Cell and Gene Therapies: From Discovery to Delivery

SPONSORED BY Developments in Point‑of‑Care Manufacture of Advanced Therapies Current Challenges in Cell Line Development for Therapeutics Why Are mRNA Vaccines Not Giving Long-Lasting Protection? CELL AND GENE THERAPIES: From Discovery to Delivery CONTENTS 5 Harnessing Cell Lines To Advance Cancer Research and Treatment 9 Why Are mRNA Vaccines Not Giving Long-Lasting Protection? 13 Improving the Efficacy and Accessibility of CAR Therapies 17 Mass Spectrometry Imaging in Pharmaceutical Development 21 Cell and Gene Therapy Manufacturing 22 Current Challenges in Cell Line Development for Therapeutics 27 New Purification Techniques in Biopharmaceuticals 31 A Bright Future for RNA Therapeutics 34 Developments in Point‑of‑Care Manufacture of Advanced Therapies CELL AND GENE THERAPIES: FROM DISCOVERY TO DELIVERY 3 TECHNOLOGYNETWORKS.COM FOREWORD Biotherapeutics are evolving rapidly, fueled by innovative science and collaborative ambition. This eBook explores some of the most exciting frontiers in modern medicine, from the molecular precision of RNA therapeutics to the logistical advancements enabling point-of-care manufacturing. These topics highlight how diverse scientific breakthroughs are converging to reshape drug development and patient care. Inside, readers will discover the nuanced challenges of maintaining long-term vaccine efficacy, the role of engineered cell lines in cancer research and how advances in CAR and NK cell therapies are expanding the promise of immunotherapy. Articles also delve into enabling technologies like mass spectrometry imaging and the latest purification techniques critical to scaling and refining biopharmaceutical production. Whether you’re an industry professional, academic researcher or healthcare innovator, this eBook provides a valuable perspective on the current state and future direction of biopharmaceutical development. The Technology Networks editorial team WHY CLINICAL RESEARCHERS TRUST ddPCR...Goals: Effective evaluation of cell and gene therapies in patientsClinical ResearchSerialMonitoringBiodistributionDose ResponseEvaluationKey Benefits Precision Accuracy Sensitivity Research Use OnlyWHY QA/QC SCIENTISTS TRUST ddPCR...Goals: Reliable assessments of purity, potency and safetyFinal QA/QCKey Benefits Precision Accuracy Sensitivity/ Specicity Time to resultsViral Titer and Empty-Full Capsid RatioMycoplasmaDetectionTransgeneCopy NumberResidual DNACell and gene therapies are continuing to gain popularity, with 431 now FDA approved and 4,000+ more in development.2 While these treatments are promising, managing safety and effectiveness in patients is complex. Around the world, groups ranging from drug discovery, development, and manufacturing to clinical laboratories are using Bio-Rad's Droplet Digital™ PCR (ddPCR™) technology as a reliable and scalable solution to myriad workflow challenges.Explore Bio-Rad's end-to-end solutions for developing and manufacturing cell and gene therapies at www.bio-rad.com/CGT1. https://bioinformant.com/u-s-fda-approved-cell-and-gene-therapies/2. https://www.asgct.org/global/documents/asgct-citeline-q1-2024-report.aspxAn End-to-End Cell and GeneTherapy Development PartnershipWHY CELL AND GENE THERAPY SCIENTISTS TRUST ddPCR....Goals: CMC submissions, efcient and scalable process developmentKey Benefits Precision Accuracy Inhibitor Tolerance/ Sensitivity MultiplexingViral Titer and Empty-Full Capsid RatioTransgene CopyNumberTransgeneExpressionIn Process TestingPlasmidIntegrity 5 CELL AND GENE THERAPIES Credit: iStock/koto_feja Harnessing Cell Lines To Advance Cancer Research and Treatment Monica Hoyos Flight, PhD Genetic variation affects the expression of genes and function of proteins. DNA sequencing technologies have uncovered millions of genetic changes associated with various human traits and disease. However, it is still largely unclear which of these changes are harmful and how they cause problems. Efforts to understand the effects of gene variants in cancer cell lines are shedding light on cancer biology and drug resistance, and are paving the way for personalized therapies. This article examines some of the cell-based approaches that researchers are using to study acquired drug resistance and the effects of specific genetic variants on cancer cell physiology. It also explores some of the challenges associated with working with cells, whether as model systems or for the production of biotherapeutics, focussing on the biological consequences of cell line engineering. Developing cell lines to understand acquired drug resistance Despite decades of medical advances, cancer is among the leading causes of death worldwide. A significant challenge in improving patient outcomes, particularly of those with metastatic disease, is the development of drug resistance. Cancers that initially respond to treatment can later recur in a resistant form. TECHNOLOGYNETWORKS.COM CELL AND GENE THERAPIES: FROM DISCOVERY TO DELIVERY 6 This is partly due to clonal evolution, a process through which cancer cells expand and diversify by accumulating mutations over time. As a result, even closely related tumor cells may respond differently to the same therapy. Moreover, anti-cancer therapies exert selective pressure, accelerating clonal evolution processes and reducing the efficacy of treatment. Studying resistance mechanisms in tumor tissue is challenging as it requires serial biopsies, which are invasive and often not feasible. The use of liquid biopsies has made monitoring cancer cell evolution easier, but translating this information into treatment decisions requires further research and clinical validation. By growing cancer cells in the presence of anti-cancer drugs, Martin Michaelis, professor of molecular medicine at the University of Kent, aims to understand what makes certain cells resistant to specific drugs and what renders them vulnerable to others. He has been looking after the Resistant Cancer Cell Line (RCCL) collection since the 80s. The collection, which originated in the laboratory of Jindrich Cinatl in Frankfurt am Main, Germany, now comprises over 3,000 cell lines. The cells are continuously exposed to increasing anticancer drug concentrations to establish readily growing sub-lines that show a clear resistance to the selection agent. “Establishing a cell line resistant to classic cytotoxic chemotherapeutics can take over a year,” Michaelis said, “It is tough, expensive and labour intensive.” Once the cells have acquired resistance to one drug, their response to other drugs can be tested. “We often find that cells that are resistant to one drug show collateral vulnerability that makes them more sensitive to other drugs,” he explained. Identifying biomarkers of these cells could eventually help guide treatment decisions. Over 100 research groups in academia and industry are using the RCCL collection, applying omics methods to characterize cells in depth and develop novel therapies for patients who currently lack effective treatment options. A lot of work is being done to understand the rapid emergence of resistance to targeted therapies. For example, patients with melanomas that harbor the BRAF V600E mutation often respond very well to BRAF inhibitors initially. But, resistance emerges rapidly due to the development of compensatory mechanisms within the cells. Combining BRAF inhibitors with MEK inhibitors, which target a related protein in the signaling pathway, can mitigate this rapid resistance.1 This is reflected in cell culture models where melanoma cells adapt to BRAF inhibitor therapy much faster than to traditional cytotoxic drugs. Deciphering the mechanisms of resistance is no mean feat. Michaelis and others have shown that every resistance formation process is different. “If we take one cell line and adapt it to the same drug 10–20 times, we get 10–20 different phenotypes and drug resistance profiles,” he said. This diversity makes it crucial to generate so many cell lines and support the infrastructure required to maintain them. High-throughput screening of cells with cancer-associated mutations Another way to study cancer cell biology and intrinsic mechanisms of drug resistance, which are present before any treatment is administered, involves systematically knocking out or editing cancer-related genes. Geneediting technologies are allowing researchers to introduce these mutations into cell lines and examine both their individual and combined effects on cancer progression, metastasis and response to therapies. Francisco Sánchez-Rivera, assistant professor of biology at the Massachusetts Institute of Technology (MIT) has been using prime editing, a refined version of CRISPR technology, to examine the effects of genetic variations on cancer cell growth and response to drugs. Prime editing enables any kind of point mutation to be introduced, as well as insertions and deletions, into the DNA of living cells without creating double-strand DNA breaks characteristic of traditional CRISPRCas9 methods. “With gene editing technologies it is technically possible to understand the impact of every single gene variant in every single coding or non-coding TECHNOLOGYNETWORKS.COM CELL AND GENE THERAPIES: FROM DISCOVERY TO DELIVERY 7 region in the human genome,” he said. “We are just beginning to scratch the surface!” His team has engineered over 1,000 variants of the TP53 tumor suppressor gene, the most frequently mutated gene in cancer patients, to investigate their impact on lung adenocarcinoma cells. By using a high-throughput prime editing sensor strategy that couples prime editing guide RNAs with synthetic versions of their cognate target sites, they can quantitatively assess the effects of genetic variants found in patients on cell function at scale.2 Their work highlights the role of gene dosage in influencing the natural proportions of proteins and their interactions with each other. Sanchez-Rivera is very enthusiastic about advances in the field. “With CRISPR-based technologies we can carry out experiments with remarkable precision and efficiency; they are already allowing us to model and dissect the aberrant circuitry of cancer cells.” Moreover, as sequencing technologies increasingly become integrated into clinical practice, this knowledge will help tailor therapies to patients with tumors that have a defined genetic makeup. “The next 5–10 years will be truly transformative,” he said. Challenges of working with cell lines In addition to the challenges associated with monitoring the viability of cells, optimizing growth conditions and avoiding contamination, the first major hurdles relate to the efficiency of introducing genetic material into cells (transfection) and controlling gene expression levels. “We often have to test various delivery modalities, because different cell types are more or less amenable to transfection, and tweak and test different types of functional editing reporters to ensure that our cell line is capable of editing at very high efficiency,” Sanchez- Rivera explained. His team recently experienced significant challenges trying to stably introduce cytosine and adenine base editors into mouse B cell acute lymphoblastic leukemia cells using lentiviruses. To overcome this issue, they developed a strategy that couples stable viral guide RNA delivery and transient mRNA electroporation. Using this approach, they have produced single gene variantbased cellular models as well as multiplexed scalable mouse models in which thousands of genetic variants can be examined in vivo.3 When working with genetically engineered cells, it is important to consider the effect that the deleted, modified or new gene will have on cellular resources as this will influence the cells’ gene expression capacity. “Every time you engineer cells with genetic constructs, they draw on the cells’ enzymes, nucleotides and ribosomes to express them,” said synthetic biologist Francesca Ceroni. She and others have shown that the introduction of genetic constructs can impose an unintended “cost” or burden on mammalian cells.4, 5, 6 The diversion of cellular resources to transcribe and translate new genes can lead to slower growth rates and reduced expression, which can limit the production of biotherapeutics. Ceroni’s group based at Imperial College London focuses on characterizing the cellular burden caused by recombinant protein production and identifying design rules for synthetic systems to achieve robust control of gene expression. They are working in collaboration with several pharmaceutical companies to optimize the production of biotherapeutics. Addressing resource competition and cellular burden is crucial for advancing cell engineering across various applications, including disease modeling, bioproduction and cell therapies. Ceroni calls for further research into the implications of resource competition in primary cells. “We need to further characterize the problem so that we can then develop tools to address it.” Approaches to precisely titrate the stoichiometry of gene expression in mammalian cells, such as the host-independent and programmable transcriptional system developed by Qin and colleagues that can improve the production of viruslike particles (VLPs),7 will contribute to improve the production of biotherapeutics. “The toolkit for controlling gene expression is limited,” Ceroni said. By using machine learning to increase the design space, her team are testing new components and optimizing construct expression. TECHNOLOGYNETWORKS.COM CELL AND GENE THERAPIES: FROM DISCOVERY TO DELIVERY 8 When it comes to the production of biologics, this could be key to reducing costs. She concluded by emphasizing the importance of a systems-thinking approach, especially when developing biotherapeutics. “You can’t only look at the cells, the interactions between different components of the process (cells, growth conditions, cell engineering) need to be carefully considered.” REFERENCES: 1. Subbiah V, Baik C, Kirkwood JM. Clinical development of BRAF plus MEK inhibitor combinations. Trends Cancer. 2020;6(9):797– 810. doi: 10.1016/j.trecan.2020.05.009 2. Gould SI, Wuest AN, Dong K et al. High-throughput evaluation of genetic variants with prime editing sensor libraries. Nat Biotechnol. 2024:1–15. doi: 10.1038/s41587-024-02172-9 3. Acosta J, Johnson GA, Gould SI et al. Multiplexed in vivo base editing identifies functional gene-variant-context interactions. bioRxiv. 2025. doi: 10.1101/2025.02.23.639770 4. Gabrielli J, Di Blasi R, Kontoravdi C et al. Degradation bottlenecks and resource competition in transiently and stably engineered mammalian cells. Nat Commun. 2025;16(1):328. doi: 10.1038/ s41467-024-55311-w 5. Di Blasi R, Gabrielli J, Shabestary K et al. Understanding resource competition to achieve predictable synthetic gene expression in eukaryotes. Nat Rev Bioeng. 2024;2(9):721–732 . doi: 10.1038/ s44222-024-00206-0 6. Jones RD, Qian Y, Ilia K et al. Robust and tunable signal processing in mammalian cells via engineered covalent modification cycles. Nat Commun. 2022;13(1):1720. doi: 10.1038/s41467-022-29338-w 7. Qin C, Xiang Y, Liu J et al. Precise programming of multigene expression stoichiometry in mammalian cells by a modular and programmable transcriptional system. Nat Commun. 2023;14(1):1500. doi: 10.1038/s41467-023-37244-y MEET THE INTERVIEWEES: Martin Michaelis is a professor of molecular medicine at the University of Kent, Canterbury, UK, and a research group leader at the Dr Petra Joh Research Institute in Frankfurt am Main, Germany. His research focuses on the investigation of acquired drug resistance in cancer using the Resistant Cancer Cell Line (RCCL) collection, a unique resource consisting of more than 3,000 drug-adapted cancer cell lines. Additional research interests include the identification of virulence mechanisms and therapeutic targets in viruses, and meta-research on practices in the life sciences, with a focus on reproducibility and ethical standards. Francisco Sánchez-Rivera, is an assistant professor of biology at the Massachusetts Institute of Technology (MIT). His group studies the cellular and molecular mechanisms by which genes and disease-predisposing mutations influence the development of diseases like cancer. To do so, they are applying CRISPR-based methods to engineer mutations with high efficiency and precision in cells and tissues of living animals to quantitatively interrogate cancer variants at scale and pave the way for novel therapeutic strategies. Francesca Ceroni is a senior lecturer in the Department of Chemical Engineering at Imperial College London, UK. She sits on the Imperial College Centre for Synthetic Biology Management Board and between 2018 and 2021 she was a member of the World Economic Forum Expert Network. Her research focuses on understanding the complexity of genetic regulation by building synthetic systems from the bottom up. Her team works on genetically engineered bacterial and mammalian cells to develop innovative approaches to mitigate resource competition and burden, and improve construct performance. 9 CELL AND GENE THERAPIES Credit: iStock/MicroStockHub Why Are mRNA Vaccines Not Giving Long-Lasting Protection? Aron Gyorgypal, PhD Why do mRNA vaccines struggle to provide lasting protection, and could the absence of bone marrow longlived plasma cells explain the waning antibody levels and provide insight for durable vaccine development? Viral infections, reinfections and immunity – SARS-CoV-2 as an example Viral infections can spread through human-to-human interaction when viral particles enter the body via mucous membranes, such as those in the nose, mouth or eyes, as well as through open wounds. Briefly, the cascade of events for respiratory viruses goes as follows: viruses infect the airway by binding to specific receptors on our cells within the respiratory tract; for example, the ACE2 receptors on the pneumocytes within the alveoli are targeted by SARS-CoV-2. The subsequent targeting allows the viral pathogen to enter the cell and release its genetic material, hijacking the cell’s machinery to cause viral replication. The immune system recognizes the immunogenic viral RNA produced through pattern recognition receptors and arms the innate immune system. Antigen-presenting cells (APCs), such as dendritic cells, take in antigens and present them to CD4+ T cells, priming them; likewise, B cells also recognize antigens, internalizing them and presenting them on MHC II receptors on their surface. The B and T cells then undergo linked recognition, producing memory B and T cells and plasma cells, leading to adaptive immunity. TECHNOLOGYNETWORKS.COM CELL AND GENE THERAPIES: FROM DISCOVERY TO DELIVERY 10 These plasma cells are required to produce antibodies against the virus that produce protection against reinfection. It is known that long-lived bone marrow plasma cells are needed to produce protective antibodies against reinfection. It is also known that patients who have recovered from COVID-19 initially have a substantially lower risk of reinfection with SARSCoV- 2.1 Nonetheless, anti-SARS-CoV-2 antibodies decrease rapidly within the first few months after infection. Literature has shown that the anti-spike proteins of SARS-CoV-2 diminish rapidly from circulation within the first four months and then more gradually in the subsequent seven months postinfection. 2 A major concern during the pandemic was that this loss in humoral antibodies would correlate to a loss in protection against the virus. However, Turner and colleagues showed that COVID-19-recovered individuals indeed had circulating memory B cells directed against SAR-CoV-2 and that the longevity of serum antibodies is not the only factor in determining durable immunity against reinfection as memory B cells could rapidly differentiate into antibody-secreting cells during virus re-exposure.3 This provided evidence that natural infection to SARS-CoV-2 indeed establishes two armed humoral immune memory through long-lived plasma B cells and memory B cells. Protecting against infections with vaccination However, what about protecting against a viral infection even before the first exposure? The first commercially produced and FDA-approved mRNA vaccine was against the SARS-CoV-2 within the United States. While the usual production of a conventional vaccine takes 10–15 years, the COVID-19 vaccine was developed in less than one year, raising concerns about safety and efficacy. A meta-analysis from 2024 concluded that of the vaccines tested, mRNA and inactivated-based vaccines had the greatest effect after the first and second dose, with mild local and systemic adverse effects and a very rare rate of adverse effects.4 What has been seen, however, is that without mRNA vaccine boosters, the rate of reinfection increases as these vaccines do not protect against new variants.5 Another concern is the efficacy of the vaccines to produce longterm durable protection, like natural immunity. How mRNA vaccines interact with the immune system There is a concern about the lack of durable protection from mRNA vaccination. To understand why, we need to delve into the engineering of the mRNA vaccine as well as the hypothesized method of immune response that produces protection. The mRNA vaccines consist of a 5’cap attached to the 5’UTR, followed by the coding sequence for the SARS-CoV-2 spike protein, terminated by a 3’UTR and a poly-A tail. This mRNA is encapsulated in a lipid nanoparticle (LNP) and stabilized by polyethylene glycol (PEG). After intramuscular injection of the vaccine, the mRNA-LNP is taken up by APCs, such as dendritic cells, and trafficked to the lymph nodes. Here, they prime both CD4+ and CD8+ T cells. CD8+ induces the production of cytotoxic T cells, which destroy infected cells, while the antigen-primed CD4+ T cell differentiates into Th1 cells or T follicular helper (Tfh) cells. The Tfh cells help to initiate a germinal center reaction, which results in the formation of affinity matured memory B cells and antibody-secreted long-lived plasma cells.6 The desired outcome of the vaccination regimen is to produce long-lived plasma B cells that can survive for years, producing neutralizing antibodies against the viral antigen as well as the memory B cells, which, when re-exposed to the antigen, give rise to new high-affinity antibody-secreting cells capable of neutralizing the antigen. Nevertheless, this is not the case. In recent years, during the COVID-19 pandemic, protective antibodies for immunized individuals waned over the months after the completion of the vaccination regimen. Should the longlived plasma B cells not be active and re-confer protection TECHNOLOGYNETWORKS.COM CELL AND GENE THERAPIES: FROM DISCOVERY TO DELIVERY 11 against the virus? Yes, that is the function of the adaptive immune system, yet this seems not to be happening. Why mRNA does not produce durable protection, a hypothesis The mechanism by which mRNA vaccination generates an adaptive immune response and subsequent immunity is unknown. Giannotta and Giannotta7 argue that there is an immune memory problem in which long-lived plasma B cells are absent. What is known is that the mRNA-LNP is taken up by APCs and is found at the site of injection and the draining lymph nodes, showing the translation of the mRNA cargo into spike protein. This confers that the mRNA is reaching the cytosol within the cells. Monocytes will express the antigen on their surface, typically done by bone marrow-derived APCs to prime CD8+ T cells using MHC class I molecules. However, mRNA-LNP is also taken up by non-immune cells as an off-target effect; these non-immune cells then generate proinflammatory cytokines and chemokines at the site of immunization, which can trigger antigen-specific antibodies and induction of CD8+ T-cell responses through cross-presentation. Antigen-specific CD8+ T cells are the main determinant for immune protection. These CD8+ cells, along with memory B cells, block the progression of infection. However, one major issue with vaccination is that CD8+ T cells produce a low concentration of CD38 versus that of natural infection.8 As CD38 regulates the infection-induced regulatory process to induce an adaptive immune response, this could be a reason for a differential response and lack of long-lived protection. Moreover, it is not known exactly how CD4+ T cells are activated. The SARS-CoV-2 mRNA-LNP induces proinflammatory and functional CD4+ and CD8+ T cells and a Th1 response. However, these mRNA-LNPs only encode a truncated version of the spike protein. T cells do not recognize the virus. CD8+ T cells recognize only virus-infected cells, and CD4+ T cells recognize viral antigens of infected cells. Conceptually, only long-lived plasma B cells can recognize the virus. However, there may be some reasons long-lived plasma B cells are not present after vaccination, in the same way as they are from natural infection: ∙ Cross-reactive T-cell immunity – SARS-CoV-2 shares a broad CD4+ and 8+ T-cell cross-reactivity with human endemic coronaviruses and evokes a secondary response to cross-reactive epitopes. This could eradicate the mRNA-LNP SARS-CoV-2 infection immediately. ∙ CD4+ cell-mediated memory – Cross-reactive T cells against common coronavirus may become activated, limiting the mRNA-LNP SARS-CoV-2 infection. In other words, pre-existing spike-cross- reactive T cells may be activated, limiting the infection. And as T-cell activation is required for germinal center development, this may intervene with the vaccination. ∙ Cross-reactive memory B cells and re-exposure – Memory B cells can defend against infection but not prevent it, and upon re-exposure to the virus/ antigen, if no long-lived plasma cells are present, the memory B cells are recalled. Cross-reactive memory B cells may become activated upon mRNA-LNP SARS-CoV-2 infection.9 ∙ Waning levels of anti-spike antibody levels – Although waning levels of antibody-mediated protection is expected. During the outbreak of the delta variant, highly vaccinated populations did not see protection, causing serious illness and, in some, death.10 mRNA vaccines do not produce long-lived plasma B Cells Their hypothesis was correct – Recently, Nguyen and colleagues published in Nature Medicine that the waning of SARS-CoV-2 specific antibodies could be accounted for by the absence of bone marrow long-lived plasma cells after vaccination.11 To do this, the team recruited 19 healthy adults between 2.5–33 months after receiving a SARS-CoV-2 vaccine and measured influenza, tetanus and SARS-CoV-2 antibody-secreting cells within the bone marrow. While the influenza and tetanus-specific TECHNOLOGYNETWORKS.COM CELL AND GENE THERAPIES: FROM DISCOVERY TO DELIVERY 12 IgG titers correlated with long-lived plasma cells, serum levels for SARS-CoV-2 did not. It is widely assumed that long-lived plasma cells come from memory B cells, yet SARS-CoV-2 vaccination fails to imprint the required B cell phenotype even 33 months after vaccination. However, it is important to note that the study only looked at 19 healthy individuals who self-reported their vaccination status. It is also important to note that bone marrow antibody-secreting cells are rare, and bone marrow aspirates may be a better sample for this type of interrogation. Nonetheless, the reasoning behind the failure to produce long-lived plasma cell response is unknown. mRNA Vaccines, what is next? There must be a way to produce durable protection using mRNA vaccines, specifically producing longlived plasma cells within the bone marrow. That may be done by altering the vaccination regimen. Studies looking at increasing the mRNA dose within the LNP to overcome the limits of anti-LNP antibodies and crossreactive memory B cells may be an option. The addition of adjuvants to augment the immune response could also be tried. Alternatively, the engineering of different spike proteins could be tried to alter the response. For the foreseeable future, it may be necessary to administer mRNA vaccine boosters for high-risk individuals every 4–6 months to provide protection. REFERENCES: 1. Lumley SF, O’Donnell D, Stoesser NE, et al. Antibody status and incidence of SARS-CoV-2 infection in health care workers. N Engl J Med. 2021;384(6):533-540. doi: 10.1056/nejmoa2034545 2. Seow J, Graham C, Merrick B, et al. Longitudinal observation and decline of neutralizing antibody responses in the three months following SARS-CoV-2 infection in humans. Nat Microbiol. 2020;5(12):1598-1607. doi: 10.1038/s41564-020-00813-8 3. Turner JS, Kim W, Kalaidina E, et al. SARS-CoV-2 infection induces long-lived bone marrow plasma cells in humans. Nature. 2021;595(7867):421-425. doi: 10.1038/s41586-021-03647-4 4. Beladiya J, Kumar A, Vasava Y, et al. Safety and efficacy of COVID-19 vaccines: a systematic review and meta-analysis of controlled and randomized clinical trials. Rev Med Virol. 2024;34(1). doi: 10.1002/rmv.2507 5. Tartof SY, Slezak JM, Puzniak L, et al. Effectiveness of BNT162b2 BA.4/5 bivalent mRNA vaccine against a range of COVID-19 outcomes in a large health system in the USA: a test-negative case–control study. Lancet Respir Med. 2023;11(12):1089-1100. doi: 10.1016/S2213-2600(23)00306-5 6. Bettini E, Locci M. SARS-CoV-2 mRNA vaccines: immunological mechanism and beyond. Vaccines. 2021;9(2):1-20. doi: 10.3390/ vaccines9020147 7. Giannotta G, Giannotta N. mRNA COVID-19 vaccines and long-lived plasma cells: a complicated relationship. Vaccines. 2021;9(12). doi: 10.3390/vaccines9121503 8. Oberhardt V, Luxenburger H, Kemming J, et al. Rapid and stable mobilization of CD8+ T cells by SARS-CoV-2 mRNA vaccine. Nature. 2021;597(7875):268-273. doi: 10.1038/s41586-021-03841-4 9. Song G, He W ting, Callaghan S, et al. Cross-reactive serum and memory B-cell responses to spike protein in SARS-CoV-2 and endemic coronavirus infection. Nat Commun. 2021;12(1). doi: 10.1038/s41467-021-23074-3 10. Shitrit P, Zuckerman NS, Mor O, Gottesman BS, Chowers M. Nosocomial outbreak caused by the SARS-CoV-2 Delta variant in a highly vaccinated population, Israel, July 2021. Eurosurveillance. 2021;26(39). doi: 10.2807/1560-7917.ES.2021.26.39.2100822 11. Nguyen DC, Hentenaar IT, Morrison-Porter A, et al. SARS-CoV- 2-specific plasma cells are not durably established in the bone marrow long-lived compartment after mRNA vaccination. Nat Med. 2025;31(1):235-244. doi: 10.1038/s41591-024-03278-y 13 CELL AND GENE THERAPIES Credit: iStock/artacet Improving the Efficacy and Accessibility of CAR Therapies Kate Harrison, PhD Chimeric antigen receptor (CAR) T cells have seen enormous success in cancer therapy, with seven therapies now approved for clinical use. In CAR T-cell therapy, T cells are harvested from patients and genetically altered to express CARs that strongly recognize cancer cell or tumor antigens. Enhanced cells are then expanded in vivo and transfused back into the patient to attack the cancerous cells. However, despite the successes of CAR T cell therapy, it still poses multiple challenges to widespread adoption, including potentially severe side effects such as cytokine release syndrome and neurotoxicity, limited efficacy for solid tumors and complex ex vivo processes. As a result of these challenges, CAR T-cell therapy can also be prohibitively expensive, often costing hundreds of thousands of dollars per patient and requiring hours of care. Technology Networks recently had the pleasure of speaking with Dr. Neil Sheppard, director of the Therapeutic Innovation in Natural Killer cells (THINK) lab at the Perelman School of Medicine, University of Pennsylvania, whose research focuses on the development of CAR NK-cell therapies and the improvement of CAR T-cell therapies. He discussed the challenges involved in developing new CAR-based treatments, what the future holds for the field and how effective and safe immunotherapeutics can be made accessible and affordable to everyone. Q: Can you give us a brief overview of your current research and where your focus lies? A: My lab is called the Therapeutic Innovation and NK cells (THINK) lab. We mostly work on natural killer cells and their application in cancer immunotherapy, although we also do still work on T cells – they’re both very interesting tools, suitable for different jobs. TECHNOLOGYNETWORKS.COM CELL AND GENE THERAPIES: FROM DISCOVERY TO DELIVERY 14 Within our focus on NK cells, we have several project categories. Our biggest project focuses on optimizing the NK cell “chassis” – for example, removing checkpoints or factors involved in exhaustion, to improve them as therapeutics. To do this, we’re using a form of gene editing called base editing, which allows you to do multiplex edits to a cell without damaging it, by changing one base into another. In comparison, older tools like CRISPR can break the backbone of DNA, causing extreme translocations or inducing death by apoptosis. We can also achieve the same end by programming the cell as it grows, for example with small molecule compounds that push the cells down a certain path or lock them into a certain beneficial state. We hope that with some programming with small molecules or cytokines and some gene editing, we can create an ideal NK cell that can deliver a therapeutic transgene, such as a CAR. We’ve also spent a lot of time working on NK cryopreservation. Unlike T cells, NK cells don’t cryopreserve very well – but if you can’t cryopreserve them, you don’t have a product. A lot of the trials that have been done so far with CAR NK cells have been single-center trials, where altered NK cells have been infused right away, as a fresh product, which can’t really be commercialized. Beyond that, I think successful immunotherapy is rarely going to be single agent. I see a lot of benefits for combination immunotherapy: CAR cells and other agents. We’re just wrapping up a clinical trial, the first in the world to use CAR T cells with oncolytic viruses together, and now we’re designing our own oncolytic viruses to use with NK cells. Cancer has a wound healing phenotype that doesn’t usually attract NK cells. Instead, tumors tend to collect macrophages, then reprogram them to make the tumor worse. They also collect T regulatory cells, which can create a barrier to successful immunotherapy. But if you can infect the tumor with an oncolytic virus, you’ve created the perfect context to attract CAR T, or CAR NK cells, maximizing exposure to the target tissue. Our final angle is radiotherapy. Radiotherapy kills cancer cells, but it also causes stress and DNA damage in the cells it doesn’t kill. Stress and DNA damage attract NK cells, so we think that that could increase engagement with CAR NK cells. Those are all the things my lab is working on, and we’re also working towards putting a CAR NK cell product in the clinic for glioblastoma in a few years. Q: What are the advantages of NK cells as therapeutics compared to CAR T cells? A: CAR T cells are potentially very powerful cells, because they’re all very highly antigen-specific, but they’re almost too good at doing their job. They produce high levels of cytokines, replicate rapidly and can cause a lot of damage, resulting in potentially life-threatening side effects. The two most common are cytokine release syndrome (when too many cytokines are produced, causing an immune cascade) and immune cell-associated neurotoxicity syndrome (ICANS) (excess central nervous system inflammation and disturbance caused by excessive cytokine production). Unfortunately, this means that CAR T-cell therapy can only be given in tertiary medical centers (of which there are only around 30 approved centers in the US), to an admitted patient with available space in an emergency room. This limits the accessibility and availability of CAR T cell treatments, as does the cost of these treatments, and the fact that the T cells must be taken from the patient’s own body. NK cells provide an alternative in several ways. Since NK cells don’t undergo large clonal expansions and produce a different cytokine profile from T cells, they are less likely to trigger such severe side effects. Additionally, NK cells don’t cause graft-versus-host disease, so they could be made into an allogenic therapeutic. There have been several ongoing projects using CAR NK cells against CD19 that have shown great results, but also show that they can be used as a safe allogenic therapeutic, without causing CRS or ICANS. Therefore they could potentially be given in outpatient centers or community hospitals, at a lower cost, making them far more accessible than CAR T-cell therapies. In addition, T cells are so antigen-specific (either via their own T-cell receptor or a CAR) that if they don’t see their specific antigen, or the cancer mutates, tumor escape can occur. TECHNOLOGYNETWORKS.COM CELL AND GENE THERAPIES: FROM DISCOVERY TO DELIVERY 15 In comparison, NK cells have evolved to recognize dozens of different stress signals, such as the consequences of DNA damage and metabolic stress, which are almost universal in cancer. This potentially gives CAR-NK cells a very broad activity that could help prevent tumor escape. Q: What are some of the challenges involved in developing novel CAR -based therapies? A: One of the shared challenges (though less relevant to NK cells because of their broad activity) is antigen selection, especially for solid tumors. We were lucky with the hematological malignancies, there were obvious targets in the B cell markers. In solid tumors, we’re targeting tumor-associated antigens that are over-expressed on tumors but are still present in healthy tissues. This leads to untargeted off-tumor toxicity, which can limit the dose of cell therapies. We also have to deal with antigenic heterogeneity and antigen escape. Whatever your target antigen, expression won’t be uniform and the likelihood is that a percentage of tumor cells won’t be destroyed and can potentially repopulate. You need to engage multiple antigens to solve this problem, but that also potentially means stacking toxicities. We haven’t seen many CAR NK cells for solid tumors enter clinical testing yet, but one antigen that’s received attention recently is claudin18.2 (CLDN18.2) in pancreatic and esophageal cancers. CLDN18.2 is usually expressed in tight junctions, not really visible to the immune system. Tumors depolarize membranes, so they’re no longer neatly stacked, and CLDN18.2 becomes visible – making it a great antigen to target. However, even this isn’t foolproof; high doses of CLDN18.2 CAR T cells can cause gastric bleeds – an on-target toxicity. We certainly have our work cut out for us to design effective CARs that don’t cause toxicity. We’re trying to make these CARs smarter, but we also need to make sure that once they’re inserted into the target cell, they don’t overload it, or domain swap with each other to cause tonic signaling (low level activation in the absence of target antigen), or cause any secondary malignancies. Q: How do you consider challenges like toxicity when moving your therapies towards a clinical trial? How do you think these safety concerns can be overcome entirely in future therapies? A: There’s a lot that the regulators expect you to do pre-clinically in order to get an investigational drug application approved in the US (or a clinical trial application in the UK and elsewhere). One very important thing is to screen your CAR for off-target toxicity, to make sure it isn’t going to cross react with a second tissue or antigen and cause damage. The clinical trial design provides a lot of levers to control safety – for example, starting with a low dose of CAR without lymphodepletion, with staggered administration and dose escalation. Lymphodepletion is usually needed to give physical space for your CAR cells to occupy, and to encourage the body to return to homeostatic immune cell levels by producing cytokines that encourage immune cell expansion. Without lymphodepletion, expansion of CAR T or CAR NK cells is very limited, reducing the chance of damaging side effects or toxicities. It’s also important to have treatment for toxicities readily available to mitigate any issues. Overall, patients are very carefully managed during trials. One potential way of overcoming safety issues like toxicity is to design solutions into the cell therapy. CARs can be designed with on- or off-switches that can be manipulated using small molecule drugs or monoclonal antibodies. Kill-switches are also possible, which – when the controlling small molecule is administered – will wipe out any CAR cells. However, there is then no chance of patient benefit then because the cell therapy is eliminated. It's much better to have safety by design, rather than an emergency off-button. There are a lot of options for safety management, but some of them are more precise than others. If our goal is to make these therapies accessible in outpatient clinics, we need something that is very smart, very safe and hopefully manages itself, without the need for a lot of expert physicians to control it. TECHNOLOGYNETWORKS.COM CELL AND GENE THERAPIES: FROM DISCOVERY TO DELIVERY 16 Q: What does the future hold for CAR therapeutics? A: Like any class of drugs, CAR therapies don’t have a right to exist, they could be swept away by future innovations. We’ll always do what's simple and what works best, and some things are just too complicated. Recently, we've seen a big pullback from gene therapy, despite the curative promises, because the drugs were prohibitively expensive. So, we need to fix the financial toxicity of CAR cells as well as the medical toxicities. One of the most exciting things happening at the moment is programming the patient’s own cells in vivo, by delivering CARs using viral vectors or mRNA and lipid nanoparticles. There are already multiple trials in progress that are looking very promising for B cell malignancies. For successful use of CAR therapries against solid tumors we need to really work on improving the cell chassis – like we’re doing in our lab – to make the immune cells more robust, more resilient to the immunosuppressive tumor microenvironment. This would be ex vivo cell therapy, but it could still be allogenic and off-the-shelf, which would greatly improve the cost and the safety. There are still some challenges to overcome, but I really see in vivo CAR therapeutics as the future. Engineered, gene-edited therapies for solid tumors are going to be necessary in order to get the kind of successful, longterm results we need. ABOUT THE INTERVIEWEE Neil C. Sheppard, D.Phil. (Oxon), leads a lab focused on natural killer (NK) cell biology and cancer therapies at the University of Pennsylvania’s Center for Cellular Immunotherapies (CCI). Educated in the UK at the Universities of Bristol and Oxford, Neil has over 20 years of international experience with immunotherapies and vaccines in academia and industry. He has led programs, team, and labs in the UK, Australia, California and Pennsylvania. He has advanced multiple CAR-T and TCR-T programs through successful INDs into Ph1 FTIH or Ph1b combination studies. Neil was commended by SITC in 2020 and Marquis Who's Who in 2022 for Innovation in Immunotherapy. 17 CELL AND GENE THERAPIES Credit: iStock/Suriphon Singha Mass Spectrometry Imaging in Pharmaceutical Development Joanna Owens, PhD Mass spectrometry imaging (MSI) technology has existed in simple form for decades, but recent advances in MS sensitivity and data analysis means it’s finally coming of age.1 In this article, we explore how MSI is evolving into a high-resolution spatial biology toolset to transform the traditional model of drug discovery and development. The evolving role of MS in drug development MS technologies play a crucial role in drug discovery and development, serving as effective tools for the swift identification and quantification of complex molecules.2 Modern MS methods have greatly enhanced the ability to analyze low levels of potential drug molecules – from oligonucleotides, to peptides, proteins and small molecules, and are now used across the drug development pipeline from target discovery, compound screening, and toxicity and quality control testing2, as outlined in Table 1. Many of these applications have traditionally involved bulk analysis of cells or tissues, but advances in MSI technologies mean it’s now possible to carry out spatial chemical analysis at the single-cell level. This advancement is paving the way for novel applications in drug discovery and development. What is MS imaging? “Mass spectrometry imaging is a way to look at metabolism in a spatial manner, which gives you much more information than just analyzing the bulk tissue,” explained Professor Brent Stockwell of Columbia University, USA. “Traditionally, you would administer a drug to an animal, analyze the bulk liver tissue and get a concentration of the drug at a particular timepoint, and TECHNOLOGYNETWORKS.COM CELL AND GENE THERAPIES: FROM DISCOVERY TO DELIVERY 18 this gives you useful information, but this cannot tell you which cells the drug accumulates in and what the impact of the drug was in terms of other metabolites and measures of the cell state.” MSI achieves this by chemically analyzing snapshots, called a pixel, at a single point within a tissue. For each pixel, molecules are extracted and introduced into the ionization source of the MS instrument, which separates every molecule by its mass-to-charge ratio and determines their relative abundance in the sample. From this, you can visualize the abundance of different molecules across the tissue. “There’s an inherent trade-off in the MS imaging approach: if you use a bigger pixel size, you cover more tissue but you get less spatial resolution to be able to see how patterns vary,” said Stockwell. “However, more sensitive MS instruments together with better ionization methods are making it possible to reliably detect minute amounts of sample. This, combined with improved software for data analysis and annotation, is expanding the uses for MS imaging.” The evolution of MS imaging MSI was first developed more than 50 years ago using secondary ion mass spectrometry (SIMS), and the most widely used MSI method today is MALDI.1 But despite its widespread use, MALDI-based MSI is not without its limitations – the fragmentation of biological molecules and careful consideration of the matrix for each study are a few examples.1 Other ionization methods have been used for MS imaging, such as desorption electrospray ionization (DESI) and nanoDESI which make it possible to analyze non-volatile molecules Table 1: Applications of mass spectrometry imaging across different stages of R&D. Stage of drug R&D Example applications Discovery ∙ MS-based proteomics and metabolomics on clinical tissue samples are used for target identification/validation and to gain insights into a drug’s mechanism of action.3 ∙ MS plays a pivotal role in determining the structure and characteristics of potential drug compounds, many of which are combinatorial-chemistry synthesis products.2,3 ∙ MS has increasingly become the method of choice in high-throughput or ultra-highthroughput screening.3,4 Development ∙ In drug development, liquid chromatography-mass spectrometry (LC-MS) is an essential workhorse technology used to identify and characterize the active drug and its metabolites in different tissue samples to establish the absorption, distribution, metabolism and excretion (ADME) profile of a drug candidate.5,6,7 ∙ MS is also important for identifying, monitoring and validating biomarkers throughout drug development.8 Quality control ∙ MS technologies are critical tools for ensuring the quality of traditional smallmolecule drugs during formulation and manufacturing. ∙ MS is increasingly used to confirm the identity, biophysical properties and purity of biopharmaceuticals such as monoclonal antibodies.9,10 TECHNOLOGYNETWORKS.COM CELL AND GENE THERAPIES: FROM DISCOVERY TO DELIVERY 19 without fragmentation.1 Still, it was not until recently that the technology matured to a point that allows for high-impact studies. “One downside to conventional MALDI methods is the chemical preparation you need to use to be able to detect certain molecules,” explained Professor David Muddiman, of North Carolina State University, USA. Muddiman has spent nearly two decades developing a less destructive ionization approach called matrix-assisted laser desorption electrospray ionization (MALDESI) which makes it possible to directly analyze a diverse range of biological molecules without requiring chemical derivatization in a tissue.1 “For example, neurotransmitters are tiny molecules that the matrix in MALDI interferes with, so you need to treat them with a reagent to bring them into a higher mass range. With MALDESI-based MS imaging, you can analyze the endogenous neurotransmitters in the tissue without any pre-treatment.” The same applies to glycans – carbohydrate groups added co- and post-translationally to proteins that play crucial roles in biology - which are easily fragmented during conventional MALDI-MS. “If you look in the literature at MS imaging using MALDI, there are very few instances where analysis has been able to identify sialic acids – terminal monosaccharides on carbohydrates – and there’s no method to recover that information through bioinformatics. This is because there’s no way of knowing what the original molecule in the sample was. With our method, we are reading back authentically the biology that’s been presented to us.” MS imaging opportunities in drug development The ability to track the single-cell pharmacokinetics and pharmacodynamics of a drug using MS imaging has been coined by Stockwell as “spatial pharmacology”.11 “Spatial biology methods such as spatial transcriptomics or proteomics can give you information about the cell types and cell communities and the state of individual cells,” he explained. “But right now, there’s a layer of information you can’t get directly from those methods – and that’s the small-molecule products of those metabolic reactions. But with MS imaging you can see the abundance of those metabolites across the tissue in a spatially defined manner.” In the long term, there could be a potential application for MSI in late-stage drug development, where MALDI-based MS is currently the gold standard for characterizing the distribution of drug candidates in development. In the short term, there is a clear role for MSI in the drug discovery pipeline. “I think the demand right now for MS imaging is in looking at disease models versus healthy normal tissues,” noted Stockwell. “It can help to understand disease mechanisms, find new targets and validate drug candidates from screening at an early stage.” As the technology becomes automated and higher throughput, it becomes a more feasible choice for use in In the long term, there could be a potential application for MSI in late-stage drug development, where MALDI-based MS is currently the gold standard for characterizing the distribution of drug candidates in development. TECHNOLOGYNETWORKS.COM CELL AND GENE THERAPIES: FROM DISCOVERY TO DELIVERY 20 drug screening. “Scientists in biopharma have adapted IR-MALDESI to measure around 22 wells per second for screens involving a simple enzymatic metabolic readout12, and around 1-3 wells per second for more complex high-content screens,” said Muddiman. “It’s potentially turning the drug discovery pipeline on its head – by starting with the experiment that really matters – the phenotype of a drug candidate, whether it hits its target and identifying any off-target effects.” Only in the past few years has it become possible to look at single-cell resolution using MSI and it’s still a relatively specialized field. “As companies start to adopt these latest advances that give single-cell resolution, they will gain more useful information and find new applications that will be commonly implemented into their workflows,” said Stockwell. “The cell is the fundamental unit of biology, it’s the building block of organisms. If you can’t see the individual cells, you’re always going to be somewhat in the dark.” REFERENCES 1. Caleb Bagley M, Garrard KP, Muddiman DC. The development and application of matrix assisted laser desorption electrospray ionization: The teenage years. Mass Spectrom Rev. 2023;42(1):35- 66. doi: 10.1002/mas.21696 2. Michnowicz J. Mass spectrometry in drug discovery and development. Nat Rev Drug Discov. 2002;1(8):651. doi: 10.1038/ nrd886 3. Dueñas ME, Peltier-Heap RE, Leveridge M, Annan RS, Büttner FH, Trost M. Advances in high-throughput mass spectrometry in drug discovery. EMBO Mol Med. 2023;15(1):e14850. doi: 10.15252/ emmm.202114850 4. Williams JD, Pu F, Sawicki JW, Elsen NL. Ultra-high-throughput mass spectrometry in drug discovery: fundamentals and recent advances. Expert Opin Drug Discov. 2024;19(3):291-301. doi: 10.1080/17460441.2023.2293153 5. Chu I, Nomeir AA. Utility of mass spectrometry for in-vitro ADME assays. Curr Drug Metab. 2006;7(5):467-477. doi: 10.2174/138920006777697954 6. Ahire D, Kruger L, Sharma S, Mettu VS, Basit A, Prasad B. Quantitative proteomics in translational absorption, distribution, metabolism, and excretion and precision medicine. Pharmacol Rev. 2022;74(3):769-796. doi:10.1124/pharmrev.121.000449 7. Santiago BG, Eisennagel SH, Peckham GE, et al. Perspective on high-throughput bioanalysis to support in vitro assays in early drug discovery. Bioanalysis. 2023;15(3):177-191. doi: 10.4155/bio- 2022-0207 8. Robinson MR, Miller RA, Spellman DS. Mass spectrometrybased biomarkers in drug development. Adv Exp Med Biol. 2019;1140:435-449. doi: 10.1007/978-3-030-15950-4_25 9. Deschamps E, Calabrese V, Schmitz I, Hubert-Roux M, Castagnos D, Afonso C. Advances in ultra-high-resolution mass spectrometry for pharmaceutical analysis. Molecules. 2023;28(5):2061. doi: 10.3390/molecules28052061 10. Zhang H, Cui W, Gross ML. Mass spectrometry for the biophysical characterization of therapeutic monoclonal antibodies. FEBS Lett. 2014;588(2):308-317. doi: 10.1016/j.febslet.2013.11.027 11. Rajbhandari P, Neelakantan TV, Hosny N, Stockwell BR. Spatial pharmacology using mass spectrometry imaging. Trends Pharmacol Sci. 2024;45(1):67-80. doi: 10.1016/j.tips.2023.11.003 12. Radosevich AJ, Pu F, Chang-Yen D, et al. Ultra-high-throughput ambient MS: Direct analysis at 22 samples per second by infrared matrix-assisted laser desorption electrospray ionization mass spectrometry. Anal Chem. 2022;94(12):4913-4918. doi: 10.1021/acs. analchem.1c04605 MEET THE INTERVIEWEES Brent R. Stockwell is chair of the department of biological sciences and a professor at Columbia University in the departments of biological science and chemistry. His research involves the discovery of small molecules that can be used to understand and treat cancer and neurodegeneration, with a focus on biochemical mechanisms governing cell death. Dave Muddiman is the Jacob and Betty Belin Distinguished Professor of Chemistry at NC State University. His group focuses on the development of innovative mass spectrometry measurements to solve important biological problems. Cell and Gene Therapy Manufacturing Getting Products to Patients Source material acquisition The quality of the initial material fundamentally impacts the entire manufacturing process and final product efficacy. Samples must be handled with care, preserved and tracked to ensure strict quality control. Genetic modification/editing Once the source material arrives at the manufacturing facility, genetic modification begins. Quality control at this stage involves multiple checks to verify the accuracy of genetic modifications and absence of off-target effects. Cell expansion & culture The cell expansion phase transforms small populations of modified cells into therapeutic quantities. This scaling process takes place in specialized bioreactors that precisely control temperature, pH, oxygen levels and nutrient concentrations. Purification & quality control Therapeutic cells must be separated from process‑related impurities while maintaining cell viability and functionality. This stage employs a combination of advanced separation technologies, including magnetic-activated cell sorting (MACS), fluorescence-activated cell sorting (FACS) and tangential flow filtration. Final product preparation The final stage focuses on preparing the therapy for patient administration, requiring careful consideration of product stability and delivery requirements. 1 2 3 4 5 A multi-step manufacturing process The CGT manufacturing process combines cutting-edge biotechnology with rigorous quality controls. Each step presents unique challenges and opportunities for innovation. Click here to view the full infographic 22 CELL AND GENE THERAPIES Credit: iStock/luismmolina Current Challenges in Cell Line Development for Therapeutics Kerry Taylor-Smith Cell lines are a crucial tool in scientific research; without them, many experiments would be impossible. Cell lines are “a culture of mammalian cells that can grow indefinitely in certain cultivation conditions in a laboratory setting, and retain a distinct phenotype and function, and can be stable over many population doublings,” explains Dr. Paula Meleady, associate professor in the School of Biotechnology at Dublin City University. Numerous cell lines have been derived over the past 70 years and are used extensively in biomedical research to help scientists understand many fundamental biological processes. “For example, a cancer cell line is derived from cancer cells from a specific tissue (e.g., lung, breast) and is used in research to study the biology of cancer and to evaluate drugs used in cancer treatments,” Meleady adds. This article explores some of the challenges in developing cell lines for therapeutics, such as stability of the cell line, and how they are being addressed. A vital part of the drug development process Cell lines are essential to drug development, allowing researchers to test the efficacy of their therapeutics in vitro before moving onto in vivo studies. Cell lines are used to test drug metabolism and cytotoxicity, study gene function and to generate a wide range of biological medicines, from proteins such as antibodies, recombinant protein products like insulin, to vaccines.1 Cell lines derive from a single original cell and are genetically identical, so they “offer a more uniform product, meaning that every molecule in a therapy is the same; or as close as can be,” explains Dr. James Budge, TECHNOLOGYNETWORKS.COM CELL AND GENE THERAPIES: FROM DISCOVERY TO DELIVERY 23 a postdoctoral research fellow with a focus on Chinese Hamster Ovary (CHO) cell lines at the University of Kent. “The biology of each individual cell can have an impact on the product’s biotherapeutic properties such as the glycan profile, which refers to sugars added to the molecule in the cell which work to prevent recognition by a patient’s immune system and improve its stability and half-life post administration.” “Over the past 30 years, the CHO cell line has been used for the production of biotherapeutics, particularly monoclonal antibodies,” says Meleady. “CHO cells are the most commonly used host cell line for biotherapeutic production, with over 70% of biopharmaceuticals produced from these cells.” “CHO cells were initially used to study molecular cell genetics; the reduced number of chromosomes compared to human cells was an appealing property for these studies,” adds Budge. These cells can be cultured for long periods and showed robust genetic stability, while also allowing for genetic manipulation. “It is this robustness, ease of culture and the ability to produce complex protein-based materials with humanlike post-translational modifications which have seen these cells become the dominant host for production of biotherapeutic materials,” he adds. Stability of cell lines The production of high amounts of biotherapeutics relies on the generation of stable cell lines. “Cell line development begins with the transfection of a suitable host cell line with the gene of interest, leading to the random integration of DNA from the gene of interest into the host genome,” explains Meleady. “The engineered cell line will start to grow and produce the product of interest, e.g., monoclonal antibody biotherapeutic, usually under selection pressure.” “Stability of cell lines presents a major challenge,” says Budge. Cell lines need to display and maintain functional features as similar as possible to primary cells, but those which have undergone many passages may harbor spontaneous mutations that affect their phenotype, native functions and responsiveness to stimuli, which could lead to unwanted variations in the end product.1 “Phenotypic instability is a very common challenge in biopharmaceutical production and is due to the ‘plasticity’ of CHO cells, which have a high propensity for genomic rearrangements, such as deletions or translocations,” explains Meleady. “This can be a source of cell line instability during the bioproduction process, resulting in the cells growing slower, a reduction in viability and a fall-off in productivity of the biotherapeutic of interest.” There is a lot of research focusing on trying to find solutions to this problem, Meleady adds. “This is required to ensure stable, long-term production of the biotherapeutic of interest, to ensure bioprocess consistency and to assure acceptable product quality for the regulatory authorities so that the product is of the highest quality and safe for patient administration.” Targeted integration techniques, including transponbased technologies, which “enable the knock-in of recombinant protein coding genes into well-defined and transcriptionally active genomic sites is an active area of research at the moment,” adds Meleady. Genetic engineering may minimize the risk of instability by knocking in or out specific genes. The production of high amounts of biotherapeutics relies on the generation of stable cell lines. TECHNOLOGYNETWORKS.COM CELL AND GENE THERAPIES: FROM DISCOVERY TO DELIVERY 24 “Gene editing techniques could be used to modify the genetic landscape of the host cell to ensure stable and consistent expression of the desired genes, for example, through the use of CRISPR-based genome editing tools,” explains Meleady. “This technology offers new opportunities to engineer the host cell line to produce proteins with specific and desirable characteristics, such as high cell densities, high viability, high productivity and highest product quality.” “For example, knocking out apoptosis-related genes has the potential to increase cell survival and culture longevity,” Meleady continues. “Knocking out host cell proteins has the potential to eliminate the production of proteins naturally produced by the host cell line that could co-purify with the desired product. By knocking out genes encoding these host cell proteins, the risk of contamination can be reduced, simplifying downstream processing and improving product purity.” Single cell cloning and monoclonality “One challenge surrounding the development of cell lines is cloning out individual cells and selecting the one with the desired properties,” says Budge. “Scientists need to ensure that the cell line is generating high titers but also producing molecules with the best product quality to meet its therapeutic potential.” Prior to cloning, cells are incubated individually and screened for the desired characteristics. Clones can be isolated using limiting dilution cloning, which is straightforward but time-consuming, with months spent obtaining results.2 Automation can speed up clone development and colony screening, reduces the chances of contamination and can be a useful in ensuring stability and product quality. “Another challenge is that regulatory authorities require substantial documentation of the methods used during cell line development, to ensure clonality which is crucial for product quality and safety,” says Meleady. “A monoclonal cell line originates from one parent cell, to try to eliminate the risk of genetic heterogeneity that could affect the characteristics of the protein being produced.” Growing cells can be subjected to genetic drifts, mutations or loss of a plasmid. By testing and documenting cells on day zero, scientists can prove the cell line came from a single cell, thus minimizing risk and ensuring a consistent production process and product quality. Emerging techniques include fluorescenceactivated cell sorting (FACS), the sorting of cells based on fluorescent characteristics; and microfluidic dropbased single-cell printers with the ability to image newly-plated wells containing single cells. 3,4 Productivity “The majority of significant improvements over recent years has been the ability to achieve higher cell densities in culture,” explains Budge. “More often than not, more cells mean more product, and improvements to culture media and feeding strategies have been central to this. Advancements in perfusion culture techniques and continuous culturing has also played a key role more recently.” Budge has had success in improving product titers by manipulating the lipid content of CHO cell membranes; “By overexpressing certain lipid modifying genes, Cell lines are vital to the drug development process and without them, we wouldn’t be able to test the efficacy of many important medicines. TECHNOLOGYNETWORKS.COM CELL AND GENE THERAPIES: FROM DISCOVERY TO DELIVERY 25 we’ve been able to change cellular properties such as membrane fluidity and/or endoplasmic reticulum size, which improves the capacity of our CHO hosts to produce biotherapeutic material.” “More recently we have been working on transposon ‘jumping gene’ technology, which is now commonly used in industry to produce cell pools,” says Budge. “These technologies allow you to ‘cut and paste’ DNA cargo into a host cell genome and ultimately can reduce the time taken to generate cell pools (leading to quicker cell line selection) and increase product yields by increasing the frequency of integration events at desired positions in the CHO genome. Furthermore, since the cell pools produced using transposons have higher titers, an increased percentage of ‘high expressers’ are present in a pool. This can reduce the requirement for intense selection processes.” Quality control Product quality is of key importance for the production of biotherapeutics, as Meleady explains: “Correct posttranslational modifications (PTMs), especially glycan structures, are crucial for the potency and control of pharmacokinetic and pharmacodynamic properties of biotherapeutics. Heterogeneity of N-glycans can be an issue during cell line development and bioproduction. This heterogeneity, which can arise due to the variability of N-glycan processing (and often linked to the ‘plasticity’ of CHO cells), may compromise the activity and safety of biotherapeutics, particularly monoclonal antibodies.” Meleady says control measures include the establishment and confirmation of clonality to ensure the stability of the host cell line. “There is also extensive stability testing carried out during cell line development, including monitoring the product for any changes to sequence and PTM modifications (e.g., glycosylation), i.e., using extensive quality control analytics.” Artificial intelligence (AI) and machine learning will also have an important role going forward in cell line development, Meleady believes. AI tools can automate cell culture experiments from start to finish, while AIpowered software could generate 2D and 3D cellular models and supervise automated feeding and media changing, for example. This could help researchers discover novel drug targets and investigate toxicity more quickly. Machine learning can make protocol development more efficient, flexible and powerful; their algorithms can prompt decision-making and allow for real time customization. It also allows for fully automated workflows to generate reproducible results across multiple experiments, expediting milestones and improving confidence in results. Other challenges Cell lines are cost effective, easy to grow and provide an unlimited supply of material, but they don’t truly represent how cells operate and interact within the body, particularly when used in biomedical research and drug development. The two-dimensional nature of cell culture systems means they lack some aspects of life within an organism, such as contact with tissue surfaces. Threedimensional biology such as organoids or spheroids grown in a scaffold would better represent the in vivo environment and give more physiologically relevant information, but these cultures can be inconsistent as there is no standardized way to grow them.1,5 Potential contamination is also a concern; some are obvious, like bacterial overgrowth, while others, such as microbial mycoplasma, are less so. Mycoplasma contamination can remain undetected in cell lines for long periods of time, highlighting the importance of scientists testing their cell lines frequently.1 “CHO and human embryonic kidney cells have been the workhorses behind the production of complex, multichain and multidomain molecules such as monoclonal antibodies and the bioprocesses and technologies surrounding them have been exquisitely optimized to produce such molecules,” Budge explains. “Newer molecules such as bispecific antibodies present new challenges, and the tried and tested bioprocesses require some redesign to tackle these.” TECHNOLOGYNETWORKS.COM CELL AND GENE THERAPIES: FROM DISCOVERY TO DELIVERY 26 These more complex therapeutic proteins can be used to increase the frequency of correct pairings and therefore the yield of the required protein but can be difficult to express in CHO cells. Cell lines are vital to the drug development process and without them, we wouldn’t be able to test the efficacy of many important medicines. But developing cell lines isn’t without challenges. Over the years, much effort has gone into improving and streamlining the process to improve the stability, purity and quality of cell lines. Technology is increasingly being used to automate processes and improve decision making, saving time and improving productivity – and it’s likely its use will increase, particularly as new molecules are developed. REFERENCES 1. Kaur G, Dufour JM. Cell lines. Spermatogenesis. 2012;2(1):1-5. doi: 10.4161/spmg.19885 2. Ye M, Wilhelm M, Gentschev I, Szalay A. a modified limiting dilution method for monoclonal stable cell line selection using a real-time fluorescence imaging system: a practical workflow and advanced applications. Methods Protoc. 2021;4(1):16. doi:10.3390/ mps4010016 3. Yeh CF, Lin CH, Chang HC, Tang CY, Lai PT, Hsu CH. A microfluidic single-cell cloning (SCC) device for the generation of monoclonal cells. Cells. 2020;9(6):1482. doi:10.3390/cells9061482 4. Liao X, Makris M, Luo XM. Fluorescence-activated cell sorting for purification of plasmacytoid dendritic cells from the mouse bone marrow. J Vis Exp. 2016;(117):54641. doi: 10.3791/54641 5. Cacciamali A, Villa R, Dotti S. 3D cell cultures: Evolution of an ancient tool for new applications. Front Physiol. 2022;13:836480. doi: 10.3389/fphys.2022.836480 MEET THE INTERVIEWEES: Dr. Paula Meleady is an associate professor in the School of Biotechnology at Dublin City University. Her research focuses on the use of proteomic and mass spectrometry technologies to characterize recombinant mammalian cells to gain insights to improving productivity and quality of biopharmaceuticals. Dr. James Budge is a postdoctoral research fellow at the University of Kent. His research focuses on engineering Chinese Hamster Ovary (CHO) cell lines and the development of the processes used to manipulate them for improved production of biotherapeutic materials. 27 CELL AND GENE THERAPIES Credit: iStock/Toshe_O New Purification Techniques in Biopharmaceuticals Aron Gyorgypal, PhD Biotherapeutics are produced using a multitude of technologies and vary depending on the indication. Monoclonal antibodies (mAbs) have been on the market for decades and have successfully treated cancers and autoimmune diseases. The use of viral vectors as gene therapies has recently gained attraction through new techniques, allowing the treatment of genetic disorders, infectious diseases and cancers. The use of new vaccine technology, such as messenger RNA (mRNA), has allowed the production of potent immune responses to mitigate infections, as seen with the COVID-19 pandemic. Purification methodologies between different biotherapeutics vary drastically and, depending on the product, require a nuanced approach to enable or more efficiently allow for the purification of the product after upstream production. New technology and workflows are looking to enable the more efficient or cheaper purification of these products for downstream processing. Purification and processing of mAbs The purification and downstream processing of mAbs is now relatively mature. "The platform technology is quite developed for mAb manufacturing. After production of the antibody by cell culture, the supernatant is captured by protein A chromatography and held at low pH for activation. Then follows a series of polishing steps through cation exchange or ionic exchange before the formulation of the final product," said Dr. Lukas Gerstweiler, lecturer in bioprocess engineering at the University of Adelaide, Australia. "I think the major challenges of today are to integrate the processes to TECHNOLOGYNETWORKS.COM CELL AND GENE THERAPIES: FROM DISCOVERY TO DELIVERY 28 work continuously as well as maybe finding ways to bring down the costs for production." Continuous bioprocessing poses unique challenges for antibody purification as a truly integrated line between upstream and downstream processing is still being investigated. "The past couple of years have seen the use of rapid cycling technology, which bolsters productivity," said Gerstweiler. Indeed, the use of protein A membrane adsorbers, however, has shown promise as an alternative to resin for process intensification. These absorbers eliminate the need for column packing, which lowers equipment cost, decreases fouling, pressure drops, challenging and stationary phase compression and has even been shown to decrease host cell protein contaminants.1,2 However, it does come at the cost of increased buffer volumes. However, this technology can help move mAb processing from batch-based purification to semicontinuous purification. Finding methodologies to decrease purification costs would also be helpful for less developed countries or companies looking to decrease the costs associated with downstream purification. One option is through precipitation in a coiled flow inverter reactor, leading to mAb capture by cation exchange multimodal chromatography that then polishes the protein in an ion exchange membrane.3,4 While mAb-based downstream processing has been around for decades and the purification modalities have matured, there is still a need for a better understanding of the processes to enable continuous processing and the nuances within the process to allow for better manufacturing economics. Viral vectors for gene therapy Downstream purification of viral vectors poses tough challenges due to technology limitations. While many different practices exist for their purification, they suffer from drawbacks such as poor scalability or inefficiencies in early purification steps, which make them costprohibitive due to yield losses.5 "The current state of the art for viral vector purification starts with density gradient centrifugation. The advantage of doing this is that not only can you purify your viral vector from everything else in your cell culture, but you can also separate empty and full capsids," said Dr. Caryn Heldt, director of the Health Research Institute, the James and Lorna Mack Chair in Continuous Processing and professor in the Department of Chemical Engineering at Michigan Technological University. "But the issue with this is that it is not feasible for commercial scale as it requires scaling out, meaning that more ultracentrifuges would be required to increase throughput. So, what can be done? Well, another approach is through chromatography, which works well on a lab scale but on the commercial scale, besides costs, it can cause issues as these chromatography columns produce large pressure drops which can affect purification." However, there are new advances in purification technology that could alter the current purification paradigm. "The vast majority of the newer techniques are now in chromatography. A major focus is on new ligand development and resin design," said Heldt. Affinity chromatography has not yet been developed for large-scale manufacturing. There currently exists only a handful of targeted affinity resins that are specific for only one type of virus serotype. To make the resins economically feasible, they must be reusable and bind to multiple viral serotypes. Some proprietary resins are currently being investigated and found to bind to multiple adeno-associated virus (AAV) serotypes.6 However, they still struggle with the copurification of empty capsids along with the product. A recent approach being investigated by multiple groups is to functionalize peptides to chromatography columns to allow for universal binding of virus serotypes, namely "serotype-agnostic" resins.7 The production of these protein ligands is based on mimicking the anti-AAV antibody A20 by abstracting target peptide sequences that target regions of the capsid, which are highly conserved across stereotypes of various clades.8 These peptide-ligands have comparable binding capacity with commercial adsorbents but also reduce host cell protein TECHNOLOGYNETWORKS.COM CELL AND GENE THERAPIES: FROM DISCOVERY TO DELIVERY 29 contaminants and increase stability as they allow for AAV elution at physiological pHs instead of acidic pHs.9 Where is the future of viral vector gene therapy going? "The two big things I would like to see in the future are continuous manufacturing and a better understanding of the viral vector when it is loaded with gene versus when it is not," said Heldt. Indeed, understanding the process in which these capsids bind to chromatography columns between full and empty columns would allow for more rational engineering of materials that would bias against the empty capsid products, whether this is done by engineering the resin material or the capsid itself. Purification technology for mRNA vaccines mRNA-based biotherapeutics are emerging as a promising technology for vaccine development. On both laboratory and manufacturing scale production, the use of chromatographic separation has been selected as a primary technique for purification based on selectivity, adaptability and scalability.10 The shift from more conventional protein-based therapeutic products towards a nucleic acid-based platform brings other challenges towards purification as the physiological properties, structure and even impurity composition vary drastically, requiring a selection of nuanced chromatographic resins. This also accounts for large inefficiencies that are present today in the downstream processing of mRNA vaccines.11 Such inefficiencies are due to limitations in the stationary phases used to purify the therapeutic. Current methodologies rely on the use of proprietary polymer resin materials and the use of monolithic columns, typically based on size, charge, hydrophilicity and affinity.12 As mRNA is negatively charged, anion exchange chromatography is typically utilized to purify mRNA from impurities. The selectivity on the column is based on molecular weight and sequence and is prone to produce aggregates, which can be mitigated with denaturing agents or organic reagents – although this is not favorable for large-scale manufacture due to safety concerns. The approach to limit this concern is through multimodal ion exchange/hydrogen bonding chromatography. An approach by modifying a monolithic column, can enable a high yield of mRNA from in vitro transcription products, enabling purification independent of construct size or the poly-A tail of the vaccine.13 Affinity chromatography is another attractive option for purification. The use of oligo-dT affinity chromatography can capture the poly-A tail of mRNA transition through A-T pairing. This can effectively remove impurities such as the DNA template, nucleotide substrates, enzymes and buffer components. The immobilization of oligo-dT to an electrospun polymer nanofiber adsorbent has been shown to increase both the yield and allow for higher flow rates to decrease processing time.14 The largest criticism of oligo-dT-based chromatography is that it cannot distinguish single- and double-stranded RNA. Nevertheless, this technology has been used for the purification of SARS-CoV-2 mRNA.15 In reality, the processing of mRNA may require the use of both chromatography and membrane filtration. Membrane systems may be investigated further to enable continuous processing in the future.16 The processing of mRNA-based therapeutics is still inefficient, and a better understanding of the impurities and separation modalities is required for more efficient purification technology. This could be through size exclusion, ion exchange, hydrophilic interaction, affinity interactions or another modality. REFERENCES: 1. Schmitz F, Minceva M, Kampmann M. Comparison of batch and continuous multi-column capture of monoclonal antibodies with convective diffusive membrane adsorbers. J Chromatogr A. 2024;1732:465201. doi:10.1016/j.chroma.2024.465201 2. Trnovec H, Doles T, Hribar G, Furlan N, Podgornik A. Characterization of membrane adsorbers used for impurity removal during the continuous purification of monoclonal antibodies. J Chromatogr A. 2020;1609. doi:10.1016/j. chroma.2019.460518 3. Kateja N, Kumar D, Sethi S, Rathore AS. Non-protein A purification platform for continuous processing of monoclonal antibody therapeutics. J Chromatogr A. 2018;1579:60-72. doi:10.1016/j. chroma.2018.10.031 4. Arakawa T, Tomioka Y, Nakagawa M, et al. Non-Affinity Purification of Antibodies. Antibodies. 2023;12(1). doi:10.3390/antib12010015 TECHNOLOGYNETWORKS.COM CELL AND GENE THERAPIES: FROM DISCOVERY TO DELIVERY 30 5. Singh N, Heldt CL. Challenges in downstream purification of gene therapy viral vectors. Curr Opin Chem Eng. 2022;35. doi:10.1016/j. coche.2021.100780 6. Florea M, Nicolaou F, Pacouret S, et al. High-efficiency purification of divergent AAV serotypes using AAVX affinity chromatography. Mol Ther Methods Clin Dev. 2023;28:146-159. doi:10.1016/j. omtm.2022.12.009 7. Shastry S, Barbieri E, Minzoni A, et al. Serotype-agnostic affinity purification of adeno-associated virus (AAV) via peptidefunctionalized chromatographic resins. J Chromatogr A. 2024;1734. doi:10.1016/j.chroma.2024.465320 8. Shastry S, Chu W, Barbieri E, et al. Rational design and experimental evaluation of peptide ligands for the purification of adeno-associated viruses via affinity chromatography. Biotechnol J. 2024;19(1). doi:10.1002/biot.202300230 9. Mietzsch M, Smith JK, Yu JC, et al. Characterization of AAVSpecific Affinity Ligands: Consequences for Vector Purification and Development Strategies. Mol Ther Methods Clin Dev. 2020;19:362-373. doi:10.1016/j.omtm.2020.10.001 10. Keulen D, Geldhof G, Bussy O Le, Pabst M, Ottens M. Recent advances to accelerate purification process development: A review with a focus on vaccines. J Chromatogr A. 2022;1676. doi:10.1016/j.chroma.2022.463195 11. Rosa SS, Prazeres DMF, Azevedo AM, Marques MPC. mRNA vaccines manufacturing: Challenges and bottlenecks. Vaccine. 2021;39(16):2190-2200. doi:10.1016/j.vaccine.2021.03.038 12. Feng X, Su Z, Cheng Y, Ma G, Zhang S. Messenger RNA chromatographic purification: advances and challenges. J Chromatogr A. 2023;1707. doi:10.1016/j.chroma.2023.464321 13. Megušar P, Miklavčič R, Korenč M, et al. Scalable multimodal weak anion exchange chromatographic purification for stable mRNA drug substance. Electrophoresis. 2023;44(24):1978-1988. doi:10.1002/elps.202300106 14. Dewar EA, Guterstam P, Holland D, et al. Improved mRNA affinity chromatography binding capacity and throughput using an oligodT immobilized electrospun polymer nanofiber adsorbent. J Chromatogr A. 2024;1717. doi:10.1016/j.chroma.2024.464670 15. Corbett KS, Edwards DK, Leist SR, et al. SARS-CoV-2 mRNA vaccine design enabled by prototype pathogen preparedness. Nature. 2020;586(7830):567-571. doi:10.1038/s41586-020-2622-0 16. Javidanbardan A, Messerian KO, Zydney AL. Membrane technology for the purification of RNA and DNA therapeutics. Trends Biotechnol. 2024;42(6):714-727. doi:10.1016/j. tibtech.2023.11.016 MEET THE INTERVIEWEES Lukas Gerstweiler is a lecturer in Bioprocess Engineering at the School of Chemical Engineering, University of Adelaide. His research interests are within improving biopharmaceutical processes using continuous manufacturing and model-based process optimization. Caryn Heldt is the Director of the Health Research Institute, the James and Lorna Mack Chair in Continuous Processing, Professor in the Department of Chemical Engineering, and an Affiliate Professor in Biological Sciences at Michigan Technological University. Her lab is focused on the purification, removal, inactivation and stabilization of viruses and gene therapy vectors. 31 CELL AND GENE THERAPIES Credit: iStock/ nopparit A Bright Future for RNA Therapeutics RJ Mackenzie The COVID-19 pandemic changed our society, and the technology that powers it, in an instant. Suddenly, remote working, telehealth and online learning boomed. Other changes took longer to set in – governments are only starting to appreciate the impact of a surge in chronic health conditions. One rapid change was in RNA biology – developing mRNA vaccines against COVID-19 helped us beat the virus into retreat faster than many could have hoped. The advances in RNA biology since the pandemic could have an enduring impact as well. They promise rapid, personalized and flexible treatments against multiple diseases. But these changes will only be realized if the technology can overcome new hurdles. In this article, we will explore whether RNA therapeutics can realize their full potential. If they do, their impact against COVID-19 will be only an overture. RNA therapeutics: a promising technology RNA therapeutics exploit the nucleic acid’s position as a middleman in the central dogma of biology. RNA is required to turn genetic DNA instructions into functional proteins. By introducing custom RNA instructions, incorrect DNA blueprints can be overwritten and new protein products can be generated. Classical drug development techniques that produce small-molecule therapeutics are time-consuming processes. The molecules’ surface structure needs to match their intended target receptors carefully. Additionally, only a tiny fraction of the human genome codes for small-molecule drug targets.1 What gives RNA therapeutics an edge is that their base ingredients are so well understood. “We know exactly how to build a piece of DNA or RNA to encode whatever we want,” says Brian Brown, director of the Icahn Genomics TECHNOLOGYNETWORKS.COM CELL AND GENE THERAPIES: FROM DISCOVERY TO DELIVERY 32 Institute at Mount Sinai. RNA can target non-coding genes and transcripts, meaning the druggable space for these compounds is vast. Extensive toolboxes for building RNA means new therapeutics can be designed rapidly. These are powerful tools, but they once seemed a scientific dead end. In early experiments, cells exposed to external RNA constructs reacted with a powerful and potentially lethal immune response.2 These issues seemed insurmountable – to the extent that mRNA researcher Katalin Karikó was academically blackballed by the University of Pennsylvania for pursuing her research in the area. Karikó would go on to find an innovative solution to the problem. By swapping out a nucleotide from mRNA’s structure for a modified analog that produced no inflammation, she created a molecule that didn’t set off immune alarms. Karikó would go on to share the 2023 Nobel Prize for Physiology and Medicine for her work. In addition to being a tale about the value of sticking to your guns, Karikó’s discovery was also the breakthrough that helped RNA biology enter a golden age, says John Cooke, medical director of the Center for RNA Therapeutics at Houston Methodist. A silver lining to COVID-19 After this immune roadblock was overcome, the technology blossomed. A variety of RNA therapeutics have since been designed. These include RNA interference (RNAi) molecules, which can block transcription of target genes; antisense oligonucleotides, which interfere with mRNA production; and RNA aptamers, which bind to intra- and extracellular targets with affinity and specificity matching that of antibodies.3,4,5 Alongside mRNA therapeutics, these technologies have been iteratively improved over the 20 years since Karikó cracked the code of safe RNA therapy. But these advancements went unheralded by the wider public until the COVID-19 pandemic. When the time came for the rapid development of a novel vaccine, RNA therapeutics stepped up to the task. Now, the technology is firmly in the public spotlight. This higher profile has turbocharged progress in the field. Of the 17 RNA therapeutics approved by the FDA, 9 have been given the green light in the last 5 years.6 It’s “a silver lining to the COVID crisis,” says Cooke. The new challenge is to maximize RNA therapeutics’ potential. A safe delivery Samir Mitragotri, a professor of bioengineering at Harvard University, says that delivering RNA therapeutics to their intended targets is now the field’s biggest challenge. “These molecules are not super stable,” he adds. “They are susceptible to degradation. They are large compared to other therapeutics.” These factors become even more important when you consider the impact of a misdirected therapeutic. Smallmolecule drugs, like aspirin, will leave the body rapidly, so their effects on off-target tissues are also short-lived. RNA therapeutics, by kickstarting or stopping gene transcription, can have longer durations of action. “The burden of making sure that it goes to the right place is really high,” says Mitragotri. But before they act on their targets, RNA therapeutics have to reach them in the first place. That’s not straightforward. The molecules are at the mercy of proteins called nucleases in the bloodstream that easily break them down. A solution for the problem came from nanoscience. Lipid nanoparticle (LNP) technology isn’t new. The first FDA-approved drug that utilized the technology – the antifungal amphotericin B – is over 30 years old. But it was only in 2018 that the RNAi-based compound patisiran, the first such drug to be encapsulated in LNPs, was approved to treat the neurodegenerative condition transthyretin-related hereditary amyloidosis.7 LNP technology traps therapeutic RNA inside lipid shells. This protects the RNA from nucleases, stabilizes the molecule and helps it pass through lipophilic membranes.8 The technology was used in Pfizer and Moderna’s COVID-19 mRNA vaccines. “I think that the unsung hero, at least of the [mRNA] vaccines, is really this delivery system,” says Brown. TECHNOLOGYNETWORKS.COM CELL AND GENE THERAPIES: FROM DISCOVERY TO DELIVERY 33 Washed away Other innovations have taken different routes toward protecting RNA. One approach is to change the linear structure of therapeutic RNA molecules, instead making them circular, as the nucleases that chew up RNA bind onto the molecule 5’ or 3’ end.9 “If you have a circular RNA, there's no end. So the exonucleases can't gain purchase and the RNA lasts longer,” says Cooke, whose lab has been experimenting with this new structure. Though technologies like LNPs have improved RNA therapeutics’ durability, they can’t bypass other challenges in drug delivery. Mitragotri says the challenge facing RNA–LNP complexes in the bloodstream is similar to that facing someone washed away down a mighty river. The complex’s target is on the blood vessel’s “bank”, but they lack an anchor that will get them close enough to bind. Mitragotri highlights a strategy in which LNPs “hitch a ride” on passing red blood cells, which bump against blood vessel walls when they diffuse oxygen into tissues.10 “We have designed ways to allow the nanoparticles to attach to red blood cells and circulate in the blood for a longer time and be able to target the tissue,” he explains. If these technologies improve RNA therapeutics’ stability and targeting, there could be rapid benefits to the health system. Cooke outlines a new program at Houston Methodist using mRNA vaccines to target cancer. This approach can create new vaccines quickly, and Cooke says that the team intend to develop, generate and administer cancer vaccines within the same hospital – something he thinks is a world first. Cooke hopes that this approach could become commonplace within a decade. “You'll have universitybased programs where you can have these deployable manufacturing units that can generate clinical-grade RNA on a desktop. [...] These desktop apparatuses will generate personalized RNA therapeutics,” he says. Mitragotri is similarly optimistic. He points out that having many different RNA tools – be it siRNA, mRNA, circular constructs or hitchhiking molecules – will be essential if the field is to tackle the grand challenges of human disease. “We need a relatively wide basket of technologies to support that variety of applications,” he concludes. REFERENCES: 1. Falese JP, Donlic A, Hargrove AE. Targeting RNA with small molecules: from fundamental principles towards the clinic. Chem Soc Rev. 2021;50(4):2224-2243. doi: 10.1039/D0CS01261K 2. Kim YK. RNA therapy: rich history, various applications and unlimited future prospects. Exp Mol Med. 2022;54(4):455-465. doi: 10.1038/s12276-022-00757-5 3. Traber GM, Yu AM. RNAi-Based Therapeutics and Novel RNA Bioengineering Technologies. J Pharmacol Exp Ther. 2023;384(1):133-154. doi: 10.1124/jpet.122.001234 4. Rinaldi C, Wood MJA. Antisense oligonucleotides: the next frontier for treatment of neurological disorders. Nat Rev Neurol. 2018;14(1):9-21. doi: 10.1038/nrneurol.2017.148 5. Keefe AD, Pai S, Ellington A. Aptamers as therapeutics. Nat Rev Drug Discov. 2010;9(7):537-550. doi: 10.1038/nrd3141 6. Curreri A, Sankholkar D, Mitragotri S, Zhao Z. RNA therapeutics in the clinic. Bioeng Transl Med. 2022;8(1):e10374. doi: 10.1002/ btm2.10374 7. Akinc A, Maier MA, Manoharan M, et al. The Onpattro story and the clinical translation of nanomedicines containing nucleic acidbased drugs. Nat Nanotechnol. 2019;14(12):1084-1087. doi: 10.1038/ s41565-019-0591-y 8. Jung HN, Lee SY, Lee S, Youn H, Im HJ. Lipid nanoparticles for delivery of RNA therapeutics: Current status and the role of in vivo imaging. Theranostics. 2022;12(17):7509-7531. doi: 10.7150/ thno.77259 9. Liu X, Zhang Y, Zhou S, Dain L, Mei L, Zhu G. Circular RNA: An emerging frontier in RNA therapeutic targets, RNA therapeutics, and mRNA vaccines. J Control Release. 2022;348:84-94. doi: 10.1016/j.jconrel.2022.05.043 10. Brenner J, Mitragotri S, Muzykantov V. Red Blood Cell Hitchhiking: A Novel Approach for Vascular Delivery of Nanocarriers. Annu Rev Biomed Eng. 2021;23:225-248. doi: 10.1146/annurevbioeng- 121219-024239 MEET THE INTERVIEWEES: Dr. Brian Brown is a molecular biologist and immunologist. He is director of the Icahn Genomics Institute (IGI). Dr. Samir Mitragotri is a bioengineer and Hiller Professor of Bioengineering and Hansjörg Wyss Professor of Biologically Inspired Engineering at Harvard University. Dr. John Cooke is director of the Center for Cardiovascular Regeneration and medical director of the Center for RNA Therapeutics at Houston Methodist. 34 CELL AND GENE THERAPIES Credit: iStock/Rafa Jodar Developments in Point‑of‑Care Manufacture of Advanced Therapies Isabel Ely, PhD Advanced therapy medicinal products (ATMPs) represent a frontier in personalized medicine and regenerative healthcare. This innovative medical treatment utilizes gene therapy, somatic-cell therapy or tissue engineering – sometimes designed based on each patient's clinical or even genetic features – aiming for long-lasting or permanent effects to treat disease. Currently, ATMPs are manufactured at a few centralized sites, meaning the starting materials and/or final products are generally frozen for transportation – often resulting in reduced therapy potency. Therefore, the therapy must be delivered to the patient some minutes or seconds after manufacture. Point-of-care (POC) manufacturing aims to overcome this limitation by producing ATMPs in hospitals. This is especially important when it needs to be delivered to the patient without delay and there is no time for storing the medicine. POC manufacturing enables hospitals to be in contact with cutting-edge products, refining the therapeutic skills that they hold and increasing the range of therapies made available to patients. POC ATMPs remain at the cutting edge of medical innovation, with ongoing advancements enhancing the ATMPs and streamlining the administration processes involved in POC manufacturing. In this listicle, we explore some of the latest trends and innovations shaping the field of POC manufacturing for ATMPs. TECHNOLOGYNETWORKS.COM CELL AND GENE THERAPIES: FROM DISCOVERY TO DELIVERY 35 POC manufacturing readiness and decentralization of manufacturing systems In 1974, NASA proposed the idea of technology readiness levels to assess the maturity of certain technologies.1 Adopted by the US Department of Defense, manufacturing readiness levels were then introduced, with the most mature stage (i.e., highest readiness level) detailing all materials, equipment, facilities and personnel are in place and have met full-rate production requirements.2 Manufacturing readiness levels The manufacturing readiness level serves as a metric to evaluate the maturity of manufacturing processes, analogous to technology readiness levels used to gauge technological development. Manufacturing readiness levels provide quantitative measures to assess the readiness of a technology, component or system specifically from a manufacturing standpoint. Now, manufacturing readiness is being applied to a range of technology fields – with recent applications in the biomedical field and POC manufacturing of ATMPs.3 Over the past few decades, UK hospitals have produced ATMPs in limited quantities. In the future, this production is expected to become more seamlessly integrated into routine clinical practices, with the technical and regulatory expertise hospitals have gained forming the basis for scaling up and increasing production frequency in the coming years – known as POC manufacturing readiness. Achieving POC manufacturing readiness is necessary to produce POC ATMPs, in which several resources, skills and institutional processes must be present for their success. The three main aspects of POC manufacturing readiness include staff and institutional procedures, infrastructure and transportation.3 Staff and institutional procedures The adverse effects of ATMPs can be severe, meaning their quality must be strictly monitored and individuals who conduct ATMP POC manufacturing need to be highly skilled. Often, this central role is played by a “qualified person” (QP) – a professional indicated on the manufacturer’s license as being legally responsible for ensuring that the product has been manufactured in line with quality and efficacy parameters. It is generally believed there is an insufficient number of QPs in the UK, especially in hospitals, with only 21 hospital-based QPs being registered since 2011.3 Hospitals have, therefore, attempted to advance other staffing aspects to improve manufacturing readiness, such as the creation and refinement of ATMP committees.3 These committees play a liaising role in ATMP manufacturing processes due to the various departments and facilities involved (i.e., testing laboratories, pharmacies and clinical wards). When ATMP POC manufacture becomes more frequent, these ATMP committees will play a decisive role, either by having their responsibilities expanded or by serving as a model for the creation of even more specialized committees in hospitals. Infrastructure Manufacturing cell and gene therapies undoubtedly incur high costs for infrastructure changes, which could ATMPs ideally need to be produced in a closed system where starting materials and reagents are processed within machines, with little human manipulation. TECHNOLOGYNETWORKS.COM CELL AND GENE THERAPIES: FROM DISCOVERY TO DELIVERY 36 be even more apparent for hospitals, as they were not originally designed to host ATMP manufacturing activities. Therefore, today’s hospital staff need to find ways of accommodating POC manufacturing activities in a clinical setting. Hospital laboratories will also play a key role in ATMP POC manufacture, as they may need to be mobilized for performing tests on tissues and cells as part of the quality control associated with therapy manufacture. There is also the obvious need for equipment. ATMPs ideally need to be produced in a closed system where starting materials and reagents are processed within machines, with little human manipulation, so the risk of contamination and human errors is reduced.4 Further, it is sometimes possible to freeze starting materials and final products, which requires equipment for accurate temperature control so that living structures are not damaged.5 Transportation The production of ATMPs in hospitals entails procuring a quite long list of products, be it for collection of starting materials, quality control or manufacture itself – meaning hospitals need to engage in a series of external relations. Starting materials and final products cannot be transported easily due to the presence of fragile living structures such as cells, meaning factors such as temperature and vibration need to be controlled if it is necessary to transport cells and tissues. Currently, hospitals procuring or shipping biological materials use commercial courier services. Evolving airline service strategies and security policies have complicated the model of product transport, so other methods, such as overnight shipping services, have been explored to assess their feasibility.6 These courier companies have designed specialized package tracking and monitoring programs developed specifically for healthcare and scientific businesses, which hospitals have also used. Further, hospitals have tried to ensure quality in their relations with companies transporting materials and samples by using ISO certifications. Automation and digital integration Currently, manufacturing processes for ATMPs are largely manual and performed in planar culture systems – processes that are highly laborious and difficult to scale up. Resultingly, such processes are prone to human error and can result in batch-to-batch variability, high manufacturing costs, high risk of contamination and batch loss.7 Manufacturing products following good manufacturing practice (GMP) guidelines will minimize process variability and variation in factors such as cell quality. However, full GMP compliance in the ATMP realm is currently challenging – particularly due to the increased difficulty in sourcing compliant starting material. Automation has been increasingly integrated into biopharmaceutical industries, particularly cell and gene therapies.8 Automation reduces human error, increases scalability and reduces production time. Digital tools such as real-time monitoring and artificial intelligence for process optimization are also increasingly being adopted. POC manufacturing can experience in-process variation from human handling which can subsequently impact product quality. Even when following stringent protocols, variation is observed between different handlers resulting from minor procedure imprecisions, such as variation in pipetting technique Automation has been increasingly integrated into biopharmaceutical industries, particularly cell and gene therapies. TECHNOLOGYNETWORKS.COM CELL AND GENE THERAPIES: FROM DISCOVERY TO DELIVERY 37 or slight deviations in incubation times. Automation can eliminate such in-process variation in multiple ways, such as through robotic arms to repeatedly and consistently perform pipetting or a mixing action. Even cell culture sub-processes (e.g., medium changes) can be automated, allowing for consistent speed, force and accuracy – thus leading to reduced variability and increased process reliability. Integration and automation of process analytics can remove the subjectivity of processing decisions, making more sophisticated processing rules possible. An example of this is the development of pattern recognition and image processing software that can be used to objectively determine confluence.8 Cell culture protocols rely on passaging adherent cells when they reach a confluence level of 80-90%. Confluency is typically estimated through microscopic visualization with the percentage estimation entirely subjective and dependent on the individual. Employing automated image acquisition and processing can allow for adaptive processing based on objective and comprehensible criteria.9 Automation integration in POC manufacturing of ATMPs faces several challenges including the development of technologically advanced interfaces, compliance with good manufacturing practices, the need to extensively validate automated systems and how to limit the effects on ATMP production if there is ever system downtime. Addressing these challenges requires a combination of advanced engineering, robust software solutions, regulatory foresight and interdisciplinary collaboration to realize the full potential of automation in POC ATMP manufacturing. Advancing ATMPs techniques: innovations in hydrogels Innovations of new techniques and products continue to emerge in ATMPs. Hydrogels – which are highly hydrated three-dimensional polymeric matrices – are one of these recent innovations that hold substantial promise in biomedical fields due to their biocompatible, chemically modifiable and physically tunable nature.10 For example, hydrogels have demonstrated the potential to support cell viability and functionalities and facilitate targeted delivery and controlled release of therapeutic agents.11,12,13 Synthesis and modification technologies used today have matured enough to advance hydrogel material significantly, moving away from the simplistic structures exhibited by early-generation hydrogels. Now, hydrogels have been developed to react to specific biological and pathological stimuli such as pH, temperature and reactive oxygen species, allowing the intricate requirements of specific diseases and heightened clinical demands to be met.14,15,16 Furthermore, the stable structure hydrogels experience upon water absorption acts as a delivery platform for bioactive substances and pharmaceuticals. Hydrogels can also mimic the extracellular matrix environment, promoting the growth and survival of encapsulated cells.17 The development of biodegradable variants of hydrogels, either allowing degradation over time or under specific stimuli, enables the release of their contents without eliciting toxic side effects to surrounding tissues.18 Injectable hydrogels further ensure sustained and controlled drug release at the targeted site, significantly reducing the adverse reactions associated with systemic drug exposure.19 Although significant advancements have been made in the use of hydrogels, which contribute to enhancing POC ATMPs, challenges remain. Many studies focused on hydrogel-based cell therapies remain in the nascent stages, with research progression hindered by technical complexities, biological intricacies, safety concerns, funding limitations and challenges of interdisciplinary collaboration.20 Importantly, extrapolation of results from in vitro and animal models to humans is complicated by biological variability, necessitating a comprehensive safety assessment of hydrogels to prevent adverse effects on patients. Injectable hydrogels are viewed as the most promising candidates for clinical translation. However, challenges in understanding biological degradation mechanisms and injection timing require further clarification. The misinjection of hydrogels into critical areas, such as major blood vessels therefore increasing the risk of embolism, TECHNOLOGYNETWORKS.COM CELL AND GENE THERAPIES: FROM DISCOVERY TO DELIVERY 38 warrants the need for protocols to mitigate such risks and highlights the complexity of clinical complexity. REFERENCES 1. Sadin SR, Povinelli FP, Rosen R. The NASA technology push towards future space mission systems. Acta Astronautica. 1989;20:73-77. doi: 10.1016/0094-5765(89)90054-4 2. Manufacturing Readiness Level (MRL) Deskbook. 2018. Available at: https://www.dodmrl.com/MRL_Deskbook_2018.pdf. Accessed February 10, 2025. 3. Bicudo E, Brass I. Institutional and infrastructure challenges for hospitals producing advanced therapies in the UK: The concept of ‘point-of-care manufacturing readiness.’ Regenerative Medicine. 2022. doi: 10.2217/rme-2022-0064 4. Gannon PO, Harari A, Auger A, et al. Development of an optimized closed and semi-automatic protocol for Good Manufacturing Practice manufacturing of tumor-infiltrating lymphocytes in a hospital environment. Cytotherapy. 2020;22(12):780-791. doi: 10.1016/j.jcyt.2020.07.011 5. Petrenko Y, Chudickova M, Vackova I, et al. Clinically Relevant Solution for the Hypothermic Storage and Transportation of Human Multipotent Mesenchymal Stromal Cells. Stem Cells International. 2019;2019(1):5909524. doi: 10.1155/2019/5909524 6. Howard A, Chitphakdithai P, Waller EK, et al. Evaluation of peripheral blood stem cell quality in products transported by traditional courier or commercial overnight shipping services. Transfusion. 2014;54(6):1501-1507. doi: 10.1111/trf.12533 7. Heathman TRJ, Rafiq QA, Chan AKC, et al. Characterization of human mesenchymal stem cells from multiple donors and the implications for large scale bioprocess development. Biochemical Engineering Journal. 2016;108:14-23. doi: 10.1016/j.bej.2015.06.018 8. 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Chemosphere. 2022;287:131956. doi: 10.1016/j. chemosphere.2021.131956 CELL AND GENE THERAPIES: FROM DISCOVERY TO DELIVERY 39 TECHNOLOGYNETWORKS.COM CONTRIBUTORS Aron Gyorgypal, PhD Aron is a postdoctoral research fellow at Harvard Medical School and Massachusetts General Hospital in Boston, Massachusetts. Following the completion of his PhD, he joined the Anthony Lab at Harvard Medical School and Massachusetts General Hospital in the immunology department. Isabel Ely, PhD Isabel is a Science Writer and Editor at Technology Networks. She holds a BSc in exercise and sport science from the University of Exeter, a MRes in medicine and health and a PhD in medicine from the University of Nottingham. Joanna Owens, PhD Joanna Owens holds a PhD in molecular toxicology from the University of Surrey. She has over 20 years’ experience writing about a wide range of scientific topics in biosciences, pharmaceuticals and biotechnology. 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