Viral vector manufacturing process: addressing bottlenecks with innovative solutions

Manufacturing of viral vectors comprises the generation of these vectors, which then have to be purified in order to meet the quality attributes required for further use as gene delivery systems. The first part of this article deals with the production of the most important viral vectors used in gene therapy protocols. In the second part, we briefly review the most current methods used for the purification of viral gene therapy products focusing on four viral vectors that have been the most extensively used in clinical trials: adenoviral, adeno-associated viral and lentiviral vectors. Traditionally, γ-retroviral vectors were not purified and clarified vectors containing culture supernatant was directly used for ex vivo gene therapy. The final section of this article reviews some of the basic biosafety considerations specific to the respective viral vectors.

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.

Advances and opportunities in process analytical technologies for viral vector manufacturing

Adeno-associated viruses (AAVs) for in vivo gene therapy are a dominant viral vector platform in the gene therapy landscape. A key challenge in viral vector manufacturing, particularly with AAVs, is effective and consistent separation of full particles (i.e. viral vector capsids containing the full recombinant cDNA of the gene of interest) from empty capsids. Monolith-based ion exchange chromatography (IEX) is a promising tool to achieve this separation but poses challenges with consistency upon scale-up. This paper outlines some of the scale-up challenges and potential mitigations to achieve consistent separation of full and empty particles. IEX is evolving to become the industry standard for downstream separations as industrialization of gene therapy manufacturing continues.

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.

One important aim in cell culture-based viral vaccine and vector production is the implementation of continuous processes. Such a development has the potential to reduce costs of vaccine manufacturing as volumetric productivity is increased and the manufacturing footprint is reduced. In this work, continuous production of Modified Vaccinia Ankara (MVA) virus was investigated. First, a semi-continuous two-stage cultivation system consisting of two shaker flasks in series was established as a small-scale approach. Cultures of the avian AGE1.CR.pIX cell line were expanded in the first shaker, and MVA virus was propagated and harvested in the second shaker over a period of 8–15 days. A total of nine small-scale cultivations were performed to investigate the impact of process parameters on virus yields. Harvest volumes of 0.7–1 L with maximum TCID50 titers of up to 1.0×109 virions/mL were obtained. Genetic analysis of control experiments using a recombinant MVA virus containing green-fluorescent-protein suggested that the virus was stable over at least 16 d of cultivation. In addition, a decrease or fluctuation of infectious units that may indicate an excessive accumulation of defective interfering particles was not observed. The process was automated in a two-stage continuous system comprising two connected 1 L stirred tank bioreactors. Stable MVA virus titers, and a total production volume of 7.1 L with an average TCID50 titer of 9×107 virions/mL was achieved. Because titers were at the lower range of the shake flask cultivations potential for further process optimization at large scale will be discussed. Overall, MVA virus was efficiently produced in continuous and semi-continuous cultivations making two-stage stirred tank bioreactor systems a promising platform for industrial production of MVA-derived recombinant vaccines and viral vectors.

Cell-culture process optimization via model-based predictions of metabolism and protein glycosylation

Gene Therapy with BMN 270 Results in Therapeutic Levels of FVIII in Mice and Primates and Normalization of Bleeding in Hemophilic Mice.

Novel therapeutic approaches for childhood parkinsonism

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

The accelerating development of gene therapy from research towards clinical trials and beyond has elevated the demand for practical viral vector-manufacturing solutions. The use of disposable upstream technology is gaining traction in clinical manufacturing. Packed-bed or fixed-bed reactors, where column is packed with immobilized biocatalyst particles providing surface to constrain the cells in a particular region of the reactor, have been widely used in bioprocessing applications since mid-1900s. However, the world's first single-use, fully integrated, high cell density, fixed-bed bioreactor was launched only approximately a decade ago. By now, several single-use, fixed-bed technology solutions have been developed in a small scale. Scaling-up the manufacturing can be challenging and for commercial-scale manufacturing, there is practically only one single-use, good manufacturing practice-compliant option available. This study reviews the latest, fully disposable, fixed-bed bioreactors; compares the virus production in the different systems; and discusses important manufacturing cost-related topics. It is predicted that single-use, fixed-bed bioreactors will receive even more attention in the field of viral vector manufacturing and commercialization, especially with the need for higher virus titers and virus yields.

Viral gene therapy intends to bring genetic materials to the cell using a modified virus. This delivery system is called a viral vector. The viral vector often takes the property of the virus in regards to delivery, so the choice of virus leads to various outputs. Viruses differ from one another depending on their tropism, hitting certain tissues better than others; immunogenicity; and size. This review compiles evidence of recent innovations in the delivery of gene therapy using viral vectors with a specific emphasis on inducible and chimeric viral vectors that address long-standing limitations of traditional viral vectors. Conventional viral vectors, despite being the current standard for gene therapy delivery, pose risks and have significant flaws. Many elicit severe immunogenic responses and often hit unintended cells and tissues. This review discusses two new categories of viral vectors: chimeric vectors, which are a combination of two or more vectors, and inducible viral vectors, which use spatio-temporal awareness to improve efficiency and targeting. Chimeric vectors adopt key features from multiple different vectors to create a hybrid. These hybrid vectors can target new cell types, lower immunogenicity, and make downstream purification easier. Meanwhile, inducible vectors rely on external stimuli to dictate their expression. Inducers include small molecules, RNA, and light. While concerns remain around scalability of these therapies, the next level targeting capabilities make them promising for further use in the gene therapy space.

Cationic Lipid Formulations Alter the In Vivo Tropism of AAV2/9 Vector in Lung

The development of new genetic tools has revolutionized our ability to study the functional role of specific neuronal populations and circuits generating behavior. Although this revolution has already taken place in small animal models such as mice, adoption of these techniques has been relatively slow for animals more closely related to humans, such as nonhuman primates. Current challenges include effective delivery to much larger structural targets in the primate brain, cell-type specific transduction, and immunological responses. In this chapter, we will review some of the challenges and considerations that are specific to using these viral technologies in the nonhuman primate brain. Ultimately, these challenges can be met with new advances in surgical technique and gene therapy that will spin out new viral vectors with enhanced features able to compensate for the limitations of current vectors. As the existing challenges are circumvented, this will lead to a revolution in primate neuroscientific research and a greater understanding of the functional role circuits play in complex behaviors relevant to human neurological and psychiatric diseases.Key wordsOptogenetics Chemogenetics Viral delivery Viral vectors Gene therapy Nonhuman primates Monkeys

Gene therapy is widely used to treat incurable disorders and has become a routine procedure in clinical practice. Since viruses can exhibit specific tropisms, effectively penetrate the cell, and are easy to use, most gene therapy approaches are based on viral delivery of genetic material. However, viral vectors have some disadvantages, such as immune response and cytotoxicity induced by a disturbance of cell metabolism, including miRNA pathways that are an important part of transcription regulation. Therefore, any viral-based gene therapy approach involves the evaluation of side effects and safety. It is possible for such effects to be caused either by the viral vectors themselves or by the delivered genetic material. Many gene therapy techniques use non-coding RNA delivery as an effective agent for gene expression regulation, with the risk of cellular miRNA pathways being affected due to the nature of the non-coding RNAs. This review describes the effect of viral vector entry and non-coding RNA delivery by these vectors on miRNA signaling pathways.