Some Wisdom Lost? Reflections on Four Decades of Genetic Manipulation by Matthew G Endrizzi Is it true that as we gain technology we lose some wisdom? temporarily? In the case of genetic engineering, I aim to lay out risk and benefit in a plain manner and offer suggestions for future directions where I am able. Hopefully, reading this will challenge opinions you currently have, inspire you to investigate more on your own, and open you to form new opinions that you will share with others. It may seem futile to try and regulate or limit biotechnology in any way, but I hope to present a pathway that is pro-technology, pro-health, pro-business, pro-environment, pro-choice, and pro-life. The scientists, leaders, and regulators involved in the business of DNA are many. They are scattered all over the world in universities, government institutions, research hospitals, military labs, and companies spanning several industries, from medical to agricultural to a budding chemical industry. I talked to the scientists I worked for at Florida State University, Harvard Medical School, and the Whitehead Institute. I have tried to publish my opinion, argument, and proposal in Nature and Science magazines for peer review. I have shared my point of view with Francis Collins and Paul Berg in person. I have written to Presidents Bush, Obama, and Trump. I have written to NIH directors, other federal-level officials, and numerous CEOs. Needless to say, leaders know what I am talking about. Whether they fully understand or agree is difficult to discern. Their response to my concerns has been overwhelmingly silent. The few comments I have received have not been embracing, but have not posed any counter-argument that would allay my concerns. I have communicated with hundreds of scientists, some friends I used to work with, and none have changed my viewpoint on risk. My efforts over the last 15 years have been to inform and persuade molecular biologists, as well as government and industry stakeholders, to consider containing recombinant DNA significantly more carefully. Specifically, I am concerned that by placing recombinant DNA into living cells, we create the possibility that a cell could translate a nucleic acid into a virus, transposon, or other replicating element that not only could kill people but could kill off multitudes of other organisms throughout our ecosystem. I feel compelled to restate that I support molecular biology research and think it should continue. However, scientists need to be forthright and truthful if they are to maintain public trust. Young people coming to the field need to be aware they will be handling material that cannot be put back into the bottle, could spread across the globe rapidly, and could cause the next plague, or possibly, far worse. Bioethically speaking, we have been in the throes of labor since 1972 when Paul Berg and his colleagues made the first recombinant DNA molecule [1]. This technology has evolved into CRISPR Cas9 today [2], which allows us to permanently alter any genome, including our own. With the ability to make whatever DNA sequence we can imagine, born unto us is a power to manipulate the very engine of evolution. I hesitate to say “control” evolution because that implies we have more knowledge than we do – specifically, the knowledge of what can go wrong and how we can avoid negative impact. Inadvertent tragedies involving engineered DNA could, and I argue we must presume WILL, happen. Such consequences will not discriminate between those who approve, disapprove, choose to use, or choose not use genetic engineering. So how certain am I that a disaster will occur? I am not really certain at all. The fact that life seems to adapt and continue despite the myriad of DNA rearrangements that happen naturally makes me think we will be okay. But is that where the analysis should end? As someone who made recombinant DNA for a living, I personally have profound concern that I might have created one or more molecules that will evolve into something that decimates life on the planet, at any point in the future. The reason I think the DNA sequence combinations I produced are inherently more dangerous than the unaccountably large number of DNA sequence combinations that nature produces is because the processes I used in the lab bypass natural barriers that might otherwise prevent certain DNA sequences. The reason I suggest we should presume a catastrophe will happen is because the knowledge we have gained by manipulating DNA has taught us that life runs on amazingly complex machinery that is both fragile and persistent. We have just begun to understand how life works at the molecular level. We know changing one nucleotide in a single DNA molecule could have potent effects, both lethal and lifesaving. We also know that our ability to predict how our fate will be affected by genetic engineering in a negative way is nearly impossible. So what is possible? More importantly, what is plausible? Possibly, all kinds of apocalyptic scenarios could play out, such as The Andromeda Strain, I Am Legend, etc. I leave it to others to create the next great science fiction story. Plausible is an entirely different conundrum. Plausible suggests something is more than just possible, but feasible or likely because we know some specific information that supports the outcome. So what are some outcomes of genetic engineering that are plausible? Let us fast forward from recombinant DNA in the early 1970’s to genome engineering today. Scientists can now guide whatever pieces of DNA they want into a chromosome by connecting their engineered DNA to specific sequences of DNA that exist in genomes already. It is a brilliant design that allows us to put DNA in a chromosome exactly where we think it will do some good. Great! This is certainly a step in the right direction if we want to fix genetic problems without creating new ones. What we do know for certain is that if we create a DNA or RNA molecule that codes for a virus and place that nucleic acid into a host cell, that cell automatically transcribes and translates the molecule into a functioning virus. [3] This fact alone should give everyone who

understands it great pause. Recombinant DNA is not destroyed in labs before disposal. The host cells containing them are sterilized, but the nucleic acids inside them are released to the environment. [4] Additionally, the nucleic acids made in labs are the same size as the nucleic acid backbones of viruses. [Figure 1] Based on these facts, I argue that eventually a nucleic acid will be synthesized that will become part of natural history, and we will not be able to determine if the origin is natural or human-made. Some genes are ubiquitous, by that I mean a critical sequence of DNA that is everywhere – in plants, animals, bacteria, fungi, etc. Scientists often talk about “conserved” DNA. These are specific DNA sequences that exist across many species. The idea is that conserved DNA is so critical to the functioning of the cell, it cannot tolerate mutation. Any change to this DNA and the proteins it codes for do not work and the organism dies. One concern I have is that a ubiquitous gene, like one that makes chlorophyll, inadvertently becomes a target for an engineered piece of DNA. In this scenario, a plant researcher studying the biology associated with the chlorophyll genetic region might make a DNA molecule for a research project that ends up fairly stable and attracted to a DNA sequence in the chlorophyll genetic region of plant (or phytoplankton) chromosomes. If this DNA molecule were viral or transposable it could spread to some, most, or all organisms that make chlorophyll. While natural selection would hopefully cull plants that can no longer photosynthesize and leave us with the hardiest of oxygen-producers, I believe it is plausible that something like this could happen to a great enough degree for a long enough period of time, especially to phytoplankton, that animals become extinct. Another example of conserved or ubiquitous DNA sequence is a set of genes that control when a cell dies so that it does not become a cancerous tumor. These apoptosis (programmed cell death) regulatory genes are in animals, plants, fungi, and even protozoa [5]. If this network of genes were disrupted, an outbreak of cancer could occur globally throughout almost all kingdoms of life, essentially rebooting evolution back to a unicellular world. There are sequences of DNA that code for the material that makes ribosomes. [6] Ribosomes are the parts of the cell that make proteins, so every cell on the planet (except a few specialized DNA-free ones) have these ribosome genes. If protein production were disrupted, evolution reboots to the primordial soup. Alu sequences are DNA sequences called transposons that have been inserting themselves into chromosomes throughout primate evolution. “Each  Alu  element is roughly 280 bp [base pairs] long, followed by a poly-A tail of variable length. Thus, the more than 1 million  Alu  elements comprise roughly 10% of the human genome. [7] Alu  elements have continued to insert in the modern human lineage as evidenced by their continued contribution to human genetic disease. It is estimated that there is about one new Alu insert per 20 human births [8], leading to about one in every 1,000 new human genetic diseases. [9]” How can we predict if a newly engineered nucleic acid will transpose itself into nature at some point in the future? How can we predict if a newly engineered nucleic acid will have a negative effect at all? We cannot fully answer either question without creating a disaster. If we cannot truly know which nucleic acids are the most dangerous, then we must consider any new nucleic acid we create to be at least as dangerous as the one that codes for the Small Pox virus. Think of engineered DNA molecules as tickets to the Megabucks Lottery. Any single ticket has nearly a zero chance of winning, but someone always wins, eventually. If at this point you are thinking people should not manipulate DNA at all, for any reason, I pose the following: We know that we can make insulin and other proteins that can be injected into people’s blood to alleviate suffering and save lives because we are currently doing it. We know much more about how life works at the molecular level because we engineered DNA to answer a lot of questions. We know we can still answer a lot more questions by manipulating nucleic acids in the future. What we do not know is if manipulating DNA has or will lead to a catastrophe. Even if it did, we may not be able to know if the origins of such a catastrophe were natural or human-made. Academia created the Precautionary Principle in response to emerging genetic technologies: if something is plausibly hazardous, then it should not be utilized until people can prove that it is safe. Proving safety related to genetic engineering is difficult at best.* So what should we do? Because we know genetic engineering can alleviate suffering in ways no other technology can, and we do not know if genetic engineering will trigger a catastrophe, we should proceed. So if this is the course the world is on anyway, why am I writing this? I suggest we contain recombinant DNA significantly more carefully. Specifically, I advocate that we utilize lunar facilities to create and contain engineered nucleic acids that are not replicas or compliments of natural DNA or RNA. Humans would be the only vectors back to Earth. ** https://sites.google.com/site/mattendrizzi

The Pros Pro-technology An incredible amount of new technologies will be created to develop lunar facilities. Meanwhile, biotechnology should push forward on Earth, employing non-recombinant technology such as PCR and DNA sequencing as much as possible. Pro-health Medical research can continue while a significant shield is put in place to protect against accidental epidemics. Standardization of protocols associated with sharing extraterrestrial resources will necessitate developing a global medical infrastructure that will be able to respond to health crises anywhere on the planet. Pro-business The current avenues to profit are still open while we prepare a new age of space economy. Pro-environment Many other industrial processes could be relocated in space where hazardous byproducts can be contained. Lunar development would necessitate innovations in massive and complex self-contained biospheres where we could learn more about how to better manage our environment on earth. Pro-choice To gain support from the public, people must learn more biology than they currently understand. As people do learn more, they will be in a much better position to use genetic screening to inform their reproductive, medical, and lifestyle choices. Pro-life I predict that one day, when a large enough number of people understand what leading scientists currently understand, people will have a greater respect for all life, will be more accepting of nature’s mutations, will be highly selective in how they use technology to change their DNA, and ultimately will be fuller participants in the lives they are living. The Cons Scientists have to admit to themselves and to the world that what they are doing is inherently dangerous. The rest of us have to try our best to understand what scientists are doing so democracy can have a chance of participating meaningfully in the regulation of science. It will likely take decades for political and technological progress to meet the economic challenges of working together as a global society to engineer nucleic acids outside of the Earth’s biosphere. We may want to pay more taxes, but it would be interesting to see what the public would donate on its own...

Epilogue: Is our genome more sacred than any other genome? We have been manipulating living creatures in our environment for thousands of years. Recently, we started engineering the genomes of plants to improve agricultural production. Now we can engineer ourselves. To think that changing our own DNA is worse than changing any other organism’s DNA might be fooling ourselves. We know how dependent we are on our ecosystem, so to think that changing ourselves will harm us more than if we harmed our ecosystem, I argue, is fallible. How well we are adapted to our surroundings today could be very different than how well our same genetic self might be adapted to a different environment in the future. To think we can switch DNA mutations back to “normal” if we discover we make a mistake in the future I also think is fallible. Because the outcome of a DNA mutation is fundamentally defined by the environment in which it exists, we can truly only add mutations and never subtract them. In other words, there is no baseline to return to, because the baseline is always changing. Recently, Eric Lander, director of the Broad Institute and former scientific advisor to President Obama, made a plea for caution. “The discussions that will begin in the fall may solidify a broad international consensus that germline editing should be banned — with the possible exception of correcting severe monogenic disease genes, in the few cases in which there is no alternative. For my own part, I see much wisdom in such a position, at least for the foreseeable future. A ban could always be reversed if we become technically proficient, scientifically knowledgeable, and morally wise enough and if we can make a compelling case. But authorizing scientists to make permanent changes to the DNA of our species is a decision that should require broad societal understanding and consent. It has been only about a decade since we first read the human genome. We should exercise great caution before we begin to rewrite it.” [10] I propose we consider this more broadly to encompass all splicing of nucleic acids. Some have said, and many more may think, genetic tinkering is playing God and we should not do it. We do not all think God or some spiritual force has a hand in any of this. Some of us, though, might conclude that God DOES want us to manipulate DNA. One thing we all have in common is that none of us chose to come into this world. Someone else made that choice for us. We are left to make the most of what we have while we are still here. One piece of advice passed down through the ages has been shared by religious and non-religious alike: peace, shalom, salaam. Likewise, mir, hépíng, heiwa. Shaanti, amani, et cetera. We are all in this together.

* In terms of addressing the Precautionary Principle’s call for evidence of safety, transplanting genetic engineering outside of the biosphere would allow us to compare health and environmental outcomes in a pre- and post- “recombinant world.” If outcomes are no different, then there is some evidence that biotechnology is not affecting us in a harmful way. Predicting a duration for such an experiment is problematic. ** By the time lunar facilities are operational, we may know considerably more about impacts of GMO agriculture. I imagine after some period of quarantine in a lunar biosphere, we may introduce certain crops or other GMOs into Earth’s biosphere.

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Figure 1. A histogram of viral genome lengths exhibits striking similarity to the distribution of recombinant DNA molecule lengths used in scientific research, such as plasmids, viroids, viruses, cosmids, BACs, and YACs. This is not surprising since recombinant DNA is designed to be put into cells and are themselves usually modified versions of naturally occurring nucleic acid vectors.

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