Process development innovations to improve gene therapy manufacturing

Viral vector systems are crucial in delivering therapeutic genetic material for gene and cell therapy in clinical settings. With new progress in viral vector-based gene therapies, healthcare professionals and pharmacists must stay informed and apply appropriate biosafety precautions when handling these products. Comprehensive guidelines covering biosafety measures for every viral vector-based gene therapy, including storage, preparation, administration, and disposal, remain scarce. As viral vector-based gene therapy advances rapidly, it is critical to equip healthcare professionals with the expertise to safely manage these agents in a clinical setting. As gene therapies become more integrated into mainstream medicine, with 43 cell and gene therapy products approved as of 2025, pharmacists, nurses, and caregivers face new safety challenges when handling these therapies. While guidelines such as United States Pharmacopeia (USP) 797 and USP 800 provide a foundation for handling viral vector-based gene therapies, no single resource consolidates all biosafety considerations for healthcare professionals. This review provides an overview of the viral vector-based gene therapy landscape. It compiles best practices from various sources to establish a potential standardized approach to guide safe handling procedures for viral vector systems in a healthcare setting. By adopting the biosafety strategies outlined in this article, healthcare staff and pharmacists can safely manage viral vectors while developing standard operating procedures tailored to their specific clinical environments. Implementing these measures will support the continued advancement of gene therapy while ensuring the safety of healthcare providers, patients, and the environment.

Elisa Manzotti speaks to Ying Cai, Nathalie Clement, Chantelle Gaskin, Matt Roach & Ashish Saksule. Ying Cai is the Sr. Director of Process Development at Ultragenyx Pharmaceutical. She heads AAV downstream process development and formulation development functions, also a CMC lead of AAV clinical programs. Prior to joining Ultragenyx, Ying worked at Sanofi, Biogen, Merck, and a few CDMOs. Ying has over 20 years’ experience in the development, validation, manufacturing and commercialization of different modalities including AAV, plasmid DNA, oligonucleotides, antibodies, antibody conjugates, and fusion proteins. Ying holds a Ph.D. in Chemical Engineering from the University of Arkansas at Fayetteville and a B.S. in Biochemical Engineering from Zhejiang University in China. Nathalie Clement has more than 25 years of experience in the field of Gene Therapy, with a strong expertise in viral vectors, specifically adeno-associated vectors, in the academic and industry settings. Her research focus has strongly been focused on optimizing processes to support large-scale production of high quality rAAV stocks and their implementation into the GMP settings. During her thesis work at the University Libre of Brussels, Belgium, she developed new recombinant viruses derived from the parvovirus Minute Virus of Mice (MVM) for cancer-selective gene therapy treatments. She then joined Dr. Michael Linden’s laboratory at Mount Sinai School of Medicine, New York, where she developed novel recombinant AAV vectors and directed the AAV Vector Core. She next joined the Powell Gene Therapy Center in 2008 as the Associate Director to supervise AAV production and testing at research, preclinical and clinical grades. She led the Process and Development Group and the Quality Control group responsible for the production and release of all AAV pre-clinical and clinical lots. During her time at UF she oversaw manufacturing, release and stability campaigns of more than 7 AAV INDs from start to finish, including CMC preparations and interactions with FDA. More recently, she spent several months at Resilience, Alachua, Florida, as the Director of Process and Development of the Viral Vaccines and Gene Therapy franchises. IN that role she oversaw viral vaccine and AAV production scales up to 200L in suspension format and in the icellis 500 platform for adherent platforms of a variety of viruses and AAV vectors. Currently Nathalie is taking a break before starting a new adventure in 2022. Chantelle Gaskin is a Field Applications Scientist, specializing in protein and viral vector purification and downstream process development. She held leadership positions at Applied Genetic Technology Corporation and Brammer Bio, prior to joining the Thermo Fisher Scientific Bioproduction Division in 2020. With over 10 years of experience in gene therapy, Chantelle has accumulated comprehensive knowledge of standard industry practices and regulatory standards, applying this knowledge to advance development of therapies for a variety of indications including ocular, CNS and systemic disease. Chantelle holds a Master’s degree in Chemistry from University of Florida and a Bachelor’s in Chemistry from Smith College. Matt Roach leads the AAV Process Development group at Precision BioSciences, which is focused on designing and implementing new strategies for the production and purification of adeno-associated virus. Matt completed his Bachelor’s degree in Biological Sciences at North Carolina State University and his Master’s degree in Microbiology and Cell Science at the University of Florida. Prior to Precision, Matt spent time at Pfizer working on the purification of AAV and the Biomanufacturing Training and Education Center training industry professionals on downstream bioprocessing operations. Ashish Saksule is the Cell and Gene Therapy process development lead and technical expert on bioprocessing platforms for viral vectors (Lentivirus and Adeno-associated virus vector) and non-viral vectors with more than 7 years of experience. Ashish has graduate degree in Chemical Engineering from Michigan Tech University, and Biotechnology graduate degree from Harvard University. His experience spans research & drug development, clinical stage and CRO/CMO settings. Ashish is currently working at Takeda within Global Gene Therapy and have previously worked at MilliporeSigma and Miltenyi Biotec.

Gene therapy is a technique involving the modification of an individual's genes for treating a particular disease. The key to effective gene therapy is an efficient carrier delivery system. Viral vectors that have been artificially modified to lose their pathogenicity are used widely as a delivery system, with the key advantages of their natural high transduction efficiency and stable expression. With decades of development, viral vector-based gene therapies have achieved promising clinical outcomes. Currently, the three key vector strategies are based on adeno-associated viruses, adenoviruses, and lentiviruses. However, certain challenges, such as immunotoxicity and "off-target", continue to exist. In the present review, the above three viral vectors are discussed along with their respective therapeutic applications. In addition, the major translational challenges encountered in viral vector-based gene therapies are summarized, and the possible strategies to address these challenges are also discussed.

Gangrene, revascularization, and limb function improved with E-selectin/adeno-associated virus gene therapy

Acute pain is a normal physiologic adaptive response to noxious stimuli that is essential for survival and maintenance of the integrity of the organism. Chronic pain, on the other hand, is a maladaptive, pathologic and persistent condition that results in a marked decrease in the quality of life and secondary symptoms such as anxiety and depression. Chronic pain is a common phenomenon. It is estimated that more than 50 million people in the United State alone are affected by chronic pain. Much of currently available clinic treatments for chronic pain are only partially effective and many are limited by off-target side effects, or have the potential of abuse.1 Furthermore, analgesics that are highly effective in the management of pain often provide only very short-term relief, and some forms of pain, for example, neuropathic pain are practically difficult to treat.2 In recent years, significant progress has been made in the fundamental understanding of the neurobiology of pain. In parallel, detailed molecular biologic characteristics of many of the receptors, neurotransmitters, neuropeptides, cytokines, chemokines, and ion channels that play significant roles in nociception have become available.3,4 Taking advantage of these advances, therapeutic gene transfer is emerging as an intriguing option for the development of novel treatment for chronic pain. Gene therapy was first proposed as a treatment for genetically determined inherited disorders5 and the first successful human gene therapy for X-linked severe combined immunodeficiency in children, was reported 6 years ago.6 Therapeutic gene transfer for treatment of chronic pain is not directed at correcting a genetic defect. Rather the regional specialization of spinal cord and/or brain function and the widespread and redundant use of a limited repertoire of neurotransmitters and receptors in diverse pathways in the nervous system means that the local production of neurotransmitters achieved by therapeutic gene transfer may be used to achieve desired outcomes while avoiding unwanted adverse side effects that would result from activation of the same receptors in other pathways by a systemically administered drug.7 In this review we summarize recent studies of recombinant virus vectors for in vivo pain treatment in animal. Viruses have the ability to infect and transduce their genome into host cells, and viral vector-based gene therapy takes advantage of the natural biology of virus to transduce exogenous genes into host cells. The general principle for creating a viral vector involves the deletion of nonessential genes from the virus, incapacitating its replication and, therefore, rendering it nonpathogenic, while retaining the structural motifs that allows the vector attach and transfer its genome into the host. Recently developed viral vectors provide a powerful vehicle for effective gene transfer to the nervous system.8 An ideal vector for gene therapy should achieve stable, tissue-specific regulated gene expression without eliciting the host immune response.9 The vector must possess sufficient capacity to carry the transgene, efficiently deliver that gene to the cell of interest, and express appropriate amounts of transgene product to provide a therapeutic effect, all without detectable toxicity resulting from the vector itself.10 It should be noted that rendering a virus replication deficient does not assure elimination of direct cytotoxicity or abolition of host immune response caused by expression of other viral proteins not critical for replication.11 The most commonly used viral vectors in studies of treatment of pain are based on recombinant adenovirus, adeno-associated virus (AAV), or herpes simplex virus. We will review the published data for each of these vectors below.

In 2001, the first large animal was successfully treated with a gene therapy that restored its vision. Lancelot, the Briard dog that was treated, suffered from a human childhood blindness called Leber's congenital amaurosis type 2. Sixteen years later, the gene therapy was approved by the U.S. Food and Drug Administration. The success of this gene therapy in dogs led to a fast expansion of the ocular gene therapy field. By now every class of inherited retinal dystrophy has been treated in at least one animal model and many clinical trials have been initiated in humans. In this study, we review the status of viral gene therapies for the retina, with a focus on ongoing human clinical trials. It is likely that in the next decade we will see several new viral gene therapies approved.

The promise of viral vector-based gene therapy (GT) as a transformative paradigm for treating severely debilitating and life-threatening diseases is slowly coming to fruition with the recent approval of several drug products. However, they have a unique mechanism of action often necessitating a tortuous clinical development plan. Expertise in such complex therapeutic modality is still fairly limited in this emerging class of adeno-associated virus (AAV) vector-based gene therapies. Because of the irreversible mode of action and incomplete understanding of genotype-phenotype relationship and disease progression in rare diseases careful considerations should be given to GT product's benefit-risk profile. In particular, special attention needs to be paid to safe dose selection, reliable dose exposure response (including clinically relevant endpoints), or creative approaches in study design targeting small patient populations during clinical development. We believe that quantitative tools encompassed within model-informed drug development (MIDD) framework fits quite well in the development of such novel therapies, as they enable us to benefit from the totality of data approach in order to support dose selection as well as optimize clinical trial designs, end point selection, and patient enrichment. In this thought leadership paper, we provide our collective experiences, identify challenges, and suggest areas of improvement in applications of modeling and innovative trial design in development of AAV-based GT products and reflect on the challenges and opportunities for incorporating MIDD tools and more in rational development of these products.

Long-Term Follow-up of Foamy Viral Vector-Mediated Gene Therapy for Canine Leukocyte Adhesion Deficiency

Novel therapeutic approaches for childhood parkinsonism

Development and qualification of a representative scale-down model of automated Ficoll-based processing of a cell-based therapeutic according to quality by design principles

Type 1 Diabetes Mellitus (T1DM) results from the autoimmune destruction of insulin-producing pancreatic β cells. Despite advances in our understanding of the pathogenesis and management of diabetes, as well as treatment options, which primarily include injectable insulin and whole pancreas transplant, these advancements remain unsatisfactory, with only a small percentage of patients achieving their glycemic goals. Insulin gene therapy refers to the development of β cell substitutes by introducing an insulin producing gene into endogenous cell populations that do not normally produce insulin. This strategy has the promise to overcome the limitations of current treatments. Adeno-associated viral (AAV) vector-based gene therapy holds great promise for the treatment of inherited and many complex acquired disorders. The success of AAV vectors is determined by two major factors: 1) cell transduction efficiency, and 2) persistence of the introduced gene with minimal or no host immune response following vector administration. Previous research has suggested that certain small molecules can improve AAV vector transduction efficiency and lower the vector dose below the immunological response threshold. We explore the use of teniposide, a chemotherapeutic agent, in improving the efficacy of gene therapy using AAV. We co-administered teniposide at a final dosage of 20 mg/kg with an AAV vector encoding an insulin gene controlled by a liver-specific promotor into streptozotocin (STZ) induced diabetic Hsd: ICR mice. Teniposide co-treatment produced up to a 3-fold decrease in AAV dose compared to vector alone while reducing diabetic hyperglycemia to comparable degrees as AAV delivery alone. There was no additional systemic toxicity. While the mechanism of action of teniposide-enhanced viral delivery of gene therapy is unknown, our data suggest that teniposide co-administration could significantly enhance AAV transduction in vivo and provide a novel, useful strategy to improve the clinical efficacy of gene therapy. Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

Gene therapy clinical trials are rapidly expanding for inherited metabolic liver diseases whilst two gene therapy products have now been approved for liver based monogenic disorders. Liver-directed gene therapy has recently become an option for treatment of haemophilias and is likely to become one of the favoured therapeutic strategies for inherited metabolic liver diseases in the near future. In this review, we present the different gene therapy vectors and strategies for liver-targeting, including gene editing. We highlight the current development of viral and nonviral gene therapy for a number of inherited metabolic liver diseases including urea cycle defects, organic acidaemias, Crigler-Najjar disease, Wilson disease, glycogen storage disease Type Ia, phenylketonuria and maple syrup urine disease. We describe the main limitations and open questions for further gene therapy development: immunogenicity, inflammatory response, genotoxicity, gene therapy administration in a fibrotic liver. The follow-up of a constantly growing number of gene therapy treated patients allows better understanding of its benefits and limitations and provides strategies to design safer and more efficacious treatments. Undoubtedly, liver-targeting gene therapy offers a promising avenue for innovative therapies with an unprecedented potential to address the unmet needs of patients suffering from inherited metabolic diseases.

Background Delivery of viral vectors as gene therapies to treat neurodegenerative diseases has been hampered by the inability to penetrate the blood brain barrier (BBB) and invasive or non-targeted delivery options prone to inducing immune responses. MR guided focused ultrasound (MR-g-FUS) and microbubbles have demonstrated safe, temporary, targeted BBB permeabilization clinically. Methods We developed clinically scalable, microbubble drug conjugates (MDCs) for the viral gene therapy, AAV.SIRT3-myc [adeno-associated virus expressing myc-tagged SIRT3], which has previously been shown to have disease modifying effects in animal models of Parkinson’s disease (PD). The lipid shells of the perfluorocarbon gas MDCs were covalently conjugated to antibodies with binding specificity to AAVs. Following systemic (iv) delivery of AAV.SIRT3-myc MDCs, MR-g-FUS was used to deliver SIRT3-myc to brain regions affected in PD. SIRT3-myc expression was determined post mortem, using immunohistochemistry. Results An in vitro, SH-SY5Y cell culture model was used to show that the localized destruction of MDCs using ultrasound exposures within biological safety limits dissociated AAV2-GFP (green fluorescent protein) from the MDCs in the targeted area while maintaining their transduction capacity. In rats, MR-g-FUS resulted in BBB permeabilization in the striatum and substantia nigra (SNc). SIRT3-myc was expressed in the striatum, but not the SNc. Conclusion These studies demonstrate that MDCs combined with MR-g-FUS are an effective method for delivery of viral vector gene therapies, such as AAV.SIRT3, to brain regions affected in PD. This technology may prove useful as a disease-modifying strategy in PD and other neurodegenerative disorders.

With increasing approvals of viral vector-based gene therapies, the industry is shifting its focus to the expansion of viral vector manufacturing to meet the demands of the growing number of therapies on the market. Continuous improvements in the efficiency of viral vector production, as well as optimization of complex bioproduction processes, are essential to meet demand and get lifesaving therapies to patients quickly. This must be carefully balanced with consistency and quality of the final product. In this article, multiple applications and technologies to address current manufacturing challenges and solutions to increase overall viral vector yield throughout development will be explored. Case studies will be presented to demonstrate the progress associated with process intensification and optimization from benchtop development to large-scale commercial manufacturing.

Strategies to Modulate Immune Responses: A New Frontier for Gene Therapy