CRISPR-phage antibacterials to address the antibiotic resistance crisis: scientific, economic, and regulatory considerations

Abstract

The COVID-19 pandemic has served as a reminder that infectious diseases are among the greatest threats to public health.1 Although the harms of many once-common infectious diseases have been dramatically reduced through the development of antibiotics and vaccines, as well as other public health interventions,2 the evolution of resistance means that these diseases may eventually reemerge with deadly new force.3 Already, >2.8 million antibiotic-resistant infections are estimated to occur each year in the USA, and these are responsible for >35,000 deaths.4 Drug-resistant bacteria kill 1.27 million people annually, more than HIV and malaria combined.5 Furthermore, deaths are disproportionately concentrated in low- and middle-income countries, especially in children under 5 years old, and some estimates suggest that the death toll may rise to 10 million annually by 2050.6 One means to address this precarious situation is the development of new antibacterials. Among the promising potential technologies is bacteriophage (‘phage’) therapy, which uses viruses (phages) that precisely target and kill bacteria.7 Although phage therapies have been studied for a century, the development of antibiotics in the 1940s led to decades of dormancy in phage research. Recently, however, the increasing threat of antibiotic resistance and improved understanding of genetics have not only reinvigorated scientific interest but also stimulated massive investments in the development of phage therapies.8 In particular, the rise of new genomic technologies, including genome editing techniques with the potential to augment phage effectiveness, have contributed to renewed interest. One of the most significant such technologies leverages genetic sequences known as ‘clustered regularly interspaced short palindromic repeats’ (CRISPR).9 CRISPR-based technologies utilize a natural defense mechanism of bacteria that, although normally directed against invading virus particles, can be repurposed to attack human pathogens.10 However, before CRISPR-phage therapy can be utilized, many interrelated challenges will have to be addressed.11 These range from regulatory and safety concerns to intellectual property issues and economic considerations.12 Although more evidence is needed before the US Food and Drug Administration (FDA) and other regulatory bodies are likely to approve phage therapies as safe and effective, there is increasing confidence that they may soon be more widely used to fight bacterial infections. This article evaluates the potential of CRISPR-enhanced phage therapy (CRISPR-phage therapy) as a means of addressing antibiotic resistance, providing an evaluation of the scientific and economic challenges to its development and regulatory implications. Phage therapy and new genomic technologies, including CRISPR technology, face challenges to their development. These obstacles include the development of bacterial resistance toward the phage and compatibility challenges between the different phage components (ie delivery vectors and CRISPR). Phage therapy has been studied since the 1920s for its ability to treat human bacterial infections.13 Amid the deprivations of World War II, Polish physicians used phage therapy when antibiotics were not available.14 Although the technology was not secret, published studies on phage as an effective antibacterial appeared predominantly in Eastern European and Russian language journals, which limited their reach in Western Europe and the USA.15 After the invention and widespread use of antibiotics, interest in commercializing phage therapy in the USA waned,16 only to rise in popularity decades later as antibiotic resistance emerged as an ever greater threat to public health.17 For around a century, human phage therapy trials have taken place in Eastern Europe. Most notably, the nonprofit Eliava Institute of Bacteriophages in Tblisi, Georgia, and the Institute of Immunology and Experimental Therapy in Wroclaw, Poland, have been pivotal in creating an evidence base for phage therapy.18 In a successful trial in 1938, 219 patients with bacterial dysentery were treated with a ‘cocktail’ consisting of multiple phage types, and 74 per cent showed improvement or were completely relieved of symptoms.19 In 1974, during a typhoid epidemic, 18,577 children were enrolled in a prophylactic intervention trial at the Eliava Institute using typhoid phage, resulting in a 5-fold decrease in typhoid incidence compared to placebo.20 Despite the use of phage therapy in humans in Eastern Europe, no phage therapy—aside from agricultural use—has yet been approved by the FDA, though many are in development.21Clinicaltrials.gov lists 58 clinical trials of various phage interventions as of September 2, 2023. Of these, 17 studies state that they are aimed at treating drug-resistant, chronic, or recurring bacterial infections, and several others are aimed at treating and/or preventing infections caused by bacterial species known to be antibiotic resistant (Figure 1).22 Clinical Trials of Phage Interventions. Phages are viruses that infect and kill bacteria.23 Phage therapy aims to leverage these natural enemies of bacteria by identifying phage that can precisely target those bacterial species that cause treatment-resistant human disease.24 The three-dimensional structure of phages includes an icosahedral head (a dice-like geometric shape with 20 faces), which contains the genetic material of the virus, and a long tail with fiber-like legs.25 Phages must have a host within which to reproduce in order to survive. Their tail fibers attach to receptors on the host bacteria, and then the phage inserts its genetic material into the bacterium. Once the bacterial cell has become infected, it produces millions of copies of the phage’s genetic material, eventually causing the cell to burst and allowing those new phage copies to infect and kill nearby host cells. Because different host bacteria have different receptors, a phage can bind only to those bacteria for which the proteins that make up the phage tail fibers (as determined by their genetic sequence) match the proteins on the surface of the host cell receptor, making each phage highly selective for particular host organisms.26 The selectivity of a phage can allow it to precisely target a pathogen of interest while leaving the remainder of the human microbiome relatively undisturbed.27 Phage therapy therefore has the potential to treat the patient while minimizing the untoward side effects sometimes caused by the use of antibiotics.28 This selectivity and narrow host range also reduce the risk for bacterial resistance since only the bacteria causing a particular infection are targeted, unlike the traditional antibiotics that target a wide-spectrum of bacteria.29 Phage therefore hold the potential to improve patient survival rates, decrease adverse effects, and offer new treatment possibilities against resistant bacteria. Already, phage therapy has emerged in the USA as an option of last resort, albeit in very limited circumstances. In 2015, infectious disease epidemiologist Steffanie Strathdee leveraged an international network of researchers to give an experimental phage therapy to her husband after other treatments had failed to cure him of a deadly antibiotic-resistant bacterial infection.30 After obtaining permission from the FDA under an expanded access protocol, he was treated with phage and recovered. To counteract the threat of viruses, bacteria have developed protection mechanisms known collectively as the CRISPR-Cas system, which acts as a bacterial immune system against invading viruses.31 Through this system, bacteria are able to systematically transcribe the DNA or RNA of invading viruses, store this genetic material using CRISPR arrays, and later use the stored nucleic acid sequences to identify similar invading viruses and destroy them. When a virus with similar DNA to a previous invader is encountered, the CRISPR array produces an RNA segment that acts as a guide for the ‘Cas’ protein, which enables the Cas protein to recognize a similar DNA complex that matches the RNA segment. The Cas protein then cleaves the invading nucleic acid, causing viral death as the necessary genes for survival have been shredded or cleaved.32 Because the CRISPR system evolved to have the ability to capture and store nucleic acid sequences, researchers can exploit this capability by exposing the Cas protein to a lab-created RNA sequence that allows the CRISPR sequence to identify a desired genetic target.33 The Cas enzyme, guided by the manufactured RNA sequence, then homes in on the corresponding DNA sequence and shreds it, disabling specific genes. Scientists are now seeking to create phages that are augmented with CRISPR to enhance their effectiveness as antimicrobial agents. In the laboratory, researchers can create novel guide RNA sequences that allow the Cas enzyme to target a desired bacterial (rather than viral) DNA sequence. Once the CRIPSR-Cas system is designed, it can be inserted into the phage’s genome to encode the CRISPR-Cas system. When the phage propagate, each phage will contain the CRISPR-Cas system sequence along with its usual genetic material that is then injected into the targeted bacteria (causing it to burst and further propagate the phage). Scientists can thus repurpose the CRISPR system and use a bacterium’s own defense mechanism against itself.34 Both Cas9 and Cas3 (two different kinds of enzymes that accompany the CRISPR mechanism) are being studied for antibacterial development. One such CRISPR-phage antibacterial is being developed by Janssen Pharmaceuticals, a part of Johnson & Johnson.35 In 2020, the product entered Phase 1 clinical trials for use against Escherichia coli, the furthest along in development for a drug of its kind.36 As of September 2022, the Phase 1 trial is complete and results have been posted.37 The experimental therapy is currently being tested against urinary tract infections,38 with plans to later test for efficacy against infections at other sites in the body, such as the lungs and abdomen.39 In laboratory studies, phage genetically engineered to contain CRISPR-Cas have been more effective than naturally occurring (‘wild-type’) phage in eliminating Clostridioides difficile—a bacterial species prone to antibiotic resistance.40 Bacterial resistance to phage occurs quickly.41 The addition of CRISPR-Cas3 to phage enhances the bactericidal effect because it produces faster elimination by several orders of magnitude,42 reducing the potential for resistance. CRISPR-phage kill host bacteria more quickly because, while phage replicate within the bacterial cell, the essential genes of the cell are being shredded by the CRISPR-Cas system, potentially leading to cell death even before phage-mediated biochemical processes cause cell lysis.43 The ability of phages to precisely target particular pathogens is one of the most important benefits of phage therapy over traditional broad-spectrum antibiotics. However, the extreme precision creates a challenge in identifying which phage to use. Among the largest hurdles is determining whether laboratory evidence that a given phage successfully targets a particular bacterial strain will translate to the clinical context. Laboratory measures of virulence can vary based on several factors, such as the viral dose used. The phage’s genome must also be sequenced, and the phage should not contain certain genes, such as those that code for integrase (an enzyme that could inadvertently integrate phage DNA into the bacterial genome, allowing the phage DNA to replicate passively as its host cell continues to divide, delaying lysis of the host and potentially strengthening host resistance to the immune system or other phage)44 or any antibiotic resistant genes (because phages can be reservoirs of antimicrobial resistance genes, wherein they can transfer these genes to the host bacteria and thereby induce the bacteria to become resistant).45 Although the sequencing of the phage itself may not be difficult, finding phage genomes without these characteristics can be. Phage can also mutate during treatment, potentially requiring further diagnosis and the time-consuming creation or identification of another appropriate phage. The use of phage cocktails, wherein multiple phage species are used, can help to mitigate these problems, but can enlarge the impact on the microbiome.46 Phage are also immunogenic, meaning that the human body may learn to neutralize their effect over time.47 Finally, research suggests that CRISPR-Cas editing may result in gross structural defects of the host nucleus, such as the formation of micronuclei (damaged chromosome fragments or whole chromosomes that are erroneously left outside the nucleus during cell division), which initiate a mutational process called chromothripsis and could potentially cause malignancy.48 Neither the level of risk to patients based on these genetic changes nor the more general risk to third parties or future generations from possible unanticipated effects on the microbial environment is well characterized. Nevertheless, as technological advances increase the safety and precision of CRISPR techniques, the regulatory system will soon have to adapt to protect the public from the potential risks while also supporting new opportunities.49 The science of CRISPR-phage therapy raises important economic and regulatory challenges. Even if CRISPR-phage therapy is safe and effective, these economic and regulatory challenges will need to be overcome to incentivize the research needed to obtain FDA approval and bring these new therapeutics to market. Despite their sophistication and therapeutic potential, CRISPR-phage therapies will face many of the same financial challenges as traditional antibacterials.50 As with pharmaceuticals generally, new CRISPR-phage therapies could experience high failure rates during development, lowering expected returns on investment.51 Compared to chronically administered drugs, courses of treatment for antibacterials tend to be short, depressing sales volume. Many older antibacterials are inexpensive and continue to be effective for the large majority of patients, so costly new products are reserved for when other options have been exhausted (a desirable practice known as "stewardship"), reducing volume and therefore profit. The high specificity of CRISPR-phage therapies means that they are likely to generate only modest revenues since each phage would be expected to treat only a very small number of patients suffering from a particular treatment-resistant pathogen. Unlike traditional antibiotics which can be stored in a pharmacy, the phage selection process may require bespoke manufacturing 52 that is both costly and time consuming, potentially reducing the circumstances in which phage treatments are used. These factors combine to reduce expected profits, diminishing the interest of pharmaceutical companies.53 Advances in biotechnology are helping to reduce the costs of phage therapy development. 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