conducted at Texas A&M University and by farmers around the world who benefited from his and his team’s dedication and personal sacrifice in the pursuit of scientific knowledge.

not completely eliminate potential federal funding for the research. In May 2013, scientists were able to reprogram human skin cells to become embryonic stem cells capable of transforming into any other cell type in the body. The research breakthrough was led by Dr. Shoukhrat Mitalipov at the Oregon Health & Science University.

The first “test tube baby” On November 10, 1977, the world’s first embryologist, Jean Purdy, observed that an embryo in a petri dish had divided into eight cells. It was implanted in Lesley Brown, and after nine years of trying and failing to conceive, she became pregnant.

Louise Brown

Thirty-eight weeks later, her daughter, Louise Joy Brown, was born on July 25, 1978, in Oldham, England. Louise was the first of nearly 7 million babies (and counting) born by in vitro fertilization (IVF). “At school kids used to say, ‘How did I fit in the test tube?’” Louise recalled. Today, the practice that seemed so controversial 40 years ago is commonplace. And over those years, IVF techniques and other assisted reproductive technologies have steadily improved.

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(rDNA) and, years later, the results are referred to as genetically modified organisms, or GMOs. News of recombinant DNA raised concerns about its ramifications on human health and the Earth’s ecosystems, and by the middle of 1974 a moratorium on genetically engineered projects was observed. In 1975, experts came together at the Asilomar Conference Center in Pacific Grove, California, for the first International Congress on Recombinant DNA Molecules. The conference marked the beginning of a new era for science and for public discussion of science policy. It was agreed that the research should continue but under stringent guidelines. Despite controversy and concern, several commercial ventures were begun to explore the potential of rDNA. Among them was Genentech, founded in 1976 by Dr. Boyer and a venture capitalist named Robert Swanson. In September 1978, Genentech and City of Hope National Medical Center announced the successful production of human insulin using rDNA technology. And in 1982, Eli Lilly signed a joint-venture agreement with Genentech to develop a process using genetically engineered bacteria to synthesize human insulin. The result, Humulin, was a monumental achievement. Previously, the only way to produce insulin for human use was with pig and cattle pancreatic glands. In 1987, the first field experiments of genetically modified food crops using rDNA began. And in 1994, the Flavr Savr tomato became the first food crop approved for commercial production by the FDA. The tomato had an added DNA sequence that increased its firmness and extended its shelf life. In 1994, Dr. Maria Jasin at Memorial Sloan Kettering Cancer Center became the first to induce a double-strand break at a specific point in mammalian DNA using a restriction endonuclease – hailed as “the first key discovery in the Age of Editing.” In 1996, Monsanto introduced “Roundup Ready” or glyphosate-resistant plants, beginning with genetically engineered soybeans and quickly followed by other crops, including corn and sugar beets. In 2000, Golden Rice was developed through rDNA in an effort to combat a vitamin A deficiency (estimated to kill 670,000 children under the age of 5 each year). In 2002, molecular biologists developed a new technology called zinc-finger nucleases or ZFNs. And in 2011, transcription activator-like effector nucleases or TALENs were introduced. Both TALENs and ZFNs have the early lead in the number of gene-editing clinical trials, but as CRISPR-Cas therapies win approval, the fate of these platforms becomes less certain. 50

THE DISCOVERY OF CRISPR-CAS9

Like many useful tools in biotechnology, CRISPR-Cas9 was first discovered in bacteria. In 1987, Dr. Yoshizumi Ishino and his colleagues at Osaka University in Japan noticed a strange pattern of DNA sequences in an E. coli gene. The gene had five short, repeating segments of DNA separated by short, unique “spacer” DNA sequences. All five of the repeating segments had identical sequences that were the same back to front and front to back – palindromes. Biologists had never seen anything like it before. In August 1993, Dr. Francisco Mojica, a microbiologist at the University of Alicante in Spain, announced he had found similar sequences in archaea. Two years later, Dr. Mojica had sequenced the whole stretch of the tandem repeats, but it wasn’t until 2003 that he made a momentous discovery: A computer found a DNA match to a known viral sequence. As other matches were found, the idea that CRISPR was a kind of primordial immune system was put forward. CRISPR and the nature of basic science research

The discovery of CRISPR is a reminder that nature is full of secrets that can lead to new technologies. CRISPR was the product of undirected, curiositydriven laboratory research out of which came initial observations that, decades later, culminated in a “eureka!” moment. Of interest, a recent study found that the lag time between basic biological research and its application as a molecular biology technique was, on average, 23 years. CRISPR came in at roughly 25 years. The lag time for return on investment from basic research means that it is all but impossible to quantify the benefit of a specific basic research project because of

the cascading benefits that can result. Basic biological research is crucial for the development of innovative molecular biology techniques that bring about major scientific advances. But currently, basic research lacks adequate funding and is in a vulnerable position. Investment into basic science – the pursuit of pure research for research’s sake – provides the foundation for the future of applied research. The practical benefits might take years to show up – or they might never materialize at all – but the long-term payoffs can be immense. And any shortsighted decisions made today can negatively impact progress for years, if not decades.

The dairy industry uses the bacterium Streptococcus thermophilus to convert lactose into lactic acid, which gels milk. If a phage attacks the bacterium, it can interrupt the process and spoil the yogurt culture. In 2005, the Cas9 protein was discovered by Alexander Bolotin of the French National Institute for Agricultural Research. In 2007, Rodolphe Barrangou and Philippe Horvath were working at Danisco, a leading maker of yogurt cultures, when they found that S. thermophilus genomes contained the repeating DNA sequences Dr. Mojica had described. The team later discovered sequences that matched a portion of the attacking phages’ DNA and theorized that it allowed the bacteria to recognize and fight off phage attacks. The insight was used to create phage-resistant S. thermophilus with “battle-proven” CRISPR sequences already in place. In the summer of 2011, two researchers – Dr. Jennifer Doudna at UC Berkeley and Dr. Emmanuelle Charpentier at Umeå University in Sweden – met at a conference, realized their interests aligned, and

decided to collaborate. And in a seminal August 2012 article in Science, they and their labs revealed the exact mechanism of the CRISPR process, showing that a custom-made RNA could be directed to slice a DNA double helix wherever they wanted and, further, by “hijacking” the CRISPR-Cas9 system, they could delete a gene or insert a new one. Martin Jinek, a postdoc working in Dr. Doudna’s lab, later led a team at the University of Zurich showing how Cas9 unwound DNA into two strands before making a cut across the invading DNA. Dr. Virginijus Siksnys, a Lithuanian biochemist at Vilnius University, demonstrated that the CRISPRCas9 system could be transferred from one bacterium to another. In January 2013, the first reports of using CRISPRCas9 to edit eukaryotic cells in an experimental setting were published simultaneously by Dr. Feng Zhang of the Broad Institute and Dr. George Church of Harvard. And with that, a rush to explore the frontiers of CRISPR genome editing officially began.

The pioneers of CRISPR Left to right: Drs. Jennifer Doudna, Emmanuelle Charpentier, Feng Zhang, and George Church.

THE STORY of the discovery

When Dr. Jennifer Doudna was in sixth grade, her father gave her a copy of The Double Helix – James Watson’s account of his discovery of DNA – and she became thrilled by the mystery and drama of scientific research as Watson described it. After earning a bachelor’s degree at Pomona College, she went to Harvard, earning her Ph.D. in 1989, focusing on ribozymes. In 2002, she accepted a professorship at UC Berkeley. In 2011, she met Dr. Emmanuelle Charpentier, and a long-distance collaboration began in which they demonstrated that RNA molecules could be programmed to cut and edit targeted DNA molecules. Dr. Doudna has won numerous awards and cofounded Caribou Biosciences, Intellia Therapeutics, and Mammoth Biosciences. She has been an advocate for robust public discussion of the ethical implications of gene editing.

Dr. Emmanuelle Charpentier was born outside Paris in 1968. She attended the Pierre and Marie Curie University for both her undergraduate and graduate studies, receiving her Ph.D. in microbiology in 1995. As a postdoc, she worked in France and the U.S. In 2002, she returned to Europe to head a research group at the University of Vienna and studied RNA molecules associated with CRISPR that interacted with a DNA-slicing enzyme called Cas9. In 2009, she moved to Umeå University in Sweden. Her seminal research with Dr. Jennifer Doudna unveiled the key mechanisms of CRISPR- Cas9 technology, laying the foundation for its use as a versatile gene-editing tool. Numerous awards have recognized her work. Dr. Charpentier co-founded CRISPR Therapeutics and ERS Genomics, and she is the Director of the Max Planck Institute for Infection Biology in Berlin.

Dr. Feng Zhang was born in China in 1982, raised in Iowa, and became a star at Harvard and Stanford, where he was obsessed with finding the perfect way to reprogram human cells. Dr. Zhang didn’t speak English when his mother first brought him to Des Moines, Iowa, in 1993, but he impressed the coordinator of Roosevelt High School’s gifted and talented program. With his help, young Zhang was allowed to volunteer at a nearby gene therapy lab. Dr. Zhang was first to develop CRISPR to edit genes in animal and plant cells. His work has substantially expanded the CRISPR toolbox through discovery of new CRISPRs. His interests extend to brain disorders, especially complex disorders such as autism spectrum disorder and Alzheimer’s disease. Dr. Zhang has co-founded Editas Medicine, Sherlock Biosciences, Pairwise Plants, and Beam Therapeutics.

Dr. George Church is often left off a list of key CRISPR pioneers, but the scientific record is clear: Dr. Church published a paper in the same issue of Science as did his former postdoc Dr. Feng Zhang on the use of CRISPR technology in eukaryotic cells. While a student at Duke, Church helped determine the three-dimensional structure of transfer RNA. He spent so much time in the lab he failed a course and was expelled from the graduate program. Dr. Church is recognized for his contributions, including molecular multiplexing, homologous recombination methods, and array DNA synthesizers. In 1984, he developed the first direct genomic sequencing method and helped initiate the Human Genome Project. His discoveries have led to the co-founding of Editas Medicine, Gen9bio, eGenesis, and Veritas Genetics.

of CRISPR briefly described here is, by necessity, greatly simplified. Among many names not listed are: Drs. Krzystof Chylinski, Rudolf Jaenisch, J. Keith Joung, Eugene Koonin, Luciano Marraffini, Sylvain Moineau, John van der Oost, and Erik Sontheimer. But any serious discussion of how CRISPR became the geneediting tool it is today would be incomplete without recognizing the contributions of hundreds of individuals and their research teams. Several books are set for release that will do just that, among them one by biographer Walter Isaacson. Scientists from around the world contributed to CRISPR’s discovery, and thousands more will continue to develop and refine CRISPR technology and the applications that will ultimately define its success.

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The Business of CRISPR

CRISPR is more than a scientific revolution – it’s also a business revolution because

there are few industries it does not have the power to affect. Although the patent battle between UC Berkeley and the Broad Institute is ongoing, several companies – among them, Caribou Sciences, Editas Medicine, CRISPR Therapeutics, and Intellia Therapeutics – were founded on the initial CRISPR intellectual property (IP) rights granted to key individuals and institutions. Through the formation of alliances with industry leaders, these and other companies are working to develop improvements to Cas9 editing and non-Cas9 CRISPR systems and to discover new, patentable technologies. CRISPR’s potential has also resulted in an outpouring of financial backing for smaller companies hoping to utilize CRISPR in ways that would benefit not only human health but society as a whole. Supporting CRISPR development is a robust backend supply chain that can build things like gene-editor design tools and custom synthetic guide RNAs and deliver them to researchers’ labs to help them stay focused on what they do best. As of February 2019, more than 1,700 patents have been filed by hundreds of institutions, companies, and researchers. Some 100 new patent families (a set of patents taken across a group of countries to protect a single invention) on CRISPR are being published each month. Beyond innovative human therapeutics for serious disease, agricultural companies are using CRISPR to develop gene-edited crops and food products. And there are diverse areas of application for energy, diagnostic, and manufacturing companies. As a medical therapy, clinical testing to cure a rare blood disease and sickle cell disease has begun, and CRISPR-edited CAR T-cell therapies have shown great promise in creating a new generation of cancer therapies. The business of CRISPR, above all, is the business of transformation.

PERSPECTIVE

Dr. Jeffrey

LEIDEN

Dr. Leiden is the Chairman, President, and Chief Executive Officer of Vertex Pharmaceuticals. He is a physician and scientist who, for the past 30 years, has dedicated his career to improving the lives of people with serious diseases. His experience spans all aspects of the biotech and pharmaceutical industries.

A REVOLUTION IN THERAPEUTIC OPPORTUNITY AND RESPONSIBILITY When I started my career in medicine, I remember the frustration of taking care of patients for whom no effective treatment was available. As a cardiologist, I cared for countless heart attack patients for whom we lacked adequate prevention or therapy. I had to tell patients that an HIV infection was likely a death sentence. And I watched patients with rheumatoid arthritis (RA) become wheelchairbound despite the then-standard of care therapy of injectable gold. But over the course of my career, I have been fortunate to witness the discovery of statins, implantable defibrillators, and drug-coated stents for the treatment of heart attacks; the development of highly effective, triple combination antiretroviral therapies for HIV; and the advent of anti-TNF monoclonal antibodies for the treatment of RA and other immune diseases. CRISPR-mediated gene editing represents yet another extraordinary scientific advance that holds the potential for revolutionizing the treatment of multiple diseases. However, as with all scientific innovations, it brings with it an extraordinary ethical responsibility for the companies developing these novel CRISPR-based therapies. Today, multiple companies, including Vertex, are working with doctors and patients to test CRISPRbased therapies in clinical trials for serious diseases such as sickle cell disease, beta thalassemia, Duchenne muscular dystrophy, and cancer. The rapid and responsible translation from bench to bedside, from discovery to drug, is not simple or inevitable. It takes a massive investment of people, money, and time at-risk to create a robust therapeutic technology with the potential to help patients. The companies fueling this work need to balance their sense of urgency for new treatments with scientific rigor and a full understanding of both the potential efficacy and safety of these new modalities. A handful of companies have now launched the first human clinical trials of CRISPR-based medicines. Together, we have spent several years and billions of dollars working diligently to expand our understanding of this scientific tool to ensure that it can be safely and effectively brought to patients. As we continue to advance this technology in the clinic, we must work just as hard to answer basic questions such as how to increase the efficiency of gene editing and in particular gene correction; how to more efficiently deliver CRISPR therapies in vivo to different tissues; how to regulate CRISPR expression in vivo; and how to minimize any risks of off-target cutting. I’m confident that if we work together in an atmosphere of collaboration and transparency, we will answer these questions over the coming years and, by doing so, unlock the tremendous value of CRISPR-based treatments for patients and society. Seventy years ago, Dr. Linus Pauling and colleagues described sickle cell anemia as the first “molecular disease.” Today, biotech companies are testing the first true molecular medicine for the treatment of this disease. It’s exciting to consider that CRISPR might soon provide a one-time cure for patients who have been waiting for decades with this life-shortening disease. S O U T H W E S T E R N M E D I C A L P E R S P E C T I V E S . 2 019

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WHO OWNS CRISPR-CAS9?

The CRISPR patent dispute is unique. While universities have historically worked together to share innovation and knowledge, in the case of CRISPR there is too much at stake. And while venture capitalists and tech transfer offices typically require a clear intellectual property position before making any investment commitment, this has not been the case with CRISPR. The three leading companies in the space – CRISPR Therapeutics, Editas Medicine, and Intellia Therapeutics – have a combined market capitalization of $5 billion and combined funding exceeding $400 million despite ongoing patent conflicts between UC Berkeley and the Broad Institute at Harvard and MIT. Both institutions claimed IP rights to CRISPRCas9 technology shortly after its initial discovery in 2012. Dr. Jennifer Doudna from UC Berkeley and Dr. Emmanuelle Charpentier, now with the Max Planck Institute, filed their patent applications with the U.S. Patent and Trademark Office (USPTO) first, but the Broad Institute paid for their applications to be expedited. In 2014, a foundational patent for the use of the CRISPR-Cas9 system for gene editing in eukaryotic cells was awarded to the Broad. UC called for an interference hearing, claiming that Broad’s patent overlapped with one that Dr. Charpentier and the University of Vienna had filed for two years earlier. In June 2019, the USPTO changed its mind and declared interference between several pending patents belonging to UC Berkeley and those already awarded to the Broad Institute. Later that month, the USPTO

initiated a new interference process involving the UC Berkeley patent estate and its claims regarding eukaryotic cell editing. The dozens of new versions of RNA guides engineered by research teams make the navigation into the CRISPR system technology landscape even more complex.

LICENSING CRISPR-CAS9

While ownership of the patents continues to be determined, the institutions behind CRISPR, early on, seized the opportunity by entering into a series of license agreements. Each of the key CRISPR patent holders granted exclusive rights to a spinoff or “surrogate” company formed by the institution and one of its principal researchers. For licenses in human therapeutics, those companies are CRISPR Therapeutics, Intellia Therapeutics, and Editas Medicine. For all other CRISPR applications, the companies are ERS Genomics and Caribou Biosciences. Licensing CRISPR-Cas9 has fallen into three main areas. The first is for noncommercial research. Scientists can utilize the technology for basic research without paying licensing fees or royalties, unless or until they want to market the results of their work. The second is for the development of things that can assist research and development, such as toolkits, which offer greater flexibility, power, accuracy, and economy, as well as reagents and other services related to CRISPR-based gene editing. The third is for the development, sale, and use of therapeutics that utilize CRISPR-Cas9.

Published CRISPR patent applications (IPStudies, January 2019) MIT (US) HARVARD UNIV (US)

BROAD INST (US)

CHINESE ACAD SCI (CHINA)

UNIV CALIFORNIA (US) CHINESE ACAD AGRI SCI (CHINA) DUPONT (US) EDITAS MEDICINE (US)

SANGAMO THERAPEUTICS (US)

MASS GEN HOSP (US) CRISPR THERAPEUTICS (SWITZ)

69 51

STANFORD UNIV (US)

CHINA AGRI UNIV ( CHINA)

DANA FARBER CANCER INST (US)

NR SYSTEMS

151 147 131

115

162

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PERSPECTIVE

Dr. Larry

SCHLESINGER

Dr. Schlesinger is a Professor and President and CEO of Texas Biomedical Research Institute, whose mission is to eradicate the threat of infectious disease. Texas Biomed Drs. Tim Anderson, Smita Kulkarni, and Chris Chen also contributed to this article.

NEW APPROACHES AND OPPORTUNITIES FOR COMBATING INFECTIOUS DISEASE Infectious diseases have not gone the way of the dodo, as was cheerfully predicted during the last century following successful eradication of smallpox, widespread rollout of mass vaccination campaigns, and a generation of a crop of highly effective antibiotics. Instead, we face a raft of new – and old – infectious disease challenges. Multidrug-resistant, hospital-acquired infections now affect 1 in 25 patients; Ebola is now responsible for vast epidemics in Africa; and even common childhood diseases such as measles are returning with the rise of vaccine skepticism. The “big three” infectious diseases – tuberculosis, HIV, and malaria – still account for more than 2.5 million deaths per year. CRISPR evolved in bacteria to detect and destroy invading viruses; hence, it is entirely logical that we can turn these Perhaps the most significant role CRISPR properties against pathogens. will play in leading to new therapies lies in the CRISPR-based approaches are being developed for widespread adaption of this technology as a prevention, diagnosis, and treatment of infectious diseases. standard laboratory tool for understanding gene Sherlock Biosciences, Mammoth Biosciences, and Chan function in both the host and pathogen. Zuckerberg Biohub ( a collaborative effort by Berkeley, UC San Francisco, and Stanford) are developing commercial applications to combat infectious diseases. Newly discovered CRISPR systems, which detect double or single-stranded DNA and RNA, form powerful tools for pathogen diagnostics. These methods, such as DETECTR and SHERLOCK, are a million times more sensitive in detecting nucleic acids compared to the traditional diagnostic tools. And another CRISPR-based diagnostic tool called FLASH can rapidly identify common drug-resistant microbes. We anticipate that these methods will be customizable to any infection, will become commonplace for point-of-care infection diagnostics, and will allow rapid responses to contain disease outbreaks. It is a short step from detection to destruction of pathogens. CRISPR-based “search and destroy” approaches can remove viral DNA and RNA for chronic or latent viral infections such as HIV and the hepatitis B virus (HBV). Perhaps the most significant role CRISPR will play in leading to new therapies lies in the widespread adaption of this technology as a standard laboratory tool for understanding gene function in both the host and pathogen. At Texas Biomedical Research Institute, and in research laboratories worldwide, CRISPR has transformed our ability to probe disease biology and will play a fundamental role in the development of the next generation of infectious disease therapeutics.

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Bioethics and Regulation

G enome editing did not appear overnight.

Ethical and regulatory questions and concerns with regard to genetic manipulation have been raised for more than 40 years. Recombinant DNA and a cloned sheep set off alarms. Patients died from early gene therapy treatments. Genetically modified organisms (GMOs) have raised concerns – especially in food crops – and 39 countries have banned the cultivation of genetically modifed crops. Meanwhile, the use of embryonic stem cells has divided scientists, politicians, and religious groups. At the same time, for many, initial concerns over genetic and medical advances such as in vitro fertilization, preimplantation genetic diagnosis, and implanting animal parts in humans (notably heart valves from pigs) have largely dissipated. Science has always been a powerful force for positive change in society. The best science challenges our belief systems – pushing the boundaries of existing knowledge and offering humanity a future different from where it might otherwise go. The discovery of CRISPR presents such a change. Albert Einstein once said, “Most people say that it is the intellect which makes a great scientist. They are wrong: it is character.” In large measure we have seen the character of CRISPR scientists and bioethicists, who have embraced the responsibility to guide societal change – brilliant men and women who have engaged in a wise, patient, mindful, and passionate discussion that includes thoughtful advocacy and careful ethical scrutiny of their behavior and that of their peers. Can they or can government protect society against rogue scientists? They cannot. We have arrived at the moment when not just scientists and lawmakers but all of society must summon our collective moral acumen in order to navigate what might be humanity’s most profound ethical and regulatory challenge, not only for ourselves but as our responsibility to future generations. Be prepared for a lot of questions. The answers remain a work in progress. 56

PERSPECTIVE

Dr. David

BALTIMORE

Dr. Baltimore is considered one of the world’s most influential biologists. Awarded the Nobel Prize at age 37, Baltimore has influenced national science policy on such issues as recombinant DNA research and the AIDS epidemic. He served as president of the California Institute of Technology from 1997 to 2006, and is currently the President Emeritus and Robert Andrews Millikan Professor of Biology at Caltech.

THE ROAD FROM ASILOMAR TO NAPA, HONG KONG, AND BEYOND I have lived through and participated in one of the great intellectual revolutions of history. It is a revolution of the human ability to control its own genetic destiny. When I was born, in 1938, we knew nothing about how human characteristics were determined, how they were passed down to later generations, and how mistakes were made that led to inherited diseases. Today, not only do we understand these basic processes of life, we have reached the point where we can begin to control and modify them. We can take heredity into our own hands. This is forcing us to ask new questions: how far do we wish to go in controlling human destiny? Who is to decide the limits? I started out as an experimental scientist in 1960 when I entered graduate school. Watson and Crick had already We can take heredity into our own hands. taken the first step towards understanding the mechanisms This is forcing us to ask new questions: how far of heredity by discovering the structure of DNA. From that structure, one could guess that DNA held the instructions do we wish to go in controlling human destiny? for life in a coded form and in 1961 that code began to be Who is to decide the limits? unraveled. Through the sixties the newly christened molecular biologists filled out the details but we still lacked the ability to engineer DNA: to isolate it in segments, to cut it, to paste fragments together, and to reinsert DNA back into cells and organisms. That all came in the early 1970s when a group of scientists – each working on their own pet projects but together providing a tool box for manipulating DNA – produced a methodology called recombinant DNA technology. By the mid-1970s, the first chimeric DNA molecules were made and the abilities rapidly spread worldwide. I made my contribution discovering an enzyme able to copy RNA into DNA, thus providing scientists the ability to capture as DNA the information that encodes individual proteins, and opening up the field of biotechnology. I shared a Nobel Prize for that work, as did others who participated in generating the recombinant DNA revolution. Those were heady times for molecular biology, but there was a concern: could joining chimeric DNA molecules – that may never in the history of life on earth have been together – create a dangerous organism, one that might cause disease and be untreatable? That was an immediate concern, but looking down the road there were other concerns: biological warfare could be facilitated, genes might be used to create new forms of life, people might wish to genetically enhance their offspring. Members of the community of molecular biologists, remembering that some nuclear physicists had earlier tried to limit the development of nuclear weaponry, came together and called for a pause in certain types of potentially dangerous experiments, and for an international meeting to consider how to safely initiate these new experiments while limiting the

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PERSPECTIVE C ONT.

Baltimore

potential danger. This meeting was held in 1975 at the Asilomar Conference Center in California and was limited to the immediate issues of chimeric molecules, leaving other issues to future consideration. Ultimately, the concerns were found to be minimal and the technology was adopted by the whole community, producing an era of remarkable achievements. Comfort with new forms of biological experimentation began to break down soon thereafter. The first big break was the discovery of embryonic stem cells in 1981. These cells could reproduce a whole organism, they were totipotent. Deriving them required culturing cells from a very early embryo. This requirement generated opposition to such research on human embryos from people who consider it morally improper to sacrifice a human embryo, even one destined to die, for the purposes of research. In 2006, Yamanaka showed that many different kinds of cells could be made into cells with the characteristics of embryonic stem cells through a relatively simple procedure. This new technique greatly expanded work on early embryonic cells in experimental animals but not humans. For many years, various methods of gene editing were tried. Some worked in mice but were very clumsy and not applicable to humans. Later techniques that were simpler evolved but still required great skill and were not precise enough to use in human embryos. Then, starting in 2013, with a ground-breaking discovery involving a protein called Cas9 and a guide RNA called CRISPR, gene editing became a relatively simple procedure. This new capability presented the opportunity to alter human heredity and raised obvious ethical issues. A meeting was held in Napa Valley, involving a small number of scientists to initiate a process of careful consideration of the implications of the technology. It was decided there that this group was neither large enough nor diverse enough to respond to this challenge. A larger meeting was called for Washington, D.C., led by the U.S. Academies. This meeting, designated a summit, was attended by 500 people from around the world. It was agreed that no work should be done involving the implantation of altered embryos into the uterus of a woman until much more animal experimentation had been done demonstrating a high degree of safety. The organizing committee released a statement saying that “It would be irresponsible to proceed with any clinical use of germline editing unless and until (i) the relevant safety and efficacy issues have been resolved, based on appropriate understanding and balancing of risks, potential benefits, and alternatives, and (ii) there is broad societal consensus about the appropriateness of the proposed application.” Three years later, a second international meeting was held in Hong Kong. A similar statement was released saying that “The organizing committee concludes that the scientific understanding and technical requirements for clinical practice remain too uncertain and the risks too great to permit clinical trials of germline editing at this time. Progress over the last three years and the discussions at the current summit, however, suggest that it is time to define a rigorous, responsible translational pathway toward such trials.” This process is underway along with an ongoing debate as to whether this is the appropriate path. This is only the beginning of a new world in which the evolving techniques of molecular and cellular biology will allow previously inconceivable diagnostic, therapeutic, and preventative medical procedures. These methodologies will give humans remarkable control over their own genetics and physiology. However, we may opt for utilizing the techniques in limited ways, or even not at all, controlled by our moral and ethical judgments. Aldous Huxley, in Brave New World, projected a world in which political leaders determine how new technologies are employed – that is perhaps the scariest scenario we can imagine; we must keep control over these capabilities in the hands of the general populous to be sure that they are used in ways consistent with our values. 58

The ethical and regulatory implications for CRISPR-Cas technology are vast, but the following stand out:

APPROPRIATENESS

For many, guiding CRISPR’s ethical use is the concept of “providing appropriate benefit.” There are two distinct applications: therapeutic and enhancement. Appropriate therapeutic application could not only save lives and improve quality of life but reduce the emotional suffering of families with loved ones afflicted by genetic disease. And yet, even within the confines of somatic cell editing it raises serious questions. Where do we draw the lines? At all genetic diseases? At life-threatening diseases? At only diseases where there are no other options? Are there special exemptions to consider? If so, who decides? On the surface, using CRISPR for enhancement seems far less appropriate, yet the lines between therapy and enhancement can become blurred. If CRISPR were able to lower a person’s cholesterol or make him or her stronger, should there be a limit? Should it ever be appropriate to change your child’s eye color or make him or her grow taller? What if a therapy that increased the likelihood of living longer or cancer-free was possible? Does the answer depend only on whether the enhancements are heritable? And how do we deal with the fact that appropriateness will vary by country?

SAFE AND EFFECTIVE

How do we determine whether a CRISPR procedure is safe and effective? Every surgery – and CRISPR is

surgery at the molecular level – comes with varying degrees of risk. So how safe is safe enough? How much can be known? And how reliable will those answers be?

ACCESS AND COST

As CRISPR’s extraordinary potential begins to be realized, important questions as to who will be or should be the beneficiaries will come into view. Patient access to CRISPR-Cas therapies will almost certainly be applied unevenly around the world. Initially, some CRISPR therapies might run as high as $1 million, or more. Will treatment be available only to the wealthy? Or those with the best insurance? On the other hand, what is a fair price? Unlike chronic treatments, CRISPR-Cas therapies could cure. One way to evaluate a fair cost might be to compare it to an organ transplant. A kidney transplant can save hundreds of thousands of dollars in dialysis costs. Or, in the case of hemophilia B, a decade’s worth of clotting factor can cost $2.5 million, so even a one-time CRISPR therapy at $1 million – giving the body the ability to make its own clotting factor – would pay for itself in four years.

REGULATION

Despite consensus emerging from international summits and conferences, and legal guidance from governments around the world, there can be neither a guarantee nor failsafe control over the use of CRISPR technology. To what degree do we allow scientists to self-regulate? To what degree should we trust government to author visionary legislation? And what is the rest of society’s role?

Bioethics involves more than just humans Centuries of inbreeding have left many dog breeds with a severely limited gene pool. Bulldogs and Pugs suffer from breathing problems; Great Danes and other large dogs from joint problems; Boxers and Shar-Peis from skin and eye problems. The most popular dogs tend to have the most disorders. Labrador retrievers, for example, are prone to some 50 inherited conditions. Breeders agree that the loss of genetic diversity and extreme changes in certain regions of the genome make it difficult to improve a

Dalmatians display a propensity toward deafness and allergies.

breed’s health from within the existing gene pool. To protect the purity of each breed’s characteristics, enthusiasts defend a highly controlled process, regulating genetic lines and creating

Is curing patients a sustainable business model? registries that stipulate which animals can be bred. But the more limited the number of potential mates, the greater the chance a dog will be paired with a mate that shares similar genes. If both parents have the “bad” gene, the puppy will likely inherit the disease. Here’s the question: Because humans have engineered the dog breeds that give us their loyalty and unconditional love but are destined to suffer health problems – if in the future the genetic technology exists to fix those problems – don’t we have the moral obligation to do so?

In 2018, Goldman Sachs released a report called “The Genome Revolution,” which discussed in detail many aspects of the new generation of genomic-editing techniques like CRISPR. Among them was the idea that many diseases might be cured permanently.

“The potential to deliver ‘one shot cures’ is one of the most attractive aspects of gene therapy, genetically-engineered cell therapy and gene editing. However, such treatments offer a very different outlook with regard to recurring revenue versus chronic therapies,” wrote analyst Salveen Richter. The choice between creating a product that cures people after one use and another that requires a lifetime’s supply is one that disrupts business models. As genomic medicine advances, more discussion will follow, both behind closed doors and in public debates.

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The Future

The Human Genome Project mapped the code of life.

CRISPR-Cas technology – driven by curiosity, collaboration, and camaraderie and fueled by the competitive spirit inherent in the race to solve one of life’s most remarkable challenges and opportunities – has given humanity the ability to decipher that map and navigate our world toward extraordinary destinations. We leave you with a quote and a final perspective.

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“...it’s important to recognize the costs and risks of failing to explore the use of new tools such as CRISPR for global health and development. The benefits of emerging technologies should not be reserved only for people in developed countries. Nor should decisions about whether to take advantage of them. Used responsibly, gene editing holds the potential to save millions of lives and empower millions of people to lift themselves out of poverty. It would be a tragedy to pass up the opportunity.”

BILL GATES Co-Chair and Trustee Bill & Melinda Gates Foundation

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Excerpt taken from “Gene Editing for Good: How CRISPR Could Transform Global Development,” an article by Bill Gates that appeared in the May/June 2018 issue of Foreign Affairs magazine.

PERSPECTIVE

Kathleen M.

Kathleen M. Gibson is President and CEO of Southwestern Medical Foundation. Gibson is the 10th President in the Foundation’s 80-year history. Prior to joining the Foundation, she spent 30 years serving in leadership positions within Citibank and Bank of America.

GIBSON INSPIRING CURIOSITY

It is impossible to imagine how the conversation on gene editing will evolve over the coming year – let alone over the next ten or fifty years. The pace and acquisition of scientific knowledge has quickened. Its implications and uses are at the intersection of immense progress and daunting challenge. Science has made possible the ability for CRISPR-based technologies to transform the way we live and work, the plants and animals that inhabit our world, and our lives as a human species. At a fundamental level, it is curiosity that drives scientific innovation. CRISPR would not exist if microbiologists had not been curious enough to explore the function of the repeated DNA sequences they found in bacteria. The Rockefeller Foundation, Howard Hughes Medical Institute, The Bill and Melinda Gates Foundation, and our own Foundation have been key advocates and funders of curiosity, contributing to scientific advancement, medical progress, and service to humanity. Southwestern Medical Foundation’s support of basic science research and advancement at The conversations to educate the public and UT Southwestern has been a hallmark of our history. the resources to fund curiosity are essential. We live today in a period of extraordinary scientific advancement – one where the implications of CRISPR are difficult to predict. We will not only witness its progress but also share a responsibility to understand and debate its societal, medical, and ethical consequences. In a world changing at a pace unlike any other in human history, we consider the question: “What is our role as leaders? Is it to engage in the broadening worldwide CRISPR debate surrounding what is possible, what is right, what is ethical, what is safe, what are downstream implications, and who determines proper use?” With the health and education of the public and the mission of Southwestern Medical Foundation linked and intertwined, we believe the answer is “we must.” We are deeply grateful and honored that so many leaders in the field of science and medicine, business and ethics have joined us and agreed. Each of our writers contributed their own remarkable insights and wisdom. The conversations to educate the public and the resources to fund curiosity are essential. Our collective and continued voices advocating scientific progress and appropriate debate will prepare us for a future that goes beyond the limits of our present-day imagination. It is in this way that the respectful relationship between innovators, visionaries, and investors will sustain the legacy of unparalleled yet responsible achievement.

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Let the future be bright.

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INTERNATIONAL RECOGNITION

The Breakthrough Prize

“when

SECOND UTSW RESEARCHER RECEIVES AWARD

I was 12 years old, there was this saying that if you learn math,

physics, and chemistry then you can explore the whole world. For a kid coming from a small village, the possibility of exploring the whole world was very tempting.”

THE BREAKTHROUGH PRIZE trophy was created by Olafur Eliasson.

Zhijian “James” Chen, Ph.D.

“this award recognizes Dr. Chen’s outstanding research that elucidates the fundamental mechanisms of innate immunity, the body’s first generalized response to infection. His work has advanced our understanding of the relationship between the body’s immune defense system and autoimmune disease, as well as how this immunity pathway is linked to the development of cancer, cellular aging, and Parkinson’s disease.”

Daniel K. Podolsky, M.D. President, UT Southwestern Medical Center

ON NOVEMBER 4, 2018, Zhijian “James” Chen, Ph.D., was one of five researchers awarded a 2019 Breakthrough Prize in Life Sciences given annually to scientists who have made “transformative advances toward understanding living systems and extending human life.” Dr. Chen was recognized for his work on “elucidating how DNA triggers immune and autoimmune responses from the interior of a cell through the discovery of the DNA-sensing enzyme cGAS.” “It is very gratifying to see our discovery, which started with a curiosity and desire to help patients suffering from cancer, autoimmune diseases, and Parkinson’s disease, receive such recognition,” Dr. Chen said. T-cells and other white blood cells form the frontline fighters of the immune system. Dr. Chen’s research uncovered the inner workings of an underlying, innate immune system – operating out of every cell in our body – that triggers the deployment or over-deployment of the fight-back response to viruses, stress, and radiation. Dr. Chen’s lab displayed how DNA brought in by an invader, or seeping out of a cell’s nucleus, is sensed by a protein, that, ultimately, activates T-cells and white blood cells to do their job. Dr. Chen and his team are now working to take what they’ve learned to stop diseases like cancer as well as control attacks on healthy cells that happen in autoimmune disorders such as arthritis and lupus. Upon receiving the award, Dr Chen added, “I also want to say, to all the young people around the globe, ‘Hey, if I can do it, you can too.’”

Dr. Chen is a Howard Hughes Medical Institute Investigator, Professor of Molecular Biology and Director of the Center for Inflammation Research at UT Southwestern, and a member of the National Academy of Sciences. He also holds the George L. MacGregor Distinguished Chair in Biomedical Science. Dr. Chen is responsible for several important contributions, including the discovery of MAVS (mitochondrial antiviral signaling) protein, which plays a pivotal role in immune defense against RNA viruses such as those that cause influenza, hepatitis C, and Zika. Being a fan of the Dallas Mavericks, Dr. Chen named his discovery after the team.

Dr. Chen is the second UT Southwestern faculty member to win a Breakthrough Prize, the first being Dr. Helen Hobbs in 2016 for research that led to the swift development of a drug that lowers LDL high cholesterol, helping prevent heart disease. Her work was also instrumental in changing the methodology used by many genetic researchers. YURI AND JULIA MILNER, Sergey

Brin, Anne Wojcicki, Jack Ma, Cathy Zhang, Mark Zuckerberg, and Priscilla Chan founded the Breakthrough Prize, dubbed the “Oscar of Science,” in 2012. The award honors achievements in the categories of fundamental physics, life sciences, and mathematics. Every year, committees of prior laureates choose the winners from candidates nominated in a process that is online and open to the public. Laureates each receive $3 million in prize money. This year’s ceremony was hosted by actor Pierce Brosnan. Dr. Chen received his award from actress Rachel McAdams and Daniel Schulman, President and CEO of PayPal.

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‘‘ What the Foundation has meant to our city over the past eight decades is remarkable, and I’m so grateful for their leadership. Innovation is critical for our community’s success, and we’re at the leading edge because we’ve supported this institution.’’

Michael S. Rawlings

‘‘ My challenge for my peers is to become educated and carefully examine what matters to you. Now is the time to find the causes that stir your heart.’’

Josie Sewell

‘‘ I was raised to believe that we should all give back to others.... [ W ]e are all in one boat, and we will ultimately sink or sail together. We benefit our own families and friends, as well as others, by contributing to building a stronger community.’’

William T. Solomon

‘‘ The research happening at UT Southwestern is not just good for Dallas or Texas. What’s coming out of here is good for the world.’’

Robert B. Rowling

We lead. We engage. We honor. We give. We inspire. We nurture. And while the mission of the Foundation has evolved over the course of its 80-year history, the core of who we are and the vision of what we see for the future has never wavered. Assured through a simple idea: that by connecting with one another, we further the cause of public health, medical innovation, and research. We take a moment to highlight those who, in recent years, have led and who will lead. People who asked, â&#x20AC;&#x153;What if?â&#x20AC;? People who joined us to celebrate achievement, support the next generation of doctors, and encourage philanthropic leaders of tomorrow.

C onn ect S O U T H W E S T E R N M E D I C A L P E R S P E C T I V E S . 2 019

L ea d

Robert B. Rowling was honored

by Foundation Trustees as outgoing Chairman after five years of dedicated leadership and vision. Mr. Rowling began his service on the Board of Southwestern Medical Foundation in 2010. Since then he has given precious time and treasure serving the Foundation and UT Southwestern Medical Center in innumerable ways. Side by side his wife, Terry, countless acts of service have been given with a tremendous heart and passion to move the Foundationâ&#x20AC;&#x2122;s mission forward.

Thank you, Bob S O U T H W E S T E R N M E D I C A L P E R S P E C T I V E S . 2 019

Welcome, James

We are honored to welcome

James R. Huffines as Southwestern Medical Foundationâ&#x20AC;&#x2122;s Chairman of the Board. Mr. Huffines has been a member of the Board since 2011 and an active member of the Foundationâ&#x20AC;&#x2122;s Executive Committee and Nominating Committee. He has been a leading advocate and mentor for The Cary Council, a group of emerging young leaders driving awareness of the missions of Southwestern Medical Foundation and UT Southwestern Medical Center and raising funds to support early-stage research.

En gag e

Southwestern Medical

Foundation launched the series Leading the Conversation on Health at Old Parkland to highlight health care leaders and share their insights in an engaging, small-group setting.

Leading the Conversation on Health Scientific Discovery, Neuroscience, Cancer and Genetics, Mental Health, Medical Education U P CO M I N G

CRISPR Gene Editing, Biomedical Breakthroughs

S O U T H W E S T E R N M E D I C A L P E R S P E C T I V E S . 2 019

Now weâ&#x20AC;&#x2122;ve evolved

Leading the Conversation on Health into an ongoing dialogue. While our event series remains, weâ&#x20AC;&#x2122;ve added rich digital content, blog posts, and opportunities to engage regularly around important health topics.

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Honor Recently, Southwestern Medical Foundation presented The Sprague Award to Hoblitzelle Foundation for its decades of tremendous partnership and support.

The Sprague Award but the seed of a tremendous idea and a visionary ideal. From it, in time, will spring the steel, concrete and stone of a great city of mercy where haven may be found by all, the rich, and poor alike.â&#x20AC;? â&#x20AC;&#x153;THIS BEGINNING IS

Karl Hoblitzelle Founder, Hoblitzelle Foundation on signing the Southwestern Medical Foundation charter in 1939 76

The Sprague Award,

established in 1991, was named after Dr. Charles Cameron Sprague, the first President of UT Southwestern Medical Center and later President of Southwestern Medical Foundation. It remains the regionâ&#x20AC;&#x2122;s most prestigious award in health care philanthropy.

S O U T H W E S T E R N M E D I C A L P E R S P E C T I V E S . 2 019

Since its founding in 1942,

Hoblitzelle Foundation has contributed more than $230 million to social service, cultural, educational, and medical organizations in Texas. In so doing, it has served as a lasting force in building the state as we know it today.

Karl Hoblitzelle Founder, Hoblitzelle Foundation on signing the Southwestern Medical Foundation charter in 1939

Karl Hoblitzelle, a co-founder

of Southwestern Medical Foundation, believed in the unlimited potential of medical science to build a better community. He donated more than 70 acres of land on Harry Hines Boulevard to the Foundation in honor of his wife. The campus would serve as the home of the medical center we know today as UT Southwestern.

S O U T H W E S T E R N M E D I C A L P E R S P E C T I V E S . 2 019

G i ve

WHAT IS your

vision of a better world?

In a world where change

WHAT ARE the

is rapid and constant, it can be difficult to focus on the long game. But it holds true to say that each of us possesses an opportunity to make an impact on families and causes we hold dear.

the causes behind the issues?

WHAT ARE

WHAT IS the

change you wish to see?

Nearly 100 guests

Philip Cubeta, CLU ®, ChFC ®, AEP ®, MSFS, CAP ®, is the Sallie B. and William B. Wallace Chair in Philanthropy at The American College of Financial Services.

attended Life, Love, and Legacy: A Conversation with Phil Cubeta on the Old Parkland campus.

Photos and video from this event can be found online at swmedical.org/lifelovelegacy. 80

obstacles?

Phil — a nationallyrenowned expert on philanthropy and planned giving — explored the concept of our lasting gifts...how each of us can plan our impact for good.

Legacy Tribute

In the early 1900s, Foundation leaders asked the question, “Why not a great medical center in

the Southwestern U.S.?” Their vision of quality medical education and research has transformed our region and provided a remarkable legacy of scientific achievement and advancement of care in our region.

LEADERS WHO HAVE A S K E D “ W H Y N O T?”

In the 1990s, Bill Solomon began leading

campaigns to benefit UT Southwestern. He knew that quality research would inform quality care – and he knew the importance of the patient experience as new hospitals were being designed. Through leadership and partnership with the Medical Center, the Solomons have helped build an extraordinary legacy of impact.

Beth Kahn was always a “why not” person.

When she faced glioblastoma, an aggressive form of brain cancer, she asserted that her diagnosis would not define her. Through her family, her legacy lives on. Her son, Michael Kahn, became the founder of “The Cary Council,” a group of emerging young leaders building the next generation of advocates for Southwestern Medical Foundation and UT Southwestern Medical Center.

ulie and Ken Hersh lead with tremendous courage and example. On something as big as mental health, they step in to ask – “Why not early diagnosis – like we have for breast cancer? Why not mental health support in general physicians’ offices and schools?” Partnering with UT Southwestern, they have provided significant advocacy and support for the founding of the Center for Depression Research and Clinical Care. Their vision helped ensure a critical component of the Peter O’Donnell Jr. Brain Institute on mental health. S O U T H W E S T E R N M E D I C A L P E R S P E C T I V E S . 2 019

The Heritage Society

The Heritage Society honors those who have named Southwestern Medical Foundation or UT Southwestern as

a beneficiary in their estate plans. Charles C. Sprague, M.D., while Chairman of the Foundation, first envisioned The Heritage Society, writing: â&#x20AC;&#x153;It is our hope and intention that our mutually beneficial partnership of the past will grow and prosper in the future; all of society will be the beneficiary.â&#x20AC;? Anonymous (54) Joyce T. Alban

Curran

Dick and Jacqueline Grote L. Ruth Guy, Ph.D.#

Mr.# and Mrs. James R. Alexander

Edwin R. Daniels*#

Rolf and Ute Haberecht

George A. Atnip#

Clarice Davis

Nancy and Jeremy Halbreich

Marilyn Augur*

Doris Russell Dealey*#

Sydney# and Wallace# Hall*

Paul M. Bass*#

Johann Deisenhofer, Ph.D. and

Nancy B. Hamon*#

Marsha and Michael Baylor Dr. and Mrs. James Harold Bearden W. Robert Beavers, M.D. Drs. Paul R. and Rebecca B.

Kirsten Fischer Lindahl, Ph.D. Kathleen M. and George N. DeMartino

John P. Harbin# Dr.# and Mrs. Thomas W. Harris* Art and Shirley Hastings

Anne and Brian Dethrow

Joyce A. Hendrickson

Mr. and Mrs. Robert M. Dickson

Helen B.# and Arthur E. Hewett

Michael H. Bertino, M.D.*

Paula Barshop Donovitz

Mr.# and Mrs. Donald R. Hibbert*

Josephine L. Biddle#

Grant A. Dove#

Lyda Hill

Harvey Birsner, M.D.#

Joyce Allison Eberts and

J. Roger# and Dorothy A. Hirl

Bergstresser

Jules Bohnn, M.D.*

John P. Eberts, M.D.

James M. Hoak

Jean# and Bill# Booziotis

Mack M. Elliott#

Edmund M. Hoffman*#

Beth Ann Borden

Gene# and Charlotte Emery

Debra Hooton

Nancy L. Branch

Harry M. (Monty) Evans Jr. and

Mr. and Mrs.# S. Roger Horchow

Mr. & Mrs. Robert R. Brockman/

Judy B. Evans#

Drs. Susan Hotz# and Michael

Brockman-Scruggs

Pamela and Roy Gene Evans*

Family Charitable Fund

Edith Rossi Fekete, M.D.

Dr. J.B. Howell#

Carol A. Brown, M.D.*

Richard Ferguson*#

William C. Huber#

Cherie Brown

Dave and Lori Folz

Keith and Cherie Hughes

H. Ray and Paula P.# Calvert

Mrs. Lee Ford

Lory Huitt-Masters

Sandra T. Campbell

Robert G. Freeman, M.D.#

Robert and Myra Hull

Antonio J. Campdera*#

Katherine L. Freiberger

Mrs. Morris I. Jaffe*#

W. Plack Carr Jr.*

Gretchen# and Gerald Fronterhouse

Berneice C. Johnson#

Bernard H. Chaiken, M.D.#

Dr. and Mrs. Norman F. Gant

Judith K. Johnson*

Dr. and Mrs. Anthony C. Chang

Mr. and Mrs. John Robert

Catherine R. Judd

Emogene B. Clardy#

Kevin Curran, M.D. and Shari

Gavlick Sr.

Shiekh

Mrs. Robert S. Junger#

Mr.# and Mrs.# Robert R. Click

Celia and Adi# Gazdar

Ken and Lynn Keefe

Phyllis M. Coit

David Ginn, M.D.*

Marjorie Kennedy#

Mimi and John Cole

Dr. and Mrs. T. Franklin Glass

Judge James W. Kerr Jr.

David W.# and Patricia M. Craig

Mr.# and Mrs.# F.B. Pete Goldman*

Mr. and Mrs. Thomas J. Kiernan

Frank Crawford, M.D.#

Mr. and Mrs. Joe M. Graham

Rollin W.# and Mary Ella# King

Dorothy R. Cullum*#

G. Thomas Graves III

Jack M. and Carole V. Kinnebrew

Christine Kumpuris*#

Patricia M. Patterson*

William T. Solomon*

Carol Kyler

Billy Joe Pendley#

Alayne W. Sprague

Wright L. Lassiter, Jr.*#

Beki J. Picus

Charles C. Sprague, M.D.*#

Mr. and Mrs. John Ridings Lee

Dr. and Mrs. David J. Pillow

Ronald G. Steinhart*

Will and Liza Lee

Kurt L. Plaut

Eleanor P. Stevens#

Sloan and Mary Lois Leonard

Shirley Pollock*#

S.C. Stewart, M.D.*#

Joan M. Lord

Doris E. Porter, PT

Marvin J. Stone, M.D.

Willis C. Maddrey, M.D. and

Mrs. Ashley (Kathryn) Priddy#

Sally Seay Stout*#

John Proffitt, M.D.

Thomas A. Sullivan

Geana Madison

Muriel Rabiner

Barbara C. and Robert P. Sypult

Jane Maryoung, P.T.

W. Paul Radman, D.D.S.

John G. Taylor

Nelson L. Mauldin

Nancy Carol Reddick*#

Dr. and Mrs. William W. Turner Jr.

Mr. and Mrs. C. Thomas May Jr.

Daniel Remahl

Douglas H. Unger, M.D.*

James M.# and Rosalee# McConnell

Tom B. Rhodes*#

Paul R. Vanatta, M.D.

John and Melinda McConnell

Frank Ribelin#

Robert W. Vaughan, M.D. and

Peter A. McCullough, M.D.,

Dr. and Mrs. Leonard Riggs Jr.

Ann Matt Maddrey, Ph.D.#

M.P.H.* Mr. and Mrs.# Thomas E. McCullough

Marjorie Sue Vaughan, Ph.D., RN

Jack D. Russell#

Howard and Marsha Vestal

Mr. and Mrs. John Carl Rutledge

Claire Elaine Vial and

Eleanor R. Salomon

Robert G. Vial#

Christopher F. McGratty

Stephen Raymond Salomon

Margaret Bright Vonder Hoya

Carmen Crews McCracken

Hortense# and Morton# Sanger

Irene Wadel#

Lorraine Sulkin Schein#

Carolyn W.# and Thomas C. Walker

Dr.# and Mrs.# John W.

Tim Wallace

McMillan Anne H. McNamara Ferd C. and Carole W. Meyer

Schermerhorn

Jean and Tom Walter

Kathryn B. Montgomery

Mr. and Mrs. William L. Schilling

Mr.# and Mrs. Richard L. Walton

William R. and Anne E.

Hans J. Schnitzler

Dr. Elgin W.# and Karen G. Ware

F. Michael Schultz, M.D.*

Dr. and Mrs.# Clark Watts*

Drs. Bert Moore# and Lynne Kirk

Bette Claire Schuttler#

Garry Weber

Kay Y. Moran

Sarah M.# and Charles E.# Seay*

Arthur G. Weinberg, M.D.

Jeff and Karen Morris

Ellen Taylor Seldin, M.D.

Pauline Weinberger*#

Mrs. Paula M. Mosle

William D. Seybold, M.D.*#

Vicki Whitman Wheeler*

Barbara and Robert Munford

George and Shirley Shafer

Mr. and Mrs. Dennis White

Robert H. Munger#

Wanda S. Shannon

Linda Poe White

Louis Nardizzi, M.D., Ph.D.*

Doyle L. Sharp, M.D.*#

Mr. Lawrence E. Whitman*#

Edward Neupert III, D.D.S.

Lynne and Roy Sheldon

Evelyn Whitman-Dunn*

Gerard Noteboom, M.D.#

Tom and Dorothy Shockley

Florence L.# and Frederic F.#

Rhea T. Oâ&#x20AC;&#x2122;Connor*#

Mr.# and Mrs. George A. (Tom)

Montgomery

Thomas F. Oâ&#x20AC;&#x2122;Toole

Shutt*

Wiedemann Dr. and Mrs. Kern Wildenthal

Pan Family

John S. Smale, M.D.#

Colleen and James Williams

Mrs. Sam Papert Jr.#

Dr. and Mrs. Neal C. Small

Karol Lynn Wilson#

Thomas J. Parr, M.D. and

Elizabeth Solender and

Terry M. Wilson*

Joannie Parr Selma L.# and I. Benjamin# Parrill

Gary L. Scott Ellen K.# and Robert L.# Solender*

Mr.# and Mrs.# Ivor P. Wold * Charter Member # Deceased S O U T H W E S T E R N M E D I C A L P E R S P E C T I V E S . 2 019

Dr. Joseph L. Goldstein

was awarded the Ho Din Award in 1966. Along with Dr. Michael Brown, Dr. Goldstein went on to win the 1985 Nobel Prize in Physiology or Medicine.

I n spir e The Ho Din Award Dr. Lauren Elizabeth Kolski with Rena Pederson, Southwestern Medical Foundation Trustee

he Ho Din Award, which exemplifies human understanding and medical wisdom, was established in 1943 at the first meeting of Southwestern Medical Collegeâ&#x20AC;&#x2122;s Board of Trustees. The award was created to distinguish those with a deep concern for the welfare of others and a boundless commitment to alleviate suffering. The Ho Din Award significantly raised the bar of excellence for students and to this day remains the highest honor that a UT Southwestern graduate can receive.

Dr. Philip Tolley with Jere Thompson Jr., Southwestern Medical Foundation Trustee

Dr. Bethany Werner with Dr. Marvin Stone, Southwestern Medical Foundation Trustee

S O U T H W E S T E R N M E D I C A L P E R S P E C T I V E S . 2 019

AN EVENING W I T H D O C S TA R S

N ur ture The Cary Council

The Cary Council is a group of

emerging young leaders supporting the missions of Southwestern Medical Foundation and UT Southwestern Medical Center and raising funds to support early-stage research.

Thanks to the success of its

signature fundraising event, â&#x20AC;&#x153;An Evening with DocStars,â&#x20AC;? The Cary Council has awarded significant grants to support research of young investigators.

S O U T H W E S T E R N M E D I C A L P E R S P E C T I V E S . 2 019

LEARNING FROM LEADERS

LEARNING F R O M I N N O V AT O R S

Acknowledgments

San Franciso, CA MARCY DARNOVSK Y, PH.D. JENNIFER A . DOUDNA , PH.D. WILLIAM HURLBUT, M.D. MARSHA SA XTON, PH.D.

Boston, MA GEORGE M. CHURCH, PH.D. JEFFREY LEIDEN, M.D., PH.D. FENG ZHANG, PH.D.

Bethesda, MD FR ANCIS S. COLLINS, M.D., PH.D. Raleigh, NC RODOLPHE BARR ANGOU, PH.D.

Pasadena, CA DAVID BALTIMORE, PH.D.

San Antonio, T X L ARRY SCHLESINGER, M. D.

Dallas, T X ERIC OLSON, PH.D. CHRISTI AND BEN DUPREE DANIEL K. PODOLSK Y, M.D.

Melbourne, Australia CHRISTOPHER GYNGELL , PH.D.

( Countries not shown in relative scale )

SPECIAL THANKS

This issue of Perspectives tackled one of science’s biggest and arguably fastest-moving breakthroughs: CRISPR-Cas technology and its potential impact on the world in which we live. It would not have been possible without the contributions of incredible people – scientists, researchers, business leaders, bioethicists, activists, and big sisters who gave of their time to share their thoughts. To each of them, we offer our sincerest thanks. WITH APPRECIATION

The Dupree Family Joseph L. Goldstein, M.D. UT Southwestern Office of Institutional Advancement UT Southwestern Office of Development UT Southwestern Office of Communications, Marketing, and Public Affairs REFERENCES Aguilera, Mario. “UC San Diego Researchers First to Use CRISPR/Cas9 to Control Genetic Inheritance in Mice: Technology Offers Powerful New Genetic Tools for Human Disease Research in Rodents.” UC San Diego News Center, 23 Jan. 2019, ucsdnews.ucsd.edu/pressrelease/uc_san_ diego_researchers_first_to_use_crispr_cas9_ to_control_genetic_inheritance_in_mice. Barber, Gregory. “A More Humane Livestock Industry, Brought to You by CRISPR.” Wired, 19 Mar. 2019, www.wired.com/story/crispr-gene-editing-humane-livestock/. Beltran, James. “CRISPR Halts Duchenne Muscular Dystrophy Progression in Dogs.” UT Southwestern Medical Center Newsroom, 30 Aug. 2018, www.utsouthwestern.edu/newsroom/ articles/year-2018/dmd-dogs.html. ---. “Scientists Now on Cusp of Solving Genetic Diseases by Snipping Defective DNA.” CenterTimes+, 26 June 2019, ct.utsouthwestern.edu/ ctplus/stories/2019/dna-genetic-disease.html.

Bier, Dan. “Mosquitoes Are the Deadliest Animals in History. Should We Wipe Them Out?” Freethink, 4 Feb. 2019, www.freethink.com/articles/ mosquitoes-are-the-deadliest-animals-in-historymaybe-we-should-kill-them-all. Bomgardner, Melody M. “CRISPR: A New Toolbox for Better Crops.” Chemical & Engineering News, vol. 95, no. 24, 12 June 2017, pp. 30-34, cen.acs. org/articles/95/i24/CRISPR-new-toolbox-bettercrops.html. Callaway, Ewen. “Controversial CRISPR ‘Gene Drives’ Tested in Mammals for the First Time: Experiments in Mice Suggest That the Technology Has a Long Way to Go Before Being Used for Pest Control in the Wild.” Nature, vol. 559, 6 July 2018, p. 164, DOI: 10.1038/d41586-018-05665-1. Chatsko, Maxx. “Where Will CRISPR Therapeutics Be in 5 Years?” Yahoo! Finance, 25 July 2019, finance.yahoo.com/news/where-crispr-therapeutics-5-years-152059555.html. Cohen, Jon. “China’s CRISPR Push in Animals Promises Better Meat, Novel Therapies, and Pig Organs for People.” Science, 31 July 2019, DOI: 10.1126/science.aay9194. Cohen, Jon. “New ‘prime’ genome editor could surpass CRISPR.” Science, 21 Oct. 2019, https:// www.sciencemag.org/news/2019/10/new-primegenome-editor-could-surpass-crispr. Columbia University Irving Medical Center. “New Gene Editor Harnesses Jumping Genes for Precise DNA Integration.” ScienceDaily, 12 June 2019, www.sciencedaily.com/releases/2019/06/190612141309.htm. Colwell, Brian. “Biotechnology Timeline: Humans Have Manipulated Genes Since the ‘Dawn of Civilization.’” Genetic Literacy Project, 18 July 2017, geneticliteracyproject.org/2017/07/18/ biotechnology-timeline-humans-manipulating-genes-since-dawn-civilization/.

Contreras, Jorge L., and Jacob S. Sherkow. “CRISPR, Surrogate Licensing, and Scientific Discovery.” Science, vol. 355, no. 6326, 17 Feb. 2017, pp. 698-700, DOI: 10.1126/science.aal4222.

De Almeida, Melanie. “CRISPR/Cas9: Could the Gene Editing Technology Be the Future of Drug Discovery?” Labiotech.eu, 2 June 2018, labiotech.eu/features/crispr-cas9-drug-discovery/.

Cribbs, Adam P., and Sumeth M.W. Perera. “Science and Bioethics of CRISPR-Cas9 Gene Editing: An Analysis Towards Separating Facts and Fiction.” Yale Journal of Biology and Medicine, vol. 90, no. 4, 19 Dec. 2017, pp. 625-634, www.ncbi.nlm.nih. gov/pmc/articles/PMC5733851/.

Dutton, Gail. “Gene Editing Startups Fabricate Industrial-Grade CRISPR Tools: The CRISPR Equivalent of Tool-and-Die Specialists Are Taking Gene Editing to the Manufacturing Scale.” Genetic Engineering & Biotechnology News, vol. 39, no. 6, June 2019, www.genengnews.com/ insights/gene-editing-startups-fabricate-industrial-grade-crispr-tools/.

CRISPR.” PubMed.gov, 23 Oct. 2019, www.ncbi. nlm.nih.gov/pubmed/?term=crispr. “The CRISPR Revolution: What You Need to Know – 5 Questions About Gene Editing with Samuel Sternberg.” Columbia U Irving Medical Center, 20 March 2019, www.cuimc.columbia.edu/news/ crispr-revolution-what-you-need-know. “CRISPR to Be Used to Fight a Type of Blindness.” WebMD, 26 July 2019, www.webmd.com/ eye-health/news/20190726/crispr-to-be-used-tofight-a-type-of-blindness. Cynober, Timothé. “CRISPR: One Patent to Rule Them All.” Labiotech.eu, 2 Nov. 2019, labiotech. eu/features/crispr-patent-dispute-licensing/. Cyranoski, David. “CRISPR-Baby Scientist Fails to Satisfy Critics: He Jiankui Gives Talk About Controversial Claim of Genome Editing Babies, but Ethical Questions Remain.” Nature, vol. 564, pp. 13-14, 28 Nov. 2018, DOI: 10.1038/d41586018-07573-w. ---. “Russian Biologist Plans More CRISPR-Edited Babies: The Proposal Follows a Chinese Scientist Who Claimed to Have Created Twins from Edited Embryos Last Year.” Nature, vol. 570, pp. 145-146, 10 June 2019, DOI: 10.1038/d41586019-01770-x.

Enzmann, Brittany. “CRISPR Editing Is All About DNA Repair Mechanisms.” Synthego, 30 May 2019, www.synthego.com/blog/crispr-dna-repair-pathways. Enzmann, Brittany L., and Ania Wronski. “How CRISPR Is Accelerating Drug Discovery.” Genetic Engineering & Biotechnology News, vol. 39, no. 1, Jan. 2019, www.genengnews.com/insights/ how-crispr-is-accelerating-drug-discovery/. “ExxonMobil and Synthetic Genomics Algae Biofuels Program Targets 10,000 Barrels Per Day by 2025.” Synthetic Genomics, 6 Mar. 2018, www. syntheticgenomics.com/exxonmobil-and-synthetic-genomics-algae-biofuels-program-targets-10000-barrels-per-day-by-2025/. Fan, Shelly. “Controversial ‘Gene Drives’ Just Worked in Mammals for the First Time.” Singularity Hub, 6 Feb. 2019, singularityhub. com/2019/02/06/controversial-gene-drives-justworked-in-mammals-for-the-first-time/. ---. “CRISPR Used in Human Trials for the First Time in the US.” Singularity Hub, 2 May 2019, singularityhub.com/2019/05/02/crispr-used-inhuman-trials-for-the-first-time-in-the-us/.

---. “The Hunt for a CRISPR Antidote Just Heated Up.” Singularity Hub,15 May 2019, singularityhub. com/2019/05/15/the-hunt-for-a-crispr-antidotejust-heated-up/. Fikes, Bradley J. “Synthetic Genomics and ExxonMobil Double Biofuel Yield from Algae.” San Diego Union-Tribune, 19 June 2017, www. sandiegouniontribune.com/business/biotech/ sd-me-genomics-biofuel-20170619-story.html. Fred Hutchinson Cancer Research Center. “Targeting a Blood Stem Cell Subset Shows Lasting, Therapeutically Relevant Gene Editing.” ScienceDaily, 31 July 2019, www.sciencedaily. com/releases/2019/07/190731145815.htm. Gallegos, Jenna. “Using CRISPR to Design Superior Foods.” Alliance for Science, 1 May 2018, allianceforscience.cornell.edu/blog/2018/05/ using-crispr-design-superior-foods/. Gardner, Heidi. “CRISPR Boosts Flavor and Nutrition in Tomatoes & More CRISPR News.” Synthego, 4 Oct. 2018, www.synthego.com/blog/ plant-science-tomatoes-crispr. Gates, Bill. “Gene Editing for Good: How CRISPR Could Transform Global Development.” Foreign Affairs, May/June 2018, www.foreignaffairs.com/ articles/2018-04-10/gene-editing-good. ---. “Test-Tube Mosquitoes Might Help Us Beat Malaria.” Gates Notes, 15 April 2019, www. gatesnotes.com/Health/Test-tube-mosquitoesmight-help-us-beat-malaria. Geggel, Laura. “Can Gene Editing Save the World’s Chocolate? Scientists Are Racing to Save Cacao Trees from Devastating Viruses and Fungi.” Scientific American, 5 Jan. 2018, www. scientificamerican.com/article/can-gene-editingsave-the-worlds-chocolate/. “The Genetic Breakthrough That Could Change Humanity, Explained.” The Week, 16 Jan. 2016, theweek.com/articles/599237/genetic-breakthrough-that-could-change-humanity-explained. Groover, Traigh. “What a Recent Breakthrough Means for Therapeutics Companies.” Clarkston Insights, 12 July 2019, clarkstonconsulting.com/ insights/crispr-breakthrough/. Harper, Rachael. “CRISPR-Cas Used to Edit Cystic Fibrosis Gene Mutations.” Drug Target Review, 8 Aug. 2019, www.drugtargetreview.com/ news/47502/crispr-cas-edits-mutations-cystic-fibrosis/. Harvard Medical School. “Saving Beethoven: Preventing Hereditary Deafness; Optimized Gene-Editing Tool Prevents Hearing Loss in Mice with Hereditary Deafness Without Detectable Off-Target Effects.” ScienceDaily, 3 July 2019, www.sciencedaily.com/releases/2019/07/190703121434.htm. “Healthcare Is Only the Beginning: 15 Big Industries CRISPR Technology Could Disrupt.” CB Insights, 1 Aug. 2018, www.cbinsights.com/ research/crispr-industries-disruption/. Hirsch, Francois, et al. “Ethics Assessment in Research Proposals Adopting CRISPR Technology.” Biochemia Medica, vol. 29, no. 2, June 2019, DOI: 10.11613/BM.2019.020202. “History of Genetic Engineering and the Rise of Genome Editing Tools.” Synthego, www.synthego. com/learn/genome-engineering-history. Houser, Kristen. “Russian Biologist Confirms He’s Working on More CRISPR Babies.” Futurism, 18 Oct. 2019, https://futurism.com/neoscope/ russian-biologist-more-crispr-babies. Lewis, Ricki. “Gene Therapy Challenge: How Much Should It Cost and How Do We Pay for It?” Genetic Literacy Project, 12 Dec. 2017, geneticliteracyproject.org/2017/12/12/gene-therapy-challenge-howmuch-cost-pay/.

Le Page, Michael. “Exclusive: Five Couples Lined Up for CRISPR Babies to Avoid Deafness.” New Scientist, 4 July 2019, www.newscientist.com/ article/2208777-exclusive-five-couples-lined-upfor-crispr-babies-to-avoid-deafness/.

Rana, Preetika. “How a Chinese Scientist Broke the Rules to Create the First Gene-Edited Babies.” Wall Street Journal, 10 May 2019, www.wsj.com/articles/how-a-chinese-scientist-broke-the-rules-tocreate-the-first-gene-edited-babies-11557506697.

Mackenzie, Ruairi J. “CRISPR Diagnostics Could Detect Any Disease on a Paper Strip.” Technology Networks, 26 Apr. 2018, www. technologynetworks.com/genomics/news/crisprdiagnostics-could-detect-any-disease-on-a-paperstrip-300175.

“Recombinant DNA.” What Is Biotechnology? www.whatisbiotechnology.org/index.php/science/ summary/rdna.

Molteni, Megan. “The Wired Guide to CRISPR: Everything You Need to Know About How Scientists Can Repurpose a Bacterial Immune System to Alter DNA, Making Everything from Cheap Insulin to Extra Starchy Corn.” Wired, 12 Mar. 2019, www. wired.com/story/wired-guide-to-crispr/. ---. “With Embryo Base Editing, China Gets Another CRISPR First.” Wired, 21 Aug. 2018, www.wired. com/story/crispr-base-editing-first-china/. Mullin, Emily. “Back to the Future: Pre-CRISPR Systems Are Driving Therapies to the Clinic.” Genetic Engineering & Biotechnology News, vol. 39, no. 2, Feb. 2019, www.genengnews.com/insights/ back-to-the-future-pre-crispr-systems-are-drivingtherapies-to-the-clinic/. ---. “CRISPR Detectives: Startups Seek to Expand Access to Diagnostics with Inexpensive CRISPR-Based Tests.” Clinical OMICs, vol. 6, no. 4, July/Aug. 2019, www.clinicalomics.com/ magazine-editions/volume-6-issue-number4-july-august-2019/crispr-detectives-startups-seek-to-expand-access-to-diagnostics-with-inexpensive-crispr-based-tests/.

Regan, Helen, et al. “The Scientist, the Twins and the Experiment that Geneticists Say Went Too Far.” CNN, 1 Dec. 2018, www.cnn.com/2018/11/30/ health/gene-edited-babies-he-jiankui-intl/index. html. Ronai, Isobel, and Paul E. Griffiths. “The Case for Biological Research.” Trends in Molecular Medicine, vol. 25, no. 2, Feb. 2019, pp. 65-69, DOI: 10.1016/j. molmed.2018.12.003. Ryan, Jackson. “Using CRISPR to Resurrect the Dead: Gene-Editing Breakthroughs Could Allow Us to Bring Extinct Species, Like the Woolly Mammoth, Back from the Dead. But Should We?” CNET, 19 June 2019, www.cnet.com/features/ using-crispr-to-resurrect-the-dead/. Sanders, Robert. “DNA Repair After CRISPR Cutting Not at All What People Thought.” Berkeley News, 30 July 2018, news.berkeley.edu/2018/07/30/ dna-repair-after-crispr-cutting-not-at-all-whatpeople-thought/. Scott, Andrew. “How CRISPR Is Transforming Drug Discovery: Gene Editing Is Quietly Revolutionizing the Search for New Drugs.” Nature, vol. 555, 7 Mar. 2018, pp. S10-S11, DOI: 10.1038/d41586018-02477-1.

Vinyard, Michael. “CRISPR-Scanning Towards New Drugs – Drug Discovery Is Difficult, but CRISPR Might Be Able to Help.” SITNBoston, 14 Mar. 2019, sitn.hms.harvard.edu/flash/2019/crispr-scanningtowards-new-drugs-drug-discovery-is-difficult-butcrispr-might-be-able-to-help/. Wanjek, Christopher. “Brain Cancer’s ‘Immortality Switch’ Turned Off with CRISPR.” Live Science, 10 Sept. 2018, www.livescience.com/63534-cancer-immortality-switch-gabp.html. Weintraub, Karen. “CRISPR Gene-Editing Will Change the Way Americans Eat – Here’s What’s Coming.” The Guardian, 30 May 2019, www. theguardian.com/us-news/2019/may/30/crispr-gene-edited-food-technology-us-produce. “What We Do: Malaria Strategy Overview.” Bill & Melinda Gates Foundation, www.gatesfoundation.org/what-we-do/global-health/malaria. Wormser, Deborah. “CRISPR Screen Identifies Gene That Helps Cells Resist West Nile, Zika Viruses.” UT Southwestern Medical Center Newsroom, 17 Sept. 2018, www.utsouthwestern. edu/newsroom/articles/year-2018/crispr-westnile-zika.html.

PHOTO CREDITS This issue contains several photos taken by photographers Dave Gresham and Steve Foxall for Southwestern Medical Foundation Photo (algae) page 4: Frank Fox/Okapia from Robert Harding Photo (Dr. Doudna) page 11: UC San Francisco Photo (Dr. Zhang) page 13 : Ken Richardson Photo

Muspratt, Adam. “A Complete Guide to CRISPR Cas9.” Pharma IQ, 4 June 2018, www.pharma-iq. com/pre-clinical-discovery-and-development/ articles/guide-to-crispr.

Scudellari, Megan. “Self-Destructing Mosquitoes and Sterilized Rodents: The Promise of Gene Drives.” Nature, vol. 571, 9 July 2019, pp. 160-162, DOI: 10.1038/d41586-019-02087-5.

Photo (Erwin Schrödinger) page 15: Wiki Commons

Nedelman, Michael, and Minali Nigam. “Trial Underway in US Uses CRISPR Gene-Editing in People with Severe Sickle Cell Disease.” CNN, 30 July 2019, www.cnn.com/2019/07/30/health/ crispr-trial-sickle-cell/index.html.

Sheridan, Cormac. “Go-Ahead for First In-Body CRISPR Medicine Testing: A CRISPR-Cas9-Based Therapeutic from Editas Is Poised to Enter Clinical Trials for Treating Blindness.” Nature Biotechnology, 14 Dec. 2018, DOI: 10.1038/ d41587-018-00003-2.

Photo (Dr. Barrangou) page 27: NC State University

Netburn, Deborah. “CRISPR Revolutionized Gene Editing. Now Its Toolbox Is Expanding.” Los Angeles Times, 4 Feb. 2019, www.latimes.com/ science/sciencenow/la-sci-sn-crisper-casx-cas12b20190204-story.html. Nightingale, Sarah. “UCR Research Advances Oil Production in Yeast: CRISPR-Cas9 Tool Expedites Production of Biofuel Precursors and Specialty Polymers in Living Systems.” UCR Today, 26 Jan. 2016, ucrtoday.ucr.edu/34308. Park, Alice. “A New Technique That Lets Scientists Edit DNA Is Transforming Science – and Raising Difficult Questions.” Time, 23 June 2016, time. com/4379503/crispr-scientists-edit-dna/. Philippidis, Alex. “Top 10 Companies Leveraging Gene Editing in 2018.” Genetic Engineering & Biotechnology News, 27 Aug. 2018, www. genengnews.com/a-lists/top-10-companies-leveraging-gene-editing/. Prabhune, Meenakshi. “CRISPR Has Expanded Transgenic Animal Research.” Synthego, 4 Dec. 2018, www.synthego.com/blog/crispr-transgenic-animals. ---. “CRISPR Leaps Forward with Jumping Gene Inserts.” Synthego, 3 July 2019, www.synthego. com/blog/transposon-crispr-jump. ---. “Top CRISPR Startup Companies Changing the Future of Biotech and Medicine.” Synthego, 21 June 2019, www.synthego.com/blog/crispr-startup-companies.

“SHERLOCK, DETECTR, CAMERA: Three New CRISPR Technologies.” CLN Stat, 15 Mar. 2018, www.aacc.org/publications/cln/cln-stat/2018/ march/15/sherlock-detectr-camera-three-new-crispr-technologies. Stein, Rob. “In a 1st, Doctors in U.S. Use CRISPR Tool to Treat Patient with Genetic Disorder.” NPR, 29 July 2019, www.npr.org/sections/healthshots/2019/07/29/744826505/sickle-cell-patientreveals-why-she-is-volunteering-for-landmarkgene-editing-st. Stitzer, Lucy M. “How Will CRISPR Impact Our Food?” Dirt to Dinner, 12 July 2018, www.dirt-todinner.com/how-will-crispr-impact-our-food/. Taylor, Ashley P. “Companies Use CRISPR to Improve Crops.” The Scientist, 1 Feb. 2019, www. the-scientist.com/bio-business/companies-usecrispr-to-improve-crops-65362/amp. Thomas, Liji. “First Ever American Gene-Editing Treatment Using CRISPR for Genetic Disease.” News Medical Life Sciences, 30 June 2019, www. news-medical.net/news/20190730/First-ever-American-gene-editing-treatment-using-CRISPR-for-genetic-disease.aspx.

Photo (mosquito) page 25: courtesy CDC, James Gathany Photo (Dr. Collins) page 33: Thomson Reuters/ Mike Blake Photos on page 37 (Dr. Olson), page 38 (Dr. Olson and Ben Dupree), and page 65 (Dr. Chen in lab coat) are courtesy of UT Southwestern Medical Center Photo (Christi and Ben Dupree) page 39: Lara Bierner Photo (Dr. Olson) page 40: The University of Calgary Photo (Dr. Hurlbut) page 43: Anastasiia Sapon Photo (Dr. Bourlag) page 49: Wiki Commons Photo (Dr. Charpentier) page 51: Peter Rigaud/ laif/Redux Photos (Drs. Zhang and Church) page 51: Ken Richardson Photo Photo (Dr. Schlesinger) page 55: Texas Biomedical Research Institute Photo (Dr. Baltimore) page 57: CalTech Photo (Kathleen Gibson) page 61: Grant Miller Photography Photo (Breakthrough Prizewinners on stage) page 64: Steve Jennings/Stringer Photo (Dr. Chen in tuxedo) page 65: Lachlan Cunningham/Stringer

Van Eenennaam, Alison L., et al. “Proposed U.S. Regulation of Gene-Edited Food Animals Is Not Fit for Purpose.” NPJ Science of Food, 20 Mar. 2019, DOI: 10.1038/s41538-019-0035-y. “Vertex Grows Gene Editing Presence, Acquiring Exonics and Expanding CRISPR Therapeutics Collaboration.” Genetic Engineering & Biotechnology News, vol. 39, no. 7, July 2019, www.genengnews. com/news/vertex-grows-gene-editing-presence-acquiring-exonics-and-expanding-crispr-therapeutics-collaboration/. S O U T H W E S T E R N M E D I C A L P E R S P E C T I V E S . 2 019

A Moment inTime

[ December 12, 2018 ] “ THERE ARE NOW over 350 Texas Historical Markers in Dallas County. Certainly, none of them are any more significant than this one. So often we take for granted the history behind major institutions like UT Southwestern Medical Center, and we aren’t aware of the years of dedication and work that went into creating such a facility. This is an important reminder of where it all began, and of what our community has made possible.” Fred Durham Chairman of the Dallas County Historical Commission

The Texas Historical Commission recognized Southwestern Medical College

as a significant part of Texas history by awarding it an Official Texas Historical Marker. The marker honors the founding of Southwestern Medical College, now known as UT Southwestern, by Southwestern Medical Foundation in 1943 as an important and educational milestone in local history. On December 12, 2018, the Foundation dedicated this marker on the Old Parkland campus, where it all began.

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At swmedical.org, you’ll find news, information, and links to follow us on social media.

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Ursula von Rydingsvard, Dumna, 2015, cast bronze

The transformative power of art. Science can inspire and provoke and heal. Art is no less powerful. Ursula von Rydingsvard’s 11-foot Dumna, with its jagged, patinaed surface, casts an impression of an enduring, archetypal monolith on the Dr. Donald Seldin Plaza. The work was one of two sculptures donated to the UT Southwestern Art Collection by Nobel Laureate Dr. Joseph L. Goldstein, Chairman of the Department of Molecular Genetics. “The most gratifying aspect of my sculpture gift has been to watch many of our faculty, our employees, and our students who stop, take a look, and put away their mundane thoughts for a minute or so. As Picasso once said, ‘Art washes away the dust of everyday life from our soul,’” Dr. Goldstein said.