In the mesmerizing world of genetic engineering, prime editing has emerged as a shining star, promising to rewrite the very fabric of life with pinpoint precision. But here’s the plot twist: a team of relentless scientists at the Broad Institute of MIT and Harvard have summoned the powers of phages and protein engineering to birth a new generation of prime editors that are not just efficient but astonishingly compact.
The Genesis of Prime Editing
Prime editing, akin to the artistry of a genetic sculptor, was unveiled in 2019 by the visionary David Liu and his team. This editing technique entails genome modification through search-and-replace mechanisms, all achieved without the need for double-strand breaks or donor DNA. This molecular wizardry combines a tweaked Cas9 protein, an engineered prime editing guide RNA (pegRNA), and a modified reverse transcriptase enzyme. Together, they form a potent triumvirate capable of performing DNA edits with laser-like precision.
Epic Evolution: Phages to the Rescue
Enter the heroes of our tale: bacteriophages, the viruses that prey on bacteria. The scientists harnessed the evolutionary prowess of these tiny warriors through a technique called phage-assisted continuous evolution (PACE), first forged by David Liu in 2011. Picture this: a battlefield in the lab where only the bacteriophages that bear the most desirable genetic traits can survive and thrive, replicating faster than they can be wiped out.
In this grand showdown, the bacteriophages carrying the secrets of prime editing faced countless generations of evolution. The result? Brand new, turbocharged reverse transcriptases that turbocharge prime editing to unparalleled heights.
Prime Editors: Not All Heroes Wear Capes
Here’s the twist in our tale: Not all prime editors are cut from the same genetic cloth. The evolved prime editors have their own unique superpowers. Some excel in adding or swapping just a few DNA letters, while others stumble in these tasks but soar when it comes to inserting long DNA sequences. This revelation shattered the notion that one prime editor could rule them all.
Expanding the Editing Universe
With their newfound superpowers, the prime editors, now known as PE6a through PE6g, have blown the lid off previous limitations. These titans are two to 20 times more efficient than their predecessors, raising the banner of hope for therapeutic applications higher than ever before.
Their versatility shines bright as they effortlessly insert lengthy DNA sequences—ranging from 38 to 108 base pairs—into the genomes of human cells. Even the once-untamable frontier of live mouse brains has succumbed to their precision, with editing efficiency soaring to astonishing heights.
Prime Editors: Travel Light, Edit Right
But there’s more to this story. These newfound prime editors have slimmed down, shedding excess genetic baggage. This means they can squeeze into delivery systems that previously dared not accommodate them. This improved agility opens the door for prospective therapies in animal models as a necessary preliminary step before human studies.
Bacteria and bacteriophage. Photo by Oxford (Article by Pete Wilton)
In recent years, the development of bioluminescence-based reporter bacteriophages has revolutionized the monitoring of microbes and their diseases. These reporter bacteriophages are genetically engineered to carry a gene that will be integrated into the host genome which will at one point reflect the fluorescence signals. Bioluminescence reporter bacteriophages have high specificity and sensitivity, making them useful tools for studying microbes and their diseases. They are also useful research tools that enable researchers to monitor the efficacy of bacteriophage/drug treatments, the spread of infection, the success of phage delivery, and the activity of phage-resistant bacteria. In this article, we provide an overview of bioluminescence reporter bacteriophages, describe their applications and advantages, and discuss how you can use them in your own research.
What is Bioluminescence?
Bioluminescence is the production and emission of light by living organisms. This light is produced by the living cell through an enzyme called luciferase. This enzyme catalyzes the transformation of the light-emitting substrate, luciferin, into oxyluciferin, which can then be converted to light by light-emitting complexes. The light-emitting complexes are also called photoproteins. The overall process of bioluminescence is called photoautotrophy. In photoautotrophs, light energy from the sun is used to synthesize chemical energy and regulate cell metabolism, in order to provide energy and materials to the organism. In photoheterotrophs, light energy is used to regulate metabolism and produce energy by photosynthesis. In photoautotrophs, the production of light energy is coupled with the production of essential chemicals in the form of macromolecules such as carbohydrates, lipids, proteins, and nucleic acids. In photoheterotrophs, the production of light energy is not coupled with the production of essential chemicals. Instead, the photosynthesis process uses the energy from light to synthesize complex organic molecules such as carbohydrates, lipids, and proteins. In addition to producing light energy and essential chemicals, photosynthesis requires the production of a supply of carbon. The production of this carbon supply is called photosynthetic carbon fixation. This process of photosynthetic carbon fixation is what produces bioluminescence in many types of organisms.
Bioluminescent reporter bacteriophages are genetically modified viruses that infect their host cells with high specificity and transduce a heterologous luciferase gene, the activity of which can be detected with high sensitivity to indicate the presence of viable target cells. Bacteriophages (phages) evolved as natural predators of bacteria to bind their hosts with unparalleled specificity and to rapidly deliver and replicate their viral genome. Not surprisingly, phages and phage-encoded proteins have been used to create a diverse set of diagnostic assays, many of which outperform traditional culture-based and molecular detection methods. While intact phages or phage-encoded affinity proteins can be used to capture bacteria, most phage-inspired detection systems rely on viral genome delivery and amplification: suitable phages are genetically reprogrammed to deliver heterologous reporter genes, the activity of which is typically detected via enzyme action on substrate conversion to indicate the presence of a viable host cell. Infection with this kind of engineered reporter phages usually results in a rapid burst of reporter protein production, allowing for highly sensitive detection.
Uses of Reporter bacteriophages
Bacterial pathogens in clinical samples can be detected quickly and reliably.
Assessing contaminations in samples (e.g water and food samples)
Monitoring spread of infection or contamination
Advantage of using reporter bacteriophages
They are specific
They are reliable
They are fast (act quickly)
Has potential for automation
Easy to make them reproducible and user friendly
How to use Reporter Bacteriophages in your research
Before discussing the applications of bioluminescence reporter bacteriophages in biology, it is important to consider how they are used in research. Although the majority of bioluminescence reporter bacteriophages are used as research tools, there are a few specific applications of reporter phages in healthcare and agriculture that researchers can also use them for.
Healthcare researchers use bioluminescence reporter phages for two main applications: monitoring the efficacy of treatments and tracking the spread of infection. The first application involves using bioluminescence reporter phages to monitor the efficacy of the anti-microbial agents in controlling the growth of disease-causing bacteria. Because the efficacy of antibiotic treatments can vary depending on the species of bacteria, monitoring the growth of bacteria in the presence of antibiotic compounds is highly important to researchers in the field of antibiotic design. For example, if researchers want to know how well an antibiotic works against bacteria in the Staphylococcus aureus group, they can genetically engineer a bioluminescence reporter bacteriophage to express luciferase in the S. aureus group and infect bacteria. When the bacteria are infected with the bioluminescence reporter phage, they will express the luciferase enzyme, which indicates that the antibiotic administered to the bacteria is having an effect on them and can be used to design better drugs.
Bioluminescence reporter bacteriophages are also used to track the spread of infections in livestock. As most animals are susceptible to infections from bacteria, researchers use bioluminescence reporter phages to monitor the spread of infectious agents within animals. This can be used to track the success of vaccination programs and the efficacy of anti-bacterial compounds and vaccination strategies. For example, a cattle ranch might want to know how effective a vaccination program is at controlling the spread of infectious bovine pneumonia (IBPP) in their animals. After vaccinating their cattle, researchers can use a bioluminescence reporter phage to track the spread of infection within their animals. When a bioluminescence reporter phage infects a susceptible bovine, they will express the enzymes luciferase and green fluorescent protein, which indicates that they have been infected with IBPP and will give off light signals until they die of infection. From this, researchers can determine the success of their vaccination program. Another application of bioluminescence reporter bacteriophages in agriculture is in aquaculture where a farmer can easily monitor the spread of infections from farm to farm until its managed.
NB:This technology (Bioluminescence-based Reporter Bacteriophages) can be used in a variety of other fields (food processing industries e.t.c) that involve the detection of bacteria.
Although bioluminescence reporter bacteriophages have been used mainly as research tools, this technology is at an early stage of development and has a wide range of applications in medicine and agriculture. They have a wide range of uses in both research and healthcare, including monitoring the efficacy of antibiotics and the spread of infection within organisms. They also have far-reaching potential in agriculture and environmental monitoring. The development of this technology has led to the discovery of new ways to track microbes and the spread of infection, and provides new tools for scientists to study how microbes interact with their environments.
Bacteriophages are viruses that have high specificity for their hosts. Given the fact that their target host is bacteria, it makes them an attractive means of eradicating bacteria infections. These entities are capable of exhibiting a lytic cycle, which means that they kill (lyse) their host cell to disperse the replicated bodies to other hosts and spread infection.
Despite having the most established means of fighting the bacteria i.e. anti-bacterial, bacteriophages are possible “last resort” strategies for treating infections. This has been proved by the ability of these viral particles on treating patients infested by “superbugs”
There are some major limitations to the use of phages for therapy
The phages are sensitive to changes in pH
They could be damaged by enzymatic activity caused due to both Bacterial hosts and human immune cells.
They have a short serum /plasma half-life. (Serum half-life of a drug is the time it takes for the drug’s concentration in blood plasma/serum to reduce to half of the original concentration.)
A novel approach to overcoming the limitations of phage therapy
A possible solution could be seen by loading the phage within a biomaterial (i.e., biopolymers, synthetic polymers, and ceramics). A good example is DendriPrep, A dendrimer innovative technology from New York City State University (NCSU) researchers that improves the delivery of nanoparticles i.e bacteriophages. It can shield the incorporated phage against many harmful environmental factors, as well as provide a controlled release for prolonged serum half-life.
Cancer cells. Photo by technology networks
Bacteriophages are specific natural enemies of bacteria although gene manipulation can lead to other purposes in drug delivery and vaccines. Filamentous phages have a huge surface bearing capacity and flexible genetic engineering property, thus, can easily be loaded with drugs and utilized for targeted drug delivery. In nanomedicine, phage-mimetic nanoparticles have as well been developed to achieve the same. In particular, chemical drugs can be conjugated on the phage surface by chemical modification, and gene drugs can also be inserted into the genome of the phage by recombinant DNA technology. This ability of filamentous phages makes them a potential system for cancer therapeutics and treatments.
The huge surface bearing capacity and flexible genetic engineering property in filamentous phages can effectively be used to load chemical and/or generic drugs. These particles can target the specific lesion location in the patient’s body. The peptides and proteins exhibited on the phage surface can moreover be applied directly as self-navigating drug delivery nanovesicles.
Interestingly, the filamentous phage is considered to be an eligible performer in antibody engineering which makes it gain a reputation in nanobiotechnology and cancer research. The property of bacteriophage which makes it an eligible candidate to fight against cancer is its specificity towards certain cells and/ or tissues.
The specificity of the target is related to the surface peptides and proteins on the phage coat, which can further be conjugated with other therapeutic nanoparticles. The genetic and chemical modification of the filamentous phages leads to a modular target drug-carrying platform of nanometric dimensions, where the targeting moieties (biomolecules) and the conjugated drugs can be delivered at will.
Researchers in the study done by Bar et al (2008) used genetically modified and chemically manipulated filamentous phages. The genetic modification endowed the phages with the ability to display a host-specificity-conferring ligand then they were loaded with a large payload of cytotoxic drugs by chemical conjugations. In their experiment, they used anti ErbB2 and anti ERGR antibodies as targeting moieties, the drug hygromycin conjugated to the phages by a covalent amide bond, or the drug doxorubicin conjugated to genetically-engineered cathepsin-B sites on the phage coat. It was observed that targeting phage nanomedicines via specific antibodies to receptors on cancer cell membranes results in endocytosis, intracellular degradation, and drug release, resulting in growth inhibition of the target cells in vitro with a potentiation factor of >1000 over the corresponding free drugs. They used “binding analysis with monoclonal antibodies complexed phage nanoparticles using whole-cell ELISA” as the technique to obtain the results.
In 2019 at the Imperial College of London research has been going on targeting a hopeful cure for Glioblastoma, a form of brain tumour to which currently the only available chemotherapy is a drug called temozolomide (TMZ). However, it has limited effect. An international team led by researchers at Imperial College London has had promising results using modified bacteriophages to target cancer cells in the brains of mice. They were able to deliver targeted therapy directly to cancer and amplify the effect with TMZ. The treatments led to tumours shrinking while leaving healthy tissues intact as well as increasing the life expectancy of the animals.
Case of Oncolytic Viruses
Oncolytic (tumour-killing) viruses a.k.a. OVs induce antitumor effects in a two-way mechanism one by direct lysis of target cells and eliciting an immunogenic response to the virus and ultimately to the target cells. They destroy tumour mass by infecting and multiplying within the mass. In addition to viral lysis, the tumour mass, due to the presence of immunogenic viruses, is subject to attack by the immune system. Partial remission of tumour mass when patients contracted viral diseases was observed by physicians as early as the beginning of the twentieth century. One of the FDA-approved oncolytic viruses is HSV-1 based T-VEC and is clinically in use. Attenuated human pathogens, which have been tested as potential OVs, include the adenovirus, the vaccinia virus, the measles virus, the mumps virus, and the influenza virus. Other viruses known to be poor human pathogens, which have been tested as OVs, include the Newcastle disease virus and the vesicular stomatitis virus. potential OVs are often not powerful enough for solid tumours and safety has not always been matched with efficacy, as OVs are known to exhibit a certain range of toxic effects. In addition, live viruses have been shown to be transferable from the primary treatment patient to healthcare workers and people inhabiting the same household.
Why are phages better?
A major class of OVs are pathogenic to humans and are thus not in use for the purpose. However, studies on bacteriophages have shown the potential of inhibiting tumours. As stated by Budynek et al (2010), In 1940, the accumulation of phages in cancer tissue and the inhibition of tumour growth was observed, and in 1958, the binding of phages to cancer cells was seen both in vitro and in vivo. Phage T4 and its substrain HAP1 were shown to bind melanoma cells and inhibit lung metastasis in a murine model.
Bacteriophages have been proven to be a natural and involuntary medicine as they are circulating in the mammalian body controlling invading pathogens and regulating our immune system naturally. Furthermore, they can block β3 integrin activity on neoplastic cells thus preventing growth and metastasis formation. Moreover, they also restrict angiogenesis in the developing cancerous tissues or organs and prevent metastasis by inhibiting the adhesion of platelets and T-cells to the fibrinogens.
In the study by Hwang et al, T7 bacteriophages were engineered to act as anti-cancer agents. In vivo, their experiment showed significant results in tumour-carrying mice with half of them being administered with wild-type T7 phage and half with engineered T7 phage intravenously. About 40% of mice treated with wild-type T7 phage survived while the stats were 100% positive for the mice treated with engineered T7 phage.
In the world of booming antimicrobial resistance, scientists worldwide struggle to find a proper method of treating superbugs. Recently, the savior has been recognized globally, and the interest has been reinstated to build on the foundation of what is already known. A virus that is the most abundant entity on this planet turned into an alternative to antibiotics. These viruses have so far saved patients who were in intensive care units where no antibiotic was able to cure them. Despite the trial on ways of commercializing them just like regular antibiotics, phages still suffer many problems provided that they are live particles. One of the significant problems is maintaining phage preparation stability during delivery. Mainly the stability will be influenced by the matrix/media used to suspend phages.
Researchers from New York City State University (NCSU) developed a soft material capable of preserving bacteriophages for therapy. With his colleague Christopher Gorman in the Department of Chemistry and students Ryan Smith and Juliana O’Brien, Stefano Menegatti, a University Faculty Scholar and an associate professor of chemical and biomolecular engineering, developed new dendrimers — highly structured macromolecules — that act as glue around nanoparticle templates. Such templates include bacteriophages, viruses that can infect and attack harmful bacteria in the body. While they can be an alternative to traditional antibiotics, they can be challenging to formulate and stabilize as a medicine. To remain safe and effective, therapeutic bacteriophages need to be protected from environmental changes.
Purple and green microscopic imaging shows how particles coated in Dendripeps separate when shear increases. Photo by NCSU
Menegatti and his team found that dendrimers combined with short chains of amino acids, or peptides, can surround nanoparticles and keep them stable in various conditions. Their team calls these dendrimers DendriPeps. As nanoparticles are suspended in water, these soluble DendriPeps keep them in a closed, stable system.
“These dendrimer coatings maintain the water content inside a virus while acting as a proton sponge, shielding the virus from salt or pH changes,” Menegatti said. “It’s a soft yet impermeable barrier. It maintains a nice environment for the virus.”
Once DendriPeps have coated nanoparticles, localized shear triggers their release and therapeutic activity. Soft materials can be designed to respond physically and chemically to stimuli like temperature, pressure, and moisture. Shear is an ideal stimulus for drug delivery applications because it is almost ubiquitous throughout the body. Eyelids cause shear when blinking; waste shears the gut wall during digestion; shear is created when one rubs something onto the skin. This allows DendriPeps to treat local infections.
Microscopic imaging shows how shear degranulates particles that have been coated in Dendripeps.
“Shear is often a forgotten stimulus,” Menegatti said. “With this invention, we wanted to fill that gap, and Dendripeps offered an amazing opportunity to do so.”
When a cluster of DendriPep-coated nanoparticles is sheared, it degranulates, releasing the single particles. These particles can, in turn, release a therapeutic payload or, like the bacteriophages, infect and kill dangerous bacteria. When therapeutic action is not needed, shear is removed, and the particles are recoated with DendriPeps and recluster. This stops the payload release.
This formulation can protect bacteriophages. This offers a way of replacing antibiotics in some critical applications: The widespread use of antibiotics has developed superbugs — bacteria resistant to standard doses of antibiotics. The research team is also developing more DendriPep-based nanomedicines to deliver new drugs like viral vectors in gene therapy.
“DendiPeps could be the missing link in material sciences to make therapeutic viruses the center of the next-generation antimicrobials and drugs,” Menegatti said.