Reprogramming bacteria saves life

Researchers working at the biotechnology start-up company Synlogic, which operates in Cambridge, Massachusetts, will give patients daily doses of a pill or drink loaded with billions of Escherichia coli bacteria. These bacteria usually live in our intestines, they rarely cause infectious inflammations, but for the most part they are harmless to anyone. What is special about these E. coli bacteria is that the researchers reconstructed parts of their DNA with the aim of turning these tiny cells into prey machines that tirelessly strive to devour huge amounts of toxic ammonia in the patients' bodies.

The patients in the trial suffer from a disorder in the urea cycle (UCD), a disease resulting from a deficiency of a certain enzyme in the liver that can lead to the death of newborns and severely injure adults. Those affected by the disease are born with a defective gene that results in the production of defective enzymes that are unable to break down nitrogen compounds formed by the digestion of high-protein foods, such as meat, eggs or cheese. Normal enzymes in the liver produce a compound called nitrogen from the excess Urea, Shitnan in Hebrew, which is removed from the body in urine. In people born with a defective enzyme, large amounts of another compound are made instead, ammonia, which accumulates in the blood circulation. Excess ammonia in the brain causes severe damage.

The researchers at Synlogic engineered the DNA of E. coli bacteria, giving them the ability to swallow large excesses of ammonia. Even without such intervention, the bacteria in the intestines use small amounts of ammonia and utilize it for reproduction. The change made by the scientists gives the bacteria a new genetic "circuit", which consists of DNA segments that are integrated into it similar to transistors in an electronic circuit. In this way, the researchers add genes and control sequences that "turn on" or "turn off" the genes or regulate the strength of their action. The new genome segment is inserted into the normal genome of E. coli, replacing the bacterium's normal mechanism, which utilizes ammonia slowly, with a much faster version, literally an ammonia-devouring prey animal, which comes into action when it senses the low oxygen concentrations typical of a human intestine.

If Synlogic's engineered bacteria succeed in swallowing ammonia in the human body as they did in mice experiments, UCD patients will be able to ingest a daily dose of bacteria for the rest of their lives and live without suffering the symptoms of the disease. The activated bacteria may lead to the cure of a severe genetic disease, the incidence of which in the US is about one hundred new patients every year, for which no cure has been found so far. "We are replacing a missing physiological function with a completely new type of therapy," says Paul Miller, Synlogic's Chief Scientific Officer. "It's a powerful way to attack disease." Miller's company designs genetic circuits with a similar approach to deal with more common diseases such as IBS, inflammatory diseases, disorders of the immune system, and even cancer.

Genetically modified bacteria have an important advantage over conventional drugs, such as chemical pills, since when using pills the only thing doctors can change is the dosage. The genetic circuits in bacteria can be fine-tuned so that it is possible to increase the intensity of their action, extend or shorten the duration of activity and also, if necessary, weaken them and make them safer. The natural ability of bacteria to sense their environment and respond to it gives them uniqueness in focusing on the goal: they can be programmed so that they release a healing substance only when they are in a place affected by the disease. This selective action may prevent side effects that are typical of pills and affect the whole body.

The bacteria are also able to multiply inside the human body, which no pill can do. They still have to pass safety tests, and the researchers admit that they must prove that their genetically engineered bacteria will not be released into the environment in a way that endangers it. The US Food and Drug Administration (FDA) gave Synologic the green light this year to move forward and try the treatment in humans only because the particular strain of E. coli used to treat UCD has long been used as an oral treatment for inflammatory bowel disease. If the clinical trials in humans are crowned with success, the treatment using bacteria developed by the company will be the first clinical application originating from a relatively new branch of genetic engineering called synthetic biology.

This branch of research is developing thanks to the great progress made in the field of DNA editing. Today, scientists have new tools to link DNA segments in a way that will have a much greater effect than changing a single gene. "Synthetic biology has recently achieved impressive achievements," he says James Collins, a professor of medical engineering at the Massachusetts Institute of Technology (MIT) and a leading researcher in the field. For example, they have installed in human cells increased DNA circuits that cause insulin to be injected into the blood with greater precision than the daily insulin injection that diabetics need today. Redesign of Salmonella, a bacterium responsible for outbreaks of food poisoning, now allows it to attack cancer cells and release a load of toxic drugs that harm them. The approach based on DNA circuits can also help diagnose diseases: researchers in Boston recently designed a bacterium that alerts doctors to early signs of an outbreak of Sepsis in patients in hospitals. The common tests rarely succeed in diagnosing the problem before the stage when the patients already suffer from an advanced infection and are more difficult to treat.

The new technology has the potential to transform not only bacteria, but medicine itself. "Biomedicine stands on the threshold of a revolution in medical care," he says Vandal to the sea, director of the Center for Systems Biology and Synthetic Biology at the University of California, San Francisco (UCSF). "Bacterial and human cells become flexible healing engines." But the picture was not always so rosy.

Biological engineering

Over the past forty years, scientists have used genetic engineering to discover genes, make changes in them, and reveal the incredibly complex machinery that governs all forms of life. But they lack the knowledge needed to understand how all the parts coordinate with each other and work together in real life. Things that worked in the test tube collapsed when they tried to run them in living cells or animals. In the early days of synthetic biology there was great early enthusiasm, Collins admits. But about 17 years ago, he and biologists who thought like him, and with the help of those included in the identification of DNA sequences and DNA syntheses, began to use the newly discovered genes and DNA segments as components that can be exchanged between them, to design and create medical applications that also work outside the test tube.

Part of the change was due to the contribution of scientists who had a tendency to "play" like engineers do. "In recent years, new ideas have begun to accumulate that drive progress in the field," he says Jeff Hastie, one of the managers Institute of Biological Circuits at the University of California at San Diego. Hastie began his scientific career 20 years ago with a PhD in physics. Now he describes himself, half jokingly, as "a hybrid between a computational biologist and a molecular biologist." In synthetic biology there are many people like Hesti, who embrace the "maker" tendency of engineers to "build all kinds of things," as he put it.

"Just as an electrical engineer uses conductors, resistors and capacitors to create all kinds of new electrical devices," says Collins, "so we connect together the components of biology: genes, proteins, RNA, transcription factors and other pieces of DNA, to create a certain function.”

Collins points out that electronic gadgets are useful models that aid in the understanding of genetic circuits. Think, for example, of an air conditioner thermostat. The thermostat senses the input: the increase in air temperature, and reacts with the output: turning on the air conditioner motor. When the air in the room cools down, the thermostat turns off the motor. Single-celled organisms such as bacteria survive in a similar way. They are always sensitive to any input, for example, the close presence of a competing bacterium, and in response release an output, for example, the secretion of an antibiotic substance that kills the competitor.

The circuit builders in synthetic biology separated from the traditional genetic engineering people and set out on their own path following similar insights formulated at the same time by Collins and another research group. In 2000, Collins' lab, then at Boston University, She reported on the production of "Offset switch", one of two synthetic gene systems published in the journal Nature in January of that year. The two similar reports (The second was by a group from Princeton University) are usually cited as the studies that launched synthetic biology, because they showed that "you can take parts of cells and link them together and make a new circuit like an engineer does," says Collins. (It is no coincidence that at that time he was surrounded by circles. He ran a bioengineering laboratory that was involved in the design of mechanical organs for people with disabilities. Today, Collins works in centers for synthetic biology in three different institutes in the Cambridge area near Boston. He has trained more than twenty scientists, Hastie is one of them , who currently manage independent projects.)

In the years after the first primitive DNA switches, the members of the synthetic biology community, which was still in its infancy, started a competition among themselves, and tried to break each other's record. They concocted circuits that harnessed the natural sensing-response mechanisms of cells in increasingly complex ways. "As we progressed, we realized how flexible the cells are in their behavior - much more than we initially thought," says UCSF's Lim. He describes the cells in terms of an adaptable car chassis ("chassis") into which scientists can integrate all kinds of genetic engines capable of performing various healing actions.

One of the first commercial applications appeared in 2006 as a result of the work of a group of researchers headed by J. Kiesling from the University of California at Berkeley. Kiessling's lab, aided by a $42.6 million grant from the Bill and Melinda Gates Foundation, reengineered the metabolic pathways of yeast cells. The group's scientists replaced the natural pathways with a network of laboratory-produced circuits, which turn sugar molecules into a critical component needed for the production of the drug Artemisinin Acting against malaria. Before that, the raw material of the medicine was extracted by manual extraction from the plant Artemisia Grown in Asia, an expensive process that made the drug too expensive for use in the poor, malaria-ridden parts of the world. "It was a breakthrough," says Collins. It was the first time that an entire array of genetic material, not just one gene at a time, could be transformed into an entire microorganism, yeast cells, to solve a real problem.

Circuits are disconnected

But this development has not yet sparked a revolution. around the same time, J. Craig Venter, a famous genome researcher and one of the founders of Synthetic Genomics In La Hoya, California, he joined the cauldron of synthetic biology and provided the technology with its first celebrity. His goal, which was widely publicized and resulted in a whopping $300 million investment from Exxon in 2009, was to create Gasoline from algae The animals on the surface of the water in stagnant bodies of water. In 2010, Kiessling received a $134 million grant from the US Department of Energy to fund research designed to force yeast cells to produce diesel from compounds found in high-sugar plants. A few years earlier Kiessling founded the biotechnology company with others amyris in Emeryville, California to promote commercial production of alternative fuel technologies.

Both ventures ended up giving synthetic biology a bad name. Four years later, Venter and Exxon, and like them Amiris, actually withdrew from the venture to produce synthetic fuel. The cost of expanding production to a commercial scale, compared to the current low price of oil and natural gas, forced Amyris, and several other start-up companies that aspired to produce fuel from microorganisms, to freeze their initiatives. These companies were a disaster for investors. But synthetic biology companies like Amyris, launched between 2005 and 2010 on the promise of making fuel from microorganisms, continue to make notable achievements in genetic circuit design, even if their new circuits don't garner such widespread fame. These former stars of synthetic biology are now reengineering microorganisms to produce compounds used in the chemical industry to make solvents and lubricants, as well as important ingredients in cosmetics, perfumes, detergents and over-the-counter health products.

While Wall Street investors and science media reporters focus mainly on the dreams that made headlines to produce biofuel and their collapse in meeting reality, other researchers, such as Collins and his colleagues, have singled out a large part of the first decade of this century, away from the spotlight, to attempts to overcome technical hurdles with this in mind Next: Better medicine. After years of tedious laboratory experiments, in 2010 Collins engineered a bacterium that, under laboratory conditions, weakened antibiotic-resistant bacteria enough to make them vulnerable to known antibiotic drugs.

Tim Lou, another of the scientists who received his postdoctoral training with Collins (and with a doctorate in electrical engineering and computer science from MIT, and a medical degree from Harvard University), around the same time integrated genetic circuits into a completely different microorganism, a virus that attacks bacteria. There are bacterial infections that are difficult to treat because the bacterium secretes around itself a thin membrane made of a protective mucous material, which prevents the penetration of viruses called Bacteriophages: bacterial predators. Lou designed his genetic circuit inside the virus, and inserted a gene that codes for the production of an enzyme that breaks down this membrane. Lu's circuit also reprograms the bacteria-attacking virus to sense the presence of a membrane, penetrate it, and respond by releasing the enzyme that breaks it down.

Lu and Collins realized that it would take many more years for their infection attackers to be perfected. But they also believed that it would take less time to prepare their bacteria for other commercial use. In 2013, Lu and Collins told a group of biotech investors from the Cambridge-based Atlas venture capital fund that their upgraded microorganisms could be turned into living sentinels, capable of providing early detection of diseases in the human body or of pollutants in the air and water.

However, the managers at Atlas were rather enthusiastic about a different idea that stemmed from the same line of thinking. They expected greater gains if the bacteria acted not only as sentinels, but as sensors that, after detecting a health problem in the intestines of humans, would also produce a drug to treat it. This is how the idea to establish Synlogic was born. In early 2015, about six months after the company hired its first researchers, it used Lu and Collins' ideas to create an early version of the UCD treatment.

"I've been in the pharmacology industry for a long time, and I've never seen a pharmacology development move so quickly from a scientist's idea to clinical trials," says Bharat Chaurira, a consultant to Synlogic.

Parts whose role has changed

The component that performs the treatment action in UCD is a highly sophisticated circuit consisting of different genetic segments of the islet, which biologists have discovered in their research over decades. The circuit of the Synlogic company changes the mechanism of the bacterium that normally breaks down ammonia and utilizes the nitrogen found in it for the purpose of culture, into a kind of factory that produces large amounts of an amino acid called Arginine. The researchers chose arginine because the process of building it in the cell requires a larger amount of nitrogen than is required for the production of other amino acids. The strong drive to obtain nitrogen for the construction of arginine makes the bacterium an ammonia devourer. With the circuit integrated into its genome, the bacterium produces "5,000 times more arginine than the normal strain of the bacterium," saysJose-Carlos Gutierrez-Ramos, CEO of Synlogic.

The operation of the circuit depends on the operation of a switch, a DNA sequence that reacts to a protein called Fnr. Like an air conditioner thermostat, the FNR is sensitive to changes in the bacterium's immediate environment. It allows the E. coli bacterium to respond to a low level of oxygen in its environment. When FNR senses that the bacterium is in a low-oxygen environment, such as the one that prevails in the large intestine, it activates genes that the bacterium needs to reproduce. When the bacterium goes outside the body, and is in an environment rich in oxygen, the FNR stops the action of these genes. This is a safety mechanism designed to prevent the arrival of microorganisms with a high culture rate into the environment. As soon as the bacteria leave the intestine in the feces and reach the oxygen-rich external environment, the whole system shuts down and the bacteria die.

Still, there was one problem in developing the mechanism, says Miller. In the genome of E. coli there is a "repressor switch", a gene called argR. This gene causes the production of arginine to stop when it senses that the bacterium has enough of this amino acid. The designers needed to insert into their new circuit a mechanism that would disable argR activity. To this end, the researchers replaced the long DNA segment that includes the argR gene with a DNA segment that is identical in its entirety, but without the argR gene.

Some synthetic biology researchers have developed other genetic circuits designed to deliver anticancer drugs deep into the cancerous tumor. Hastie of the University of California, San Diego inserted a special set of genetic instructions into a strain of salmonella bacteria that is not harmful to humans. Hestie's experimental cancer treatment was aided by recent research that discovered that certain bacteria are sometimes found inside cancerous tumors. Scientists speculate, but are not sure, that bacteria naturally found in the blood are attracted to cancerous tumors "because the environment that prevails in them provides them with a safe haven from the immune system," Hastie says.

The genetic array that Hesti injects into Salmonella forces the bacterium to carry out a two-step process. First, the genetic circuit is programmed to produce a specific anti-cancer drug inside the bacterial cell. Later, he directs the bacterium to penetrate deep into the tumor, as it is carried with the blood stream that the tumor needs for its growth. At a point in time determined by the genetic circuit, the salmonella bacteria "suicide" and break themselves down. When the bacterial cells break down, the drug load in them is released. "It reminds a bit of a pilot kamikaze,” says Hastie.

Through another clever design, Hesti added a few more genetic components that allow this means of treatment to regenerate on its own. "We introduced a system bug density sensing able to sense when the salmonella bacteria multiplying inside the tumor have reached a certain population density," he says. When the bacterial population in the tumor reaches the threshold density, the sensor triggers the release of a protein that breaks down the bacterial cell from the inside and results in the release of the drug. This "suicide" action kills most bacteria, but not all. The remaining ones continue to multiply and the cycle starts again and again.

The idea of ​​attacking a cancerous tumor from the inside is a particularly attractive idea, because most chemotherapy drugs work on the tumor from the outside. They first attack the cells found in the scope of the tumor and their penetration is slower. In a study conducted in mice, it was found that when the treatment was given using genetically engineered bacteria alone, without chemotherapy, it did not work any better than regular chemotherapy, Hastie says. "But when we gave this treatment together with chemotherapy, we saw a shrinking of the tumors and a 50% increase in the lifespan of mice with metastatic cancer," he says.

Search for approval

The research on salmonella bacteria continues and undergoes improvements. Synlogic's treatment of UCD is at a much more advanced stage. The treatment is in the approval processes of the FDA, which examines it very strictly because it is the first treatment that involves the use of genetically modified bacteria. The administration published regulatory rules for microorganism-based treatments within a new category called "Live products for biological treatment". Unlike other drugs (with the exception of some vaccine components), the new treatments contain living organisms, which have the potential to mutate during their culture. For this reason, the Food and Drug Administration demands that it be guaranteed that the drugs will not change and will be the same in all the batches that will be prepared. On top of that, the administration requires proof that the microorganisms are unable to survive on their own in an environment outside the body, as Synlogic claims. "We are monitoring the conduct of the regulators towards Synlogic," says Hastie. "If they don't get their treatment approved, we're all going to be in trouble."

It is likely that the FDA's approval process for cells engineered for the purpose of detecting diseases, rather than creating new compounds in the body, will be faster and less expensive than the approval process for medical treatment. Many new ventures in synthetic biology are aimed at engineering bacteria so that they diagnose diseases at the earliest stage of their appearance. "Intestinal bacteria can be engineered so that they sense, remember and report experiences they had when passing through the intestine," she says Pamela Silver, the co-founder of the Department of Systems Biology at Harvard University. Silver's laboratory has developed a proof-of-principle diagnostic tool, consisting of a genetic circuit that gives bacteria the ability to detect the presence of an antibiotic substance in the digestive system of mice. The genes in the circuit encode the production of a fluorescent signal that can be seen in the feces, if an active antibiotic substance is found in the intestine.

"This synthetic circuit illustrates our ability to produce a living diagnostic tool for, in this case, antibiotic exposure," says Silver. The ultimate goal is to use the technology to detect disease activity within the gut. "The human intestine is a 'dark' place: it is difficult to check what is happening in it, even though it is a site of a lot of activity that affects our health, and also a site of serious diseases, the most common of which are various intestinal infections," she says. The diagnostic methods available today for intestinal diseases are invasive and expensive.

A live diagnostic tool, Silver says, offers a cheaper and possibly more sensitive approach. And if it passes the tests, it will be possible to add more functions to it. "We also believe that it is possible to continue to engineer diagnostic genetic circuits further, and add to them the ability to treat intestinal diseases, right at the center of the disease," she says. "The power of the new circuits creates many and varied possibilities."