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Dissertation
An Integrated, Scalable Platform for rAAV Vector Development Designed for Translational Success
Doctor of Philosophy (Ph.D.), Drexel University
Mar 2024
DOI:
https://doi.org/10.17918/00002016
Abstract
Recombinant adeno-associated virus (rAAV) is a leading viral vector modality for gene therapy, with seven approved treatments and hundreds of ongoing clinical trials. rAAV manufacturing, while sufficient to support preclinical studies and clinical trials, is challenged at the scale-up due to the translational gap between vector development and manufacturing. Vector development is often limited to the early stage, small scales, using basic purification processes and analytical tools. It generally focuses on expression, target specificity, and immunogenicity, neglecting the crucial aspects of vector manufacturability (productivity and purity), which are essential for the clinical success of novel therapeutic vectors. I hypothesized that early identification of vector manufacturability is critical and necessary to assist with the translation of novel vectors. To address this, I proposed creating an integrated, scalable, high throughput rAAV screening and optimization platform to overcome vector development and manufacturing challenges at the discovery and pre-clinical stages. This research proceeded in three stages: (1) Develop a microscale rAAV production method for high-throughput productivity screening, enabling simultaneous testing of multiple constructs and conditions at a reduced cost and time without needing special equipment and expertise. It also provides a viable option to optimize vector design, modulate the effects of packaging plasmids, and improve production efficiency with a design of experiments (DOE) approach. The platform is scalable, reflecting productivity propensity at scale. (2) Integrate a novel UV-VIS/DLS/SLS analytic platform and additional methods like CsCl density banding, capillary electrophoresis, Nanopore sequencing, and multiplex ddPCR to screen vector purity. The platform, capable of quantifying and characterizing rAAV vectors in high throughput, eliminates the need for reference standards, large amounts of purified vectors, and intensive training, and is suitable for vector development at the early discovery stage. The combinatorial analytic platform identifies product-related impurities and optimizes vector purity early on. (3) Demonstrate the platform utility by developing a novel rAAV human factor VIII vector for Hemophilia A, ensuring optimized manufacturability for clinical success. This integrated, scalable screening platform will bridge the gap between discovery and clinical manufacturing, improving vector manufacturability throughout therapeutic development and moving novel therapies closer and faster to patients.
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Details
- Title
- An Integrated, Scalable Platform for rAAV Vector Development Designed for Translational Success
- Creators
- Xueyuan Liu
- Contributors
- Beverly Davidson (Advisor)Lin Han (Advisor)
- Awarding Institution
- Drexel University
- Degree Awarded
- Doctor of Philosophy (Ph.D.)
- Publisher
- Drexel University; Philadelphia, Pennsylvania
- Resource Type
- Dissertation
- Language
- English
- Academic Unit
- School of Biomedical Engineering, Science, and Health Systems (1997-2026); Drexel University
- Other Identifier
- 991021867712604721