The Scientific Observer Issue 36

Electronic Pulses Could Reduce the Need for High Doses in Gene Therapy Delivery How Is CRISPR Gene Editing Being Used in Infectious Disease Research? ISSUE 36, JUNE 2024 HOW CRISPR GENE THERAPY GAVE A NEW LIFE BREAKING VICTORIA GRAY CHAINS: the Sponsored by 2 CONTENT FROM THE NEWSROOM 05 ARTICLE Electronic Pulses Could Reduce the Need for High Doses in Gene Therapy Delivery 07 Kate Robinson ARTICLE Cell Therapy Targets, Clinical Applications, Manufacturing and Regulatory Considerations 11 Laura Lansdowne FEATURE ARTICLE Breaking The Chains: How CRISPR Gene Therapy Gave Victoria Gray A New Life 16 Molly Campbell ARTICLE How Is CRISPR Gene Editing Being Used in Infectious Disease Research? 26 Blake Forman ARTICLE How To Enter a New Chapter in Academic Publishing 30 Molly Campbell 07 30 14 FEATURE Breaking the Chains: How CRISPR Gene Therapy Gave Victoria Gray a New Life Molly Campbell Victoria Gray modified, Dear Readers, Welcome to the 36th issue of The Scientific Observer. This issue, we’re focusing on a field poised to change the landscape of medicine forever: cell and gene therapy. Our feature article tells the compelling story of Victoria Gray, the first patient to receive a CRISPR cell-based gene therapy for sickle cell disease (SCD). Throughout her early years, Victoria’s dreams were crushed by frequent, excruciatingly painful crises caused by her disease. After years spent in and out of hospital, she bravely chose to partake in a clinical trial that completely changed her life. Through Victoria's eyes, we learn about the daily struggles faced by SCD patients, the historical mistreatment of minority patients in medical settings and the broader implications of gene therapy accessibility. Delivery is one aspect of cell and gene therapy that contributes to their high cost. In this issue, Kate Robinson speaks with the research team behind a new technique that utilizes electricity to enhance the body’s receptivity to gene therapy. This innovative approach has the potential to improve the efficiency and effectiveness of gene delivery, opening new avenues for the treatment of a variety of genetic disorders. Continuing on the theme of innovation in gene editing, Blake Forman provides a summary of recent research utilizing CRISPR technology in infectious disease research. This summary captures how CRISPR-Cas technology is being harnessed to combat a range of infectious diseases, providing new hope for rapid and precise treatment options. We also delve into the intricacies of cell therapy with an article focused on its targets, clinical applications, manufacturing and regulatory considerations. Laura Lansdowne’s article provides a comprehensive overview of the current landscape, addressing both the promise and the challenges faced by this rapidly evolving field. Finally, we revisit the innovative world of academic publishing through an interview with Alex Freeman, the founder of Octopus. This novel publishing platform, launched in 2022 with the support of UK Research and Innovation (UKRI), represents a radical approach to scholarly publication. In our follow-up interview, Alex discusses the progress made since the platform's launch and the ongoing challenges of establishing a new paradigm in academic publishing. We hope you enjoy our exploration into these pertinent topics – and many more – in issue 36 of The Scientific Observer. The Technology Networks Editorial Team 3 Kate Robinson Kate is an Assistant Editor for Technology Networks. EDITORS’ NOTE CONTRIBUTORS Laura Elizabeth Lansdowne Laura is the Managing Editor for Technology Networks. Molly Campbell Molly is a Senior Science Writer for Technology Networks. Blake Forman Blake is a Senior Science Writer for Technology Networks. 4 iStock 5 Want to learn more? Check out theTechnology Networks newsroom. iStock, freestocks/ Umsplash, Scientists from the Allen Institute applied BARseq to interrogate gene expression patterns over four million cortical neurons across nine mouse forebrain hemispheres, at cellular resolution. They found that the transcriptomic signature of cortical neurons is highly predictive of their cortical area identity. JOURNAL: Nature BARSeq Reveals the Brain Is Like a Pointillism Painting MOLLY CAMPBELL Researchers have created the first panoramic view of infection pathways in the human placenta using ex vivo explant models, or “mini placentas” from human samples. This placenta map could highlight potential drug targets to develop pregnancysafe therapies for diseases that can cause severe pregnancy complications. JOURNAL: Cell Systems Placenta Map Reveals Source of Infection-Related Pregnancy Complications BLAKE FORMAN The first participant in a new psychedelic study has received a dose of a synthetic formulation of 5-MeO-DMT. The study is set to evaluate its neurophysiological effects on the human brain and perceived “mystical experiences.” First Patient Dosed in Study To Unravel “Mystical Experiences” of Psychedelic Derived From Toad Skin SARAH WHELAN 5 FROM THE NEWSROOM From the Newsroom 6 From the Newsroom FROM THE NEWSROOM Want to learn more? Check out theTechnology Networks newsroom. Brita Seifert/ Pixabay, iStock, leezathomas099/ Pixabay New analysis of two locks of hair belonging to Ludwig van Beethoven proves that the composer did have high levels of lead in his system when he died – but not high enough to be considered the sole cause of the great man’s death. The discovery rules out one of the most popular theories explaining the composer’s prolonged illness and eventual early death. JOURNAL: Clinical Chemistry Beethoven’s Hair Confirms He Had Lead Poisoning – But It Didn’t Kill Him ALEXANDER BEADLE After reassessing 48 previously published papers on the health effects of plant-based diets, researchers from the University of Bologna concluded that such lifestyles reduce the risk of cardiovascular diseases and certain cancers. JOURNAL: PLOS One Yes, Plant-Based Diets Really Are Better for Your Health, Review Finds LEO BEAR-MCGUINNESS With interest in women's sports at an all-time high, researchers from UCL and the University of Bath are investigating the menstrual cycle's potential impact on injury risk among elite footballers in the Women's Super League. The study monitored injury risk in female footballers at different points of their menstrual cycle. JOURNAL: Medicine & Science in Sports & Exercise Female Athletes Six Times More Likely To Get Injured in the Days Leading Up to Their Period RHIANNA-LILY SMITH 7 While gene therapy has proven to be promising for diseases ranging from cancer to diabetes, the challenge of getting the right dose of genetic material into target cells has caused a bottleneck in the application of such therapies. In new research published in PLOS ONE, researchers from the University of Wisconsin–Madison have reported on the development of a technique employing electric pulses to make the human body more receptive to certain gene therapies. We spoke to two of the study authors, Professors Susan Hagness and John Booske, to learn more about the benefits of direct delivery of gene therapy materials, the challenges associated with gene therapy delivery and the use of electronic pulses to encourage uptake of genetic material. Q: What are the benefits of direct delivery of gene therapy material? A: Direct delivery may reduce the total dose needed for treatment because it eliminates the attrition of material during circulation through the body and other organs prior to arrival in the targeted tissue/organ (compared to, say, systemic, peripheral injection into a remote blood vessel). Systemic delivery typically requires large(r) doses to compensate for losses during passage through the circulatory system and other organs. Manufacturing the genetic material delivered via virus vectors is expensive – prohibitively so for many treatments of inherited metabolic diseases (such as hemophilia, diabetes, etc.). Reducing the required dose is critical to practical, affordable treatments. Direct delivery may also minimize the time the material spends in the circulatory system before uptake in the liver cells. This reduces the likelihood that the immune system will mount an attack on the gene therapy “foreign” material and destroy or inElectronic Pulses Could Reduce the Need for High Doses in Gene Therapy Delivery KATE ROBINSON iStock 8 activate it. The larger doses required with systemic, peripheral injection, along with the associated immunological response (e.g., cytokine storms) present a heightened risk. Direct delivery is a pathway to reduce the doses required and bypass the immune counter-response, thereby reducing cost and increasing safety. Q: What are the challenges associated with gene therapy delivery, and how do these affect patients? A: The challenges of conventional gene therapy delivery approaches (peripheral injection, e.g., in a remote blood vessel) include the requirements for large doses (to ensure enough of the dose finally arrives at the targeted site and is taken up by the targeted tissue cells) and the risk of immune counter-response to the injection of foreign genetic material. The former leads to a prohibitively expensive cost of treatment, making it impractical as a widespread treatment option. The latter includes loss of genetic material that may result in ineffective treatment or a dangerous overreaction that can threaten the health of the patient. Q: How does the application of electronic pulses increase the uptake of gene therapy material into hepatocytes? A: We are not certain what the specific underlying physical mechanisms are that result in enhanced uptake of the gene therapy material. We have some hypotheses, but no definitive identification of a mechanism currently. Our findings, reported in the PLOS ONE article, help to rule out some of the possible mechanisms. For example, we know that electric pulses do not modify the genetic material directly before it is absorbed into the cells, and we know that the electric pulses do not modify the culture medium or environment that the cells reside in. Future experiments are being designed and conducted to pin down the mechanism(s) that are at play. We know that electric pulses induce (nano)pores into the cell membranes, and this effect has been exploited in other investigations to permeabilize the membranes and allow small(er) molecules to be taken up by those cells. However, the larger size of the virus vector (A AV8) capsids used in our experiments lends doubt to the likelihood that this is the mechanism responsible for enhanced uptake in our experiments. It is a subject for further investigation. Q: What impact could efficient delivery of gene therapies have on patients in the future? A: Efficient delivery could make many gene therapy cures affordable and safe for a large population of patients. To elaborate, genetic mutation-based metabolic diseases significantly reduce the quality of life for hundreds of millions of people in the world. There are 100s of such diseases, including diabetes, cystic fibrosis, sickle cell anemia and hemophilia. Many of them involve the liver due to its central role in metabolism. Developed countries spend trillions of dollars each year on patient care, with nearly a trillion dollars spent annually on type 1 diabetes (T1D) alone. Cures for many of these diseases could be attainable if practical, cost-effective methods existed to modify the gene(s) of the liver cells, sufficient to correct the inherited metabolic discrepancy. Some success with systemic injection gene therapy has been reported, but only in small mammals or with prohibitive costs (~$1 M/ treatment in the case of hemophilia). In other words, a famous statement in 1999 by Salk Institute Professor Inder Verma, one of the foremost recognized leaders in gene therapy, still remains relevant today: “There are only three iStock 9 iStock problems in gene therapy: delivery, delivery, delivery.” Q: What are the next steps in translating this research into clinical trials? A: Next steps include in vitro investigations of optimal electric pulse parameters and in vivo studies to determine how the phenomenon (which we have observed in vitro) manifests in living tissues. To date, we have only had the opportunity to investigate electric pulsing with a single choice of electric field strength and pulse length, and only with single pulses. An important question to answer is whether other treatment parameter combinations–i.e., different electric field strengths, different pulse lengths, and/or multiple pulses–produce a stronger effect. Of course, it is important to identify the maximum treatment parameter thresholds above which cell or tissue damage occurs, to ensure that parameter choices are always safely below those thresholds. Meanwhile, our experiments to date were conducted on cells in well culture plates (i.e., in vitro). Although research has shown that cells in vivo (in living tissues) can have similar responses to electric pulsing as cells in vitro, the magnitude of the responses and the parameter values that produce the maximum safe response can be expected to be different. So, experiments with animal models are the next critical step, especially with larger mammals. Q: Do you have plans to explore the use of electronic pulses in the delivery of gene therapies to other cell types? A: The potential impact of translating this to clinical practice in liver/ hepatocytes has considerable impact value, so that is our primary focus for now. But certainly, expanding the scope of these investigations is of interest in the longer term. ⚫ Direct delivery is a pathway to reduce the doses required and bypass the immune counterresponse, thereby reducing cost and increasing safety. 10 iStock From selecting instrumentation, building a team, managing day to day operations and helping to shape the future of the organization you work for, running a lab presents many challenges. Leadership experts will provide practical tips and guidance for current and aspiring lab managers working in industry and academia. REGISTER NOW Brought to you by the publication August 14–15, 2024 8AM PDT | 11AM EDT | 4PM BST Sean Tucker Director of Laboratory Services North Kansas City Hospital Antoni Lacinai Workplace Communication Expert Lacinai Communication & Performance Development Kabrena Rodda, PhD Research Line Manager Pacific Northwest National Laboratory Rigoberto Advincula, PhD Governor’s Chair and Professor Oak Ridge National Laboratory Oluwatoyin Asojo, PhD Associate Director for Strategic Initiatives Dartmouth Cancer Center Bamidele Farinre Chartered Biomedical Scientist Institute of Biomedical Science SPEAKERS MORE SPEAKERS TO BE ANNOUNCED! INTERESTED IN SPONSORING EVENTS LIKE THIS? CONTACT OUR FRIENDLY SALES TEAM HERE Modern Lab Leader 2024 ONLINE SYMPOSIUM 11 iStock Cell therapy is a therapeutic strategy that involves the transfer of autologous (patient-derived) or allogeneic (donor-derived) cells into a patient’s body. Cell therapies can be divided into two broad categories – stem-cell therapies and non-stem-cell therapies. Within these two groups, a variety of mechanisms of action are employed, tailored to the specific type of cell therapy and the disease or condition it’s designed to target. For example, in regenerative medicine, stem cell therapies work by replacing damaged or diseased cells with new, functional ones to restore organ and/or tissue function. Whereas adoptive cell therapies for cancer treatment exploit immune cells by either expanding the number of cells, or by genetically modifying them to boost their cancer-fighting abilities, before being administered to the patient. The three main cell therapy modalities are outlined in Figure 1. In 2023, the global market value for cell therapy was estimated to be USD 4.74 billion. This market is expected to experience rapid growth due to increasing demand for innovative cell therapies, which tend to have fewer adverse effects compared to traditional modalities. This, coupled with their wide range of clinical applications, is anticipated to propel the market to approximately USD 20 billion by 2030. In this listicle, we explore new therapeutic targets and clinical applications, and discuss challenges and key considerations related to the manufacture and regulation of cell therapies. CLINICAL APPLICATIONS AND NOVEL RESEARCH While the oncology segment led the overall cell therapy market in 2023, Cell Therapy Targets, Clinical Applications, Manufacturing and Regulatory Considerations LAURA LANSDOWNE From selecting instrumentation, building a team, managing day to day operations and helping to shape the future of the organization you work for, running a lab presents many challenges. Leadership experts will provide practical tips and guidance for current and aspiring lab managers working in industry and academia. REGISTER NOW Brought to you by the publication August 14–15, 2024 8AM PDT | 11AM EDT | 4PM BST Sean Tucker Director of Laboratory Services North Kansas City Hospital Antoni Lacinai Workplace Communication Expert Lacinai Communication & Performance Development Kabrena Rodda, PhD Research Line Manager Pacific Northwest National Laboratory Rigoberto Advincula, PhD Governor’s Chair and Professor Oak Ridge National Laboratory Oluwatoyin Asojo, PhD Associate Director for Strategic Initiatives Dartmouth Cancer Center Bamidele Farinre Chartered Biomedical Scientist Institute of Biomedical Science SPEAKERS MORE SPEAKERS TO BE ANNOUNCED! INTERESTED IN SPONSORING EVENTS LIKE THIS? CONTACT OUR FRIENDLY SALES TEAM HERE Modern Lab Leader 2024 ONLINE SYMPOSIUM 12 Technology Networks the potential of cell therapy in other areas is increasingly evident. Here we highlight recent research that illustrates the diverse potential of cell therapy across different therapeutic areas. Neurology UC San Diego Health became one of the first facilities in the United States to administer the experimental neural cell therapy, NRTX-1001, to subjects with drug-resistant epilepsy. This cell therapy involves the delivery of interneurons that secrete the inhibitory neurotransmitter gamma-aminobutyric acid (GABA), into the epileptic region of the brain. In December 2023, the biotherapeutics company developing NRTX-1001 announced that the initial two trial subjects consistently reported a decrease in seizure frequency (> 95% reduction from baseline), more than one year after receiving treatment. Regenerative medicine Using mRNA technology encapsulated in nanoparticles, researchers designed a novel stem cell therapy that aims to stimulate the liver's natural repair processes. The study was published in Cell Stem Cell. The therapy works by delivering vascular endothelial growth factor A (VEGFA) mRNA via lipid nanoparticles. This promotes the conversion of biliary epithelial cells into hepatocytes, the liver's functional cells. The research was conducted using mouse and zebrafish disease models. Immunology Researchers developed a novel T-cell therapy to treat a specific form of autoimmune encephalitis (NMDAR encephalitis), an immune-mediated condition whereby antibodies attack healthy brain cells, causing inflammation and various neurological symptoms. The team genetically modified T cells to selectively eliminate anti-NMDAR B cells and autoantibodies against the NMDA receptor. This preclinical study was published in Cell. They now plan to test the therapy in human subjects with NMDAR encephalitis. Inherited blood disorders The US Food and Drug Administration (FDA) approved two cell-based gene therapies – Casgevy™ (exagamglogene autotemcel) and Lyfgenia™ (lovotibeglogene autotemcel) – to treat sickle cell disease in patients ≥ 12 years. Both therapies are created using patients’ hematopoietic stem cells, which are genetically modified and then reintroduced as a single-dose infusion. Casgevy is the first FDA-approved therapy that exploits CRISPR-Cas9 gene editing. CRISPR-Cas9 is used to reduce the expression of BCL11A, which in turn, boosts the synthesis of γ-globin and reactivates fetal hemoglobin production, preventing sickling. Lyfgenia uses a lentiviral vector to genetically modify the stem cells to produce a specific hemoglobin called HbAT87Q. Cells containing this hemoglobin have a reduced risk of sickling. Oncology A preclinical study published in Nature Communications describes a novel allogeneic CAR T-cell therapy targeting T-cell malignancies. This therapy focuses on eliminating cancerous T cells that express a dominant T-cell receptor called Vβ2. Unlike traditional treatments that risk depleting all of a patient's T cells, this CAR T-cell therapy selectively kills the diseased cells, preserving Vβ2-negative healthy cells. CRISPR gene-editing technology was used to specifically target the Vβ2-positive cancer cells. Cell therapies Cells are administered to a patient as a therapeutic modality. Genetically modified cell therapies Patient/donor cells are genetically modified to perform a unique function they wouldn't typically do that provides therapeutic benefit to a patient. Tissue-engineered products Cells and/or biologically active substances are designed to restore, maintain or replace damaged tissues/organs. Figure 1: Overview of cell therapy modalities. 13 iStock The FDA approved Amtagvi™ (lifileucel), a tumor-derived autologous T-cell immunotherapy, via its Accelerated Approval Program. Amtagvi is indicated for use in adult patients with a melanoma (unresectable or metastatic), that has failed to respond to/stopped responding to other specific therapies. A portion of the patient’s tumor is surgically removed. Tumor-derived T cells are then isolated from the excised tumor tissue, expanded at a manufacturing site and then administered as a single-dose intravenous infusion to the same patient. UNDERSTANDING THE REGULATORY LANDSCAPE OF CELL THERAPY The pharmaceutical industry is one of the most regulated industries globally, and regulatory authorities play a crucial role in overseeing various steps related to the development of new therapies. Several different regulatory authorities exist worldwide and each issues specific guidelines relating to the development, registration, manufacturing, licensing, marketing and labeling of medicines. As such, it is vital cell therapy developers understand regulatory variations and consider how these differences will impact the development of a novel cell therapy, depending on where in the world they are seeking marketing authorization. Here we take a closer look at regulatory guidance in the UK, Europe and the US. The European Medicines Agency (EMA) classes cell therapies as a type of advanced therapy medicinal product (ATMP). As such, they are governed by medicinal product regulatory frameworks (Regulation (EC) No 1394/2007, Directive 2001/83/ EC). The manufacturing of these products must comply with Good Manufacturing Practice (GMP) principles (EudraLex Volume 4, Part IV). Similar to the EMA, the UK’s Medicines and Healthcare products Regulatory Agency (MHRA) classes cell therapies as a type of ATMP. The donation, procurement and testing of cells is covered by the EU Tissues and Cells Directive (2004/23/EC). Under this directive there are two authorities of note – the Human Fertilisation and Embryology Authority (HFEA) and the Human Tissue Authority (HTA). The HFEA oversees the use of gametes and embryos in the development of ATMPs and the HTA is responsible for the licensing and inspection for all other tissue and cell types. In the US, cell therapies are regulated by the FDA’s Center for Biologics Evaluation and Research (CBER). They fall under Title 21 of the Code of Federal Regulations (CFR), Part 127.3(d), and are defined as, “Articles containing or consisting of human cells or tissues that are intended for implantation, transplantation, infusion or transfer into a human recipient". The FDA recommends that “product testing for cellular therapies include, but not be limited to, microbiological testing (including sterility, mycoplasma and adventitious viral agent testing) to ensure safety and assessments of other product characteristics such as identity, purity (including endotoxin), viability and potency.” The FDA has numerous cellular and gene therapy guidance documents, addressing specific aspects of cell and gene therapy development – for example, potency assurance, manufacturing, trial design and general regulatory considerations. ADDRESSING MANUFACTURING AND SCALABILITY CHALLENGES While the demand for cell therapies is undeniable, their production comes with key challenges and regulators require manufacturers to conduct extensive testing. Cell therapy manufacturing methods range in complexity. Some therapies require significant manipulation of cells (e.g., genetic modification), while others may require comprehensive cell cultivation steps. Here we discuss several manufacturing and scalability considerations. Sourcing high-quality cells The initial challenge in cell therapy production lies in sourcing high-quality biological materials. The exact geographical region and regulatory authority will influence how starting material must be obtained, for example collection/apheresis best practices. Progress is being made to refine the cell extraction and separation processes. For example, researchers recently 14 iStock developed a technology capable of extracting mesenchymal stem cells (MSCs) directly from bone marrow – without the need for dilution. By continuously sorting and isolating stem cells from blood cells using a novel microfluidic platform, it was possible to double the number of MSCs obtained from bone marrow samples and reduce the extraction time to approximately 20 minutes. In the case of allogenic (donor-derived) cell therapies, specific donor eligibility requirements will also need to be considered as these may differ depending on country. Establishing an appropriate shelf life Once obtained, the shelf life of fresh cells is often short. For example, hemopoietic stem cells can be stored unprocessed at 4 °C or room temperature for approximately 72 hours post-collection. However, after this time they begin to degrade, resulting in compromised product quality and potency. To address this, developers typically opt to cryogenically freeze cells, increasing their shelf life to months or years, but this relies on specialist facilities and procedures to ensure the cells remain viable and stable. The optimal cryopreservation and freeze thawing process will differ depending on the particular cell product. Regulators require manufacturers to conduct stability testing to confirm the product's integrity and efficacy over time. Addressing temperature sensitivity Increased temperature sensitivity is another key challenge faced by cell therapy manufacturers. Good distribution practice guidelines stress that medicinal products must not be exposed to conditions during transport, “that may compromise their quality and integrity.” Temperature changes must be tracked to confirm that products stay within "defined limits" while in transit. The difficulty lies in establishing these limits, as they must be broad enough to allow transfer of the product, but not so broad they jeopardize its quality. Achieving quality bioproduction at scale A product’s critical quality attributes (CQAs) must be well characterized early on in development before scaling bioproduction to ensure the correct quality control metrics are being monitored. Developers want to avoid the need for major manufacturing changes late in development as this could impact commercial viability of the therapy. The term “technology transfer” describes the transfer of a process from small scale (a laboratory setting) to a commercial manufacturing facility. If employing a contract manufacturing organization (CMO) or contract development and manufacturing organization (CDMO) to support the scale-up of a therapy, it’s important to limit technology-transfer risk by ensuring clear knowledge transfer, adequate training and ensuring equipment/process commonalities. Depending on the type of cell therapy, developers will choose whether to scale-up (increase batch size) or scale-out (increase the number of batches). Allogenic therapies are– typically scaled up whereas autologous therapies are scaled out. Automated integration of multiple manufacturing steps in a closed system environment is vital to ensure GMP compliance and process reproducibility, and reduce contamination risk. CONCLUSION Continued exploration and investment in cell therapies is ushering in a new era of medical interventions. The discovery of novel targets and mechanisms, refinement of manufacturing processes and creation of detailed regulatory guidance are helping to accelerate the approval of cell therapies, offering hope and improved outcomes for patients worldwide. ⚫ 15 EDITORS’ NOTE CONTRIBUTORS Have an idea for a story? If you would like to contribute to The Scientific Observer, please feel free to email our friendly editorial team. Giving talks and posters at conferences can be great but can also be expensive and time consuming. Your reach is also limited to the people in the room. What if you could get your paper in front of 250,000 scientists located across the globe, without leaving your desk, for free and with minimal work? Sound too good to be true? Find out more NEED TO DISSEMINATE YOUR RESEARCH? We want to help connect scientists with great research and have created an easyto-complete form and guidance to help distil the essence of your paper into a short, punchy article that will then be shared: on our site, which receives over 1 million page views every month with our 4 million + social media followers in our 18 newsletters and may be included in our regular digital magazine The Scientific Observer HOW CRISPR GENE THERAPY GAVE A NEW LIFE MOLLY CAMPBELL BREAKING VICTORIA GRAY CHAINS: the 17 Veronica Gray modified, iStock L ike many young girls in elementary school, Victoria Gray wanted to be a cheerleader, until she was told by her doctor that this simply wouldn’t be possible; the exertion placed on her body by the training regimen could have devastating consequences. “That was my first disappointment as a kid,” she recalls. Sadly, it wouldn’t be her last. Tall for her age, Victoria later thought about trying out for a spot on the school basketball team. Her uncle had a great love for the game, which inspired her. Plus, everyone around her said she would probably have a natural knack for shooting hoops. Why not give it a go? She thought. But once again, her enthusiasm was shot down by another firm “no” from her pediatrician. If you’re lucky enough to grow up having a healthy childhood, it’s hard to imagine the cruel realities of experiencing a sick one. Victoria describes the motions of her life as though it was a distressing movie being played out in front of her, and someone – somewhere – kept hitting the pause button. Her days were shaped not by the whimsical imagination of a young child, but by her illness. That ’s because at just three months of age she had been diagnosed with sickle cell disease (SCD). SCD is a group of inherited blood disorders that cause red blood cells to become hard and sticky. In healthy individuals, red blood cells are a disc shape, allowing them to f low easily through our blood vessels. In SCD, they form a “C” shape known as a sickle, which causes the blood cells to die quickly or cause blockages in blood vessels. According to the National Institutes of Health, over 20 million people worldwide are affected by SCD. SCD stole Victoria’s childhood, including her right to an education. “I had to alter everything I dreamed about every step of the way from childhood to adulthood. W hen I started college, I wanted to be a cardiologist. But doctors explained that the stress involved with studying medicine wouldn’t be good for me. So, I put myself on pause – once again,” she says. Undeterred and eager to help care for others, Victoria started to explore a nursing degree, until “I had one the worst pain crises of my life,” she says. Crises refer to acute conditions, such as the blockage of a blood vessel by “My life was constantly full of ‘noes’, and people telling me ‘you can’t do this’ – it was limitation after limitation,” she says. “I had to alter everything I dreamed about, every step of the way from childhood to adulthood.” “I really gave up on becoming anything else. I thought sickle cell was just going to be my life from beginning to end.” sickled cells, caused by SCD. They are the main clinical hallmark of the disease, causing severe, debilitating pain and extreme fatigue, among other symptoms. Oftentimes, they strike at random, and can persist for long periods of time requiring extended stays in hospital. “That crisis put me in the hospital for three months. I lost the ability to use my arms and my legs,” Victoria describes. Perhaps most devastatingly, she says, “I also lost my ability to dream.” After undergoing comprehensive rehabilitation, Victoria eventually regained her strength and her ability to walk. But despite the physical improvements, mentally, she had to resign herself to the fact that a career maybe wasn’t on the cards for her. It was yet another blow, but one that she handled with the grace and determination she carried from a young age to find fulfillment in life. Victoria chose to focus her time, and the energy she could muster between crises, on being a wonderful mom to her four children, and a strong partner to her husband, Earl. Then, in 2018, an opportunity came around that finally awarded Victoria the opportunity to say “yes” for the first time in her life. It was, she would come to learn, an opportunity that marked not only a major milestone in the history of medicine, but one that freed her from the chains of SCD – most likely, forever FINALLY A "YES” FROM VICTORIA Patients with SCD are often prescribed drugs, such as painkillers, to try and curb the symptoms associated with crises. These medications fail to target or cure the underlying cause of the disease, and while they offer symptomatic relief, they can have harmful side effects. There are few authorized therapeutics on the market for SCD patients. In 2018, Victoria, who lives in Forest, Mississippi, was under the care of Dr. Haydar Frangoul, waiting to learn of her eligibility for one such treatment – a haploidentical “haplo” stem cell transplant using cells donated from her brother. Dr. Frangroul is the director of the Pediatric Stem Cell Transplant program at Tristar Centennial Children’s Hospital, and an investigator at the Sarah Cannon Research Institute in Nashville. W hile at his clinic in Nashville, Victoria experienced a crisis that would ultimately change her life. “I had to be admitted, and during that hospital stay Dr. Frangoul approached me at my bedside. He knew that I was feeling really down, and he offered me a second option besides the Haplo transplant,” she recalls. Victoria had some reservations about the Haplo transplant, largely due to the risk of graft versus host disease, a complication that can occur when a donor’s cells attack the recipient’s: “Dr. Frangoul told me that they were starting a new trial soon using CR ISPR gene therapy. I hadn’t heard about it, so he showed me a small video on his phone and sent me a link so I could review it later.” As with any clinical trial, there were risks involved. Given that Victoria would be the first patient to ever receive this experimental therapy, these risks felt somewhat heightened. She had a difficult decision to make. “I'm a woman of faith – I pray a lot. I went to God, in private, about graft versus host disease, because I really didn’t want to experience that. So, when gene therapy came along, I felt like it was my answer from God, as though he was saying to me ‘I remove your fears now. This opportunity is for you’,” Victoria says. Ultimately, the possibility of a life that would not be plagued by pain outweighed any doubts she might have had about being patient one. Within 24 hours of speaking to Dr. Frangoul, Victoria took the courageous decision to volunteer for the trial. 18 The clinical trial that Victoria participated in was testing a cell-based gene therapy known as CasgevyTM. The therapy utilizes CRISPR-Cas9 gene-editing technology, which is directed to cut DNA in specific locations and enable the removal, addition or replacement of DNA. During treatment, the SCD patient’s blood stem cells are extracted from their body and edited in a laboratory. CRISPR-Cas9 is used to create an edit in the BCL11A gene within the patient’s cells, which triggers the production of fetal hemoglobin. Once these cells are re-inserted into the patient, they settle back into the bone marrow and the increased fetal hemoglobin facilitates the delivery of oxygen around the body. 19 In July 2019, she became the first patient to receive CRISPR gene therapy for SCD. AM I DEAD? Eight months after the therapy was administered, Victoria woke one morning and felt “different ”, though she couldn’t quite place what that meant. “I remember wak ing up, and the room was really bright. I didn’t feel any thing, which was strange. I didn’t have any shortness of breath when I stood up, as had been the case most mornings,” she says. Having lived most of her life in excruciating pain and extreme fatigue, the experience of wak ing up in its absence was so downright bizarre, Victoria had convinced herself that she must be dead. Pinching the sk in on her face, and her thighs, she was reassured to feel the sharp, physical sensations. “I shouted to my k ids, ‘Hey y ’all, come in here!’, and as they entered the room, their faces lit up. I k new in that moment, they could see me, and I realized that I was very much alive. I cried tears of joy, because I k new then that the gene therapy had worked,” Victoria describes, visibly emotional. That bright, beautiful morning was four years ago, and it marked Victoria’s new beginning. She describes her life now as one of freedom. She is free from constant pain and exhaustion. Free from having to stare at a hospital room’s four walls while experiencing a crisis episode. Free from relying on medication just to make it through the day. Free from all the ways that SCD stole her autonomy and chained her to a life burdened by illness. Victoria can now play with her children and embody the parent she always dreamed of being. She can immerse herself in typical mom activities, often taken for granted as mundane, but that were once out of her reach. She can make choices. She can travel abroad – even f lying to London last year to speak at the Third International Summit on Human Genome Editing. Eventually, Victoria’s health improved to the extent that she could realize her ambition of working fulltime, securing a position as a cashier at her local Walmart. It was during a particularly busy shift on December 8, 2023, when she received news that the US Food and Drug Administration (FDA) had decided to approve Casgevy for the treatment of SCD. Casgevy’s approval was based on data submitted from the trial that Victoria herself had bravely participated in. The trial’s primary outcome had been freedom from severe vaso-occlusive crises for at least 12 months during the study’s 24-month follow-up period. In total, 44 patients were dosed with Casgev y. Upon submitting the trial data, 31 patients had sufficient follow-up time to be Veronica Gray “Dr. Frangoul explained CRISPR therapy to me like this: he said, ‘just imagine a textbook with thousands and thousands of words, and there are a few words in there that are incorrect. The CRISPR technology could go into the cells, find the incorrect word and edit it without changing the story’.” 20 evaluated. Of these, 29 patients reached the primary efficacy outcome, with 0 patients suffering from graft failure or graft rejection – the trial had proven a success. Casgev y became the first gene therapy utilizing CR ISPR-Cas9 to receive FDA approval, marking a historic moment for the SCD community, as lovotibeglogene autotemcel (LyfgeniaT M) – another cell-based gene therapy for SCD, developed by Bluebird Bio Inc. – also received a green light from the agency. SCD PATIENTS FACE MEDICAL AND RACIAL DISCRIMINATION, HINDERING CLINICAL RESEARCH PROSPECTS Victoria received many phone calls that day, including one from a nurse who treated her at Dr. Frangoul’s clinic in Nashville. “ We both cried,” she says, “It was tears of pure joy, because all of the pain, disappointment and judgement that I had faced from childhood now felt worth it. This was going to bring about change for other people who feel alone and feel overlooked.” While reminiscing, Victoria takes a moment to pause. It’s clear that the emotions attached to these memories are a complex mixture of elation and pain. “I want to emphasize that, had it not been for the positive way in which I was treated by Dr. Frangoul and his team, I might not have accepted the opportunity to take part in this trial that has saved my life,” she says. Discrimination is an issue encountered by many SCD patients across the globe during their lifetime. In a perspective piece published in 2020, Power-Hays and McGann expressed that there may be “no population of patients whose health care and outcomes are more affected by racism than those with SCD.” During the 1970s, many African A merican people were deprived of jobs, educational opportunities, marriage licenses and insurance in the US if they carried the SCD “The trial was a different experience to anything I’ve had before, because for the first time, I felt hopeful. I was fighting for my life, and for my family.” Sure, I had to travel back to Nashville a lot for testing, and there were times [during the trial], especially after the chemotherapy, that it was hard. But my dad was with me, and he kept reminding me, ‘Vicky, you’ve seen worse, you are strong enough to get through this’. It helped to lift me back up and remind me of why I was doing this. Veronica Gray 21 trait. This grim picture was mirrored across the pond in the UK . “In the 20th century, I'd say that racism showed itself in the lack of willingness on the part of statutory services, such as education, social services and housing, to take account of the needs of those living with SCD,” says Professor Simon Dyson, director of the Unit for the Social Study of Thalassaemia and Sickle Cell at De Montfort University in the UK . “In terms of sickle-cell screening, SCD had to meet prevalence thresholds not demanded of other rarer conditions, before newborn screening to save the lives of black infants was eventually made universal in England in 2004.” A 2018 systematic review by Dr. Dominique Bulgin and colleagues explored the extent of health-related stigma in adolescents and adults living with SCD, analyzing data from 27 studies published between 2004–2017. They found that people with SCD experience health-related stigma based not only on their race, but on their disease status, socioeconomic status, delayed growth and puberty and having chronic and acute pain, requiring opioid treatment. “Individuals with SCD reported being stigmatized as drug seeking or drug addicts and having their experiences of pain discredited by healthcare providers,” the review states. Sadly, this experience is one that resonates with Victoria. “I was once in the emergency room when a nurse said to me, ‘ You know, I feel so sorry for you sicklers’ – this was a term he used – ‘because you guys just get addicted to these pain meds, and then you can’t tell the difference between withdrawal and a real crisis’,” she says. “He then said that it’s just inevitable that all SCD patients become drug addicts. I couldn’t believe it. I was in crisis. I was in pain, and I was crying out to a person who was supposed to help me. Instead, he judged me,” she continues. This, sadly, was not an isolated incident. Victoria recounts another crisis episode, where she felt as though her symptoms of pain were starting to improve. She asked the nurse treating her to avoid administering any excessive pain medication that might make her feel drowsy, as she wanted to sit up and try to move around the room. Instead, the nurse pressed the button on her medication dispenser, and Victoria fell asleep. W hen she woke, she learned that instead of pain medication, the nurse had given her a different type of drug because they “wanted to see what it would do”. Unfortunately, there was little consequence for the nurse, but the ramifications for Victoria were heartbreaking, she explains, and led her to question her worth as a SCD patient: “I felt as though the nurse had been given the right to experiment on me without my consent. W hat if that drug had taken my life? I couldn’t trust the staff that was treating me, I felt as though I was a burden, like they were trying to get rid of me when I was coming to them for help.” “There is no way,” Victoria adds, “that I would have accepted an experimental treatment like gene therapy, if I had been offered it at this facility.” Research shows that she is not alone in feeling this way. In 2020, Cho et al. conducted a study examining the motivations and decision-making processes of enrollees and decliners of high-risk trials for SCD. Of the 26 SCD patients interviewed, the majority reported negative interactions with health care providers. These experiences were so bad that many individuals had chosen to avoid hospitals during significant pain crises. If SCD patients are afraid to even ask for help when they are in pain, how can they be expected to partake in high-risk clinical research? It’s an almost impossible decision, but it’s one that Victoria chose to make for herself, for her family and for the SCD community. It’s a decision that, now, “When I saw the text from my husband, that the FDA had approved Casgevy, I had to rush off the floor at Walmart because I felt the tears coming,” she says. “I got in my car, and I just cried. I was so, so happy because I knew what a relief this would be for other sickle cell patients that were in a dark place like I had once been. Now, there was hope.” “It had felt as though nobody was coming to save us. Then suddenly we had a novel therapy for the SCD community. I felt so happy and grateful,” she adds. 22 iStock in hindsight, she is thrilled about, but she remains eager to warn the medical community of the risks they pose to the health of SCD patients – and the future of SCD research – by forgetting that the patient in front of them is a human being. PATIENT ADVOCACY AND INCREASING ACCESS TO GENE THERAPIES Now, five years after the trial commenced, Victoria is undertaking her follow-up appointments – which last 15 years from the study enrollment date – as per the study protocol. These appointments monitor her health and assess the long-term efficacy and safety of Casgev y. As gene therapies are emerging drugs, whether or not they are effective for the duration of a patient’s life is yet to be determined. “I pray that it is a forever change, and I believe that it is,” Victoria says. She is enjoying applying her newfound energy to spread the word about her experience as an SCD patient, and a CR ISPR gene therapy recipient, through advocacy work. Once the news broke that she was the first patient to receive CR ISPR gene therapy for SCD, Victoria was invited to speak with SCD organizations, meet other patients – or “warriors”, as she refers to them – and attend international events to share her story. “ W hen I was f lown to London to speak at the summit on human genome editing, I was amazed, because people really cared about my experience,” she says. A recent highlight, she says, was her appearance on Good Morning America, where Victoria was able to meet a fellow SCD patient – Jamie – who had been inspired by her story and made the decision to receive CRISPR gene therapy. “It was truly a fulfilling moment,” she recalls, “because it was one thing that I hoped for – to be able to affect someone else's life in a positive way. To see [Jamie] looking so healthy after not being able to leave the house, or struggling to take care of his children, it was just incredible.” Victoria also had the opportunity to visit the production facility that created her CR ISPR gene therapy. “I was like a kid, looking at the machines and listening to how everything works. I know that this [therapy] was many years in the making, and it was an honor to meet the scientists that were working so hard when I thought no one cared.” “Please, just treat us how you want to be treated,” Victoria asks. “We did not choose to have SCD. We did not choose the amount of medication we require to ease our pain. We just want to be cared for, and we want to feel normal. Please, treat use with respect.” 23 iStock Her advocacy work has most recently turned to the costs associated with CR ISPR-based, and other gene therapies, for SCD. A major challenge for patients and clinicians in accessing such emerging therapies will undoubtedly be their price. W hile information on the exact cost is currently limited, Casgev y will reportedly be priced at $2.2 million per patient in the US, and over £1. 5 million per patient in the UK . Granted, it’s designed to be a “oneand-done” treatment, which could override economic burden of a lifetime of prescriptions, hospital stays and other associated costs with SCD management. But very few people – especially marginalized patients – have $2.2 million at their disposal, and in the US particularly, there’s lingering uncertainty as to whether most insurance companies will cover the therapies. “ W hen I heard the price after the approval, it made the moment bittersweet,” Victoria says. “I knew that, for me, if I would have had to pay for it, I wouldn't have been able to afford it. Because I couldn't even work. Speaking to SCD patients through my work, it’s clear that other patients and their families feel the same way.” There is major research work going on across the globe in an attempt to reduce the costs associated with gene therapies, including those that are CR ISPR-based. One example is a proposed movement towards in vivo delivery of gene therapies, which could reduce the costs associated with extracting cells, editing them in a laboratory and then infusing them back into the patient. At present, this research is in the laboratory stage, rather than clinical testing. The Innovative Genomics Institute (IGI) is a non-profit academic institution that was founded by Professor Jennifer Doudna, who became a Nobel Laureate in 2020 in recognition of her research discovering CRISPR gene editing technology. It’s a joint effort between leading research institutes in the Bay A rea, including the University of California (UC) Berkeley and UC San Francisco, with affiliates at UC Davis, Lawrence Berkeley National Laboratory, Lawrence Livermore National Laboratory, Gladstone Institutes and and other institutions. Dr. Melinda Kliegman, director of the Public Impact at the IGI, recently led the IGI’s Affordability Task Force in generating a report, titled “Making Genetic Therapies Affordable and Accessible”. The task force assembled in 2021 to start work on the report, which explores the key drivers of the high prices associated with gene therapies and identifies approaches to increase their accessibility. “The IGI develops the underlying technology used to make these [gene] therapies, but we are not involved in commercialization and pricing. We wanted to understand more about what goes into setting these prices, and what, if anything, we could do to lower them,” says K liegman. “The process of assembling the task force and developing the report 24 iStock took over a year and involved 35 task force members. The breadth of expertise of task force members helped us cover the many different complex issues that lead to high prices of gene therapies. It was a huge effort, but interesting, since we were all learning new things,” Kliegman adds. The report is 75 pages long and provides a comprehensive overview of the various factors contributing to the current pricing of gene therapy. K liegman summarizes the key “take home points” of the task force’s findings: “The issues affecting affordability are multifaceted and system wide. I would like to acknowledge that these therapies are expensive and difficult to manufacture, and there are small patient populations from which to recover costs,” she says. “Companies also need to make a profit and adequate returns for investors. Given this, a for-profit company may not be the best business model for developing bespoke gene therapies. We need a paradigm shift in the way these therapies are commercialized, for example utilizing non-profit medical research organizations and public benefit corporations running on moderate-cost capital from social impact investors and government and philanthropic grants,” K liegman continues. As part of the report, the IGI team built a model that shows it could be possible to commercialize a gene therapy for 10x less than they are currently marketed at today, roughly ~$250,000 per patient for a therapy that could treat 2000 patients per year. This model is a departure away from the traditional methods used to develop gene therapies, and so, responses to the report have been mixed, Kliegman says. “Our proposal is oriented towards access, not profit maximization. Many people think it's naive to expect anyone to ‘leave money on the table’, while others agree with the need to improve access and affordability.” There is change on the horizon, though: “ We have had the privilege of meeting with organizations aligned with the suggestions in the report. Unsurprisingly, some of them had representatives on our task force,” Kliegman emphasizes. “The 90-10 Institute recently launched, which is a nonprofit working to establish an impact investment fund for public benefit pharmaceutical companies. There are also organizations like Odylia Therapeutics and Caring Cross, which are nonprofits developing and delivering gene therapies and public benefit manufacturing organizations like Landmark Bio and Vector BioMed.” In Victoria’s mind, the high price of gene therapy is “just another hurdle to overcome”. She remains optimistic that the industry will reach a collective decision on how to ensure all patients can access the therapies they so desperately need. One day, she hopes that there will be no SCD patients having to attend emergency rooms, receive transfusions or rely on pain medication. “I can live with the title of being a sickle cell warrior – I think we all can. But I want everyone in this community to be free from the hold that the disease places on our lives.” That is her dream, she says, and unlike the dreams that SCD denied her in childhood, hopefully, science can make this one come true. ⚫ It’s simple, just choose the groups and click the bubble. Our Facebook groups allow you to keep up to date with the latest news and research and share content with like-minded professionals. With three diverse groups there is something for everyone. Hi. How are you? Have you seen our Facebook groups? Thanks! I’ll come join the conversation. Come join the Neuroscience, News and Research Group here. Discover the Analytical Science Network Group here. Explore The Science Explorer Group here. No I haven’t. Could you tell me more? Sounds great! How do I join? 26 iStock CRISPR gene editing has enabled scientists to rewrite the genetic code of living organisms and is revolutionizing medicine. In 2023, Casgevy became the first US Food and Drug Administration (FDA)-approved therapy that utilizes CR ISPR-Cas9, offering new hope for patients with sickle cell disease. Alongside its cell and gene therapy applications, CR ISPR technology is becoming an increasingly popular tool for infectious disease research. It has allowed scientists to improve their understanding of the biology and genetics of human pathogens, and is being explored as a technique for diagnosing and treating diseases such as human immunodeficiency virus (HIV). Here, we highlight some of the latest applications of CR ISPR gene editing in infectious disease research. COULD CRISPR GENE EDITING PROVIDE A CURE FOR HIV? Retroviruses like HIV cleverly integrate their genetic material into host genomes and are notoriously difficult to treat. Even with effective treatment, some immune cells go into a resting state but still contain HIV DNA. Infection with HIV is currently treatable with lifelong antiviral therapy to reduce viral load to undetectable levels, but it is not curable. CRISPR has provided new hope in the search for a HIV cure, and researchers are working towards using this technology to completely excise the viral DNA from the genome of host cells. Scientists at Temple University published evidence last year showing that a single injection of a novel CRISPR gene-editing treatment safely and efficiently removes simian immunodeficiency virus (SIV) from the genomes of rhesus macaque monkeys. The outcomes of this study set the stage for an ongoing Phase 1/2 clinical trial of EBT-101, a HIV-specific CRISPR-Cas9 gene-editing therapy, which was granted FDA Fast Track Designation in July 2023. The preclinical study, published in the journal Gene Therapy, represented a significant advance in the generation of a cure for HIV in humans. How Is CRISPR Gene Editing Being Used in Infectious Disease Research? BLAKE FORMAN 27 In a proof-of-concept study (the results of which are yet to be peer reviewed) presented at the 2024 European Congress of Clinical Microbiology and Infectious Diseases (ECCMID), researchers claimed they removed HIV from lab-cultured cells using CRISPR-Cas9 gene editing.* According to the scientists, they adopted a broad-spectrum approach, using CRISPR technology to edit two regions of the HIV genome that are conserved across all known strains of the virus. They found that the size of the vector used to transport the cassette encoding the therapeutic CRISPR-Cas reagents into the cells was too large. Another challenge was reaching the HIV reservoir cells that rebound when HIV antiretroviral treatment is stopped. To overcome these challenges, the authors tried various techniques to reduce the size of the cassette and therefore the vector system itself. They successfully minimized the size of the vector, enhancing its delivery to HIV-infected cells, and were able to target HIV reservoir cells by focusing on specific proteins found on the surfaces of these cells. The researchers hope to advance to preclinical models to study the safety and efficacy of a therapeutic strategy combining CRISPR therapeutics and receptor-targeting reagents. CRISPR ENZYMES SUPPORT PROACTIVE PLANNING AGAINST FUTURE PANDEMICS Searching for ways to improve CRISPR-based solutions to RNA viruses, which could help combat future pandemics, is an active area of research. CRISPR-Cas13 systems have become indispensable tools for various RNA targeting applications, including antiviral development to combat viruses such as SARS-CoV-2. Within the Cas13 family, Cas13d is the most active subtype in mammalian cells. However, it is inefficient in the cytoplasm of cells, where many RNA viruses replicate. What is EBT-101? EBT-101 is an in vivo CRISPR-based therapeutic candidate designed to excise HIV pro-viral DNA from HIV-infected cells. The treatment employs CRISPR-Cas9 and two guide RNAs that target three sites within the HIV genome, thereby excising large portions of the HIV genome. Examples of Cas enzymes Many CRISPR associated proteins (Cas) possess nuclease activity and play a vital part in the bacterial and archaeal defense system. In 2005, the first Cas protein with nuclease activity was discovered while studying the genome of Streptococcus thermophilus. The protein is now known as Cas9 and is one of the most common Cas proteins used in CRISPR gene-editing of DNA. Other popular systems include the CRISPR-Cas13 system, which is used for precise RNA manipulation without permanent changes to the genome. Cas12 and Cas14 enzymes are also being explored in genome editing technologies. The authors stated, “We have developed an efficient combinatorial CRISPR attack on the HIV virus in various cells and the locations where it can be hidden in reservoirs, and demonstrated that therapeutics can be specifically delivered to the cells of interest.” 28 iStock Researchers from Helmholtz Munich and the Technical University of Munich overcame this obstacle by engineering nucleocytoplasmic shuttling Cas13d (Cas13d-NCS). This system can transfer nuclear CRISPR RNA into the cytosol. The scientists showed that Cas13dNCS outperforms its predecessors in degrading mRNA targets and neutralizing self-replicating RNA, including replicating sequences of several variants of SARS-CoV-2. This achievement represents a significant step in strengthening our defenses against future outbreaks of RNA viruses. DISEASE VECTORS: CRISPR ADDRESSES THE ROOT OF THE PROBLEM Many human pathogens such as malaria are vector-borne. Vectors are living organisms that can transmit infectious pathogens between humans. Vector-borne diseases are human illnesses caused by parasites, viruses and bacteria that are transmitted by vectors. These diseases are often transmitted from blood-feeding arthropods like mosquitoes. CRISPR gene editing provides an opportunity to control the spread of these animal vectors, thus preventing the transmission of the pathogens they carry. Chagas disease can be transferred to humans by insects such as triatomine bugs, also known as kissing bugs. As treatment options are limited, strategies for Chagas disease control have focused on ways to manipulate the organisms that carry the parasite. The application of CRISPR technology in kissing bugs has proven difficult. Traditional gene editing methods involve injecting the CRISPR gene-editing material directly into embryos, which, due to the hardness of kissing bugs' eggs, has proven challenging. In a recent paper published in The CRISPR Journal, researchers demonstrated the application of CRISPR-Cas9 gene editing in kissing bugs for the first time, creating new possibilities for using genetic technologies to control vector-borne Chagas disease. The research team from Penn State College of Agricultural Sciences have developed an approach called Receptor-Mediated Ovary Transduction of Cargo or “ReMOT Control”. This technology enables the injection of materials directly into the mother's circulatory system and guides that material to the developing eggs. CRISPR IMPROVES THE TIME TO RESULT IN DISEASE DIAGNOSIS In addition to treating and preventing infectious diseases, CRISPR technol- “Here, we showed that you could genetically modify this vector insect. Our technology has the potential to make gene editing more efficient, easier and cheaper in a wide range of animals,” said Dr. Jason Rasgon, Dorothy Foehr Huck and J. Lloyd Huck Endowed Chair in Disease Epidemiology and Biotechnology at Penn State College of Agricultural Sciences. 29 iStock ogy has been widely used in research developing novel diagnostic tools for diseases such as SARS-CoV-2. A rapid test for diagnosing melioidosis, a rare tropical disease, was recently described in a study published in The Lancet Microbe. Melioidosis is caused by the bacterium Burkholderia pseudomallei. Present in soil and water in tropical and subtropical regions, the bacterium enters humans via inoculation through skin abrasions, ingestion or inhalation. “Melioidosis has been neglected despite its high mortality rate and high incidence in many parts of Asia. Early diagnosis is essential so that the specific treatment required can be started as soon as possible,” said Professor Nick Day, senior author and director of the Mahidol-Oxford Tropical Medicine Research Unit . Diagnosis of melioidosis requires culturing bacterial samples, which takes three to four days. In this study, the team set out to develop a new rapid test to reduce patient diagnosis time. Their test, called CRISPR-BP34, involves rupturing bacterial cells and using a recombinase polymerase amplification reaction to amplify the bacterial target DNA. Additionally, a CRISPR reaction is used to provide specificity, and a simple lateral flow read-out is employed to confirm cases of melioidosis. The team collected clinical samples from 114 patients with melioidosis and 216 patients without the disease at a hospital in northeast Thailand, where the disease is endemic. The CRISPR-BP34 test was then applied to these samples. The new test showed enhanced sensitivity at 93%, compared to 66.7% in the current gold-standard culture-based method. It also delivered results in less than four hours for urine, pus and sputum samples, and within one day for blood samples. ELIMINATING ANTIMICROBIAL-RESISTANT BACTERIA WITH GENE EDITING Widespread misuse and overuse of antimicrobials have led to antimicrobial resistance (AMR) being declared one of the top global public health and development threats. Scientists across the globe are now rapidly searching for viable alternatives to antibiotics. CRISPR has not only been used to identify AMR genes but has potential as a therapeutic tool to treat antibiotic-resistant bacteria and other pathogens. At North Carolina State University, researchers showed that the CRISPR-Cas system can target and eliminate the gut bacteria Clostridioides difficile (C. difficile) in vivo. The results were published in the journal mBio. Antibiotic use is a major risk factor for C. difficile infection because broad-spectrum antimicrobials disrupt the indigenous gut microbiota, decreasing colonization resistance against C. difficile. The study showed that the CRISPR-Cas system in C. difficile can be repurposed as an antimicrobial agent through the expression of a self-targeting CRISPR that redirects endogenous CRISPR-Cas3 activity against the bacterial chromosome. The researchers tested this approach in mice infected with C. difficile. Two days after the CRISPR treatment, the mice showed reduced C. difficile levels, however, those levels began to increase again two days later. The researchers explained that future work will include retooling the phage to prevent C. difficile from returning after the initial effective killing. THE FUTURE OF CRISPR IN INFECTIOUS DISEASE RESEARCH The versatility of CRISPR gene editing has resulted in its application in many facets of infectious disease research. As CRISPR technology continues to undergo technical improvements, the prospects for its application in treating incurable diseases such as HIV are becoming increasingly promising. The notable applications discussed here merely offer a glimpse into the evolving landscape of how CRISPR gene editing can be harnessed to improve human health. ⚫ * These research findings are yet to be peer-reviewed. Results are therefore regarded as preliminary and should be interpreted as such. Find out about the role of the peer review process in research here. For further information, please contact the cited source. 30 iStock I n 2016, Dr. Alexandra Freeman returned to a career in academic research after several successful years working in media. Shortly after this transition, she observed concerning parallels between the industry that she had left behind, and that which she had re-joined. It seemed as though, all around her, academics held a firm desire to tell what Freeman describes as “neat, short, easy-to-read and persuasive” stories in their papers. But good research isn’t about storytelling, she thought – it’s about evidence communication. Inspired to create change, Freeman sat down one evening and got to work developing Octopus, a novel and radical publishing platform for scholarly research. She penned the idea in a single night, and with funding from UK Research and Innovation (UKRI), officially launched Octopus in the summer of 2022. “In Octopus, there are eight types of publication, each reflecting a part of the research process,” Freeman told Technology Networks in an interview ahead of Octopus’ launch. “This allows researchers to specialize in one or two of these parts of the process (with their very different skills) and to publish their work literally in collaboration with the rest of the research community – in time, as well as in space! Someone might publish a theoretical idea now, which someone else, 20 years in the future, will collect data to test. Then, another researcher, 20 How To Enter a New Chapter in Academic Publishing MOLLY CAMPBELL RADICALLY RETHINKING SCIENTIFIC PUBLICATION: THE "OCTOPUS" MODEL READ MORE 31 Alexandra Freeman years further in the future, might again choose to analyze that data using new techniques.” As Octopus approaches its second birthday, Technology Networks reconnected with Freeman to learn about the academic community’s response to the platform, how it has evolved and her current stance on the publishing landscape. Molly Campbell (MC): Can you provide our readers with an update on how the launch of Octopus went, and how the scientific community has reacted to it? Alexandra Freeman (AF): The launch itself was a great event, but it was just the lighting of the fuse. Since then, we’ve been continuing to develop all the features that we need to in order to fulfil the vision that I originally had for Octopus – and that will continue for another year or more. The reception has been great – we’ve got over 1,000 users, and publications on Octopus get viewed a lot – far more than I had anticipated! W hen we show Octopus to people, they are almost entirely positive about it too – but that isn’t to say that everything’s plain sailing, as I knew it wouldn’t be. MC: Octopus evolved from your feelings of frustration towards researchers’ desire to tell “neat, short, easy-to-read persuasive stories in academic papers.” Has anything changed? AF: In the big picture, no – not yet. The incentive structure for researchers is still mostly geared towards a paper in a journal (or a monograph). To get the biggest readership (and hence market) for those, the pressure is for brevity, simplicity and readability. Very few “casual” readers want highly detailed methods, complete results or analytical code etc., and it’s not commercially viable to cater for those who do, (even though that is what is needed to be able to assess and build on research that’s been done). However, the ground is shifting – funders are beginning to make moves that could change the entire landscape. Funders really care about Figure 1: A branching chain of research publications on Octopus.ac. The pressures that researchers feel come from institutions and funders – the people who will employ them and pay them. 32 the quality of research work and have very few competing interests, so it’s them that needed to take the first steps – and that is happening. I think we’re going to see the pace of change pick up now. MC: Almost two years after launch, is Octopus having the impact that you hoped for? Have there been any challenges? If so, how did you overcome them? AF: Not yet. It is having an effect, in many different ways, but I’m very ambitious for the change needed, so there’s a long way to go. W hen we introduce the platform and explain why it is designed the way it is, researchers understand it. A lmost all say they would like to try it and support its aims. Most, though, say that they don’t feel able to use it regularly because of the pressures they’re under – or feel they’re under – to publish “the traditional way.” Until fairly recently, there weren’t that many alternatives to the traditional article formats, and it was understandable for the research assessment system to be built around these formats. But now, there are better alternatives for sharing work in enough detail for it to be fully assessed and built on (Octopus being one example) and so the whole landscape can change. We need to make funders and institutions aware of these new alternative publishing platforms and their advantages (to them, to researchers and to the whole research landscape) so that they can change the incentive system that researchers feel trapped within. That is something I’m very much working on. MC: Do you feel that the research community is aware of Octopus and how they can use it? AF: “The research community” is huge! A lot of people have heard about it, although we know from research that many have some misconceptions about it – it is quite a different way of working. I have published on it myself and know some of the questions that arise, like “what should be in a Results publication, as opposed to an Analysis publication?”, so I think there’s quite a lot of work still to do helping people with their first publications. But there are also going to be huge numbers of people – around the world – who haven’t yet even heard of it. Many of those will, I think, be mid-career researchers who feel there is a set career path that they are on. They likely feel very under pressure to publish in certain ways, and don’t even have the time and energy to look at anything other than getting the next publication in the specific journal that they think they have to, in order to secure their next contract or grant. The only way these researchers can be freed to think about the quality of the work they’re doing – and how useful what they’re sharing is for people other than themselves – is if institutions and funders make it very clear indeed that “the system” has changed and demonstrate that the old rules no longer apply. MC: There have been many notable changes to the publishing landscape since our last conversation, such as the introduction of large language models (LLMs). What are your thoughts on the current scientific publishing landscape? AF: There have been some big changes. As you say, generative A I is a huge one. We were dealing with a system where people were incentivised to get their names on as many publications as possible – that was bad enough. Now those publications can even be generated artificially within minutes, at scale, by computers. I can’t see any way in which the old system, with a small group of volunteer peer-reviewers and editors, can deal with sifting through an almost infinite volume of papers, to try and recognize the ones that are created by humans and based on actual research, and then pick out the good research. Rather than resorting to “easy” cues that inevitably lead to bias (such as previous reputation of authors or institutions), I think we’ll need to move to systems that demand greater evidence of the work done and its trustworthiness – such as open data, analytical code, etc. I’ve always thought that the old system of editorial approval is unsustainable: that we cannot rely on “peer reviewed” as being a stamp of trustworthiness. I think that has become increasingly obvious as the volume of published A I-generated papers and A I-generated reviews has been revealed. As readers, we The old system of editorial checking was already breaking down under the volume of publication, and that pressure hose of publications has just been turned right up. 33 are going to have to judge things for ourselves more carefully and not take things “on trust”, outsourcing the judgement to anonymous reviewers; just as we have to with any information we read online these days. On a more positive note, the Gates Foundation has signaled the start of another major change in the publishing landscape – it has announced that it will no longer pay publishers to publish papers. Instead, it will be supporting more free, alternative ways of sharing work, such as the use of pre-print servers. I think this is exactly the kind of leadership that funders need to take. They have the power to change the status quo, and someone needs to! MC: UKRI provided the funding to launch Octopus, and for several years thereafter. Has funding been secured for the future? AF: UKRI, through Research England, has released two more years of funding for Octopus. However, we do need to look ahead to the future. Most importantly we want to keep our costs minimal – it’s only a tiny staff and enough to cover the technical costs of keeping the platform running. We want to make the back-end database distributed so that institutions can volunteer to host a portion of it. This will keep our hosting costs low, and will ensure that all the data is safely mirrored across different geographical locations. MC: Are there any misunderstandings or misconceptions about Octopus that you would like to address? AF: A few have come up, and it’d be great to set the record straight. 1. Octopus is like a pre-print server in that you can publish work on it – and get peer reviews – and then submit it to a journal. However, since Octopus doesn’t publish papers, you need to format your work differently. 2. It is like a pre-registration platform, in that you can publish your research questions, hypotheses and methods before collecting any data (and there is a marker to specifically highlight that you are pre-registering). But you do have to make these public before they get DOIs and dates. On the plus side, like a registered report, you can get peer review of these before you go and collect data. 3. It is a bit like using GitHub, in that you can “fork” a chain of research and take it in a different direction, but unlike GitHub (or ResearchEquals, or Jupyter notebooks) it’s not a place for day-to-day work, which is constantly changing or being updated. It’s designed to be where you publish finished work (this can be a smaller chunk of work than you might think of when you’re used to publishing papers). 4. It is like a repository, in that you can put work on Octopus that has been published in journals, but again, the format is different so you will need to do a bit of work to turn it into Octopus publications. The benefit, of course, is that Octopus is open for others to read, so your work can get a broader readership than a paywalled article, and you don’t have word or format limits so you can go into more detail too. MC: You run Octopus in your spare time – you must be busy! How are you balancing everything? AF: I’m used to being busy! But all the day-to-day work is done by a team based at Jisc now, so although I keep across everything with meetings – and I still give quite a few talks – I can fit it in. Octopus is so important – I’ll make as much time as it needs for as long as I possibly can. MC: Is there anything else that you would like our readers to know about Octopus? AF: If you agree with the principles of Octopus, the best way that you can help support us as a researcher is to publish on it. It will probably take you about an hour or two to take one of your published papers and put the work up on Octopus, depending on how quick your co-authors are to approve publications If you have less time than that, see whether there is a publication you can write a peer review of – these take a much shorter time than reviews of whole papers. If you have the power to change policy at an institution or a funder, check whether your policies support people who use these alternative publishing platforms. W hat can you do to help change the tidal stream that good, careful researchers currently feel is against them? We all have our parts to play. We can all make a positive difference. ⚫ Keeping our costs minimal means we shouldn’t need too much to keep going. But it’s not nothing, so I am going to be doing a lot of talking to funders, philanthropists and institutions. iStock, Alexandra Freeman, Victoria Gray 34 Meet the interviewees whose insights featured in issue 35 of The Scientific Observer: John Booske is the Keith and Jane Morgan Nosbusch emeritus professor in electrical and computer engineering at University of Wisconsin–Madison. His research focuses on plasmas, metamaterials, metasurfaces and media that have a strong interaction with electromagnetic radiation, electromagnetic field effects and microwave vacuum electronics. John holds a PhD in Nuclear Engineering from the University of Michigan. Victoria Gray was the first sickle cell anemia patient in the world to be treated with CRISPR gene editing in 2019. After a lifetime of pain, treatments and hospitalizations for sickle cell disease, she is now symptom-free and working as a patient advocate and international speaker to spread the word about CRISPR and rare disease to clinicians, scientists, patients and students. Susan Hagness, is the Philip Dunham Reed Professor and department chair of electrical and computer engineering at the University of Wisconsin–Madison. Her group’s research spans computational and experimental applied electromagnetics, with an emphasis on bioelectromagnetics. Susan golds a PhD in Electrical Engineering from Northwestern University. Alexandra Freeman, PhD started with a 16-year career at the BBC, working on various television series. Her work won a number of awards, from a BAFTA to a AAAS Kavli gold award for science journalism. She then joined the University of Cambridge to lead the Winton Centre, where she had a particular interest in helping professionals such as doctors, journalists or legal professionals communicate numbers and uncertainty better, and in whether narrative can be used as a tool to inform but not persuade. In 2024 she was chosen to be a crossbench peer in the House of Lords through a process of selection. She is an advocate of Open Research practices and the reform of the science publishing system, and in her spare time leads the Octopus platform for primary research publication. Melinda Kliegman, PhD, is director of public impact at the Innovative Genomics Institute (IGI). In this role, she leads the Public Impact team, which works to align IGI’s genome-engineering innovations with societal values by engaging in public dialogue, original research, and policy creation through outreach to key stakeholders to ensure that genome-editing technology benefits everyone equitably. 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