The European Society of Gene and Cell Therapy, Madrid, 2013
Current serum-free lentiviral production methods for gene therapy generally generate disappointing titres. As a consequence, multiple production enhancers have been tested in an attempt to improve these yields, including a number of media supplements. Of these, cholesterol in particular has been shown to significantly improve viral titres in established cell lines.
In this study, media supplementation with cGMP compliant cholesterol is shown to also be effective in the HIV transient transfection of suspension cultured 293T cells using branched PEI. Importantly, in this context, the time point at which cholesterol is added is crucial. It was found that supplementation with cholesterol pre-transfection drastically impairs lentiviral production. However, when added post-transfection the resulting titres more than doubled.
This effect was observed when using a number of permutations of lentiviral vector constructs (different transgenes or surface proteins). However, issues of biomaterial compatibility need to be evaluated if such measures are to be used in large-scale lentivirus production. Methyl-beta-cyclodextrin, a component of cholesterol supplements, can interact with both cholesterol and the plastic surface of disposable bioreactors, leading to cell death.
Post-transfection addition of cholesterol is therefore an effective supplementation in a transient transfection to enhance lentiviral vector production, but care must be taken in the selection of the growth parameters.