Throughout the life and death (and nonlife) struggle between bacteria and bacteriophages, bacteria try to cut apart the genetic material that bacteriophages deploy when they try to commandeer bacterial resources. And now the bacterial weapon of choice, the immune system known as CRISPR, is starting to cut both ways. CRISPR components are being engineered into bacteriophages, arming them against pathogenic bacteria, including antimicrobial-resistant bacteria. In phage therapies, CRISPR-wielding bacteriophages may kill bacteria or compel them to carry out useful functions. For example, after suffering a few thrusts of the CRISPR sword, otherwise recalcitrant bacteria may have no choice but to express therapeutic proteins.

The swashbuckling ways of engineered phages are possible only though the patient work of highly disciplined researchers. For example, two research teams—one based at Rockefeller University, and one at the Massachusetts Institute of Technology (MIT)—began independent investigations of phage therapy that culminated in a close and productive collaboration.

Antibacterial teamwork

Five years ago, Luciano Marraffini, PhD, was leading work at Rockefeller to undercut the Gram-positive Staphylococcus aureus, while Timothy K. Lu, MD, PhD, was heading a group at MIT that was taking stabs at the Gram-negative Escherichia coli. In both labs, bacteria were being set upon by phages that had been weaponized with CRISPR-Cas technology.

The similarity in the projects was discovered when Xavier Duportet, PhD, an MIT graduate student familiar with Lu’s lab, talked to his friend David Bikard, PhD, a postdoctoral fellow in Marraffini’s lab. When Lu’s Boston area lab and Marraffini’s New York lab started sharing data, they formed a bond that withstood the usual interlab rivalries (as well as the divisions that must have emerged during sports season).

The labs produced a pair of papers showing, for the first time, that a Cas system targeting a bacterial chromosome could efficiently kill bacteria. Published back to back in Nature Biotechnology, the papers described work that not only delivered scientific value, but also held commercial potential. With Duportet and Bikard at the helm, the work led to the birth of the Eligo Biosciences, a Paris-based company that has assigned itself the task of developing targeted, CRISPR-based antimicrobials to kill antibiotic-resistant bacteria.

Using phages to kill bacteria is a century-old idea that has been slow to come to fruition. To date, no prospective phage therapy has become an FDA-approved drug. But phage therapy continues to inspire research because it could overcome antibiotic resistance, which is reaching dire proportions. It has been estimated that by 2050, 10 million people will die each year from antimicrobial-resistant infections.

Research into phage therapy is exciting, Marraffini tells GEN, because it could “generate a new antimicrobial that will prevent the worst-case scenario from happening.” In the past, people used phages without modifying or engineering them. At present, however, researchers are going farther. For example, scientists at Eligo are adding payloads into phages that directly target bacteria in an orthogonal way to rapidly kill the bacteria.

“Leveraging phage-based killing of microbes is more complicated than it may sound,” notes Lu. Natural phages can kill bacteria, but in many cases, bacteria have evolved resistance to phage activity. One way to circumvent bacterial resistance is through the genetic modification of exterior phage components—an art in which Lu is a “master,” says Marraffini. There are also intracellular barriers such as phage restriction mechanisms and, as noted earlier, the bacterial immune system called CRISPR (clustered regularly interspaced short palindromic repeats).

In the CRISPR realm, Marraffini’s expertise is invaluable. He helped pioneer CRISPR research by collaborating with Feng Zhang, PhD, to illustrate CRISPR’s capacity in human cells. A microbiologist by training, Marraffini notes that it is particularly exciting to see CRISPR advances being applied to antimicrobial-resistant bacteria.

Eligo has developed technology that avoids indiscriminate killing. Associated with current broad-spectrum antibiotics, this sort of killing can eliminate commensal bacteria and accelerate the evolution of drug resistance. The company’s targeted approach is emphasized in the company’s name, which includes “eligo,” a Latin word meaning to choose or select.

Duportet says that while a phage may be able to bind to all the bacteria in a species, by using a guide RNA that is specific for a genetic sequence present in one strain of bacteria and absent in another, it will kill only the bacteria that carry that specific genetic target. In addition, CRISPR-Cas9 can be programmed to cut in 10 different regions of the bacterial genome, which would challenge the bacteria to evolve resistance in 10 different places—a highly unlikely event.

Banking on Cas3

Another company that is engineering CRISPR-wielding delivery vehicles is Locus Biosciences, a 2015 spinout of North Carolina State University (NCSU). Locus is working to introduce Cas3, a nuclease that completely obliterates the cell’s DNA, to bacteria.

Locus’ technology emerged from the NCSU labs of Rodolphe Barrangou, PhD, and Chase Beisel, PhD. Barrangou, distinguished professor at NCSU, pioneered Cas3 research. Beisel, currently an assistant professor at the Helmholtz Centre for Infection Research in Germany, contributed to (and continues to work on) phage packaging.

Paul Garofolo, co-founder of Locus and currently the company’s CEO and Joe Nixon, senior vice president of business development, knew that Cas enzymes in addition to Cas9 represented untapped potential, and they were particularly interested in Cas3. Not only did it “seem to have the best mechanism of action,” Garofolo recalls, but it also avoided the sort of intellectual property drama surrounding Cas9. Barrangou adds that building the company on Cas3 was “obviously valuable, creative, different, inventive, and novel” while being “not obvious to others.”

Locus sources a panel of clinically relevant clinical isolates and then performs high-throughput screening (through their acquisition of EpiBiome last summer) to identify a block of phages that have infectivity to those clinical isolates. That cocktail of phages will be the starting point for engineering. The company also sequences the clinical isolates to identify conserved genes. These genes are used to build the CRISPR targets.

CRISPR allows selective and precise removal of the one genotype of interest by targeting one distinct locus in that strain. Barrangou explains that being precise at this point in the process is of paramount importance. In fact, it is the cornerstone of the company’s technology. It is no coincidence that the company name includes the word “locus.”

“You have to pick the right place,” Barrangou insists. “You can take two strains that are 99.9% identical, but the locus of interest—the locus that is different for each of them—enables you to segregate the two.” He adds that CRISPR is like a sniper in its ability to target the locus of interest.

Another application that interests both Locus and Eligo is the microbiome. The companies hope to manipulate complex bacterial populations in a sequence-specific manner. With the growing understanding that certain resident bacteria cause disease, the ability to kill bacteria in a targeted way may lead to treatments for microbiome-specific alterations. This fits nicely with the overall goal that Marraffini described. That is, to create a smarter antimicrobial that does not wipe out all the bacteria in the body. Such an antimicrobial preserves the good bacteria while targeting the bad ones.

“This will help us develop new technologies to edit the gut microbiota to treat many gastrointestinal diseases,” asserts Casey Theriot, PhD, an assistant professor at NCSU College of Veterinary Medicine and a scientific advisory board member at Locus. “[It will also advance the] rational design of the gut microbiota, which will aid in precision medicine.”

Any advances in this realm, according to Duportet, “rely on the advance of the understanding of the microbiome and moving from a correlation between specific strains and a disease to a causative relationship.” To that end, Eligo is working on some targets through undisclosed partnerships with microbiome companies.

It’s not all about killing

In addition to killing bacteria, Eligo is focused on developing “microbiome gene therapy” which involves putting nonlethal payloads into phages to functionalize the microbiome. The goal is to harness the bacteria resident in the body to express proteins that may serve as biotherapeutics, degrading toxins or otherwise promoting health.

A similar approach has already achieved this by producing genetically modified probiotics. For example, the work being done at Synlogic to lower levels of phenylalanine in people with phenylketonuria. However, most of these probiotics do not colonize the gut for a very long time. So, after these probiotics stop being introduced, they disappear in a matter of weeks. Eligo would rather target resident microbes that are already adapted to the environment.

Beyond bacteria

Cas3, which shreds rather than snips DNA, is already being loaded into bacteria-killing phages. Adapting Cas3 (Type I CRISPR) technology to eukaryotic cells is a long-range goal for Locus. Charles Gersbach, PhD, a Locus scientific co-founder and an associate professor of biomedical engineering at Duke University, is helping the company reach this goal through the development of highly targeted technologies for editing eukaryotic genome sequences, altering epigenomic regulation, and rewiring cellular gene circuits.

“Type I CRISPR systems could potentially be used for all of the same applications that other DNA-targeting CRISPR technologies are used for, plus more,” notes Gersbach. The Cascade complex that targets DNA serves as a scaffold to which one may add nuclease enzymes that cut DNA, or transcriptional modulators that control gene expression. The addition of Cas3 to Cascade could potentially serve as a means for the elimination of unwanted DNA such as viral infections, cancerous gene sequences, and chromosomal abnormalities. Gersbach adds that this is something that current CRISPR systems cannot do.

Alternative means of delivery are needed, however, if Cas3 is to target eukaryotic cells with any efficiency. Type I CRISPR-Cas systems can be delivered by any of the same delivery vehicles that other CRISPR systems and gene therapies use, including viral vectors, nanoparticles, and electroporation.

“The Cas9 systems that have dominated our attention make up only a small fraction of total CRISPR systems in nature,” Gersbach points out. “The type I CRISPR systems that we are now working with actually make up the vast majority, and it is incredibly exciting to open up that area of biology for the genome engineering community to explore all the different ways these additional diverse systems could be used in biotechnology and medicine.”

Phage therapies for a new century

Both Locus and Eligo issued Series A funding announcements in the fall of 2017. Since then, Eligo has been laying low, building the company and working with a focus on research. Locus, however, recently announced a collaboration and license agreement with Janssen Pharmaceuticals, part of Johnson & Johnson, to develop, manufacture, and commercialize Locus’ CRISPR-Cas3-enhanced bacteriophage (crPhage™). The partners intend to target two undisclosed bacterial pathogens. Locus will receive $20 million in initial payments and is eligible for up to a total of $798 million in potential future development and commercial milestones, as well as royalties on any product sales.

Offering a scientific prediction for 2019 to STAT, Steffanie Strathdee, PhD, associate dean of global health sciences, University of California, San Diego, and co-director of the Center for Innovative Phage Applications and Therapeutics, cited advances in CRISPR gene editing and phage therapy. “[These] will coalesce,” she said, “and we will witness the first genetically modified phage cocktails being used to cure patients with multidrug-resistant bacterial infections.” Strathdee added that “this will attract new players in the biotech and pharma space and will provide new momentum to bring phage therapy into clinical trials in the United States.” Indeed, both Eligo and Locus report that they are starting clinical trials soon.

However, Lu predicts that antibiotics will always be a key component in the fight against bacteria because, despite their limitations, antibiotics are cheap and easy. Even if the source of the infection is unknown, it can be effectively treated with antibiotics. Treatments that rely on phages are more complicated. Although phage therapies are more complicated, they must be pursued, Lu maintains, because they promise to counter antimicrobial-resistant infections, a task that may soon exhaust our current armamentarium.

Opening a New Phase of Phage Discovery with Microbiologist Martha Clokie GEN caught up with Martha Clokie, PhD, professor of microbiology at the University of Leicester, UK, to ask her about the future of phage therapy. Just days earlier, Clokie had agreed to serve as the editor-in-chief of PHAGE: Therapy, Applications, and Research, a journal that Mary Ann Liebert, Inc. plans to launch later this year. Clokie started her career studying the molecular evolution of plants, but she migrated to the world of cyanobacteria so that she could work on “something that evolved a bit faster.”

The world of cyanobacteria, she came to appreciate, is profoundly influenced by phages, which are vast in number. Research into cyanobacteria/phage links deepened when Clokie discovered that marine phages contain photosynthesis genes. She showed that a phage can do more than exert selective pressure on a cyanobacterium’s infection-survival mechanisms. It can also acquire genes from bacterial prey, extending to its host a valuable characteristic—ensuring that energy (and therefore more phages) are produced. Phages, then, aren’t just parasites. They can be partners in physiology. “Getting phages to the clinic has been a tough road,” notes Clokie. A stable, high-titer phage therapy is both hard to produce and tricky to regulate. Encouragingly, Clokie adds that there is a new level of engagement from the regulatory agencies. She observes that more people from the FDA are attending phage meetings than ever before. “When you look at it,” notes Clokie, you see that “there is very little money spent on phage research—dribs and drabs” compared to other research. However, the pressing need for novel ways to combat antimicrobial resistance may change research priorities. She points to the example of the funding being directed toward Locus Biosciences, surmising that their interesting approach coupled with having the CRISPR-Cas3 string in their bow likely proved useful when seeking funding. When asked to choose this moment’s most exciting phage research, Clokie responds, “It is much too hard to narrow it down.” She adds that even though people have been studying phages for a century, “we are just now entering a new phase of phage discovery.”

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