Ibrahim Irfanullah

This project sought to develop a synthetic cell line with increased Recombinant Adeno-Associated Virus (rAAV) titer by downregulating GFP production. Recombinant Adeno-Associated Virus (rAAV) is a gene delivery platform that has shown considerable promise for use in gene therapy. However, difficulty producing rAAV presents a significant challenge to its therapeutic application. There are a number of rAAV production methods, including transient triple transfection, which involves the plasmid transfection of viral genes into host cells, and synthetic transfection which involves the integration of recombinant viral genes into host cell genomes. Preliminary data collected in Hu Lab has shown that synthetic transfection is a promising method for rAAV production. However, certain aspects of the production process, such as the replication of GFP, can be in direct competition for cellular resources required for more essential processes in viral production. The purpose of this project was to develop a synthetic cell line in which GFP production processes are inhibited in order to reduce intracellular competition and thus increase viral titer. Cell lines were produced using a LacSwitch II inducible element in order to modify GFP production. Minor variations in the concentrations of inducible elements were introduced, and multiple clones of each cell lines were developed using techniques in cell-culture and single-cell cloning. Analytical techniques including qPCR and cellular assays were used to measure factors such as DNA/RNA concentration and viral titer in order to isolate the most efficient cell lines.