10/29/2024
By Danielle Fretwell

The Francis College of Engineering, Department of Biomedical Engineering and Biotechnology, invites you to attend a Doctoral Dissertation defense by Qiang Fu on, "Development of an Inducible Stable Cell Line for rAAV Production."

Candidate Name: Qiang Fu
Degree: Doctoral
Defense Date: Monday, Nov. 4, 2024
Time: Noon - 2 p.m.
Location: Southwick 240

Committee:
Advisor: Seongkyu Yoon, Ph.D., Chemical Engineering, UMass Lowell

Committee Members*
1. David McNally, Ph.D., Chemical Engineering, UMass Lowell
2. Maurizio Cattaneo, Ph.D., Chemical Engineering, UMass Lowell
3. Michael Graves, Ph.D., Biological Sciences, UMass Lowell

Brief Abstract:
AAV-mediated gene therapy is a quickly growing segment of the pharmaceutical market; however, the current transient transfection process to produce rAAV has several challenges. The stable cells are ideal for large-scale continuous production, overcoming the drawbacks in the current transient transfection and streamlining rAAV production. In this study, we proposed to use synthetic inducible promoters to control the viral component expression and develop the baseline of HEK293T stable cells via site-specific integration mediated with CRISPR-Cas9, targeting safe harbor sites of human genome (ROSA26, AAVS1, and CCR5 locus). With a total of three round integrations, stable cell pools were developed and evaluated at each round of integration. Single clones were further characterized for each integration round. Regarding the stable pools, the 5’ and 3’ junction PCR results confirmed the site-specific integration to each locus. The genome copy result showed that AAV components, including Rep78/68, E2A, E4orf6, Cap, and Rep52/40, were successfully integrated into the host cell genome. Genome and capsid titer after induction confirmed rAAV production for stable cell pools in each round. The packaging cell line (after second round integration) was able to produce rAAV. However, it was observed that the genome titer was ten-fold lower than the that of rAAV products done with triple plasmids transfection. The out-to-out PCR and qPCR assay results further confirm the site-specific integration. This research demonstrates the feasibility of developing the inducible stable cell line with the refactored viral vectors via a
site-specific integration.