The late 2010s marked a defining moment for bespoke bacteria. In 2017, a team from the SynBio Lab at the National University of Singapore (NUS) engineered a bacterium to produce antimicrobial proteins in response to a pathogenic bacterium’s communication signals. In 2018, the same team engineered gut bacteria to bind to colon tumor cells and secrete a tumor-inhibiting molecule.^1,2 A research group at Synlogic, a biotech company based in Massachusetts, had a similar idea; in 2020, they engineered a common gut strain of Escherichia coli to produce the proteins needed to correct rare metabolic deficiencies.³ 

The researchers at Synlogic were first to the finish line for bringing engineered therapeutic bacteria to clinical trials. They currently have two therapeutic bacteria in early-stage clinical development: SYNB1618 for the management of phenylketonuria and SYNB1891 for immuno-oncology. “In addition, the company is developing SYNB8802 for enteric hyperoxaluria and expects to initiate a Phase 1 clinical study in early 2021,” said Liz Wolffe, former head of investor relations and corporate communications at Synlogic, via email.

Researchers have known for decades that bacteria living in the gut affect human health.⁴ Synthetic biologists now focus on the gut microbiome, engineering non-harmful bacterial species to not only deliver therapeutics, but also to monitor and respond to specific environmental signals in the gut to treat disease. 

Inspired by nature

The human gut microbiome contains thousands of different species of bacteria; choosing one to become a therapeutic producer is easier said than done. The NUS team was inspired by bacterial communication processes that are common in nature, and by observations of bacteria producing killing molecules to defend their own ecological niche. 

“While we were investigating these two independent mechanisms separately, with a particular interest in infectious disease, recent advances in the field of synthetic biology motivated us to integrate the two mechanisms,” said Matthew Wook Chang, head of the SynBio Lab at the NUS and lead author of the 2017 and 2018 papers, by email. “Based on the synthetic biology framework, [we designed] a novel, unconventional approach of attacking infectious pathogens.” 

Isolating a beneficial bacterial species from the human host and then developing it for therapeutic purposes is one way to develop potential drugs. Both the Singapore team and the Synlogic team, however, wanted to take a more rational approach to drug design. The researchers carefully studied the human gut microbiome before settling on the bacterial strain E. coli Nissle 1917 as a target. 

E. coli Nissle 1917, originally isolated by Alfred Nissle from the Hygiene Institute of the Albert-Ludwigs-University of Freiburg, Germany in 1917, prevented its host from developing infectious diarrhea from Shigella during World War I. E. coli Nissle 1917 has since been used as a probiotic, so Chang, Wolffe, and their teams knew it had a record of safety.⁵

Targeting the gut

Chang’s team engineered E. coli Nissle with prophylactic and therapeutic activity against P. aeruginosa. Using a previously developed synthetic genetic system in 2017,⁶ they enabled the E. coli to sense small molecules that pathogenic P. aeruginosa produce to communicate with each other. They also incorporated genes encoding an anti-biofilm enzyme and antibiotic proteins specific for P. aeruginosa into the bacterial genome. “Pseudomonas aeruginosa is a leading cause of hospital-acquired infections, and is becoming more difficult to eradicate because of increasing antibiotic resistance,” Chang said. 

In 2018, the team genetically engineered the same strain of E. coli to bind the heparin sulfate proteoglycan of colorectal tumors. Once bound, the bacteria secrete an enzyme that converts dietary glucosinolate—abundant in cruciferous vegetables—to sulforaphane, which inhibits tumor growth and promotes apoptosis. 

Concurrently, the Synlogic research team modified E. coli Nissle to treat the metabolic diseases phenylketonuria (PKU) and enteric hyperoxaluria. In both conditions, a metabolite—phenylalanine in the case of PKU and oxalate in the case of enteric hyperoxaluria—accumulates in the blood and tissues to toxic levels. Both diseases are well understood, have unmet medical needs, and are affected by dietary intervention, which makes them suitable for a gastrointestinal-based approach. “We can engineer bacteria in the gastrointestinal tract to enable [affected patients] to consume these metabolites, with the goal of lowering levels in the blood and tissues,” Wolffe said. 

“We developed a platform that enables us to engineer the relevant therapeutic functions into the E.coli Nissle ‘chassis’ with the desired potency and under control of inducible switches,” Wolffe explained. The team also engineers additional safety features so that the strains do not grow outside of the manufacturing process, allowing them to be safely cleared by the patient. This feature allows scientists to monitor the effects of particular “doses” of the engineered strain, without worrying about differential colonization in different individuals.

Overcoming challenges

Scientists must overcome various hurdles when engineering potential therapeutic-producing bacteria, including producing reliable yet safe and robust microbes. “There is a naïve tendency for some to try to engineer a very abundant commensal that may have a low residency time,” explained Pamela Silver, a professor in the Department of Systems Biology at Harvard Medical School, by email. However, better strategies might include engineering a commensal species that is stable in the gut and can seek out sites of inflammation to deliver a targeted therapeutic. 

Silver and her team also engineer bacteria to function in the mammalian gut long term as live diagnostics of inflammation.⁷ Their long-term goal is to further engineer these sensor bacteria to release a therapeutic to treat the inflammation. “We feel that our sensor bacteria are half the story,” she said. 

Determining how much of a therapeutic needs to be delivered, and whether appropriate levels can be achieved with the engineered bacteria, is another difficulty. “This is fundamentally a big-systems biology question that would benefit from more quantitation and modeling,” Silver explained.

Opening up research avenues 

Synthetic biologists potentially can use therapeutic-producing bacteria for a vast array of diseases, including gut disorders such as inflammatory bowel disease, solid tumors and even viruses. Alternative routes of administration are also an option. “We also have an initiative to develop a bacterially based vaccine against SARS-CoV-2 that could be intranasally administered to generate mucosal immunity,” Wolffe said. 

The brain-gut connection is also a potential target. “Engineered gut bacteria may be used for neurologic and behavioral disorders,” Silver explained. Skin and oral bacteria also offer potential routes for therapies.

Chang’s team now hopes to follow in the footsteps of Synlogic to evaluate the efficacy, safety, and stability of their engineered probiotic bacteria through clinical trials. Silver’s group hopes to turn their sensor bacteria into therapeutic-producers.

Although engineering bacteria as therapeutics is still in its infancy and there are challenges to getting it into the clinic, the technology is already proving to be a powerful tool with the potential to treat conditions that other therapies cannot touch. 

References

1. I.Y. Hwang et al., “Engineered probiotic Escherichia coli can eliminate and prevent pseudomonas aeruginosa gut infection in animal models,” Nat Commun, 8:15028, 2017. 

2. C.L. Ho et al., “Engineered commensal microbes for diet-mediated colorectal-cancer chemoprevention,” Nat Biomed Eng, 2(1):27-37, 2018. 

3. M.R. Charbonneau et al., “Developing a new class of engineered live bacterial therapeutics to treat human diseases,” Nat Commun, 11:1738, 2020. 

4. Y-J. Zhang et al., “Impacts of gut bacteria on human health and diseases,” Int J Mol Sci, 16(4):7493-519, 2015. 

5. F. Scaldaferri et al., “Role and mechanisms of action of Escherichia coli Nissle 1917 in the maintenance of remission in ulcerative colitis patients: An update,” World J Gastroenterol, 22(24):5505–11, 2016.

6. N. Saeidi et al., “Engineering microbes to sense and eradicate Pseudomonas aeruginosa, a human pathogen,” Mol Syst Biol, 7:521, 2011.

7. D.T. Riglar et al., “Engineered bacteria can function in the mammalian gut long-term as live diagnostics of inflammation,” Nat Biotechnol, 35:653-58, 2017.