Optimisation of transfection efficiency for maximum vector production

Project ID: 2228cd1260 (You will need this ID for your application)

Research Theme: Manufacturing The Future

UCL Lead department: Biochemical Engineering

Project Summary:

Adeno-associated virus (AAV) is a leading platform for gene delivery for therapy and vaccination as it had an excellent safety profile and efficient transduction to various target tissues. However, the large-scale production and long-term storage of viral vectors is not efficient resulting in lower yields, moderate purity, and shorter shelf-life compared to recombinant protein therapeutics. The transfection efficiency and the high cost of the DNA and transfection reagents are one of the major challenges in AAV vector production. Typically, AAV vectors are produced in HEK293 cells following transfection of three DNA plasmids. Successful transfection of all three plasmids is necessary to produce AAV vectors, which otherwise results in a) low titre and b) incomplete/empty viral packaging. Methods for plasmid transfection include calcium phosphate, liposome and polyethyleneimine (PEI). Each method has its own limitations that range from cost to pH sensitivity and cell toxicity. This study will investigate all three routes of transfection to elucidate the impact of optimised transfection conditions at small scale with the view to inform scale- up. Key characteristics that need to be addressed are a) titre; b) kinetics of transfection c) percentage /distribution of different capsids d) size and density distribution and e) the impurity profile post lysis in each system.