The case studies presented in this thesis explored the autologous supply chains and automation, a range of viral vector manufacturing flowsheets and viral vector process changes. In addition, a cost of drug development model and a cash flow model were built to evaluate the impact of process changes at different stages in the drug development pathway and evaluate the profitability of different manufacturing strategies. From the cost perspective, models were built to compute manufacturing costs, namely cost of goods (COG) and fixed capital investment (FCI), and coupled with brute force optimisation to identify optimal manufacturing strategies. The decisional tools employed in this thesis included bioprocess economics models tailored to autologous CAR T-cell therapy and viral vector products.
This thesis aims to explore these avenues by developing and applying advanced decisional tools that analyse the gene therapy supply chain systematically whilst capturing multiple stakeholder perspectives. Given the relative infancy of the sector, there is a strong drive towards adopting technologies that minimise costs and supply chain complexity. chimeric antigen receptor T-cell therapy – CAR T), hence the supply chain of these products is highly complex. Most of today’s gene therapy products are viral vector-based, typically relying on plasmid DNA supply for their production, and many are autologous ex vivo applications (e.g. Gene therapy products have tremendous therapeutic potential for indications such as cancer and even curative potential for some genetic diseases. Neither liver sections of AAV2-DJ/8-shGlrx-mVenus injected mice, incubated with secondary antibody alone (middle row), nor saline-injected mice exhibited a significant mVenus signal at identical exposure time settings. The mVenus signal was most intense around the triad (T) and faded towards (arrow) the central vein (C), showing zoning across the liver acinus. Nuclei were counterstained with Hoechst 33342. (C) Liver sections from an AAV2-DJ/8-shGlrx-mVenus injected mouse showed a strong fluorescence signal of mVenus (top row, red color) in cytoplasm and nuclei. The membrane (dotted line indicates the cut) was probed for mVenus and Glrx expression 2 weeks post AAV injection. (B) Western blot analysis of the liver samples described in (A). Statistical differences (n = 6-7 P < 0.05 SD) were compared to shControl. Gene expression was measured by RT-qPCR and the statistical analysis was performed using the two-tailed Mann-Whitney test. (A) Glrx mRNA expression levels in livers of C57BL/6J mice two weeks after administration of AAV2-DJ/8-shControl-mVenus (shControl) or AAV2-DJ/8-shGlrx-mVenus (shGlrx). MRNA and protein expression of Glrx in mouse liver. Our study provides an improved protocol for a more economical and efficient purified AAV preparation. AAVs coding for glutaredoxin-1 (Glrx) shRNA successfully inhibited Glrx expression by ~66% in the liver and skeletal muscle. For proof of concept, we verified in vivo transduction via Western blot, qPCR, luminescence, and immunohistochemistry. Of note, we achieved titers of 1010–1011 viral genome copies per µl with a typical production volume of up to 1 ml while requiring five times less than the usual number of HEK293T cells used in standard protocols. Furthermore, we then implemented an iodixanol gradient purification, which resulted in preparations with purities adequate for in vivo use. Using a helper-free AAV system, we purified AAVs from HEK293T cell lysates and medium by polyethylene glycol precipitation with subsequent aqueous two-phase partitioning. Here, we report an improved protocol to produce serotype-independent purified AAVs economically. However, major obstacles remain for widespread AAV utilization, such as impractical purification strategies and low viral quantities. Combined with modern gene technologies, such as cell-specific promoters, the Cre/lox system, and genome editing, AAVs represent a practical, rapid, and economical alternative to conditional knockout and transgenic mouse models. Among viral delivery systems, adeno-associated viruses (AAVs) are relatively safe and demonstrate high gene transfer efficiency, low immunogenicity, stable long-term expression, and selective tissue tropism. Delivering and expressing a gene of interest in cells or living animals has become a pivotal technique in biomedical research and gene therapy.