Harnessing Synthetic Biology for Phage Therapy and Probiotic Development
Researchers are using synthetic biology techniques to develop more targeted antimicrobials.
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Our toolbox for understanding and manipulating biology has developed substantially in the last decade, culminating in novel therapies for combating disease. However, pathogens have been forging defense systems of their own. The evolution of drug-resistance mechanisms has led to antimicrobial resistance (AMR) being declared one of the top global public health threats. To combat AMR, researchers are turning to antibiotic alternatives such as phage therapies for a potential solution.
Synthetic biology techniques have emerged as transformative tools in the tug-of-war between humans and drug-resistant bacteria. This multidisciplinary field, involving the engineering of new or modified living systems, can be used to engineer new antimicrobials in the fight against superbugs.
Today, researchers are utilizing synthetic biology techniques to modify antibiotics, improve antibiotic production and engineer more targeted phages, to name just a few examples.
The scale of the AMR crisis
Antibiotic resistance has been declared a global health crisis, with estimates suggesting that bacterial AMR was directly responsible for 1.27 million deaths in 2019.1 The increase in clinical cases of antibiotic-resistant infections has been attributed to factors including overuse, inappropriate prescribing and lack of innovation in antibiotics.
Despite the need for new antimicrobial drugs, many major pharmaceutical companies are abandoning the field. This decline is believed to be due to low return on investment, the difficulty of identifying new compounds using traditional methods, speculation that resistance will emerge for new antimicrobials and the tough regulatory requirements for bringing new antimicrobials to market.2
The development of synthetic biology approaches is now aiding the search for novel agents and more targeted strategies.
The phage therapy renaissance
Bacteriophages, or phages, have emerged as a powerful alternative to traditional antibiotics. In contrast to many antibiotics, which simultaneously attack the gut microbiota, each phage has evolved to target a specific bacterial strain or species.
Phage therapies first emerged over 100 years ago but fell out of favor in the West in preference of antibiotics. In most Western countries, their application has been limited to the fringes of medicine, being used mostly in compassionate use cases.3 This is often attributed to the absence of clinical evidence showing that phage therapies are efficacious. With the growing threat of AMR, interest has been rekindled in this antimicrobial approach that predates antibiotics.
What is phage therapy?
Lytic bacteriophages, or phages, are viruses that specifically target and kill bacteria. Phage therapy involves administering virulent phages to a patient to lyse a bacterial pathogen causing an infection. The FDA has not yet approved any bacteriophage products for human clinical use. Therefore, US researchers or clinicians intending to administer phages to patients must first submit an investigational new drug application.
“Bacteriophages, they’re not new, they have a hundred years’ worth of isolation and characterization and history associated with trying to use them,” Prof. Martha Clokie, a professor of microbiology at the University of Leicester and the director of the Centre for Phage Research, previously told Technology Networks. “But essentially, previously it [phage research] was done blindly but now we can delve into the genomics. This can help us find phages for many new organisms.”
Ongoing clinical trials are hoping to increase clinical evidence supporting the use of phage therapies. One early-stage clinical trial is evaluating the use of phage therapy in adults with cystic fibrosis who carry Pseudomonas aeruginosa (P. aeruginosa) in their lungs.
To help bridge the translational phage research gap, scientists at the Geisel School of Medicine at Dartmouth recently investigated phase-host responses in human cells. They found that therapeutic phages can be detected by epithelial cells of the human respiratory tract, eliciting proinflammatory responses. The researchers suggest that it may be possible to harness immune responses to phages to improve phage therapy efficacy on a case-by-case basis.4
“Even though phages cannot replicate in mammalian cells, phages can display pathogen-associated molecular patterns that can be recognized by immune pattern recognition receptors on human cells,” Dr. Jennifer Bomberger, professor of microbiology and immunology at the Geisel School of Medicine at Dartmouth, previously told Technology Networks.
“Our study, as well as the few others that have evaluated phage therapy effects in the human host, agree that the immune responses appear to be self-limiting and without causing debilitating effects in the people that have received this therapeutic.”
Enhancing phages with synthetic biology
Alongside a lack of clinical data supporting their use, natural phages often suffer from restricted host range and can suffer from the emergence of phage resistance. These constraints can be overcome by leveraging synthetic biology to provide phages with additional therapeutic capabilities.
Using techniques such as recombineering, CRISPR-Cas-assisted selection or synthetic in vitro genome assembly, specific genes or gene clusters within a phage genome can be precisely engineered to modify a phage's function.5
SNIPR Biome has engineered phages with CRISPR–Cas machinery to specifically target Escherichia coli (E. coli), a frequent cause of bloodstream infections resulting from the translocation of gut bacteria.6
The researchers screened a library of 162 wild-type phages, identifying 8 phages with broad coverage of E. coli that could stably carry inserted cargo. These phages were subjected to tail fiber engineering and CRISPR–Cas arming.
Subsequent tests revealed that the engineered phages reduced the emergence of phage-tolerant E. coli and out-competed their ancestral wild-type phages in coculture experiments.
These findings led to the development of SNIPR001, a combination of the four most complementary CRISPR–Cas-armed phages that target a diverse spectrum of E. coli strains. SNIPR001 has now entered clinical development.
Combining phage’s natural abilities with synthetic tools can expand the range of bacteria they target and help eliminate multi-resistant bacteria. Coupled with advances in genomics and artificial intelligence that can help more accurately identify phages that target particular strains of bacteria, phages have once again become a promising alternative to antibiotics.
Reacting to the 2023-2024 UK House of Commons Science, Innovation and Technology Committee report on the antimicrobial potential of bacteriophages, Dr. Ken Bruce, senior lecturer at King’s College London, said: “The renewed interest in the use of phage therapy represents a positive step in terms of dealing with clinical infection. Phage therapies have been used for many years and interest in the UK is now growing within the scientific community.”
Modifying probiotics for antimicrobial activity
Another promising alternative to antibiotics is engineered probiotics.7 These “beneficial” bacteria can be reprogrammed to recognize specific molecules, triggering the secretion of antimicrobials that disrupt bacterial pathogens.
Researchers at Gyeongsang National University have genetically engineered probiotics capable of detecting and eradicating P. aeruginosa.8 For this, the researchers created a plasmid-based system that produces a P. aeruginosa-selective antimicrobial peptide (AMP). The plasmid-based system was then transferred to the probiotic E. coli Nissle 1917 (EcN).
The researchers demonstrated the ability of the engineered probiotics to express and secrete the AMP, resulting in the inhibition of P. aeruginosa in vitro. “In addition, in a mouse model of intestinal P. aeruginosa colonization, the administration of engineered EcN resulted in reduced levels of P. aeruginosa in both the feces and the colon,” the researchers said.
While engineered probiotics are beneficial in their ability to detect specific molecules, more research in humans is needed to determine the impact of these therapies. The antimicrobials released by the probiotic may cause intestinal dysbiosis, metabolic disorders or other side effects. In addition, AMPs are usually toxic to the probiotic strains and can even kill the producing cells.9
Detecting pathogens before they become an issue
To communicate and coordinate cellular behavior with each other, and other species, bacteria utilize a process known as quorum sensing. Synthetic biologists have attempted to harness the properties of quorum sensing as a tool to construct microbial control systems for detecting harmful pathogens before they can spread and become an issue.
Scientists from the University of Notre Dame created a novel whole-cell biosensor to detect water contamination by P. aeruginosa and Burkholderia pseudomallei (B. pseudomallei), both of which are recognized as common causative agents for waterborne diseases.10 This study constructed and characterized the biosensor based on a quorum-sensing signal system.
Engineering synthetic circuits into intestinal bacteria to sense, record and respond to in vivo signals is a promising new approach for the diagnosis, treatment and prevention of disease.
Synthetic biology meets personalized medicine
Antimicrobial-resistant pathogens have evolved to overcome many antibiotics commonly used to treat infection. Synthetic biology can offer new options to fight AMR through the engineering of phages and bacteria for targeted antimicrobial activity.
There are still significant challenges to overcome before many of these synthetically created therapies can be translated into human medicine. These include the need for more controlled trials of sufficient scale and repeatability to affirm efficacy and safety.
Successful antimicrobial therapies must have little to no impact on an individual's microbiome to reduce negative side effects. Towards this end, increasing effort is being directed toward developing precision antimicrobial therapeutics that target harmful pathogens while leaving host microbiota undisturbed.11 In the future, it may be possible to harness individual microbiome data to tailor antimicrobial therapies to an individual.12
