Cell and Gene Therapies: Innovations, Challenges and Clinical Breakthroughs

CELL AND GENE THERAPIES: Innovations, Challenges and Clinical Breakthroughs Credit: iStock SPONSORED BY CHALLENGES AND SOLUTIONS IN CELL THERAPY DEVELOPMENT ADVANCES IN BIOPHARMACEUTICAL ANALYSIS GENE THERAPY: A NEW FRONTIER IN DISEASEMODIFYING THERAPIES CONTENTS 04 Cell Therapy: Clinical Applications, Manufacturing and Regulation 08 Microbial Monitoring for Biopharma Manufacturing 12 Challenges and Solutions in Cell Therapy Development 16 Removing the Glycocalyx Armor of Cancer Cells for Treatment 20 Electronic Pulses Could Reduce the Need for High Doses in Gene Therapy Delivery 23 Gene Therapy: A New Frontier in Disease-Modifying Therapies 27 Advances in Biopharmaceutical Analysis 31 How To Run a Successful CRISPR Experiment 35 Breaking the Chains: How CRISPR Gene Therapy Gave Victoria Gray a New Life CELL AND GENE THERAPIES: INNOVATIONS, CHALLENGES AND CLINICAL BREAKTHROUGHS 3 TECHNOLOGYNETWORKS.COM FOREWORD The rapid advancement of cell and gene therapies is transforming the landscape of modern medicine. These groundbreaking treatments harness the power of biological mechanisms, offering innovative solutions for some of the most challenging diseases. Covering a broad spectrum of topics, this eBook examines cutting-edge approaches like CRISPR-driven gene editing, stem cell-based regenerative medicine and advancements in cellular immunotherapies. It highlights personalized medicine’s growing role, demonstrating how tailored treatments are revolutionizing care for conditions like cancer and inherited disorders. Readers will also explore breakthroughs in manufacturing and regulatory processes that are improving accessibility and scalability. Whether you’re a researcher, healthcare professional or simply curious about the future of medicine, this eBook offers an insightful exploration into the world of cell-based and genebased interventions. The Technology Networks editorial team. Cell therapy is a therapeutic strategy that involves the transfer of autologous (patient-derived) or allogeneic (donor-derived) cells into a patient’s body. Cell therapies can be divided into two broad categories — stem-cell therapies and non-stem-cell therapies. Within these two groups, a variety of mechanisms of action are employed, tailored to the specific type of cell therapy and the disease or condition it’s designed to target. For example, in regenerative medicine, stem cell therapies work by replacing damaged or diseased cells with new, functional ones to restore organ and/or tissue function. Whereas adoptive cell therapies for cancer treatment exploit immune cells by either expanding the number of cells, or by genetically modifying them to boost their cancer-fighting abilities, before being administered to the patient. The three main cell therapy modalities are outlined in Figure 1. In 2023, the global market value for cell therapy was estimated to be USD 4.74 billion. This market is expected to experience rapid growth due to increasing demand for innovative cell therapies, which tend to have fewer adverse effects compared to traditional modalities. This, coupled with their wide range of clinical applications, is anticipated to propel the market to approximately USD 20 billion by 2030. In this listicle, we explore new therapeutic targets and clinical applications, and discuss challenges and key considerations related to the manufacture and regulation of cell therapies. Clinical applications and novel research While the oncology segment led the overall cell therapy market in 2023, the potential of cell therapy in other areas is increasingly evident. Here we highlight recent research that illustrates the diverse potential of cell therapy across different therapeutic areas. WOMEN IN SCIENCE Cell Therapy Targets, Clinical Applications, Manufacturing and Regulatory Considerations Laura Elizabeth Lansdowne 4 Credit: iStock TECHNOLOGYNETWORKS.COM Neurology UC San Diego Health became one of the first facilities in the United States to administer the experimental neural cell therapy, NRTX-1001, to subjects with drug-resistant epilepsy. This cell therapy involves the delivery of interneurons that secrete the inhibitory neurotransmitter gamma-aminobutyric acid (GABA), into the epileptic region of the brain. In December 2023, the biotherapeutics company developing NRTX1001 announced that the initial two trial subjects consistently reported a decrease in seizure frequency (> 95% reduction from baseline), more than one year after receiving treatment. Regenerative medicine Using mRNA technology encapsulated in nanoparticles, researchers designed a novel stem cell therapy that aims to stimulate the liver's natural repair processes. The study was published in Cell Stem Cell. The therapy works by delivering vascular endothelial growth factor A (VEGFA) mRNA via lipid nanoparticles. This promotes the conversion of biliary epithelial cells into hepatocytes, the liver's functional cells. The research was conducted using mouse and zebrafish disease models. Immunology Researchers developed a novel T-cell therapy to treat a specific form of autoimmune encephalitis (NMDAR encephalitis), an immune-mediated condition whereby antibodies attack healthy brain cells, causing inflammation and various neurological symptoms. The team genetically modified T cells to selectively eliminate anti-NMDAR B cells and autoantibodies against the NMDA receptor. This preclinical study was published in Cell. They now plan to test the therapy in human subjects with NMDAR encephalitis. Inherited blood disorders The US Food and Drug Administration (FDA) approved two cell-based gene therapies — Casgevy™ (exagamglogene autotemcel) and Lyfgenia™ (lovotibeglogene autotemcel) — to treat sickle cell disease in patients ≥ 12 years. Both therapies are created using patients’ hematopoietic stem cells, which are genetically modified and then reintroduced as a single-dose infusion. Casgevy is the first FDAapproved therapy that exploits CRISPR-Cas9 gene editing. CRISPR-Cas9 is used to reduce the expression of BCL11A, which in turn, boosts the synthesis of γ-globin and reactivates fetal hemoglobin production, CELL AND GENE THERAPIES: INNOVATIONS, CHALLENGES AND CLINICAL BREAKTHROUGHS 5 Cell Therapies Cells are administered to a patient as a therapeutic modality. Genetically modified cell therapies Patient/donor cells are genetically modified to perform a unique function they wouldn't typically do that provides therapeutic benefit to a patient. Tissue-engineered products Cells and/or biologically active substances are designed to restore, maintain or replace damaged tissues/organs. Figure 1: Overview of cell therapy modalities. Credit: Technology Networks. CELL AND GENE THERAPIES: INNOVATIONS, CHALLENGES AND CLINICAL BREAKTHROUGHS 6 TECHNOLOGYNETWORKS.COM preventing sickling. Lyfgenia uses a lentiviral vector to genetically modify the stem cells to produce a specific hemoglobin called HbAT87Q. Cells containing this hemoglobin have a reduced risk of sickling. Oncology A preclinical study published in Nature Communications describes a novel allogeneic CAR T-cell therapy targeting T-cell malignancies. This therapy focuses on eliminating cancerous T cells that express a dominant T-cell receptor called Vβ2. Unlike traditional treatments that risk depleting all of a patient's T cells, this CAR T-cell therapy selectively kills the diseased cells, preserving Vβ2-negative healthy cells. CRISPR gene-editing technology was used to specifically target the Vβ2-positive cancer cells. The FDA approved Amtagvi™ (lifileucel), a tumor-derived autologous T-cell immunotherapy, via its Accelerated Approval Program. Amtagvi is indicated for use in adult patients with a melanoma (unresectable or metastatic), that has failed to respond to/stopped responding to other specific therapies. A portion of the patient’s tumor is surgically removed. Tumor-derived T cells are then isolated from the excised tumor tissue, expanded at a manufacturing site and then administered as a single-dose intravenous infusion to the same patient. Understanding the regulatory landscape of cell therapy The pharmaceutical industry is one of the most regulated industries globally, and regulatory authorities play a crucial role in overseeing various steps related to the development of new therapies. Several different regulatory authorities exist worldwide and each issues specific guidelines relating to the development, registration, manufacturing, licensing, marketing and labeling of medicines. As such, it is vital cell therapy developers understand regulatory variations and consider how these differences will impact the development of a novel cell therapy, depending on where in the world they are seeking marketing authorization. Here we take a closer look at regulatory guidance in the UK, Europe and the US. The European Medicines Agency (EMA) classes cell therapies as a type of advanced therapy medicinal product (ATMP). As such, they are governed by medicinal product regulatory frameworks (Regulation (EC) No 1394/2007, Directive 2001/83/EC). The manufacturing of these products must comply with Good Manufacturing Practice (GMP) principles (EudraLex Volume 4, Part IV). Similar to the EMA, the UK’s Medicines and Healthcare products Regulatory Agency (MHRA) classes cell therapies as a type of ATMP. The donation, procurement and testing of cells is covered by the EU Tissues and Cells Directive (2004/23/EC). Under this directive there are two authorities of note — the Human Fertilisation and Embryology Authority (HFEA) and the Human Tissue Authority (HTA). The HFEA oversees the use of gametes and embryos in the development of ATMPs and the HTA is responsible for the licensing and inspection for all other tissue and cell types. In the US, cell therapies are regulated by the FDA’s Center for Biologics Evaluation and Research (CBER). They fall under Title 21 of the Code of Federal Regulations (CFR), Part 127.3(d), and are defined as, “Articles containing or consisting of human cells or tissues that are intended for implantation, transplantation, infusion or transfer into a human recipient". The FDA recommends that “product testing for cellular therapies include, but not be limited to, microbiological testing (including sterility, mycoplasma and adventitious viral agent testing) to ensure safety and assessments of other product characteristics such as identity, purity (including endotoxin), viability and potency.” The FDA has numerous cellular and gene therapy guidance documents, addressing specific aspects of cell and gene therapy development – for example, potency assurance, manufacturing, trial design and general regulatory considerations. Addressing manufacturing and scalability challenges While the demand for cell therapies is undeniable, their production comes with key challenges and regulators require manufacturers to conduct extensive testing. Cell therapy manufacturing methods range in complexity. Some therapies require significant manipulation of cells (e.g., genetic modification), while others may require comprehensive cell cultivation steps. Here we discuss several manufacturing and scalability considerations. TECHNOLOGYNETWORKS.COM Sourcing high-quality cells The initial challenge in cell therapy production lies in sourcing high-quality biological materials. The exact geographical region and regulatory authority will influence how starting material must be obtained, for example collection/apheresis best practices. Progress is being made to refine the cell extraction and separation processes. For example, researchers recently developed a technology capable of extracting mesenchymal stem cells (MSCs) directly from bone marrow – without the need for dilution. By continuously sorting and isolating stem cells from blood cells using a novel microfluidic platform, it was possible to double the number of MSCs obtained from bone marrow samples and reduce the extraction time to approximately 20 minutes. In the case of allogenic (donor-derived) cell therapies, specific donor eligibility requirements will also need to be considered as these may differ depending on country. Establishing an appropriate shelf life Once obtained, the shelf life of fresh cells is often short. For example, hemopoietic stem cells can be stored unprocessed at 4 °C or room temperature for approximately 72 hours post-collection. However, after this time they begin to degrade, resulting in compromised product quality and potency. To address this, developers typically opt to cryogenically freeze cells, increasing their shelf life to months or years, but this relies on specialist facilities and procedures to ensure the cells remain viable and stable. The optimal cryopreservation and freeze—thawing process will differ depending on the particular cell product. Regulators require manufacturers to conduct stability testing to confirm the product's integrity and efficacy over time. Addressing temperature sensitivity Increased temperature sensitivity is another key challenge faced by cell therapy manufacturers. Good distribution practice guidelines stress that medicinal products must not be exposed to conditions during transport, “that may compromise their quality and integrity.” Temperature changes must be tracked to confirm that products stay within "defined limits" while in transit. The difficulty lies in establishing these limits, as they must be broad enough to allow transfer of the product, but not so broad they jeopardize its quality. Achieving quality bioproduction at scale A product’s critical quality attributes (CQAs) must be well characterized early on in development before scaling bioproduction to ensure the correct quality control metrics are being monitored. Developers want to avoid the need for major manufacturing changes late in development as this could impact commercial viability of the therapy. The term “technology transfer” describes the transfer of a process from small scale (a laboratory setting) to a commercial manufacturing facility. If employing a contract manufacturing organization (CMO) or contract development and manufacturing organization (CDMO) to support the scale-up of a therapy, it’s important to limit technology-transfer risk by ensuring clear knowledge transfer, adequate training and ensuring equipment/process commonalities. Depending on the type of cell therapy, developers will choose whether to scale-up (increase batch size) or scale-out (increase the number of batches). Allogenic therapies are typically scaled up whereas autologous therapies are scaled out. Automated integration of multiple manufacturing steps in a closed system environment is vital to ensure GMP compliance and process reproducibility, and reduce contamination risk. Continued exploration and investment in cell therapies is ushering in a new era of medical interventions. The discovery of novel targets and mechanisms, refinement of manufacturing processes and creation of detailed regulatory guidance are helping to accelerate the approval of cell therapies, offering hope and improved outcomes for patients worldwide. CELL AND GENE THERAPIES: INNOVATIONS, CHALLENGES AND CLINICAL BREAKTHROUGHS 7 Microbial contamination Biomanufacturing relies on the use of living cells as factories to produce biotherapeutics, or as therapeutics themselves. Most biologic drugs are highly specific in their mechanistic function, making them popular for the treatment of cancers, autoimmune diseases and infections. Production and characterization of these drugs are exceedingly more complex than conventional small molecule drugs that are chemically synthesized. Cells must be grown and expanded under aseptic conditions. The use of good manufacturing practices (GMP) and strict quality control are applied to mitigate the risk of product contamination.1 The World Health Organization (WHO) defines adventitious agents as contaminating microorganisms of cell culture or source material that have been unintentionally introduced into the manufacturing process of a biological product. This includes bacteria, fungi, mycoplasma, parasites and viruses.2 These contaminants can be introduced through starting materials, such as cell substrates, or by environmental exposure, such as equipment, handling or personnel. Contamination is a significant issue for bioprocessing as it results in loss of time, money and effort. Yearly revenue losses are estimated between $100–300 million due to contamination.3 Contaminated products that are not detected during production must also be pulled off shelves, which may result in shortages of life-saving drugs and vaccines.4 The risk of contamination necessitates the need to monitor for microbial contamination during bioprocessing to mitigate both product lot failures as well as adverse reactions to patients. Detecting contamination during bioprocessing Bioprocess monitoring is a critical aspect of manufacturing as it ensures the final product meets the desired specifications, quality and safety requirements. Monitoring of contamination is typically achieved by the WOMEN IN SCIENCE Microbial Monitoring for Biopharma Manufacturing Aron Gyorgypal, PhD 8 Credit: iStock TECHNOLOGYNETWORKS.COM analysis of process parameters such as pH, temperature, dissolved oxygen, nutrient levels and cell density and viability. These variables are already monitored during processing as optimized conditions are required to produce quality products.5 Multiple established methods exist to measure microbial contamination: • Enzyme-linked immunosorbent assay (ELISA): assesses the presence and concentrations of specific proteins or antibodies and can also be used to indicate the presence of bacterial contamination. • Polymerase chain reaction (PCR): can be used to amplify specific DNA sequences of interest, particularly for those of microbial contaminants.6 • Process Analytical technology (PAT): may be able to detect contamination by statistical or mathematic techniques if process deviations are detected.5,7 • Limulus Amebocyte Lysate (LAL): used to test for endotoxins that are released by gram-negative bacteria.8 Contamination’s influence on product quality and safety What are some of the key signs of cell culture contamination? “Because microbes multiply much faster than mammalian cells, the culture could quickly become significantly more turbid with a high concentration of bacteria. The microbes would also rapidly consume the sugar and oxygen in the culture, effectively starving the mammalian cells,” says Dr. Erica Berilla, senior research scientist in the Office of Pharmaceutical Quality Research (OPQR) within the Food and Drug Administration’s (FDA) Center for Drug Evaluation and Research. Berilla’s research focuses on new manufacturing technologies used in the production of biologics and the methods used to monitor those manufacturing processes. Such projects include, but are not limited to, end-to-end continuous manufacturing, automated in-process sampling and microbial monitoring during biomanufacturing processes. This research can help guide and inform the FDA’s product quality assessors who may be encountering these new technologies during their assessments of protein drug substance products. Berilla’s team wanted to understand microbial contamination in terms of product quality while also building an understanding of early contamination detection. To do this, they intentionally contaminated a Chinese hamster ovary (CHO) cell bioreactor that was used to produce a model monoclonal antibody (mAb). 9 Near-infrared (NIR) spectroscopy was used to analyze the culture in 15-minute intervals to generate a partial least square (PLS) model. Though the model was not sensitive to detect low-level contamination it could detect contaminant in the later stages. “We found that cell cultures contaminated with Mycoplasma arginini (M. arginini) plateaued in their growth once the mycoplasma concentration was maximal, which usually took two to three days after mycoplasma introduction… We noted significant increases in ammonium and depletion of arginine that was not seen in the control.” This result was perhaps to be expected, given that there is a competition for nutrients within the cell culture between the mammalian and the bacterial cells. Also, because bacteria are more metabolically efficient, there will be an increase in their proliferation and production of metabolic byproducts that are toxic to mammalian cells. These experiments also gave insights into how the quality of mAbs is affected by M. arginini contamination. Principal component analysis revealed a correlation CELL AND GENE THERAPIES: INNOVATIONS, CHALLENGES AND CLINICAL BREAKTHROUGHS 9 Methods to detect microbe contamination in a bioprocess. Credit: Aron Gyorgypal, Adobe Illustrator. TECHNOLOGYNETWORKS.COM between contamination and increased antibody charge variants and hypoglycosylation – qualities that influence therapeutic efficacy and clearance.10 To mitigate the lost time and cost associated with manufacturing, there needs to be more advances in detection methods, Barilla says: “Technologies such as next-generation sequencing (NGS) and spectroscopy have the potential for use in earlier detection of adventitious agent events during bioprocessing. Further development and validation of these technologies could lead to more rapid/real-time testing.” NGS hold potential for contamination monitoring as indicated by the 2024 ICH-Q5A(R2) guidance document published by the FDA, which has encouraged the use of the technology. In a recent study by Hiarai et al., the performance of NGS was evaluated for in vitro contaminant detection in biologic products.11 Here the authors inoculated a model adenovirus into Vero cells and checked for contamination with increasing dilutions to find the limit of detection (LOD). The authors found that the LOD for this assay was roughly 2.49x10-4 copies/ngRNA, or about 1:107 dilution from their original inoculate. This proved to be extremely sensitive, however there were some drawbacks such as the risk for false positives that may arise when multiplexing the assay. While these results are promising, the technology will need to be validated for contaminant monitoring before it can be implemented for routine use. Emerging methods for detecting microbial contaminations There are many different methods currently used to detect contamination. The method of choice will vary depending on the type of culture that is being run, such as CHO-cellproducing mAbs, human-derived cells for cell therapies or the production of viral vectors. “The first key factor to look at is the unit operation along with the sensitivity required of the assay,” says Dr. Richa Pandey, assistant professor of biomedical engineering at the University of Calgary. Pandey's research group is focused on point-of-care diagnostics, but she has also taken an interest in using her expertise in biosensor technology, applying it to biomanufacturing. She recently published a review article that highlights the current methods for the analysis of biological contaminants within the biomanufacturing industry.12 Pandey argues the need for continuous monitoring systems for microbe detection that can be enabled by optical and electrochemical sensors and biosensors equipped with microfluidics. “Optical sensors work by interacting light with thin films that are functionalized with nanoparticles that, once interacted with by the antigen of interest, produce a signal. These sensors are quite advantageous as they are sensitive and require low volumes of analyte,” says Pandey. An example of this technology was showcased by Zandieh et al., who demonstrated that the use of silver nanocolumns functionalized with a polycationic peptide, polymyxin B (PmB), to recognize and interact with lipopolysaccharide (LPS) a highly toxic endotoxin produced by gram native bacteria.13 The methodology allowed sensitivity as low as 340 pg/mL of endotoxin. Although the conventionally used LAL assay may be more sensitive, it suffers from the need for sample prep, which makes this optical sensor methodology more attractive for a streamlined and fast bioprocess contamination screening. Electrochemical-based sensors are another methodology that have been used to detect bioprocess contaminants. “Unlike the optical sensor, an electrochemical sensor measures binding events through either change in conductance, resistance or capacitance on its surface,” says Pandey. CELL AND GENE THERAPIES: INNOVATIONS, CHALLENGES AND CLINICAL BREAKTHROUGHS 10 Representation of an optical (left) and electrochemical (right) biosensor for detecting contamination such as toxins produced by microbes in a bioprocess. Credit: Aron Gyorgypal, Adobe Illustrator. TECHNOLOGYNETWORKS.COM The use of aptamer-based electrochemical sensors has gained interest in recent years. Their popularity is derived from their high affinity to a variety of targets, such as proteins, small molecules and living cells. Kim et al. used an endotoxin-specific aptamer to detect bioprocess microbial contaminants.14 This methodology showed reduced processing time against the conventional LAL, although with less sensitivity. Studies like this one showcased the use of aptamers for the detection of not only endotoxins, but also whole pathogens, and can be used to measure different compounds in a bioprocess culture.15,16 Indeed, the use of optical and electrochemical sensors does have some advantages over current practices, given that they allow for faster detection with little to no processing. “Of course, there is merit to the current methods used for contaminant detection, but there still needs to be advances to make them more sensitive, as well as with automation to allow for at-line or in-line monitoring,” says Pandey. Moving towards automated monitoring Although the current gold standard technologies for contamination detection are sensitive and provide specific detection, they are still limited to batch monitoring. The current priority is to detect contamination quickly and accurately, without the need for off-line timely assays. The production of a real-time monitoring system capable of continuous contaminant monitoring would allow for early intervention that would prevent the spread of adventitious agents and reduce the risk for product recalls. ABOUT THE INTERVIEWEES Dr. Erica Berilla is a senior research scientist in the Office of Pharmaceutical Quality Research (OPQR) within the Food and Drug Administration’s (FDA) Center for Drug Evaluation and Research. She obtained a PhD in Biomedical Sciences from the University of South Florida in 2014. Dr. Richa Pandey is an assistant professor of biomedical engineering at the University of Calgary. She obtained a PhD in physical electronics from Tel Aviv University in 2018. Her research is focused on wearable biosensors, point of care technologies, biosensors and lateral flow assays. REFERENCES 1. Geigert J. Risk Management of the Minimum CMC Regulatory Compliance Continuum. In: The Challenge of CMC Regulatory Compliance for Biopharmaceuticals. Switzerland: Springer, Cham; 2023:77-119. doi: 10.1007/978-3-031-31909-9_4. Accessed April 10, 2024. 2. World Health Organization. Annex 2 WHO Good Manufacturing Practices for Biological Products. https://www.who.int/publications/m/item/ annex-2-trs-no-999-WHO-gmp-for-biological-products. Published August 19, 2016. Accessed April 10, 2024. 3. Shiratori M, Kiss R. Risk Mitigation in Preventing Adventitious Agent Contamination of Mammalian Cell Cultures. In: New Bioprocessing Strategies: Development and Manufacturing of Recombinant Antibodies and Proteins. Springer, Cham; 2017:75-93. doi: 10.1007/10_2017_38. Accessed April 10, 2024. 4. Ramanan S, Grampp G. Preventing shortages of biologic medicines. Expert Rev Clin Pharmacol. 2014;7(2):151-159. doi: 10.1586/17512433.2014.874281. 5. Gomes J, Chopda VR, Rathore AS. Integrating systems analysis and control for implementing process analytical technology in bioprocess development. JCTB. 2015;90(4):583-589. doi: 10.1002/jctb.4591. 6. Morris C, Lee YS, Yoon S. Adventitious agent detection methods in bio-pharmaceutical applications with a focus on viruses, bacteria, and mycoplasma. Curr Opin Biotechnol. 2021;71:105-114. doi: 10.1016/j. copbio.2021.06.027. 7. Wehbe K, Vezzalini M, Cinque G. Detection of mycoplasma in contaminated mammalian cell culture using FTIR microspectroscopy. Anal Bioanal Chem. 2018;410(12):3003-3016. doi: 10.1007/s00216-018- 0987-9. 8. Novitsky TJ. Biomedical Applications of Limulus Amebocyte Lysate. In: Biology and Conservation of Horseshoe Crabs. Springer US; 2009:315- 329. doi: 10.1007/978-0-387-89959-6_20. Accessed April 10, 2024. 9. Fratz-Berilla EJ, Faison T, Kohnhorst CL, et al. Impacts of intentional mycoplasma contamination on CHO cell bioreactor cultures. Biotechnol Bioeng. 2019;116(12):3242-3252. doi: 10.1002/bit.27161. 10. Das TK, Narhi LO, Sreedhara A, et al. Stress factors in mab drug substance production processes: critical assessment of impact on product quality and control strategy. J Pharm Sci. 2020;109(1):116-133. doi: 10.1016/j.xphs.2019.09.023. 11. Hirai T, Kataoka K, Yuan Y, et al. Evaluation of next-generation sequencing performance for in vitro detection of viruses in biological products. Biologicals. 2024;85:101739. doi: 10.1016/j. biologicals.2023.101739. 12. Janghorban M, Kazemi S, Tormon R, Ngaju P, Pandey R. Methods and analysis of biological contaminants in the biomanufacturing industry. Chemosensors. 2023;11(5):298. doi: 10.3390/chemosensors11050298. 13. Zandieh M, Hosseini SN, Vossoughi M, Khatami M, Abbasian S, Moshaii A. Label-free and simple detection of endotoxins using a sensitive LSPR biosensor based on silver nanocolumns. Anal Biochem. 2018;548:96-101. doi: 10.1016/j.ab.2018.02.023. 14. Kim SE, Su W, Cho M, Lee Y, Choe WS. Harnessing aptamers for electrochemical detection of endotoxin. Anal Biochem. 2012;424(1):12-20. doi: 10.1016/j.ab.2012.02.016. 15. Weaver S, Mohammadi MH, Nakatsuka N. Aptamer-functionalized capacitive biosensors. Biosens Bioelectron. 2023;224:115014. doi: 10.1016/j.bios.2022.115014. 16. Robin P, Gerber-Lemaire S. Design and preparation of sensing surfaces for capacitive biodetection. Biosensors. 2022;13(1):17. doi: 10.3390/bios13010017. CELL AND GENE THERAPIES: INNOVATIONS, CHALLENGES AND CLINICAL BREAKTHROUGHS 11 Cell therapy involves the transplantation of live human cells into a patient to repair damaged tissue or cure disease. A variety of cell types either belonging to the patient, in the case of autologous therapies, or from a donor – known as allogeneic cell therapies – can be used. Although cell therapies in the form of bone marrow transplants have been used to treat rare forms of blood cancer since the 1950s, most commercially available cellbased therapies today have been approved within the past decade. To date, 27 cell therapies have been approved by the United States Food and Drug Administration (FDA) for the treatment of multiple indications, including various forms of cancer, inherited metabolic disorders and immune system disorders.1 Many more are likely to be submitted for approval soon, given the over 1,600 ongoing clinical trials for cell therapies registered with ClinicalTrials.gov.2 In this article, we take a look at how researchers are tackling some of the challenges that are preventing cell therapies from being more widely used, paying particular attention to cellular immunotherapies for cancer and induced pluripotent stem cells (iPSC) for regenerative medicine. We also outline some advances in manufacturing and regulatory approval processes that will help expand patient access to innovative, lifechanging treatments. Non-stem cell-based therapies These types of therapies involve somatic cells from a patient or a donor, such as fibroblasts, pancreatic islet cells or immune cells. Examples include allogeneic cultured keratinocytes for the treatment of adults with thermal burns and allogeneic pancreatic islet cells for people with type 1 diabetes who are unable to maintain healthy blood sugar levels despite thorough diabetes management and education. WOMEN IN SCIENCE Challenges and Solutions in Cell Therapy Development Monica Hoyos Flight, PhD 12 Credit: iStock TECHNOLOGYNETWORKS.COM CELL AND GENE THERAPIES: INNOVATIONS, CHALLENGES AND CLINICAL BREAKTHROUGHS 13 The most commonly used somatic cells are T cells, white blood cells that are critical for triggering an immune response to pathogens, allergens and tumors. The first chimeric antigen receptor (CAR) T cell to be approved by the FDA in 2017 was invented by Professor Carl June at the University of Pennsylvania. This CAR T-cell therapy – known as tisagenlecleucel (Kymriah®) – can be used for the treatment of certain pediatric and young adult patients with advanced chronic lymphocytic leukemia. Currently, there are six FDA-approved CAR T-cell therapies for the treatment of various lymphomas and myeloma. These cellular immunotherapies involve collecting T cells from the patient’s blood and modifying them genetically in the laboratory so that they make CARs that track down cancer-associated antigens. When re-infused into the patient, the modified T cells attach to and kill the cancer cells, thereby helping to clear the cancer from the body. Dr. Neil Sheppard, a director in Prof. Carl June’s laboratory, highlights the advantages of cellular immunotherapies for cancer: “We know it is possible for the immune system to completely cure us of even advanced diseases, including cancer, under the right circumstances and potentially without damaging healthy tissues,” he explains. While he acknowledges that a lot of work remains to be done to realize the full potential of these therapies, he is optimistic about progress. “With various genetic, epigenetic and RNA-editing tools the promise of cellular immunotherapies is within our grasp,” he says. Next-generation cellular immunotherapies Despite the remarkable efficacy of CAR T-cell therapies against B-cell malignancies, patients can experience recurrence if the cancer cells stop expressing the targeted antigen, and CAR T-cell therapies have also shown limited efficacy against solid tumors.3,4 To address these challenges, Sheppard and others are pursuing several approaches. These include developing CAR T cells that target multiple antigens to prevent immune escape and using alternative cell types such as natural killer (NK) cells, which do not mediate graftversus-host disease and might be easier to use in an allogeneic setting.5 “Solid tumors remain a challenge,” says Sheppard. “To achieve CAR T efficacy in solid tumors, target antigens such as claudin 6 and claudin 18.2 are being utilized, sometimes in conjunction with vaccination with the antigen to boost CAR T-cell proliferation and activity.” There is growing evidence that intratumoral injection of CAR T cells can have a greater effect compared to intravenous infusion. Moreover, combining CAR T cells with oncolytic viruses that attract CAR T cells to the tumor aids tumor infiltration and strengthens resistance to the immunosuppressive tumor microenvironment.6 The recent approval of the first tumor-infiltrating lymphocyte (TIL) therapy raises new hope for cell therapies in solid cancers.7 TILs have an innate ability to seek out cancer neoantigens, fragments of mutated intracellular proteins that are presented on the surface of cancer cells. One such treatment for unresectable or metastatic melanoma – lifileucel (Amtagvi™) – involves the removal of TILs from patients’ tumor tissue, growing them in bioreactors and re-infusing them into patients to destroy cancer cells expressing these patient-specific antigens. Stem cell-based therapies Stem cell treatments play a key role in regenerative medicine as their ability to self-renew and differentiate into specialized cells can be used to replace damaged or diseased cells and restore normal function. Dr. Shinya Yamanaka’s groundbreaking discovery that adult cells can be reprogrammed into pluripotent stem cells through the expression of embryonic transcription factors offers researchers the opportunity to generate patient-specific regenerative cell therapies without having to rely on controversial embryonic stem cells or worry about immune rejection.8 Since then, great progress has been made in generating and differentiating iPSCs. “iPSCs are remarkably useful, and in a class by themselves,” says Professor Jeanne Loring, founding director of the Center for Regenerative Medicine and emeritus professor at the Scripps Research Institute in La Jolla, California. “They are the only noncancer cells that can continue to divide indefinitely in a culture dish, and retain the ability to give rise to every cell type in the body.” TECHNOLOGYNETWORKS.COM Loring’s research focuses on developing iPSC-based therapies for Parkinson’s disease, multiple sclerosis and autism. She believes these therapies hold great promise for conditions known to be caused by the loss of specific cell types. However, there are still several obstacles associated with iPSC-based therapies that need to be addressed before they can enter clinical trials. “The development of iPSC-derived cell therapies requires cutting-edge technologies in multiple areas – from making the stem cells and differentiating them into the right cell type in culture, to determining the best stage of differentiation for transplanting the cells and sophisticated genomics and bioinformatics, with machine learning to make sure the cells are at the right stage and that they don’t contain dangerous mutations,” Loring explains. In her opinion, technology is key to overcoming these challenges. “This is a field where all of the work has been hands-on with highly skilled people, which makes it very expensive,“ she says. “The use of AI to make decisions about the culture of the cells and new genomics methods that ensure the integrity of the genomes of cells before they are transplanted, are just a couple of examples of ways to advance cell therapies to the clinic.” Manufacturing innovations In addition to sharing the main challenges of drug development, such as the need to demonstrate safety, target engagement and pharmacological activity, cell therapies face unique challenges. “The manufacturing process for CAR T-cell therapy greatly affects the potency of cell therapy product,” says Sheppard. His team is exploring how to scale up their cell manufacturing process in the laboratory into a large-scale good manufacturing process (GMP) needed for clinical trials or even a commercial scale process designed to serve thousands of patients each year. So far, they have learned that, with patient-specific immune cells, the longer the duration of the cell manufacturing process and the more manipulation steps involved, the lower the potency of the emerging cell. “The evidence shows that CAR T-cell products made in manufacturing processes as short as one to three days have superior activity once inside the human body compared to those made over nine days or more.” In the not-too-distant future, Sheppard envisages the creation of self-contained GMP machines where a patient’s T cells are loaded and the chosen type of CAR T-cell therapy is produced at the press of a button in a hospital setting. “I imagine the future of CAR T-cell therapy production being as simple from the prescriber’s perspective as using the office coffee machine – load the desired sachet (gene vector), water (reagents) and a cup (cells), press the button and your work is done.” Speeding up production of stem cell-based therapeutics Since 2003, the UK’s Stem Cell Bank (UKSCB) has been supporting the development of pluripotent stem cell-derived therapies and driving improvements in the manufacturing process to produce quality-controlled pluripotent stem cell lines (iPSCs and human embryonic stem cells, or hESCs) that can be differentiated into specialized cell types for tissue regeneration. At the 20th anniversary of the cell repository last October, Dr. Lee Carpenter, the head of the UKSCB, highlighted automation and machine learning as being key to accelerating the safe manufacture of stem cell-derived therapies and giving patients faster access to potentially life-saving treatments.9 The UKSCB has demonstrated, using a robotic system, that clinical-grade pluripotent stem cell production is possible and is currently comparing the automated process to manual production. The use of automation, robotics and modular production platforms will not just help reduce reliance on manual operations and variability in process performance, it will also enable scalability and drive down costs. “There is a race to develop the instrumentation to automate stem cellderived cell therapy manufacturing,” says Loring. Streamlining the approval process The final hurdle when developing cell therapies is obtaining regulatory approval. The review of a New Drug Application (NDA) by the FDA can take up to a year, significantly delaying the commercialization of a new therapy.10 Cell therapies are evolving quickly, and so too must the regulations. Regulatory agencies are adapting to the unique challenges of cell therapies by implementing CELL AND GENE THERAPIES: INNOVATIONS, CHALLENGES AND CLINICAL BREAKTHROUGHS 14 TECHNOLOGYNETWORKS.COM pathways aimed at expediting assessments, such as the FDA's Regenerative Medicine Advanced Therapy (RMAT) designation and the European Medicines Agency (EMA)'s Priority Medicines (PRIME) scheme.11 In addition, the FDA is on a recruitment drive to hire more staff in its Office of Therapeutic Products to tackle the increasing workload.12 Efforts to increase the dialog between regulators, industry and academia to harmonize regulatory standards are also starting to bear fruit. The benefits of international programs to build regulatory capacity and share information are evidenced by multinational approvals of CAR T-cell therapies.13 As the cell therapy pipeline continues to expand, increasing the alignment of regulatory pathways across countries will be crucial to facilitating access to new transformative treatments to patients around the world. ABOUT THE INTERVIEWEES Dr. Neil Sheppard is the director of the Therapeutic Innovation in Natural Killer cells (THINK) lab, which focuses on improving the therapeutic application of NK cells in cancer. As part of Prof. Carl June's team, he is also overseeing the development of combination strategies for clinical-stage CAR T-cell therapies. Sheppard joined Penn Medicine’s Center for Cellular Immunotherapies in 2019 and has over 20 years experience of working on immunotherapies in academia, pharma and biotechnology companies. Dr. Jeanne Loring is professor emeritus at The Scripps Research Institute, where she was the founding director of the Center for Regenerative Medicine. She is an internationally recognized authority in stem cell research and has received multiple awards for her accomplishments as a scientist, educator and advocate for evidence-based patient therapies. In 2018 she co-founded the biotechnology company Aspen Neuroscience to develop personalized cell therapies for Parkinson’s disease. REFERENCES 1. Approved Cellular and Gene Therapy Products. FDA. Published online March 18, 2024. Accessed April 26, 2024. https://www.fda. gov/vaccines-blood-biologics/cellular-gene-therapy-products/ approved-cellular-and-gene-therapy-products 2. Active clinical trials of cellular therapies. clinicaltrials. gov. https://clinicaltrials.gov/search?intr=cell%20 therapy&aggFilters=status:act 3. Cappell KM, Kochenderfer JN. Long-term outcomes following CAR T cell therapy: what we know so far. Nat Rev Clin Oncol. 2023;20(6):359-371. doi: 10.1038/s41571-023-00754-1 4. Hou AJ, Chen LC, Chen YY. Navigating CAR-T cells through the solid-tumour microenvironment. Nat Rev Drug Discov. 2021;20(7):531-550. doi: 10.1038/s41573-021-00189-2 5. Myers JA, Miller JS. Exploring the NK cell platform for cancer immunotherapy. Nat Rev Clin Oncol. 2021;18(2):85-100. doi: 10.1038/s41571-020-0426-7 6. Daei Sorkhabi A, Mohamed Khosroshahi L, Sarkesh A, et al. The current landscape of CAR T-cell therapy for solid tumors: Mechanisms, research progress, challenges, and counterstrategies. Front Immunol. 2023;14. doi: 10.3389/ fimmu.2023.1113882 7. Mullard A. FDA approves first tumour-infiltrating lymphocyte (TIL) therapy, bolstering hopes for cell therapies in solid cancers. Nat Rev Drug Discov. 2024;23(4):238-238. doi: 10.1038/d41573- 024-00035-1 8. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006;126(4):663-676. doi: 10.1016/j.cell.2006.07.024 9. Patients could have faster access to ground-breaking stem cell treatment with manufacturing innovation. GOV.UK. Published online October 11, 2023. Accessed April 29, 2024. https://www. gov.uk/government/news/patients-could-have-faster-accessto-ground-breaking-stem-cell-treatment-with-manufacturinginnovation 10. Rotolo A, Chabannon C, Gramignoli R. Identification of hurdles in the development of cell-based therapies. Cytotherapy. 2020;22(2):53-56. doi: 10.1016/j.jcyt.2019.12.009 11. Drago D, Foss-Campbell B, Wonnacott K, Barrett D, Ndu A. Global regulatory progress in delivering on the promise of gene therapies for unmet medical needs. Mol Ther Methods Clin Dev. 2021;21:524-529. doi: 10.1016/j.omtm.2021.04.001 12. Establishment of the Office of Therapeutic Products. FDA. Published online March 16, 2023. Accessed April 29, 2024. https://www.fda.gov/vaccines-blood-biologics/cellular-genetherapy-products/establishment-office-therapeutic-products 13. Arcidiacono J. International Harmonization for Cell and Gene Therapy Products. In: Galli MC, ed. Regulatory Aspects of Gene Therapy and Cell Therapy Products: A Global Perspective. Springer International Publishing; 2023:235-240. doi: 10.1007/978-3-031-34567-8_14 CELL AND GENE THERAPIES: INNOVATIONS, CHALLENGES AND CLINICAL BREAKTHROUGHS 15 Cancer has claimed more than eight million lives worldwide each year since 2012.1 While mortality rates of major cancer types like lung, breast and colorectal have decreased, the overall number of cancer patients is not seeing a decline. Cell-based immunotherapy is an emerging and promising treatment using engineered immune cells such as cytotoxic T cells and natural killer (NK) cells to eliminate cancer cells.2 However, there are challenges for effective use of immune cells against solid tumors including the immunosuppressive tumor microenvironment and antigen escape by cancer cells. In addition, cancer cells can also construct a glycocalyx barrier against attack by patrolling immune cells and engineered cell therapies. A glycocalyx barrier is a carbohydrate or sugar coating that can biochemically downregulate the activities of immune cells and physically shield molecular interactions between immune cells and their target cancer cells. One can consider the glycocalyx as an armor of cancer cells. Mucins are an important component of the glycocalyx that, at high cell surface densities, can form a thick and brush-like layer to protect immune cells from being recognized by immune cells.3 Park and colleagues recently showed that enhanced stimulation of NK and T cells with chimeric antigen receptors (CARs) can improve cytotoxicity against mucin-bearing cancer cells.4 CARs are engineered proteins that enable immune cells to recognize specific cancer antigens and enable cancer cell destruction. In addition, by endowing the surfaces of effector immune cells with glycocalyx-editing enzymes such as mucinases, such immunoengineering approaches can overcome the glycocalyx armor of cancer cells. A model of glycocalyx armor To understand how the thickness of the glycocalyx layer protects cancer cells from destruction by immune cells, the authors created a breast cancer cell line that stably WOMEN IN SCIENCE Removing the Glycocalyx Armor of Cancer Cells for Treatment Andy Tay, PhD 16 Credit: iStock TECHNOLOGYNETWORKS.COM expresses a green fluorescent protein-mucin (GFP-Muc1) protein under the control of a tetracycline-inducible promoter. This meant that at higher doses of doxycycline (an antibiotic), there was stronger induction of mucin expression. The authors also made sure there was minimal cell-to-cell variability in Muc1 surface levels on their breast cancer cells. The higher the expression of GFP-Muc1, the weaker the killing efficacy by NK-92, a NK cell line which is being tested for anti-cancer therapy. However, removal of Muc1 ectodomain with StcE, a mucin-specific metalloprotease, and genetic deletion revoked the protection. Ectodomain is the domain of a membrane protein that extends into the extracellular space. They are usually the parts of proteins that initiate contact with surfaces, which leads to signal transduction. A change of glycocalyx thickness from ~100 nm to ~60 nm made a huge difference to whether NK-92 cells were able to eliminate the breast cancer cells. The authors further proposed that the mucin-rich glycocalyx can be modelled as a polymer brush whose properties can be tuned by glycosylation and crosslinking. For instance, breast cancer cells with mucins that are truncated and contain less glycosylation (at the O-glycan sites) were more vulnerable to NK-92-medicated cytotoxicity. Synergy between chimeric antigen receptors and glycocalyx-editing enzymes To assess the utility of engineered immune cells to eliminate cancer cells, the authors introduced NK-92 cells and primary T cells with CARs against human epidermal growth factor receptor 2 (HER2) and CD19. Interestingly, even at high GFP-Muc1 induction and expression levels, CAR NK and T cells were able to eliminate the cancer cells, suggesting that protection mediated by a thicker glycocalyx layer can be at least partially overcome with stronger receptor-mediated stimulation between CARs and their targets. To further enhance the cancer killing efficacy of CAR immune cells, the authors considered other immunoengineering approaches to enable immune cells to kill target cancer cells that express high levels of mucin. They first tethered the surfaces of NK-92 cells with StcE mucinase that is known to break down gut mucosal mucin barriers and found that surprisingly, StcE would readily and stably adhere to the surfaces of NK-92 cells for a prolonged period even after stringent buffer washing. Further experiments with a mutated mucinase i.e. StcE ΔX409, which was catalytically active but did not tether to the NK-92 cell surface after rinsing, found that there was no enhanced cytolytic ability, indicating the importance of tethering. When the authors performed brightfield imaging, they found that while both unmodified and StcE-modified NK-92 cells could form viable mechanical connections with cancer cells, a high density of GFP-Muc1 and cancerassociated microvilli persisted in the contact zone. On the other hand, with StcE-modified NK-92 cells, there were lower GFP-Muc1 levels and fewer microvilli, suggesting that surface-tethered StcE locally cleaved the mucin and disrupted cancer-associated microvilli. The synergistic effects of StcE-modifications with HER2 CAR NK-92 cells were demonstrated as the treated cancer cells had 50% (120 nm to 60 nm) reduction in glycocalyx thickness and there was close to 300% and 40% better cytolytic outcomes compared to unmodified NK-92 cells and HER2 CAR NK-92 cells, respectively. Facile method to equip immune cells with mucinase To improve the conjugation of StcE to the cell surfaces of NK cells, the authors finally developed a modular strategy by anchoring glycocalyx-editing enzymes onto surfaces of NK-92 cells through leucine-zipper-mediated coupling. First, the RR leucine zipper was genetically expressed onto the surfaces of NK-92 cells as a fusion protein with an ALFA-tag and sfGFP fluorescent marker to form ZIPNK-92 cells. StcE was also modified with an EE leucine zipper, enabling it to bind strongly to ZIP-NK-92 cells and cleave Muc1 from surfaces of cancer cells. "This is an interesting study that suggests a new angle for attacking cancer cells by trying to attack its the protective glycocalyx," Dr. Pakorn Kanchanawong, associate professor at the National University of Singapore, who is not involved in this paper said. "In terms of future therapeutic research, especially in vivo, I would imagine that as the glycocalyx is probably easily replenishable by CELL AND GENE THERAPIES: INNOVATIONS, CHALLENGES AND CLINICAL BREAKTHROUGHS 17 TECHNOLOGYNETWORKS.COM cells, very likely this will have to be pursued in conjunction with perturbing glycosylation mechanisms," he added. In conclusion, immunoengineering of immune cells with surface-displayed or secreted enzymes that can disrupt the glycocalyx armor of cancer cells is an attractive strategy for cancer treatment. This approach is particularly powerful if it can be used to specifically cleave cancer-associated mucins. Additionally, pharmacological and metabolic agents can be employed to disrupt the synthesis of glycocalyx in cancer cells, for synergy with CAR immune cell therapies. Such a combined strategy effectively leverages on advances in biomolecular and biophysical understanding of interactions between effector immune cells and their target cancer cells. ABOUT THE INTERVIEWEE Dr. Pakorn Kanchanawong is an associate professor at the National University of Singapore. His research interests include superresolution microscopy, optical bioimaging, force generation, force transmission and force sensing, and mechanobiology of cell–cell and cell–matrix interaction. REFERENCES 1. Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, Jemal A. Global cancer statistics, 2012. CA Cancer J Clin. 2015;65(2):87- 108. doi: 10.3322/caac.21262 2. Mellman I, Coukos G, Dranoff G. Cancer immunotherapy comes of age. Nature. 2011;480(7378):480-489. doi: 10.1038/nature10673 3. Paszek MJ, DuFort CC, Rossier O, et al. The cancer glycocalyx mechanically primes integrin-mediated growth and survival. Nature. 2014;511(7509):319-325. doi: 10.1038/nature13535 4. Park S, Colville MJ, Paek JH, et al. Immunoengineering can overcome the glycocalyx armour of cancer cells. Nat Mater. 2024;23(3):429-438. doi: 10.1038/s41563-024-01808-0 CELL AND GENE THERAPIES: INNOVATIONS, CHALLENGES AND CLINICAL BREAKTHROUGHS 18 While gene therapy has proven to be promising for diseases ranging from cancer to diabetes, the challenge of getting the right dose of genetic material into target cells has caused a bottleneck in the application of such therapies. In new research published in PLOS ONE, researchers from the University of Wisconsin–Madison have reported on the development of a technique employing electric pulses to make the human body more receptive to certain gene therapies. We spoke to two of the study authors, Professors Susan Hagness and John Booske, to learn more about the benefits of direct delivery of gene therapy materials, the challenges associated with gene therapy delivery and the use of electronic pulses to encourage uptake of genetic material. Q: What are the benefits of direct delivery of gene therapy material? A: Direct delivery may reduce the total dose needed for treatment because it eliminates the attrition of material during circulation through the body and other organs prior to arrival in the targeted tissue/organ (compared to, say, systemic, peripheral injection into a remote blood vessel). WOMEN IN SCIENCE Electronic Pulses Could Reduce the Need for High Doses in Gene Therapy Delivery Kate Robinson 20 Credit: iStock TECHNOLOGYNETWORKS.COM Systemic delivery typically requires large(r) doses to compensate for losses during passage through the circulatory system and other organs. Manufacturing the genetic material delivered via virus vectors is expensive – prohibitively so for many treatments of inherited metabolic diseases (such as hemophilia, diabetes, etc.). Reducing the required dose is critical to practical, affordable treatments. Direct delivery may also minimize the time the material spends in the circulatory system before uptake in the liver cells. This reduces the likelihood that the immune system will mount an attack on the gene therapy “foreign” material and destroy or inactivate it. The larger doses required with systemic, peripheral injection, along with the associated immunological response (e.g., cytokine storms) present a heightened risk. Direct delivery is a pathway to reduce the doses required and bypass the immune counter-response, thereby reducing cost and increasing safety. Q: What are the challenges of gene therapy delivery, and how do these affect patients? A: The challenges of conventional gene therapy delivery approaches (peripheral injection, e.g., in a remote blood vessel) include the requirements for large doses (to ensure enough of the dose finally arrives at the targeted site and is taken up by the targeted tissue cells) and the risk of immune counter-response to the injection of foreign genetic material. The former leads to a prohibitively expensive cost of treatment, making it impractical as a widespread treatment option. The latter includes loss of genetic material that may result in ineffective treatment or a dangerous overreaction that can threaten the health of the patient. Q: How does the application of electronic pulses increase the uptake of gene therapy material into hepatocytes? A: We are not certain what the specific underlying physical mechanisms are that result in enhanced uptake of the gene therapy material. We have some hypotheses, but no definitive identification of a mechanism currently. Our findings, reported in the PLOS ONE article, help to rule out some of the possible mechanisms. For example, we know that electric pulses do not modify the genetic material directly before it is absorbed into the cells, and we know that the electric pulses do not modify the culture medium or environment that the cells reside in. Future experiments are being designed and conducted to pin down the mechanism(s) that are at play. We know that electric pulses induce (nano)pores into the cell membranes, and this effect has been exploited in other investigations to permeabilize the membranes and allow small(er) molecules to be taken up by those cells. However, the larger size of the virus vector (AAV8) capsids used in our experiments lends doubt to the likelihood that this is the mechanism responsible for enhanced uptake in our experiments. It is a subject for further investigation. Q: What impact could efficient delivery of gene therapies have on patients in the future? A: Efficient delivery could make many gene therapy cures affordable and safe for a large population of patients. To elaborate, genetic mutation-based metabolic diseases significantly reduce the quality of life for hundreds of millions of people in the world. There are 100s of such diseases, including diabetes, cystic fibrosis, sickle cell anemia and hemophilia. Many of them involve the liver due to its central role in metabolism. Developed countries spend trillions of dollars each year on patient care, with nearly a trillion dollars spent annually on type 1 diabetes (T1D) alone. Cures for many of these diseases could be attainable if practical, cost-effective methods existed to modify the gene(s) of the liver cells, sufficient to correct the inherited metabolic discrepancy. Some success with systemic injection gene therapy has been reported, but only in small mammals or with prohibitive costs (~$1 M/treatment in the case of hemophilia). In other words, a famous statement in 1999 by Salk Institute Professor Inder Verma, one of the foremost recognized leaders in gene therapy, still remains relevant today: “There are only three problems in gene therapy: delivery, delivery, delivery.” CELL AND GENE THERAPIES: INNOVATIONS, CHALLENGES AND CLINICAL BREAKTHROUGHS 21 TECHNOLOGYNETWORKS.COM Q: What are the next steps in translating this research into clinical trials? A: Next steps include in vitro investigations of optimal electric pulse parameters and in vivo studies to determine how the phenomenon (which we have observed in vitro) manifests in living tissues. To date, we have only had the opportunity to investigate electric pulsing with a single choice of electric field strength and pulse length, and only with single pulses. An important question to answer is whether other treatment parameter combinations–i.e., different electric field strengths, different pulse lengths, and/or multiple pulses–produce a stronger effect. Of course, it is important to identify the maximum treatment parameter thresholds above which cell or tissue damage occurs, to ensure that parameter choices are always safely below those thresholds. Meanwhile, our experiments to date were conducted on cells in well culture plates (i.e., in vitro). Although research has shown that cells in vivo (in living tissues) can have similar responses to electric pulsing as cells in vitro, the magnitude of the responses and the parameter values that produce the maximum safe response can be expected to be different. So, experiments with animal models are the next critical step, especially with larger mammals. Q: Do you have plans to explore the use of electronic pulses in the delivery of gene therapies to other cell types? A: The potential impact of translating this to clinical practice in liver/hepatocytes has considerable impact value, so that is our primary focus for now. But certainly, expanding the scope of these investigations is of interest in the longer term. ABOUT THE INTERVIEWEES John Booske is the Keith and Jane Morgan Nosbusch emeritus professor in electrical and computer engineering at University of Wisconsin–Madison. His research focuses on plasmas, metamaterials, metasurfaces and media that have a strong interaction with electromagnetic radiation, electromagnetic field effects and microwave vacuum electronics. John holds a PhD in Nuclear Engineering from the University of Michigan. Susan Hagness is the Philip Dunham Reed Professor and department chair of electrical and computer engineering at the University of Wisconsin–Madison. Her group’s research spans computational and experimental applied electromagnetics, with an emphasis on bioelectromagnetics. Susan golds a PhD in Electrical Engineering from Northwestern University. CELL AND GENE THERAPIES: INNOVATIONS, CHALLENGES AND CLINICAL BREAKTHROUGHS 22 There are over 6,000 genetic disorders and the list grows continually. Gene therapy works to treat or prevent disease of monogenic cause by modifying an individual's genes. In 2012, alipogene tiparvovec was the first to receive European regulatory approval for treating lipoprotein lipase deficiency by delivering a functional copy of the enzyme to muscle cells using an adeno-associated viral (AAV) vector. This approval (now withdrawn due to low demand) was followed closely in 2017, in the US, by voretigene neparvovec, for treating Leber congenital amaurosis, an inherited eye disease of progressive blindness, in biallelic carriers of mutations to the RPE65 gene. Since these landmark approvals, there has been several additional approved gene therapies. Aside from treating monogenic disorders, AAV-based gene therapies have also been approved for treating certain cancers, through engineered chimeric antigen receptor T cells, and they are being explored against infectious diseases via delivered recombinant antibodies. The gene therapy landscape is dynamic and constantly shifting. The technology holds the promise of potential disease modification, providing life-altering benefits for patients that otherwise would have faced dire outcomes. In the midst of this shifting landscape, the gene therapy field has celebrated victories and faced obstacles. Herein, we will review major advances in disease modification for illnesses that presently still lack effective treatments, as well as some challenges and approaches to overcome them. Gene therapy: Panacea for neuromuscular and neurodegenerative disease? Neuromuscular diseases (NMDs) and neurodegenerative diseases (NDDs) are generally progressive illnesses that often shorten lifespan. Unfortunately, many also lack disease-modifying therapies. This has made NMDs and NDDs of genetic etiology prime candidates for gene therapy, by addressing the root cause of disease, i.e., the WOMEN IN SCIENCE Gene Therapy: A New Frontier in Disease-Modifying Therapies Masha G. Savelieff, PhD 23 Credit: iStock TECHNOLOGYNETWORKS.COM defective gene. The 2019 approval by the US Food and Drug Administration for onasemnogene abeparvovec, an AAV therapy for spinal muscle atrophy (SMA), marked a turning point in the treatment of NMDs. “SMA is a NMD caused by mutations to the gene SMN1, which codes for survival of motor neuron, a protein essential to neuronal health,” explained Kathrin Meyer, assistant professor at the University of Missouri, who was involved in the preclinical and clinical studies that led to the approval of onasemnogene abeparvovec. SMA patients exhibit gradual loss of motor neurons, leading to progressive muscle weakness, although the rate of decline varies across individuals. In the most severe cases, respiratory failure occurs. “The gene therapy treatment for SMA involves delivering a functional copy of SMN1 using an AAV9 carrier by intravenous injection. In the clinical trial, recipients experienced improved functional and survival outcomes compared to natural history data of the disease progression,” Meyer elaborated. Meyer is presently assessing the feasibility of gene therapy for additional NMDs and NDDs. One focus is Batten disease and the family of neuronal ceroid lipofuscinoses, which are NDDs caused by mutations to lysosome storage genes called ceroid-lipofuscinosis neuronal (CLN). “We found that delivery of AAVs carrying a functional CLN copy improves lifespan and brain and behavioral pathology in CLN6 and CLN8 animal models of Batten disease,” Meyer summarized of this line of work. Guangping Gao, director of the Horae Gene Therapy Center at the University of Massachusetts Medical School, is also actively researching gene therapy as a potential treatment avenue for NMDs and NDDs. “We are especially interested in Canavan disease, a fatal rare recessive NDD caused by loss-of-function mutations to aspartoacylase (ASPA),” Gao described of his research. Loss of aspartoacylase activity prevents breakdown of N-acetylaspartate, which accumulates in the central nervous system and interferes with neuron myelin sheaths. Disease onset occurs in infancy and manifests with poor motor function with reduced head control, lack of visual fixation and muscular hypotonia, which develops to include spasticity, ataxia and seizure. “We have shown that AAV-mediated delivery of a functional ASPA copy to ASPA–/– mice drastically improves neuropathology, myelination and motor function. Moreover, using our Canavan disease model and gene therapy, we have been able to shed insight into disease pathophysiology, such as peripheral involvement, would could help further refine our therapeutic approach,” Gao explained. Gene therapy can replace genes with defective loss-offunction mutations with a functional copy of the gene. Alternatively, the AAV vehicle can be used to deliver short hairpin (shRNA) or interfering RNAs (RNAi) to knock down expression of a gene with toxic gain-of-function mutation. This is a viable approach, for example, for cases of the motor neuron disease amyotrophic lateral sclerosis caused by mutations to the gene superoxide dismutase 1 (SOD1). In animal models of ALS, AAV-mediated delivery of shRNA or RNAi slows disease progression in SOD1G93A mice. Rather than targeting a specific mutation, gene therapy can also be used to support damaged tissues, e.g., delivering a copy of gene that strengthens muscles to treat NMDs with muscle weakness, or a growth factor to regenerate injured nerves. Ultimately, a gene therapy with a base-editing CRISPR-Cas cargo could correct genes, with either lossor gain-of-function mutations. Gene therapy has already celebrated some successes, with approved therapies that tangibly improve outcomes for patients, and potential developments in place for additional diseases. Like all technologies, however, it also faces some obstacles. Overcoming the immune response A prominent challenge facing gene therapies is that AAVs and their transgene cargo can elicit an immune response, innate or adaptive, which diminishes transduction efficiency and long-term gene expression and increases the chances of adverse events. “Ideally, gene therapy was envisioned as a one-shot treatment, but in practicality, re-dosing is likely needed depending on the target tissue and age of treatment. Moreover, although AAVs are quite effective, high systemic doses are sometimes needed. Both these factors potentially provoke adverse events associated with AAV and transgene immunogenicity,” Gao discussed. CELL AND GENE THERAPIES: INNOVATIONS, CHALLENGES AND CLINICAL BREAKTHROUGHS 24 TECHNOLOGYNETWORKS.COM One route is to change the injection method for AAV administration. “It is possible to reduce a systemic immune response by employing intrathecal injection. Although this delivery route is safer, local immune responses in highly targeted regions, so called “hotspots” can still be of concern,” Meyer explained. “However, this injection method preferably targets central nervous system tissue, such as the brain and spinal cord, which will not be the optimal tissue target for many diseases.” Another option is to eliminate preexisting AAV antibodies, resulting either from prior AAV gene therapy doses or from natural infection to wild-type AAV, which is endemic to humans. “There are several ways to accomplish this,” Gao outlined. “Pharmacological agents, for instance rituximab to deplete B cells, or physical methods, such as plasmapheresis and AAV serotype-specific IgG absorption to remove circulating antibodies. We can also block the innate response through complement deactivation with antibodies against complement components, such as anti-C5 eculizumab.” Moreover, it is possible to engineer the AAV capsid, vector genome and transgene, e.g., by “cloaking”, to lower their immunogenicity. The transgene expression from AAV vector can also be engineered to evade immune cells, such as antigen presenting cells (APCs), an arm of the adaptive immune response. Gao developed a microRNA (miR)- based strategy to evade APCs. In a proof-of-concept study using an AAV expressing the highly immunogenic ovalbumin, Gao demonstrated that incorporating the target sites of the APC-specific miR142 and other miRNAs into the transgene detargeted ovalbumin expression from APCs, i.e., they did not display ovalbumin antigens, blunting their immune response against it. In tandem, the cytotoxic T-cell response against ovalbumin was also blunted. More recently, Gao extended this approach by evaluating combinatorial microRNAs to better block the APC and cytotoxic T-cell response. Towards tailored tissue targeting and transduction Another major goal of gene therapy research is improving AAV tissue specificity and augmenting tissue transduction, i.e., that the AAVs target the intended tissue and disseminate widely across it. Wild-type AAVs occur in several serotypes, distinct variations in the capsid, that differ in their tissue specificity and ability to cross the blood–brain barrier, impacting their tissue targeting and transduction efficiency. “Widespread transgene delivery is a major challenge,” Meyer elaborated. “There are AAVs, such as AAV9, with good ability to cross the blood–brain barrier and access the brain and spinal cord, which are optimal target tissues for NMDs and NDDs.” Using intrathecal AAV9 injection, Meyer successfully showed widespread transgene expression in mouse and nonhuman primate animal models, which achieves up to 80% targeting to spinal motor neurons as well as across various brain areas. Transduction can be further enhanced by placing the animal in the Trendelenburg position – lain flat on the back with the head tilted 15 to 30 degrees below the feet. “Although we’ve developed injection methods that enhance transgene distribution across neural tissue, penetration to the deep layers remains relatively limited, but we are actively researching better methods,” Meyer added. An alternative to wild-type AAVs is to rationally engineer or evolve novel capsids with distinct or improved tissue targeting and blood–brain barrier and trans-vascular penetrance. “Improved capsid specificity and transduction efficiency will mean lower doses, which will mean fewer possible complications arising from immune responses to high dose AAV gene therapy,” Gao explained. “Further refinement may be achieved by tissue-specific codonoptimized and CpG motif-reduced transgenes, to enhance translation within the target tissue versus other tissues and blunt innate immune response.” Gene expression in moderation and in context Another facet of gene therapy research is modulating transgene expression. The overarching goal is stable and durable expression, but to levels commensurate with physiological expression. “You need to be able to physiologically regulate transgene expression because uncontrolled expression, even of endogenous proteins, becomes toxic at excessive levels,” Gao elaborated. “Furthermore, you want to be able to turn some gene therapies off. For example, if using an AAV vehicle to deliver CRISPR-Cas cargo, you don’t want the Cas nuclease CELL AND GENE THERAPIES: INNOVATIONS, CHALLENGES AND CLINICAL BREAKTHROUGHS 25 TECHNOLOGYNETWORKS.COM expressed in perpetuity because it will increase off-target cleavage and cause unintended genomic damage.” Meyer suggested that the field needs to understand expression of the target gene in its native environment better. “Animal models are great and have helped us test the efficacy of potential gene therapies for rescuing disease phenotypes. However, oftentimes we don’t understand the disease mechanisms in detail. On paper it looks simple; it seems like you might need to deliver a functional gene copy for diseases with loss-of-function mutations, and deliver a shRNA or RNAi for diseases with gain-of-function mutations. In practice, it isn’t always this straightforward.” In amyotrophic lateral sclerosis, for example, the most common known monogenic cause are repeat expansions to the gene C9orf72, which cause both toxic gain-offunction as well as haploinsufficiency and loss-offunction. “Generating models using patient-derived cells allows us to investigate disease pathophysiology and gene expression patterns of genetic mutations in context, and to dissect contributions from various cellular compartments. Failing to understand the complex mechanisms in context in patient cells could lead to patients not responding to a gene therapy.” A hefty price tag Although gene therapies have profoundly improved outcomes for some patients, their hefty price tags limit access. Moreover, some NMDs and NDDs are caused by rare mutations, so patients are not always diagnosed early. “There are three major hurdles that are currently impacting translation of gene therapies to patients,” Meyer explained. “On the first front, we need to streamline manufacturing, which is currently expensive and variable. On the second front, regulatory aspects are difficult to navigate because methods to assess gene therapies, such as dosage, are not standardized, making it problematic to compare efficacy between different gene therapy products. This was the scenario with therapeutic antibodies a while back, which has since been streamlined, so we should be able to achieve the same for gene therapies. On the third front, we need to make sequencing technologies more accessible to patients with rare diseases to identify mutations and diagnose patients earlier. We will need to be able to treat patients earlier, pre-emptively, before too much tissue damage has already occurred to truly modify the disease course.” Gao echoed similar sentiments regarding manufacturing when asked about future directions for gene therapy. “I think if we look to the future, we have to identify what the current problems are, the challenges we are presently facing,” he replied. “If we are to bring gene therapies to patients more readily down the drug development pipeline, we will need to develop better manufacturing platform technologies and lower costs.” ABOUT THE INTERVIEWEES Kathrin Meyer is an assistant professor in the department of physical medicine and rehabilitation at the University of Missouri. She studied molecular and cellular biology at the University of Bern, Switzerland followed by a post-doctoral fellowship at Nationwide Children’s Hospital, Columbus Ohio, USA developing different therapeutic approaches for spinal muscular atrophy. Guangping Goa is a professor in microbiology and physiological systems, the director of the Horae Gene Therapy Center and the Viral Vector Core and the Penelope Booth Rockwell Professor in Biomedical Research at the University of Massachusetts Medical School. CELL AND GENE THERAPIES: INNOVATIONS, CHALLENGES AND CLINICAL BREAKTHROUGHS 26 Biopharmaceuticals – also known as biological therapies or biologics – are usually derived from some form of living system, be it cell culture, animal products or human cell or tissue samples. They are complex and expensive to produce and highly vulnerable to degradation and contamination, requiring careful analysis during research, development and manufacturing stages to ensure quality and integrity. In this article we highlight some of the analytical techniques employed in biopharmaceutical analysis, and how the field is evolving to meet new challenges. . Biopharmaceutical characterization and quality The field of biopharmaceuticals not only includes antibody-based drugs, but also the expanding fields of cell and gene therapies. Across the spectrum of biologics, the most common analytical technologies for characterizing biopharmaceuticals are mass spectrometry (MS) and liquid chromatography (LC), either alone or in tandem. “Some of the most important properties you’re testing for with biologics are post-translational modifications (PTMs) and protein aggregation, said Jared Auclair, director of bioinnovation at the Biopharmaceutical Analysis and Training Laboratory (BATL), Northeastern University, USA. “For PTMs, you might use reverse-phase LC in tandem with MS, whereas size exclusion chromatography might be used to detect aggregation in the development of antibody-based drugs.” However, although there exists an entire toolbox of methods required to characterize protein and antibodybased products, method development for cell and gene therapy is lagging, says Auclair. “I liken it to different stages of construction of a house: for antibodies, the house is built, albeit not 100% complete, and you can live in it. For gene therapies, we have a basic structure, but for cell therapies, we’ve only just got the building blocks. Cell-based therapies are about 5–10 years behind where antibody-based biologics are in terms of analytics.” WOMEN IN SCIENCE Advances in Biopharmaceutical Analysis Joanna Owens, PhD 27 Credit: iStock TECHNOLOGYNETWORKS.COM What’s universal to all biopharmaceuticals, though, is that whichever characterization method is used, it needs to be carried through the entire development process from research to commercialization. “This doesn’t only include collecting the same data, if you’re going to monitor PTMs, for example, you should be using as close to the same instrumentation as possible,” said Auclair. “And if the quality of the product is paramount to its efficacy, then continued monitoring is key and we need to be looking at opportunities to monitor in real-time, so we can kill products that are below quality before they advance too far towards commercialization.” Process innovation for biopharmaceuticals While developing and improving methods to monitor impurities is a major focus of the research at BATL, it sits alongside a focus on process analytics. Here, method development goes hand-in-hand with engineering to determine the pain points of biopharmaceutical production processes and find solutions, such as new in-line sensors that can monitor different aspects of the process. But there are challenges. “One of the major challenges is integration, especially where different equipment manufacturers and software providers are involved. The technologies are there, but it’s just a matter of implementation and automation,” said Auclair. “In our lab, we have instrumentation that pulls samples from the bioreactor so we can test different quality checks and try to make that process better and more robust.” Another headache is data analysis. “The hardware is always ahead of the data analysis tools, in terms of capability, speed and ease of use,” says Auclair. “In the research space, it’s OK to have a really complex process, but as you move down the commercialization space, you need technologies and processes that don’t require 30 years’ experience in informatics to run.” One area that is gaining momentum is the multi-attribute method (MAM), an MS-based protocol that monitors several properties, such as PTMs, sequence coverage and new peak detection, in the same experiment.1 Auclair’s team is looking at how they can optimize MAM as development processes change, how to integrate it inline with bioreactors and how to apply the approach to gene and cell therapies. “The ultimate goal of our work is to innovate these product development and commercialization processes, so that more good medicines are discovered and don’t fail because the quality of the product wasn’t considered throughout the process.” Microbial monitoring in biopharmaceutical development Another major area of biopharmaceutical analysis is monitoring for contamination by microbes and viruses. It is essential to ensure that biopharmaceuticals are not contaminated with pathogenic or environmental bacteria, CELL AND GENE THERAPIES: INNOVATIONS, CHALLENGES AND CLINICAL BREAKTHROUGHS 28 “In drug development, especially academic drug development, many people don’t think about product quality from the beginning. And if you don’t think about that from the beginning then your product doesn’t get to market, it dies in the valley of death,“ says Auclair. TECHNOLOGYNETWORKS.COM fungi or viruses, which, if introduced even in small amounts, can potentially multiply to high levels in the bulk material during manufacturing or in the final drug product. Pathogens can potentially be introduced via cell lines, raw materials, use of contaminated human or animal products during manufacture or from the environment – such as human contact during sampling.2 Many recombinant protein therapeutics, vaccines and plasma products are produced using cell culture, which is susceptible to contamination. Although a rare occurrence, this can reduce the yield of the drug, affect the quality of the end product and can have considerable financial costs for industry and potentially serious safety implications for patients.3 “As the manufacture of biomedicines is a complex, multi-step process, typically involving potentially non-sterile biological ingredients, a sound microbial and viral safety strategy is required,” said Dr. Oleg Krut, head of the microbial safety section at the Paul-Ehrlich-Institut, Federal Institute for Vaccines and Biomedicines, Germany. “The use of sensitive methods to detect bacteria or fungi themselves is an integral part of this strategy. All ingredients must be tested using the most sensitive assays to ensure that any microbes and viruses are detected before use and that contaminated material will not be used during manufacture.” Bacteria and fungi are usually detected by growth-based methods, that is, detection of turbidity in liquid microbial culture or the enumeration of colonies on agar plates. “The main methodological challenges are the time required to obtain a valid result (up to 14 days), and the high sensitivity required to detect a single microorganism,” explains Krut. “A further challenge is that introducing more advanced microbiological techniques that maintain the highest sensitivity with shorter time-to-result is slow, because of the necessary validation requirements.” Viruses have traditionally been detected by inoculating samples into animals or indicator cell cultures that are monitored for signs of infection.2,3 However, only a limited range of viruses can be detected through animal testing.3 “Replacement of animal testing is being considered through the revision of regulatory guidelines such as ICH Guideline Q5A(R2) on viral safety evaluation of biotechnology products derived from cell lines of human or animal origin, in line with the 3R principles for animal welfare,” said Dr. Johannes Blümel, head of the viral safety section, at the Paul-Ehrlich-Institut, Federal Institute for Vaccines and Biomedicines, Germany. “The use of indicator cells is still a very valuable method for testing cell cultures for viral contamination as it has an excellent sensitivity for many viruses. However, the range of viruses that can be detected is still limited by the ability of the indicator cells to support viral replication and to show signs of infection that are easily visible under a light microscope.” Virus testing using indicator cells is complemented by nucleic acid amplification technologies such as polymerase chain reaction (PCR). However, viral variants or newly emerging viruses may pose a threat. “Viruses can be specifically detected by very sensitive methods if their genomes or host cells are known,” said Blümel. “However, although PCR has excellent sensitivity, the technology remains limited towards detection of specific viruses. A battery of several different PCR tests therefore needs to be designed according to a risk assessment of the virus species that could contaminate.” Advances in microbial monitoring methods There have been two major trends in microbial safety in recent years, says Krut. “The first is the automation of classical cell culture methods, such as automated liquid culture and automated colony counting. These improvements shorten the time to results, reduce handson time and operator bias and increase throughput and data integrity. As they are modifications of classical methods, they are easier to validate.” The second trend is the emergence of rapid microbiological methods (RMMs), such as nucleic acid-based techniques, adenosine triphosphate (ATP)-based contamination detection, solid-state cytometry and MS. “Unlike classical microbial culture, which takes two weeks, RMMs can detect microbial contamination within days or even hours,” says Krut. “Such methods are needed for biomedicines with short shelf-lives where classical methods are simply too slow. However, they require extensive validation, which is often seen as a major barrier to their implementation in biopharmaceutical production.” CELL AND GENE THERAPIES: INNOVATIONS, CHALLENGES AND CLINICAL BREAKTHROUGHS 29 TECHNOLOGYNETWORKS.COM Dr. Krut’s team is evaluating culture-independent flow or solid-state cytometry for bacterial detection and enumeration, which it is hoped will allow faster time to results, more rapid Official Medicines Control Laboratories (OMCLs) provision of certificates and earlier governmental batch release of biomedicine batches to the market. In viral safety, using next-generation sequencing (NGS) or high-throughput sequencing (HTS) in a metagenomic approach (i.e. detecting all viral sequences in a sample) has the potential to overcome the limitations of current PCR-based assays.4 Research at the Paul-EhrlichInstitut is focused on optimizing HTS technology for the sensitive detection of human viruses in blood samples. “Our multidisciplinary team of medical virologists, molecular biologists and bioinformaticians is particularly interested in reducing host cell background and optimizing library construction in order to increase the sensitivity of the method, as well as in applying artificial intelligence and machine learning methods to advance the bioinformatics pipeline further,” says Blümel. “Our method allows us to address any type of emerging agent, using technology that meets regulatory requirements, and will ensure we keep pace with emerging viruses and are prepared.” ABOUT THE INTERVIEWEES Jared Auclair is the director of bioinnovation and the Vice Provost Research Economic Development at Northeastern University. He holds a PhD in Biomedical Sciences from the University of Massachusetts. Oleg Krut is head of the microbial safety section at the PaulEhrlich-Institut, Federal Institute for Vaccines and Biomedicines, Germany. REFERENCES 1. Millán-Martín S, Jakes C, Carillo S, et al. Multi-attribute method (MAM): An emerging analytical workflow for biopharmaceutical characterization, batch release and cGMP purity testing at the peptide and intact protein level. Crit Rev Anal Chem. 2023;1-18. doi :10.1080/10408347.2023.2238058 2. Valiant WG, Cai K, Vallone PM. A history of adventitious agent contamination and the current methods to detect and remove them from pharmaceutical products. Biologicals. 2022;80:6-17. doi:10.1016/j.biologicals.2022.10.002 3. Barone PW, Wiebe ME, Leung JC, et al. Viral contamination in biologic manufacture and implications for emerging therapies. Nat Biotechnol. 2020;38(5):563-572. doi:10.1038/s41587-020- 0507-2 4. Khan AS, Mallet L, Blümel J, et al. Report of the third conference on next-generation sequencing for adventitious virus detection in biologics for humans and animals [published online ahead of print, 2023 Jul 19]. Biologicals. 2023;83:101696. doi:10.1016/j. biologicals.2023.101696 CELL AND GENE THERAPIES: INNOVATIONS, CHALLENGES AND CLINICAL BREAKTHROUGHS 30 CRISPR is a powerful technique that can enhance your research. If you are getting to grips with a CRISPR protocol for the first time, optimizing and planning your experiment can make things easier and can be the key to a successful CRISPR experiment. Optimize CRISPR delivery in your chosen cell line With every new cell line that we use in the lab, we first do an optimization experiment. We trial a few different transfection conditions (lipofection, nucleofection, etc.) delivering Cas9 and a control guide RNA that we know consistently work in different cell types. We routinely use a guide targeting the AAVS1 locus for human cells and tubulin beta for mouse cells. Then, we quantify the editing efficiency using PCR, Sanger sequencing and inference of CRISPR edits (ICE) analysis and determine the best possible delivery method for the cell type. Surprisingly, there is a lot of variation in CRISPR efficiency between delivery methods and cell lines. You might think that this is too time-consuming, but ultimately you will be saving time and money. At the end of a CRISPR optimization experiment, you will have a protocol specific for your cell line that you can use to CRISPR different regions of the genome. Most labs work only with a handful of cell lines, usually derived from similar tissues making this step a valuable resource for future work. Design your experiment - CRISPR knockout Once you have established the best CRISPR delivery for your cell line of interest, you can select your desired modification. CRISPR knockouts are the easiest experiments, and they require two components: Cas9 protein and guide RNA. When I first started doing CRISPR cell lines, we used to deliver Cas9 and guide RNAs as plasmids. Now, we WOMEN IN SCIENCE How To Run a Successful CRISPR Experiment Samantha Carrera, PhD 31 Credit: iStock TECHNOLOGYNETWORKS.COM know that the most efficient CRISPR method is using Cas9 proteins and chemically synthesized guide RNAs, which are preassembled into a ribonucleoprotein (RNP) and delivered into the cells.1 Even though they are more expensive to buy, it will be more cost-effective in the end as you will save time and effort not having to repeat your experiment. The most important part to design is the guide RNA; there are numerous websites where you can input your sequence of interest, and they will give you a list of guide RNAs that you can use. The tricky part is deciding which one to choose. First you must decide which exon of your gene you want to target. I recommend avoiding the first exon if possible. When you create insertions/deletions (indels) here, the splicing machinery can skip the exon and still produce a truncated version of the protein.2 We usually pick exons that are as upstream as possible and that are common to all isoforms. The most common modification introduced by Cas9 is +/- 1 bp so directing a guide to exons with an asymmetric splicing phase makes it more likely for the transcript to be knocked out of frame. With every CRISPR design you should pick guides that have low off-targeting potential. Even though some guides are labelled as inefficient, it is difficult to predict the efficiency of the guide RNA. For this reason, I would recommend designing three guides and delivering them in parallel alongside the control guide RNAs (either AAVS1 or tubulin). Once you have transfected cells for a gene knockout, you can isolate genomic DNA and use primers designed around the cutting area to determine the percentage of indels you have in your cell population using Sanger sequencing and ICE/TIDE analysis. Once you have determined this, you will have to do some functional validations to determine whether your protein of interest has been knocked down; this can be done by western blotting. Design your experiment - CRISPR knock-in I believe that CRISPR knock-ins are where you really realize how powerful this technique is. You can add functional tags to your gene of interest, including fluorescent proteins, luminescent reporters, proximity labelling enzymes, degron tags and pull-down tags. You can also insert transgenes in safe harbor loci, so-called because they aren’t easily silenced. These transgenes are used in technology platforms that allow you to control the expression of your protein of interest by adding small molecules. These include degron systems and tetOn/off compatible cells. You can also introduce point mutations into your gene of interest or into enhancer sequences to study gene regulation. A CRISPR knock-in experiment is harder to plan than a knockout experiment and decisions need to be made about the site of insertion and the need for additional tags and linker sequences. If you are introducing functional tags you might need to first assess if N- or C- terminal tagging is better for preserving the function and expression of your protein. Once you have decided this, you must design a guide that is close to the site (within 20 bp) where you want your insertion. The next step is to design your homology directed repair (HDR) template, which will contain your insert of interest flanked by homology arms. The homology arms are sequences of DNA that correspond to your gene of interest on either side of your insertion site. It is worth remembering that the larger the insert, the more inefficient the HDR process is going to be and the longer homology arms you will need. If your insertion is small (<100 bp) the best thing to use is a single-stranded DNA template with ~80 bp-long homology arms on each side. These are commercially available and usually have modifications that make them more likely to get integrated in the genome. For larger insertions we prefer using plasmids where we clone the homology arms and our insert. The homology arms in this case are around 800 bp. It is important to make sure to mutate the protospacer adjacent motif (PAM) site in the HDR template to avoid Cas9 cutting it (causing insertions or deletions). Once you have designed your guides and your template, you can use your optimized protocol to deliver them into your cells and create a polyclonal population. At this point, you will have many cells that have not been modified and it is worth spending some time considering how to enrich your pool of cells with your desired modification. CELL AND GENE THERAPIES: INNOVATIONS, CHALLENGES AND CLINICAL BREAKTHROUGHS 32 TECHNOLOGYNETWORKS.COM Enrich your modification The next thing that you need to consider is if a polyclonal cell line is sufficient for your experiments or if you need to go through the labour-intensive process of generating a monoclonal cell line. If your desired edit contains a sortable marker, like a fluorophore, you can first enrich your population of cells by fluorescence-activated cell sorting (FACS). This will result in a pool of cells that are slightly different from each other, but which all express your fluorophore. It is good to bear in mind that the levels of fluorescence will depend on the expression of your gene of interest, so it might be hard to distinguish a positive population if the levels are too low. Another way of enriching your population is by introducing an antibiotic selection cassette and treating the cells with that antibiotic. This will result in the elimination of the cells without the insert, leaving behind a positive pool of cells. Sometimes you don’t want your protein to have a fluorophore or an antibiotic cassette that could potentially interfere with its function. In this case, we co-integrate a removable antibiotic selection cassette which allows us to select for HDR-positive cells. A promoter and the desired antibiotic are flanked by loxP sites that allow their removal once the cells have been verified by expressing Cre-recombinase (Figure 1). With the fluorophores you can introduce a cleavable signal like T2A, in that way your protein of interest and the CELL AND GENE THERAPIES: INNOVATIONS, CHALLENGES AND CLINICAL BREAKTHROUGHS 33 HOW TO RUN A SUCCESSFUL CRISPR EXPERIMENT 3 ppysites that allow their removal once the cells have been verified by expressing Cre-recombinase (Figure 1). With the fluorophores you can introduce a cleavable signal like T2A, in that way your protein of interest and the fluorophore will be cleaved when transduced, making it less likely that the insertion interferes with the function of the protein of interest. Gene of interest gRNA_1 5'H 3'H gRNA_2 Cas9 HDR template Gene of interest 5'H with PAM mutations mScarlet-I 3'H 5'H with PAM mutations mScarlet-I PGK promoter bGH poly(A) signal PuroR 3'H Cre recombinase 3'H IoxP IoxP T2A 7500 IoxP 7500 10,000 2000 3000 Figure 1: CRISPR knock-in enrichment by removable antibiotic selection. Cas9 is delivered into the cells along with two guide RNAs (gRNA) and an HDR template. The HDR template includes a Puromycin resistance cassette that will allow the selection of cells that had integrated the fluorophore (mScarlet-I). Once the cells have been selected, the antibiotic selection cassette can be removed by the addition of Cre recombinase. Credit: Technology Networks TECHNOLOGYNETWORKS.COM fluorophore will be cleaved when transduced making it less likely that the insertion interferes with the function of the protein of interest. If you want to produce monoclonal cell lines you can use your enriched population and either FACS sort into single cells or use the serial dilution method. This method consists in diluting your cells until you have one cell for each 100 µl and seed this volume into a single well on a 96-well plate. It is important to note that not all cell lines are happy growing as single cells and you will struggle to create monoclonal populations out of these cell lines. In my experience with these cells, it is worth seeding the single cells into conditional media – this is media taken from a growing population of cells that has been filtered to avoid carrying any cells but with the advantage of carrying any secreted proteins. Finally, you need to verify that your cells have the desired modification. For this, there are a few things you must consider. First, designing your primers outside of the homology arms to discard the possibility that your template has integrated in another area of the genome. You also must remember that your PCR will preferentially amplify any unmodified alleles, as it requires less time to copy a smaller DNA product than a bigger one. I usually design a handful of primers including internal ones and insert-specific ones that I can use in different combinations. This will give a good indication that the cells have the desired insert in them. I will always send these PCR products for sequencing to check that there are no insertions, deletions or mutations that will interfere with the expression of my gene. You will also need to do some functional validations, for example using western blotting to see if your protein has a size shift that corresponds to your insert. Conclusion Creating CRISPR cell lines doesn’t have to be difficult process. If you take advantage of the tips above you can create all the tools you need to advance your research opening different possibilities for studying your prefered cellular pathway. With all these things in mind, enjoy CRISPRing! REFERENCES 1. Kim S, Kim D, Cho SW, Kim J, Kim JS. Highly efficient RNAguided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res. 2014;24(6):1012-1019. doi: 10.1101/gr.171322.113 2. Sharpe JJ, Cooper TA. Unexpected consequences: exon skipping caused by CRISPR-generated mutations. Genome Biol. 2017;18(1):109. doi:10.1186/s13059-017-1240-0 CELL AND GENE THERAPIES: INNOVATIONS, CHALLENGES AND CLINICAL BREAKTHROUGHS 34 Like many young girls in elementary school, Victoria Gray wanted to be a cheerleader, until she was told by her doctor that this simply wouldn’t be possible; the exertion placed on her body by the training regimen could have devastating consequences. “That was my first disappointment as a kid,” she recalls. Sadly, it wouldn’t be her last. Tall for her age, Victoria later thought about trying out for a spot on the school basketball team. Her uncle had a great love for the game, which inspired her. Plus, everyone around her said she would probably have a natural knack for shooting hoops. Why not give it a go? She thought. But once again, her enthusiasm was shot down by another firm “no” from her pediatrician. WOMEN IN SCIENCE Breaking the Chains: How CRISPR Gene Therapy Gave Victoria Gray a New Life Molly Coddington 35 “My life was constantly full of ‘noes’, and people telling me ‘you can’t do this’ – it was limitation after limitation,” she says. “I had to alter everything I dreamed about, every step of the way from childhood to adulthood.” TECHNOLOGYNETWORKS.COM If you’re lucky enough to grow up having a healthy childhood, it’s hard to imagine the cruel realities of experiencing a sick one. Victoria describes the motions of her life as though it was a distressing movie being played out in front of her, and someone – somewhere – kept hitting the pause button. Her days were shaped not by the whimsical imagination of a young child, but by her illness. That’s because at just three months of age she had been diagnosed with sickle cell disease (SCD). SCD is a group of inherited blood disorders that cause red blood cells to become hard and sticky. In healthy individuals, red blood cells are a disc shape, allowing them to flow easily through our blood vessels. In SCD, they form a “C” shape known as a sickle, which causes the blood cells to die quickly or cause blockages in blood vessels. According to the National Institutes of Health, over 20 million people worldwide are affected by SCD. SCD stole Victoria’s childhood, including her right to an education. “I had to alter everything I dreamed about every step of the way from childhood to adulthood. When I started college, I wanted to be a cardiologist. But doctors explained that the stress involved with studying medicine wouldn’t be good for me. So, I put myself on pause – once again,” she says. Undeterred and eager to help care for others, Victoria started to explore a nursing degree, until “I had one the worst pain crises of my life,” she says. Crises refer to acute conditions, such as the blockage of a blood vessel by sickled cells, caused by SCD. They are the main clinical hallmark of the disease, causing severe, debilitating pain and extreme fatigue, among other symptoms. Oftentimes, they strike at random, and can persist for long periods of time requiring extended stays in hospital. “That crisis put me in the hospital for three months. I lost the ability to use my arms and my legs,” Victoria describes. Perhaps most devastatingly, she says, “I also lost my ability to dream.” After undergoing comprehensive rehabilitation, Victoria eventually regained her strength and her ability to walk. But despite the physical improvements, mentally, she had to resign herself to the fact that a career maybe wasn’t on the cards for her. It was yet another blow, but one that she handled with the grace and determination she carried from a young age to find fulfillment in life. Victoria chose to focus her time, and the energy she could muster between crises, on being a wonderful mom to her four children, and a strong partner to her husband, Earl. Then, in 2018, an opportunity came around that finally awarded Victoria the opportunity to say “yes” for the first time in her life. It was, she would come to learn, an opportunity that marked not only a major milestone in the history of medicine, but one that freed her from the chains of SCD – most likely, forever. Finally, a “yes” for Victoria Patients with SCD are often prescribed drugs, such as painkillers, to try and curb the symptoms associated with crises. These medications fail to target or cure the underlying cause of the disease, and while they offer symptomatic relief, they can have harmful side effects. There are few authorized therapeutics on the market for SCD patients. In 2018, Victoria, who lives in Forest, Mississippi, was under the care of Dr. Haydar Frangoul, waiting to learn of her eligibility for one such treatment – a haploidentical “haplo” stem cell transplant using cells donated from her brother. Dr. Frangroul is the director of the Pediatric Stem Cell Transplant program at Tristar Centennial Children’s Hospital, and an investigator at the Sarah Cannon Research Institute in Nashville. While at his clinic in CELL AND GENE THERAPIES: INNOVATIONS, CHALLENGES AND CLINICAL BREAKTHROUGHS 36 “I really gave up on becoming anything else. I thought sickle cell was just going to be my life from beginning to end.” TECHNOLOGYNETWORKS.COM Nashville, Victoria experienced a crisis that would ultimately change her life. “I had to be admitted, and during that hospital stay Dr. Frangoul approached me at my bedside. He knew that I was feeling really down, and he offered me a second option besides the Haplo transplant,” she recalls. Victoria had some reservations about the Haplo transplant, largely due to the risk of graft versus host disease, a complication that can occur when a donor’s cells attack the recipient’s: “Dr. Frangoul told me that they were starting a new trial soon using CRISPR gene therapy. I hadn’t heard about it, so he showed me a small video on his phone and sent me a link so I could review it later.” The clinical trial that Victoria participated in was testing a cell-based gene therapy known as CasgevyTM. The therapy utilizes CRISPR-Cas9 gene-editing technology, which is directed to cut DNA in specific locations and enable the removal, addition or replacement of DNA. During treatment, the SCD patient’s blood stem cells are extracted from their body and edited in a laboratory. CRISPR-Cas9 is used to create an edit in the BCL11A gene within the patient’s cells, which triggers the production of fetal hemoglobin. Once these cells are re-inserted into the patient, they settle back into the bone marrow and the increased fetal hemoglobin facilitates the delivery of oxygen around the body. As with any clinical trial, there were risks involved. Given that Victoria would be the first patient to ever receive this experimental therapy, these risks felt somewhat heightened. She had a difficult decision to make. “I'm a woman of faith – I pray a lot. I went to God, in private, about graft versus host disease, because I really didn’t want to experience that. So, when gene therapy came along, I felt like it was my answer from God, as though he was saying to me ‘I remove your fears now. This opportunity is for you’,” Victoria says. Ultimately, the possibility of a life that would not be plagued by pain outweighed any doubts she might have had about being patient one. Within 24 hours of speaking to Dr. Frangoul, Victoria took the courageous decision to volunteer for the trial. In July 2019, she became the first patient to receive CRISPR gene therapy for SCD. “The trial was a different experience to anything I’ve had before, because for the first time, I felt hopeful. I was fighting for my life, and for my family.” Sure, I had to travel back to Nashville a lot for testing, and there were times [during the trial], especially after the chemotherapy, that it was hard. But my dad was with me, and he kept reminding me, ‘Vicky, you’ve seen worse, you are strong enough to get through this’. It helped to lift me back up and remind me of why I was doing this. Am I dead? Eight months after the therapy was administered, Victoria woke one morning and felt “different”, though she couldn’t quite place what that meant. “I remember waking up, and the room was really bright. I didn’t feel anything, which was strange. I didn’t have any shortness of breath when I stood up, as had been the case most mornings,” she says. CELL AND GENE THERAPIES: INNOVATIONS, CHALLENGES AND CLINICAL BREAKTHROUGHS 37 “My“Dr. Frangoul explained CRISPR therapy to me like this: he said, ‘just imagine a textbook with thousands and thousands of words, and there are a few words in there that are incorrect. The CRISPR technology could go into the cells, find the incorrect word and edit it without changing the story’.” TECHNOLOGYNETWORKS.COM Having lived most of her life in excruciating pain and extreme fatigue, the experience of waking up in its absence was so downright bizarre, Victoria had convinced herself that she must be dead. Pinching the skin on her face, and her thighs, she was reassured to feel the sharp, physical sensations. “I shouted to my kids, ‘Hey y’all, come in here!’, and as they entered the room, their faces lit up. I knew in that moment, they could see me, and I realized that I was very much alive. I cried tears of joy, because I knew then that the gene therapy had worked,” Victoria describes, visibly emotional. That bright, beautiful morning was four years ago, and it marked Victoria’s new beginning. She describes her life now as one of freedom. She is free from constant pain and exhaustion. Free from having to stare at a hospital room’s four walls while experiencing a crisis episode. Free from relying on medication just to make it through the day. Free from all the ways that SCD stole her autonomy and chained her to a life burdened by illness. Victoria can now play with her children and embody the parent she always dreamed of being. She can immerse herself in typical mom activities, often taken for granted as mundane, but that were once out of her reach. She can make choices. She can travel abroad – even flying to London last year to speak at the Third International Summit on Human Genome Editing. Eventually, Victoria’s health improved to the extent that she could realize her ambition of working full-time, securing a position as a cashier at her local Walmart. It was during a particularly busy shift on December 8, 2023, when she received news that the US Food and Drug Administration (FDA) had decided to approve Casgevy for the treatment of SCD. Casgevy’s approval was based on data submitted from the trial that Victoria herself had bravely participated in. The trial’s primary outcome had been freedom from severe vaso-occlusive crises for at least 12 months during the study’s 24-month follow-up period. In total, 44 patients were dosed with Casgevy. Upon submitting the trial data, 31 patients had sufficient follow-up time to be evaluated. Of these, 29 patients reached the primary efficacy outcome, with 0 patients suffering from graft failure or graft rejection – the trial had proven a success. Casgevy became the first gene therapy utilizing CRISPR-Cas9 to receive FDA approval, marking a historic moment for the SCD community, as lovotibeglogene autotemcel (LyfgeniaTM) – another cellbased gene therapy for SCD, developed by Bluebird Bio Inc. – also received a green light from the agency. SCD patients face medical and racial discrimination, hindering clinical research prospects Victoria received many phone calls that day, including one from a nurse who treated her at Dr. Frangoul’s clinic in Nashville. “We both cried,” she says, “It was tears of pure CELL AND GENE THERAPIES: INNOVATIONS, CHALLENGES AND CLINICAL BREAKTHROUGHS 38 “When I saw the text [from my husband], that the FDA had approved Casgevy, I had to rush off the floor at Walmart because I felt the tears coming,” she says. “I got in my car, and I just cried. I was so, so happy because I knew what a relief this would be for other sickle cell patients that were in a dark place like I had once been. Now, there was hope.” TECHNOLOGYNETWORKS.COM joy, because all of the pain, disappointment and judgement that I had faced from childhood now felt worth it. This was going to bring about change for other people who feel alone and feel overlooked.” “It had felt as though nobody was coming to save us. Then suddenly we had a novel therapy for the SCD community. I felt so happy and grateful,” she adds. While reminiscing, Victoria takes a moment to pause. It’s clear that the emotions attached to these memories are a complex mixture of elation and pain. “I want to emphasize that, had it not been for the positive way in which I was treated by Dr. Frangoul and his team, I might not have accepted the opportunity to take part in this trial that has saved my life,” she says. Discrimination is an issue encountered by many SCD patients across the globe during their lifetime. In a perspective piece published in 2020, Power-Hays and McGann expressed that there may be “no population of patients whose health care and outcomes are more affected by racism than those with SCD.” During the 1970s, many African American people were deprived of jobs, educational opportunities, marriage licenses and insurance in the US if they carried the SCD trait. This grim picture was mirrored across the pond in the UK. “In the 20th century, I'd say that racism showed itself in the lack of willingness on the part of statutory services, such as education, social services and housing, to take account of the needs of those living with SCD,” says Professor Simon Dyson, director of the Unit for the Social Study of Thalassaemia and Sickle Cell at De Montfort University in the UK. “In terms of sickle-cell screening, SCD had to meet prevalence thresholds not demanded of other rarer conditions, before newborn screening to save the lives of black infants was eventually made universal in England in 2004.” A 2018 systematic review by Dr. Dominique Bulgin and colleagues explored the extent of health-related stigma in adolescents and adults living with SCD, analyzing data from 27 studies published between 2004–2017. They found that people with SCD experience healthrelated stigma based not only on their race, but on their disease status, socioeconomic status, delayed growth and puberty and having chronic and acute pain, requiring opioid treatment. “Individuals with SCD reported being stigmatized as drug seeking or drug addicts and having their experiences of pain discredited by healthcare providers,” the review states. Sadly, this experience is one that resonates with Victoria. “I was once in the emergency room when a nurse said to me, ‘You know, I feel so sorry for you sicklers’ – this was a term he used – ‘because you guys just get addicted to these pain meds, and then you can’t tell the difference between withdrawal and a real crisis’,” she says. “He then said that it’s just inevitable that all SCD patients become drug addicts. I couldn’t believe it. I was in crisis. I was in pain, and I was crying out to a person who was supposed to help me. Instead, he judged me,” she continues. This, sadly, was not an isolated incident. Victoria recounts another crisis episode, where she felt as though her symptoms of pain were starting to improve. She asked the nurse treating her to avoid administering any excessive pain medication that might make her feel drowsy, as she wanted to sit up and try to move around the room. Instead, the nurse pressed the button on her medication dispenser, and Victoria fell asleep. When she woke, she learned that instead of pain medication, the nurse had given her a different type of drug because they “wanted to see what it would do”. Unfortunately, there was little consequence for the nurse, but the ramifications for Victoria were heartbreaking, she explains, and led her to question her worth as a SCD patient: “I felt as though the nurse had been given the right to experiment on me without my consent. What if that drug had taken my life? I couldn’t trust the staff that was treating me, I felt as though I was a burden, like they were trying to get rid of me when I was coming to them for help.” “There is no way,” Victoria adds, “that I would have accepted an experimental treatment like gene therapy, if I had been offered it at this facility.” Research shows that she is not alone in feeling this way. In 2020, Cho et al. conducted a study examining CELL AND GENE THERAPIES: INNOVATIONS, CHALLENGES AND CLINICAL BREAKTHROUGHS 39 TECHNOLOGYNETWORKS.COM the motivations and decision-making processes of enrollees and decliners of high-risk trials for SCD. Of the 26 SCD patients interviewed, the majority reported negative interactions with health care providers. These experiences were so bad that many individuals had chosen to avoid hospitals during significant pain crises. If SCD patients are afraid to even ask for help when they are in pain, how can they be expected to partake in high-risk clinical research? It’s an almost impossible decision, but it’s one that Victoria chose to make for herself, for her family and for the SCD community. It’s a decision that, now, in hindsight, she is thrilled about, but she remains eager to warn the medical community of the risks they pose to the health of SCD patients – and the future of SCD research – by forgetting that the patient in front of them is a human being. Patient advocacy and increasing access to gene therapies Now, five years after the trial commenced, Victoria is undertaking her follow-up appointments – which last 15 years from the study enrollment date – as per the study protocol. These appointments monitor her health and assess the long-term efficacy and safety of Casgevy. As gene therapies are emerging drugs, whether or not they are effective for the duration of a patient’s life is yet to be determined. “I pray that it is a forever change, and I believe that it is,” Victoria says. She is enjoying applying her newfound energy to spread the word about her experience as an SCD patient, and a CRISPR gene therapy recipient, through advocacy work. Once the news broke that she was the first patient to receive CRISPR gene therapy for SCD, Victoria was invited to speak with SCD organizations, meet other patients – or “warriors”, as she refers to them – and attend international events to share her story. “When I was flown to London to speak at the summit on human genome editing, I was amazed, because people really cared about my experience,” she says. A recent highlight, she says, was her appearance on Good Morning America, where Victoria was able to meet a fellow SCD patient – Jamie – who had been inspired by her story and made the decision to receive CRISPR gene therapy. “It was truly a fulfilling moment,” she recalls, “because it was one thing that I hoped for – to be able to affect someone else's life in a positive way. To see [Jamie] looking so healthy after not being able to leave the house, or struggling to take care of his children, it was just incredible.” Victoria also had the opportunity to visit the production facility that created her CRISPR gene therapy. “I was like a kid, looking at the machines and listening to how everything works. I know that this [therapy] was many years in the making, and it was an honor to meet the scientists that were working so hard when I thought no one cared.” Her advocacy work has most recently turned to the costs associated with CRISPR-based, and other gene therapies, for SCD. A major challenge for patients and clinicians in accessing such emerging therapies will CELL AND GENE THERAPIES: INNOVATIONS, CHALLENGES AND CLINICAL BREAKTHROUGHS 40 “Please, just treat us how you want to be treated,” Victoria asks. “We did not choose to have SCD. We did not choose the amount of medication we require to ease our pain. We just want to be cared for, and we want to feel normal. Please, treat use with respect.” TECHNOLOGYNETWORKS.COM undoubtedly be their price. While information on the exact cost is currently limited, Casgevy will reportedly be priced at $2.2 million per patient in the US, and over £1.5 million per patient in the UK. Granted, it’s designed to be a “one-and-done” treatment, which could override economic burden of a lifetime of prescriptions, hospital stays and other associated costs with SCD management. But very few people – especially marginalized patients – have $2.2 million at their disposal, and in the US particularly, there’s lingering uncertainty as to whether most insurance companies will cover the therapies. “When I heard the price after the approval, it made the moment bittersweet,” Victoria says. “I knew that, for me, if I would have had to pay for it, I wouldn't have been able to afford it. Because I couldn't even work. Speaking to SCD patients through my work, it’s clear that other patients and their families feel the same way.” There is major research work going on across the globe in an attempt to reduce the costs associated with gene therapies, including those that are CRISPR-based. One example is a proposed movement towards in vivo delivery of gene therapies, which could reduce the costs associated with extracting cells, editing them in a laboratory and then infusing them back into the patient. At present, this research is in the laboratory stage, rather than clinical testing. The Innovative Genomics Institute (IGI) is a non-profit academic institution that was founded by Professor Jennifer Doudna, who became a Nobel Laureate in 2020 in recognition of her research discovering CRISPR gene editing technology. It’s a joint effort between leading research institutes in the Bay Area, including the University of California (UC) Berkeley and UC San Francisco, with affiliates at UC Davis, Lawrence Berkeley National Laboratory, Lawrence Livermore National Laboratory, Gladstone Institutes and and other institutions. Dr. Melinda Kliegman, director of the Public Impact at the IGI, recently led the IGI’s Affordability Task Force in generating a report, titled “Making Genetic Therapies Affordable and Accessible”. The task force assembled in 2021 to start work on the report, which explores the key drivers of the high prices associated with gene therapies and identifies approaches to increase their accessibility. “The IGI develops the underlying technology used to make these [gene] therapies, but we are not involved in commercialization and pricing. We wanted to understand more about what goes into setting these prices, and what, if anything, we could do to lower them,” says Kliegman. “The process of assembling the task force and developing the report took over a year and involved 35 task force members. The breadth of expertise of task force members helped us cover the many different complex issues that lead to high prices of gene therapies. It was a huge effort, but interesting, since we were all learning new things,” Kliegman adds. The report is 75 pages long and provides a comprehensive overview of the various factors contributing to the current pricing of gene therapy. Kliegman summarizes the key “take home points” of the task force’s findings: “The issues affecting affordability are multifaceted and system wide. I would like to acknowledge that these therapies are expensive and difficult to manufacture, and there are small patient populations from which to recover costs,” she says. “Companies also need to make a profit and adequate returns for investors. Given this, a for-profit company may not be the best business model for developing bespoke gene therapies. We need a paradigm shift in the way these therapies are commercialized, for example utilizing non-profit medical research organizations and public benefit corporations running on moderate-cost capital from social impact investors and government and philanthropic grants,” Kliegman continues. As part of the report, the IGI team built a model that shows it could be possible to commercialize a gene therapy for 10x less than they are currently marketed at today, roughly ~$250,000 per patient for a therapy that could treat 2000 patients per year. This model is a departure away from the traditional methods used to develop gene therapies, and so, responses to the report have been mixed, Kliegman says. “Our proposal is oriented towards access, not profit maximization. Many people think it's naive to expect anyone to ‘leave money CELL AND GENE THERAPIES: INNOVATIONS, CHALLENGES AND CLINICAL BREAKTHROUGHS 41 TECHNOLOGYNETWORKS.COM on the table’, while others agree with the need to improve access and affordability.” There is change on the horizon, though: “We have had the privilege of meeting with organizations aligned with the suggestions in the report. Unsurprisingly, some of them had representatives on our task force,” Kliegman emphasizes. “The 90-10 Institute recently launched, which is a nonprofit working to establish an impact investment fund for public benefit pharmaceutical companies. There are also organizations like Odylia Therapeutics and Caring Cross, which are nonprofits developing and delivering gene therapies and public benefit manufacturing organizations like Landmark Bio and Vector BioMed.” In Victoria’s mind, the high price of gene therapy is “just another hurdle to overcome”. She remains optimistic that the industry will reach a collective decision on how to ensure all patients can access the therapies they so desperately need. One day, she hopes that there will be no SCD patients having to attend emergency rooms, receive transfusions or rely on pain medication. “I can live with the title of being a sickle cell warrior – I think we all can. But I want everyone in this community to be free from the hold that the disease places on our lives.” That is her dream, she says, and unlike the dreams that SCD denied her in childhood, hopefully, science can make this one come true. ABOUT THE INTERVIEWEES Victoria Gray was the first sickle cell anemia patient in the world to be treated with CRISPR gene editing in 2019. After a lifetime of pain, treatments and hospitalizations for sickle cell disease, she is now symptom-free and working as a patient advocate and international speaker to spread the word about CRISPR and rare disease to clinicians, scientists, patients and students. Melinda Kliegman is director of public impact at the Innovative Genomics Institute (IGI). In this role, she leads the Public Impact team, which works to align IGI’s genome-engineering innovations with societal values by engaging in public dialogue, original research, and policy creation through outreach to key stakeholders to ensure that genome-editing technology benefits everyone equitably. Melinda holds a PhD in Biology from Stanford University. Before joining the IGI, she worked at the Bill & Melinda Gates Foundation, the world’s largest philanthropic organization. CELL AND GENE THERAPIES: INNOVATIONS, CHALLENGES AND CLINICAL BREAKTHROUGHS 42 TECHNOLOGYNETWORKS.COM CELL AND GENE THERAPIES: INNOVATIONS, CHALLENGES AND CLINICAL BREAKTHROUGHS 43 CONTRIBUTORS Andy Tay, PhD Andy Tay received his PhD from the University of California, Los Angeles, focusing on neuromodulation and engineering. He subsequently completed his postdoctoral training at Stanford University where he developed nanotechnologies for immuno-engineering. Andy Tay is currently a Presidential Young Professor at the National University of Singapore. Aron Gyorgypal, PhD Aron Gyorgypal is a postdoctoral research fellow at Harvard Medical School and Massachusetts General Hospital in Boston, Massachusetts. Traditionally trained as an engineer, Aron received his PhD in chemical and biochemical engineering from Rutgers University in New Jersey, where he studied antibody glycosylation in the context of bioprocessing to produce more homogeneous biologics. Kate Robinson Kate Robinson is a science editor for Technology Networks. She joined the team in 2021 after obtaining a bachelor's degree in biomedical sciences 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. Laura Lansdowne Laura Lansdowne is the managing editor at Technology Networks, she holds a first-class honors degree in biology. Before her move into scientific publishing, Laura worked at the Wellcome Sanger Institute and GW Pharma. Masha Savelieff, PhD Masha Savelieff is a professional freelance scientific writer. She began her science career in chemistry at the American University of Beirut in Lebanon, where she earned her BSc and MSc degrees. She then went on to receive her PhD, also in chemistry, from the University of Illinois in 2008. TECHNOLOGYNETWORKS.COM CELL AND GENE THERAPIES: INNOVATIONS, CHALLENGES AND CLINICAL BREAKTHROUGHS 44 CONTRIBUTORS Molly Coddington Molly Coddington is a Senior Writer and Newsroom Team Lead for Technology Networks. She holds a first-class honors degree in neuroscience. In 2021 Molly was shortlisted for the Women in Journalism Georgina Henry Award. Monica Hoyos Flight, PhD Monica is a freelance science communications consultant providing writing and editing services for researchers, research organizations and companies. She has a PhD in neuroscience from Imperial College London and extensive experience as an editor for Springer Nature journals and research communications manager at the University of Edinburgh. Samantha Carrera, PhD Samantha Carrera is an experimental officer in the Genome Editing Unit at the University of Manchester. Originally from Mexico, she completed her PhD in Biochemistry at the University of Leicester in the UK where she studied the effect of the microenvironment on the response to apoptosis.