September 1, 2017 (Vol. 37, No. 15)
In a world where market-oriented thinking reigns supreme, the word “guild” may seem quaint, a reminder of an organizational form that is not only dead, but unmourned. The word, however, still has its uses, as the founders of the Genome Writers Guild would likely argue. They would say that a guild may be understood as a body that oversees a craft, inculcating expertise, maintaining standards of quality, and advancing the development of tools and techniques. A guild, they would add, may serve its members’ interests while promoting the greater good.
The test of a guild’s beneficence comes down to straightforward question: Is the guild devoted to openness or secrecy? The answer, in the case of the Genome Writers Guild, is clear: openness. The organization recently held its first annual meeting, “Realizing the Future: Genome Engineering 2017.” As the meeting’s agenda makes clear, the future to be realized is not just the future for genome engineers. It’s everybody’s future.
Twenty years ago, several colleagues at the University of Minnesota’s Arnold and Mabel Beckman Center for Transposon Research—now the Center for Genome Engineering—got together to discuss the future of genome engineering and gene editing. These forward-looking genome engineers included Stephen Ekker, Ph.D. (professor of biochemistry and molecular biology at the Mayo Clinic), David A. Largaespada, Ph.D. (professor of genetics, cell biology, and development at the University of Minnesota), and Perry B. Hackett, Ph.D. (professor of genetics, cell biology, and development at the University of Minnesota). Subsequently, they were joined by many others, including Dan Voytas, Ph.D. (director of the Center for Genome Engineering) and Aron Geurts, Ph.D. (associate professor of physiology at Medical College of Wisconsin).
Over the years, discussions drew additional participants to the guild and ranged ever more widely, necessitating a more deliberate organizational approach. Hence, the founding of the Genome Writers Guild, which tries to help genome engineers make sense of a potentially confusing field of discovery.
“This is the 2.0 version,” said Dr. Ekker, echoing the assessment offered by Dr. Hackett at the annual meeting, which was held in the architecturally fantastic McNamara Alumni Building at the University of Minnesota. “The issue is not the imagination,” he explained, noting that the exercise of the imagination has already yielded a vision. “Our goal now is realizing that vision.”
At the annual meeting, Dr. Ekker and his colleagues assembled over 200 people—scientists, engineers, industry investors, corporate leaders, regulators, artists, communicators, and entrepreneurs—with a unifying goal. All were challenged to put the ideas of genomic engineering and gene editing into formalized practice.
“If you are going to impact the world, you are not going to do it just from a small scientific community,” Dr. Ekker declared. “You are going to have to have the whole ecosystem represented. This meeting represents our first attempt at getting that whole ecosystem.”
Incidentally, many of the ecosystem’s participants have direct relationships with Dr. Ekker himself. Some of the relationships are apprenticeships; others, partnerships. At the meeting, he updated novices and professionals alike on the editing of mitochondrial DNA.
The Tools and the Trade
While several of the gene-editing researchers, such as Drs. Ekker and Hackett, have been staunch supporters of zebrafish models for knockout and replacement genomics, Dr. Geurts has spent over a decade working to produce rodent models of disease through genome engineering. His laboratory utilizes the Sleeping Beauty transposon system, which was developed by Dr. Hackett and others back in 1997. Originally, the system was used to add new genes to the fish genome. In 2006, Dr. Geurts’ laboratory adapted the system to accelerate transgenic and gene-knockout studies in rats.
A few years later, in 2009, Dr. Geurts’ laboratory was the first to first to demonstrate that zinc finger nucleases (ZFNs) could be applied to rat embryos to generate the world’s first targeted gene-knockout rats. “The zinc finger nuclease,” Dr. Geurts elaborated, “allowed us to go into small regions and change even a single nucleotide.” Earlier work had shown that genetic mapping could be used to narrow disease culprits to a small list of genes, Dr. Geurts pointed out, but this approach, unlike ZFN, was seldom capable of focusing on just one gene at a time.
Several nuclease technologies are being employed by an ever-expanding group of investigators. “All the technologies have the same basic function,” said Dr. Geurts. “They enter a cell, find a piece of DNA, and cause a double-stranded break or maybe a single-stranded break.” Depending on the gene and its location, you might pick from several currently available techniques, including ZFNs, transcription activator-like effector nucleases (TALENs), or the CRISPR/Cas9 system.
“I think the real exciting thing is seeing all kinds of scientists coming into the field,” he commented, adding that these investigators don’t want to limit the toolkit at this time. Rather, they want to expand it “There are always going to be limitations with each tool,” he noted, “so I think there is good reason to keep exploring all three technologies.”
One of the disease models that the Geurts laboratory has developed makes use of the Dahl Salt Sensitive rat, which develops high blood pressure and renal damage when it is fed a high salt diet. The laboratory crossed this rat with a Brown Norway rat, which does not develop high blood pressure, so that the rats would exchange specific chromosomes. This work allowed the laboratory to show that chromosome 13 replacement reduces blood pressure.
After the locations of potential disease-related genes were narrowed down to four specific regions in the chromosome, the investigators used ZFN technology to disrupt individual candidate genes. As a result, the investigators were able to evaluate, at a functional level, whether these genes are involved in hypertension. The investigators were able to significantly enhance understanding of this complex disease, which affects 30% of American adults.
Subsequently, Medical College of Wisconsin researchers founded the Gene Editing Rat Resource Center (GERRC), which has produced over 100 investigator-suggested rat strains for use in understanding disease. “Human genetics and rat genetics are often pointing us in the same direction,” commented Dr. Geurts. “Now it’s getting down to, what does the gene actually do? If we can figure that out, we can design a drug that either enhances or prevents the gene’s function.”
At the meeting, it was clear that gene-editing tool usage has multiplied robustly and that the number of groups involved has increased. A group led by Jeffrey J. Essner, Ph.D., professor of genetics, development, and cell biology at Iowa State University, has developed several plasmid vector-based tools for exon disruption. These tools, which Dr. Essner calls pGTAG and pPRISM, incorporate fluorescent reporters within the exon. Both tools can be used for gene editing when they are used in combination with CRISPR/Cas9 or other nuclease technologies. pGTAG, the simpler of the two systems, allows for the incorporation of 2A-fusion tags of RFP, GFP, and Gal4/Vp16, providing for a variety of colored alleles.
“They can go into any gene,” Dr. Essner pointed out, “but what’s really important is that both vector sets are designed to drop into an exon, disrupt that exon, and give a report of the transcriptional activity of that gene.” The vector sets work by essentially knocking out the function of the gene allele while simultaneously inserting a reporter that suggests incorporation. More important, Dr. Essner’s vector sets provide a methodology for allelic replacement of a locus with a user-designed targeting cassette. When the pPRISM vector is used, two reporters and/or two cassettes can be added to a single gene locus.
“What I am really trying to push toward,” stated Dr. Essner, “is to use these vectors to do in vivo cell biology.” Dr. Essner and colleagues have used these vector systems to interrogate everything from developmental cancers to vasculature formation in zebrafish. The zebrafish models developed by Dr. Essner and others allow the effects of allelic alteration to be assessed at the single-cell level but in the context of the whole organism.
Hind Fadel, M.D., Ph.D., a senior associate consultant in the department of internal medicine at the Mayo Clinic, is utilizing gene-editing tools to derail the infectivity of one of the most notorious viruses on the planet. “We and other groups are applying this technology to reengineer the immune system to make it again resistant to human immunodeficiency virus (HIV),” said Dr. Fadel. “This has really been inspired by the Berlin patient,” she elaborated. After receiving bone marrow transplants in 2007 and 2008, this patient was functionally cured of the disease because the transplants introduced a mutant version of the CCR5 protein, a surface receptor important for incorporation of the R5 tropic version of the virus.
As HIV’s mechanisms were being clarified, gene-editing technology was being developed, Dr. Fadel pointed out. “The thought was that we could use some of this technology to reengineer people’s own hematopoietic stem cells and make them resistant to HIV.”
Dr. Fadel and others working in this area use gene-editing tools such as TALEN, CRISPR, and others to remove, or knock out, factors that HIV requires for infectivity. One such factor, a nuclear protein called LEDGF, was described in Dr. Fadel’s presentation. LEDGF binds HIV integrase on one end and the host DNA on the other to aid in HIV integration into the host genome.
“If I take away a factor like CCR5 or LEDGF,” Dr. Fadel proposed, “I take away something that HIV needs.” Dr. Fadel’s initial findings indicate that HIV replication could be severely inhibited if important HIV targets such as LEDGF are knocked out. Such work suggests that gene editing could be used to reduce or eliminate disease.
Karl Clark, Ph.D., assistant professor of biochemistry and molecular biology at the Mayo Clinic, is another investigator working to pinpoint gene variants that contribute to specific diseases. A former member of the Ekker and Hackett laboratories, Dr. Clark now works with the Mayo Clinic’s Center for Individualized Medicine (CIM), where his functional validation laboratory uses a combination of genome engineering and cellular and zebrafish models to interrogate rare patient-specific diseases. The CIM’s mission is to help patients find solutions to these rare diseases, which can affect nearly 6% of the population, in a more efficient time frame.
In his presentation, Dr. Clark suggested that patients afflicted with rare diseases may endure so-called diagnostic odysseys. “There can be a gap of 5 to 30 years,” he said, “before a patient gets an indication of what is going on.”
Patients that cannot be diagnosed immediately are often offered whole-genome or whole-exome sequencing, which can provide clues to genetic variation in their personal genome. But 75% of the time, noted Dr. Clark, there is not a direct link to a genetic variant associated with the disease etiology.
The CIM developed a three-year pilot program to look into these cases of unknown disease etiology. CIM investigators start with individual patients’ DNA sequences and other omics data and progress through targeted functional genetic approaches.
The CIM investigators are postdoctoral fellows that collaborate with clinicians and work with bioinformatics, protein modeling, and the functional validation data to provide improved diagnostic evidence. For example, CIM investigators are using a functional validation laboratory to evaluate the potential of predictive microhomology repair. This work, which is currently focused on the repair of targeted double-strand breaks in somatic cells of zebrafish, is yielding insights that could reduce genetic testing to as little as two months. Such discovery-expediting work suggests how gene-editing technology may be relevant to individual patients.
Ian Clift Ph.D. is Scientific Communications Consultant, Biomedical Associates and Clinical Assistant Professor, Indiana University
Sci-Fi Visions of Genomic Engineering: An Interview with David Brin
The futurist, astrophysicist, and science-fiction author David Brin, Ph.D., gave the keynote presentation at the first annual conference of the Genome Writers Guild in Minneapolis in July 2017. After completing his presentation, he sat down with GEN to discuss the role science fiction plays in influencing genetic engineering. Excerpts from the edited discussion appear below.
GEN: One of the subjects I see you addressing is the speed with which life forms change to adapt to their environments. If we were to continue adapting the old-fashioned way, naturally, we would likely acquire changes slowly and gradually. But if were to sharpen our gene-editing skills, we could gain the ability to make changes immediately. The question is, might we choose to enhance our fitness for any particular environment?
Dr. Brin: If we live on a dimmer world, we’ll have to make bigger eyes, as if we were lemurs. There have long been arguments in science fiction on what the effects of living on a lighter gravity planet would be. Would we develop into tall, gracile beings, or would we gain the ability to carry around more fat? We don’t know what that effect would be.
The trade-offs for genetic engineering are huge, and our ability to manage these trade-offs may depend on our capacity for imaginative or delusional thinking. One of the main points I made in my speech is that we are inherently delusional beings.
Each of us may be deluded, but others don’t necessarily share our delusions; they have their own. Others can see our delusions, and we can see theirs. We live in a gift economy where I will happily point out your mistakes, and you will happily point out mine. It’s called reciprocal criticism, and we foolishly hate it when, it is, in fact, one of the greatest gifts.
GEN: You also talk about the failure modes emphasized by science fiction. What are some of the failure modes for genome engineering? Which of these modes warrant special attention?
Dr. Brin: Genome engineering is attempting to replicate skills that took several billion years to evolve. When nature created these skills, it was unafraid of error and death. We are trying to learn, within a decade, how to meddle in these things, and our value system says that we must minimize the errors and keep to a minimum the blood on the floor. We don’t want there to be side-effects.
This is exceptionally hard to do, especially when a third of the DNA has unknown purposes and diverse and nonlinear qualities. Meddling in one chromosome can easily amplify or deregulate something on another chromosome.
The danger that I fear is that if something is unleashed from the genetic labs, then we could see a major clampdown. This would not stop genetic research. The lords and the mighty would still finance it because they would seek immortality. But when these sciences dive into secrecy, you lose the cleansing effect of criticism, and the number of mistakes multiply.
GEN: That’s quite a dystopian vision. Could you elaborate?
Dr. Brin: If the rich and the mighty get a chance to monopolize genome engineering, you might see a real nightmare. Those at the top of the pyramid have always told themselves that they are inherently better than everyone below, when in fact, desirable traits such as height and intelligence have a lot to do with access to protein and better education. If genome engineering is monopolized by the rich and powerful, then their children may be better than our children in actual fact. The mythology of genetic superiority of the nobility, which has always been a lie, might actually come true. We would be locked into feudalism forever.
Continue reading the complete set of Genome Engineering articles.