An integrated manufacturing platform for engineering cellular therapies
Creators: name: Timmins, Lauren Marie
Publisher: name: No Publisher Supplied
Year: 2022

description: Cell and gene engineering has transformed the landscape of treatment for patients. Viral vectors such as lentiviruses (LVs) are widely used in the cell therapy field as an effective and permanent method of gene delivery to cells for clinical use. This has resulted in the design and development of therapeutics such as chimeric antigen receptor (CAR)-T cells, the blockbuster therapy approved by the FDA in 2017 that has since successfully treated cancer patients that were unresponsive to other treatments. Despite the success and transformative nature of cell therapies, a major obstacle for continued success has been the large-scale production of uniform viral vector batches that meet clinical scale demands. The issue has been the scale-up of downstream manufacturing methods for the instable and fragile vector without compromising high titer and minimizing loss of functionality.The growing demand for LVs for clinical use has created a gap to address in process scalability given the unique nature of LVs. To satisfy the demands of engineered cell therapy, 1011 to 1014 particles are required per patient. Additionally, the complex biology of viral vectors makes them unstable and subject to damage during traditional processing and purification steps. Given what is known about the production of viral vectors, the most likely route to achieving a consistent and reproducible manufacturing process is through an automated, closed system process.In this thesis, we investigate a novel method for production of viral vectors that addresses concerns with downstream loss of infectious particles and increasing the volumetric production of lentivirus in a fixed compartment of volume. We also address concerns surrounding the stability and productivity of human embryonic kidney cells (HEK293) which are the gold standard vehicles for transient expression of viral vectors. We first developed a Transwell® engineering coculture system, which combines the key processes of transient transfection and lentiviral-based transduction in a single step system. We achieved this by using porous membrane technology to separate producer HEK293 cells and target cells while allowing the passage of nanometer sized lentiviral vectors. This method addresses the issues with handling and freezing large volumes of delicate vector and exposes target cells to be engineered with freshly produced virus, which is when the lentivirus is most infective.Next, we sought to address the critical issue of maximizing vector production in a fixed compartment of volume while maintaining high titer, as well as improving transduction efficiency through improved virus production and increased interaction between vector and target cells. We utilized 3D technologies, because large scale cell expansion is not suitable in 2D, to increase the surface area in Transwell inserts for which HEK293 cells can be seeded using microcarrier beads. In these studies, we discovered that HEK-carriers increased volumetric productivity (TU/cell), stabilized HEK cells and improved transduction in Transwells at low densities. We also looked at other engineering variables in the Transwell system that could affect transduction of target cells to improve the platform.Finally, we took a look at producer HEK293 cells themselves, and some of the challenges that impede stable and high viral vector production yields in high-cell density cultures. For these studies, we investigated a variety of methods to enhance and stabilize transient protein expression in HEK293 cells by studying the secretion dynamics over time based on uncontrolled parts of the transient transfection process in HEK293T cells. We used an inflammatory reporter cell line, NFκB, which when activated up front with a pro-inflammatory cytokine TNF-α provides a read out on the inflammatory status of cells in culture through secretion of reporter gene G-Luciferase (GLuc). Non-stimulated NFκB cells had similar viral production levels at equivalent time points to stimulated groups, and the production of gene therapy LVs in HEK cells weakly triggered NFκB activation but did not impact viral vector yields. Non-stimulated NFκB cells had higher transduction efficiency in earlier time point fractions compared to stimulated cell groups, which could result from the high inflammation state immediately after stimulation which caused poor quality virus production. We next looked at cell cycle arrest molecule sodium butyrate (NaBut) to control for unsynchronized cells in culture and saw increased production of virus to control at all time points, and significantly at later time points. We last looked at metabolic engineering of HEK cells with sodium dichloroacetate (DCA), a pyruvate dehydrogenase kinase inhibitor (PDK) to study impact on duration and production of virus. We focused on the first 60 hours of culture during which the majority of transient transfection takes place. These studies were performed using a one-way perfusion system with downstream collection to cleanly study dynamics of HEK secretion without retrotransduction to understand the duration of bioreactor use.Collectively, these experiments established the groundwork to control for cell mass and duration for use for a closed system bioreactor that can be theoretically scaled to achieve human doses of engineered cell products.