Enhancement of Lentiviral vector efficiency by modification of cis- and transacting factors affecting genomic RNA encapsidation.

Lentiviral vectors have been established as highly useful laboratory tools and for therapeutic use in humans for many years. Through continuous improvements, HIV-1 derived vectors have evolved into safer and more effective gene delivery vehicles. This vector system offers great advantages including sustained gene expression through stable vector integration, the capability of infecting both dividing and non-diving cells, broad tissue tropism, and a potentially safer integration site profile. More than two decades have passed since lentiviral vectors started being explored for gene therapy use. Despite further modifications, the lentiviral vector production system has not drastically changed since the introduction of the 3rd generation four plasmid system. Typically they comprise a transgene flanked by cis-acting sequences including the CMV promoter for transcription, the 5' leader of HIV containing the packaging signal sequences and a second sequence the Rev Response Element (RRE).
Improvement of our understanding of the HIV-1 life cycle is a prerequisite for optimization of the current vector design. Our knowledge about the RNA packaging process has increased significantly since these vectors were first developed. We have a clearer knowledge of the 2D and 3D structure of the viral RNA in the packaging signal region and we understand more about how the RNA is selected and transported to the site of encapsidation into virions. There are still many unanswered questions that restrict us from improving viral titres and gene transfer efficiency. The following questions, including: Which are the important sequences that influence HIV-1 vectors to manage to traffic RNA to the budding site to become its genome? Which are the critical host cell proteins contributing to this process? Which are the finer details of RNA structure elsewhere in the virus that may influence packaging.
Using recent findings about packaging the first aim is to make an informed mutational analysis of the packaging signal region to establish the minimal sequences required for efficient packaging. Mutational analysis will also be used to stabilize the RNA structure so that it favourites the formation of the U5-AUG interaction that promotes transcription and dimerization, but supresses translation. The infectious HIV-1 strain has to acquire flexible structures that serve various functions, which the HIV-1 derived vectors don't have to perform. The cellular chaperone proteins which accompany the viral RNA genome through the cell to the budding site will also be sought by RNA pull down and cross linking techniques and Mass Spectroscopy of proteins found linked to the genomic RNA. Their importance will be investigated using interfering RNA knockdown and/or cDNA overexpression experiments. Lastly, another aspect of this project will be the modification of the current cell lines used to produce lentiviral vectors. This will be achieved by either introducing vital replication factors for the virus or by identifying restriction factors whose genes will be knocked down.
The overall aim of the project is to increase our understanding of the RNA encapsidation process from many angles and use the knowledge to optimise design of vectors to generate highly efficient versions with maximised transgene carrying and delivery capacity for treatment of human disease