GEN BiotechnologyVol. 2, No. 3 News FeaturesFree AccessNature's Needles: Adapting Bacterial “Syringes” into Programmable Protein Delivery DevicesJonathan D. GrinsteinJonathan D. GrinsteinE-mail Address: jgrinstein@liebertpub.comSenior Editor, GEN Media Group.Search for more papers by this authorPublished Online:19 Jun 2023AboutSectionsPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail Discovered in parasitic bacteria nearly two decades ago, contractile injection systems have been redesigned by Feng Zhang and colleagues to efficiently target human cells with protein payloads and have possible applications in gene therapy, cancer therapy, and biocontrol.Joseph “Joe” Kreitz has a background in studying bacterial viruses, which have a molecular structure for injecting their genomes into target cells. When Kreitz, a graduate student in Feng Zhang's laboratory at the Broad Institute, found that there are systems in bacteria reminiscent of bacteriophage tails capable of delivering proteins (rather than nucleic acids) into eukaryotic organisms, he was immediately hooked. Could these systems be engineered for the delivery of payloads into human cells as a therapeutic delivery device?It is a monumental question. “We really need new approaches in the delivery space,” Zhang told GEN Biotechnology. “So much emphasis has been put on viral vectors or LNPs, and new approaches are really important.” Earlier this year, Zhang launched a new company called Aera Therapeutics that focuses on moving the cargo of genetic medicines—RNAi, antisense RNA, mRNA, or a genetic editing payload.Joseph Kreitz, Graduate Student in Feng Zhang's laboratory at the Broad InstituteKreitz's curiosity took him and his colleagues down a productive road that ended up in the generation of a new technology that could be a game changer for the therapeutic delivery of biomolecules.1 As reported recently in Nature, Kreitz led a group of colleagues into reprogramming these bacterial “syringes”—called contractile injection systems (CIS)—to target eukaryotic cells not natively targeted by these systems, including mice and human cells, with efficiencies approaching 100%. CIS can load diverse protein payloads, such as the Cas9 nuclease, base editors, and toxins. With further optimization, these programmable devices could be used for a host of applications in gene therapy, cancer therapy, and biocontrol.The work has been hailed by commentators including Rodolphe Barrangou, distinguished professor at North Carolina State University and chief editor of The CRISPR Journal. Barrangou said that the ability to reprogram these molecular machines for cell- and tissue-specific delivery is very intriguing and potentially very useful to target editing strategies to specific cell types and tissues. “Being able to deliver Cas9 and expand this to other genome effectors is exciting and useful at a time when delivery is the challenge for genome editing's next step into the clinic,” Barrangou told GEN Biotechnology.Back to the FutureIndeed, the re-engineering of CIS follows a story much like that of CRISPR, in which unheralded research on natural microbial systems ultimately gave rise to a next-generation genome editing technology. Across the biosphere, there is incredible diversity in CIS used to mediate symbiosis between bacteria and their eukaryotic hosts. This seems to be a general strategy among bacteria that live within larger organisms (i.e., symbionts) and was uncovered by several independent lines of research in the United States, United Kingdom, and New Zealand, each group studying different pathogen–host systems dating back almost two decades.In 2004, Waterfield et al. from the University of Bath in England reported the identification of regions in the bacterial genome responsible for unique aspects of bacterial behavior, such as host symbiosis and pathogenicity.2 A few years later, Yang et al. in Waterfield's laboratory showed that the insect–pathogenic bacterium Photorhabdus—the same organism used by Kreitz et al. to engineer CIS—has phage-related loci containing putative toxin effector genes, which they designated the “Photorhabdus virulence cassettes” (PVCs) (Fig. 1).FIG. 1. Photorhabdus virulence cassette.To inject different proteins (called effector proteins) into specific insect cells, Photorhabdus bacteria secrete an extracellular structure (gray). These payloads (red) are directed toward the hollow tube of the structure by a packaging domain (green). Specificity for the intended cell is provided by the binding protein (blue) in the tail fibers. The sheath tightens after the fibers contact the target cell and bind to surface receptors there. This mechanically forces the tube through the specific membrane, allowing for the intracellular delivery of proteins. (Courtesy of Joseph Kreitz).In that 2006 article, the researchers showed that recombinant Escherichia coli expressing a PVC from Photorhabdus has injectable insecticidal activity against larvae of the wax moth.3 Using electron microscopy, they revealed that the structure of the PVC products was similar to the structure of the antibacterial R-type pyocins—those archetypal viral structures consisting of a tube, feet, and a tail spike. A comparison of the genomic organizations of several PVCs showed that they have a conserved phage-like structure with a variable number of putative anti-insect effectors encoded at one end.While this was happening in the United Kingdom, Mark Hurst at the Canterbury Agricultural and Science Centre in New Zealand was studying how an Enterobacteriaceae (Serratia entomophila) that had been made commercially into a biopesticide worked to infect an important grass grub and pasture pest in New Zealand Costelytra zealandica. Hurst wanted to understand how this host-specific parasite, affecting only the larvae of this species of New Zealand scarab, was causing infected larvae to cease feeding within days of being ingested, turning the grub's gut, which is normally dark amber, clear, and decreasing the levels of the major gut protease digestive enzymes, such as trypsin.The larvae infected with this “amber disease” then remained in this state for a prolonged period (1–3 months) before bacteria eventually invade the insect's body cavity (hemocoel), resulting in rapid death of the larvae.In 2004, Hurst et al. demonstrated the identification of an Serratia entomophila gene encoding a potential prophage necessary for antifeeding activity toward larvae of C. zealandica that appeared to be highly conserved remnants of prophages. This, Hurst wrote, suggested the presence of a “novel toxin delivery system that utilizes a phage-type structure through which to mediate transfer of the toxins,” and claimed that, to their knowledge, this was the “first example of a phage-type element located on a mobile plasmid.”4Three years later, Hurst published the first electron microscopy photographs of a purified version of the antifeeding prophage protein. These images showed that the bacterial protein “resembled a phage tail-like bacteriocin, exhibiting two distinct morphologies: an extended and a contracted form.”5 In this article, Hurst tipped his hat to Yang's work on PVCs, acknowledging the similarities in the structures.Then there is a third group of researchers from the laboratory of John Mekalanos in the department of microbiology and molecular genetics at Harvard Medical School. This group was studying the virulence mechanisms from a strain of Vibrio cholerae—which caused a cholera-like outbreak in Sudan in 1967. Led by Stefan Pukatzki and Amy Ma, the group found a set of genes that encode a unique protein secretion machinery that they named “VAS” (virulence-associated secretion).6 The analysis by Mekalanos's team indicated that this strain had a unique mechanism for secretion, which they called “type VI secretion” (T6SS) to clarify its relationship with previously described secretion systems involved in microbial pathogenesis.A year later, Pukatzki et al. presented evidence that the V. cholerae T6SS likely builds a structure similar to the tail spike protein complex of the E. coli bacteriophage T4, which, similar to PVCs, contain the archetypal, alien-looking tube, feet, and tail spike of viruses.7 The predicted complex was hypothesized to serve the purpose of puncturing host membranes as well as serving as a channel for export of macromolecules out of the bacterial cell and into a target cell.Over the next decade, a new class of CIS were recognized—R-type pyocins, the bacterial T6SS, and the PVCs—to describe a class of syringe-like nanomachines resembling bacteriophage tails.8–10Reimaging Injectors for Medical InterventionCISs have since been found to target mouse cells, raising the possibility that these systems could be harnessed as protein delivery tools. Kreitz, who has a background in bacteriophages and particularly how phages used molecular “syringes” to inject their genomes into cells, was keenly following work showing that CIS, especially PVCs, could target mice. He began thinking about how to use CIS as therapeutic delivery devices in humans.Working with colleagues in Zhang's group, Kreitz first cloned the naturally occurring PVC, expressed it in E. coli, and then used the purified protein to demonstrate its activity in insect cells, which they then tailored to kill human cells. “These PVCs naturally target insect cells, so they don't target human cells at all,” Zhang explained. “This is beneficial for us because that's how Joe [Kreitz] is able to make it very specific—you can remove the part that binds to an insect cell and replace it with something that binds to a specific thing on a human cell surface and get it to go into a human cell.”As it is unclear what part of the insect cell the PVC binds to, Zhang's group used AlphaFold—the powerful artificial intelligence-based protein structure prediction system—to 0 in on the tail fiber region mediating this interaction.“[AlphaFold] gave us the information we needed to make a new delivery strategy that can be changed to target different cells,” said Kreitz. “All it takes is the addition of a very simple modification to its tail fiber protein that extends from the base to the part near the spike that actually gets driven through the membrane. We can add a novel binding domain to this tail fiber that would trick the syringe into binding a human cell instead of an insect.” In this way, PVCs can be repurposed by attaching binding domains from human viruses, allowing them to bind to human cells.By altering the binding domain, Kreitz et al. demonstrated various ways to reprogram the tail fibers. For example, they customized the tail fibers to target the epidermal growth factor receptor (EGFR), demonstrating the system's ability to generate activity in EGFR-positive cell lines.In a targeted way, this system kills cancer cells extremely well. PVCs can specifically target cancer epitopes, and then the system can be programmed to inject toxins, killing the cell with minimal side effects. “It doesn't seem to kill cells that don't contain the receptor that you're targeting,” said Kreitz. “You could imagine an application where you're giving patients a PVC that targets a cancer epitope and then having the system go and deactivate cells that express that cancer epitope.”The Broad group also demonstrated that their redesigned CIS could transport payloads other than toxins. “The system is actually quite powerful,” said Kreitz. “It can load and deliver a really versatile set of protein payloads. The system naturally delivers toxins because, of course, it's trying to kill the insect. Those are around 300 amino acids [in size].”In a demonstration of the system's payload versatility, Zhang's group reprogrammed it to transport a range of proteins, from the smallest proteins to Cas9, which is several multiples of magnitude larger than a standard PVC toxin. Kreitz speculates that protein unfolding may play a role in the ability of the CIS of Photorhabdus to deliver such a diverse array of proteins. “This system likely unfolds these proteins to some extent because it needs to fit the protein into this tube,” said Kreitz. “It is quite remarkable that the system can load such large payloads and then actually deliver them and retain the function of those payloads in target cells.”Protein Payloads and BeyondIn the Nature article, Kreitz et al. also described the system's efficiency and specificity in reaching their intended cells while minimizing collateral damage. They injected the modified CIS intracranially into a living mouse brain, but it is not yet known where the PVC travels, how well it penetrates tissues, or whether it is simply cleared. “Because this is quite a large complex, it would be interesting to study exactly whether it's able to move through tissues and reach cells that are farther away from where you're actually injecting,” said Kreitz.“In our paper, we're injecting it into a very defined, small region of the brain that we're interested in. But for some diseases, you might want it to go in a more systemic way throughout the body. The PVC's behavior in vivo is a place that will require more work in the future.”This is just the tip of the iceberg, when it comes to the diversity of CIS options available. For example, Zhang was excited to see that they can be found in the human gut. “It would be very interesting to see what [the CISs in the human gut] may do inherently to target human cells,” said Zhang. “There may also be invading properties that they may have because they've been living in the human gut. Those are just some examples, but there's a lot that we can do.”Zhang speculates that cells have appropriated a variety of natural mechanisms for molecule transport. “There are also many other versions of this in other bacteria, so you can either engineer this one or explore all the others, which will probably have different properties, to achieve a set of capabilities, and I think we'll have to do both,” said Zhang.“It's pretty trivial to re-engineer and is similar to producing any recombinant protein, but in terms of developing this into a biomedical tool, there's still a lot of work to be done across the board, from discovery to manufacturing. You can grow a lot of bacteria using fermentation processes, and we'll have to make sure that they're pure because we need to get really endotoxins and other bacterial contaminants, which could be immunogenic. That could be part of the challenge, but that's more about the manufacturing. It's still early days for this as a technology.”Michael Mitchell, an assistant professor at the University of Pennsylvania who was a key contributor to the development of the lipid nanoprotein (LNP) platform while a postdoc in Bob Langer's laboratory at Massachusetts Institute of Technology, says that although LNPs are ideal for administering RNAs, they present significant difficulties when it comes to delivering proteins for applications such as genome editing. “It is difficult to encapsulate proteins into LNPs and deliver them efficiently into cells,” said Mitchell. “This PVC platform can potentially overcome those challenges.”However, there is a potential stumbling block: at least in its current form, this CIS is only a protein delivery system. Further engineering will be required if researchers want to entertain delivery of different biomolecules. When Zhang's team initially tried to reprogram the PVC to carry nucleic acids, they ran into problems.“Right now, it's a protein delivery system only, so it'll be great to get it to deliver RNA or DNA,” Zhang told me. “There are a lot of different phage or secretion system mechanisms that deliver things like RNA or DNA, so we'll be trying to engineer that.”Future studies, according to Barrangou, should investigate increasing the protein payloads to other genome editing effectors beyond Cas9 and determining payload capacity and capability for multiplexing. Barrangou and Mitchell are also in agreement that the system needs more widespread testing. Mitchell wants to know whether PVCs can be given intravenously to transport proteins to the cells and tissues that need them for treatment. As Mitchell put it, “they show an initial in vivo proof of concept as a direct injection into the brain,” but demonstrating IV delivery could open many therapeutic opportunities in organs such as the liver and lungs. Barrangou wants to see the system tested in other cell types and organisms, including plants, given the challenges of deploying genome editing in plants.And that is the unlikely story of how the study of a couple of bacteria pathogenic to different insects and a rare variant that caused a cholera-like outbreak in Sudan more than half a century ago helped inspire the development of a novel technology platform that could be a game changer for the delivery of biomolecules. Time will tell whether this nature-inspired tool will, like CRISPR, reverberate through the world of biotechnology and, perhaps, even change the course of countless lives for the better.