THE YEAST SACCHAROMYCES CEREVISIAE AS CELLULAR EVIRONMENT TO STUDY ADENO-ASSOCIATED VIRUS REPLICATION, INTEGRATION AND ENCAPSIDATION

The ideal approach of gene therapy is to replace the defective gene with a functional copy. The most promising viral vectors for gene therapy studies are those based on the adeno-associated virus (AAV), a non pathogenic single stranded DNA virus. To date, the most extensive clinical application of AAV is for cystic fibrosis which is a monogenic disease. We believe that a more profound knowledge of the AAV biology may give a great contribution for AAV therapeutic application. Two main limitations block the usage of AAV vectors for gene therapy, are the high variability of transduction efficiency depending on cell type, and the lack of a safe and efficient production and purification methods. The most used methods to produce AAV vector are time consuming and very expensive. Therefore, to find a more simple eukaryotic system where AAV replicates and integrates, provides an excellent opportunity for studying AAV biology and, more importantly, for AAV vector encapsidation. Recently, the yeast Saccharomyces cerevisiae has been shown to be a very useful genetic tool for virus research. Our final goal is to use the yeast Saccharomyces cerevisiae as cell recipient where AAV may be assembled. When AAV infects host cells in the absence of helper virus it integrates in chromosome 19 at the level of a sequence called AAVS1. The mechanism of site specific integration involves AAV proteins which are absent in the vectors. Accordingly, the AAV vectors do not integrate in AAVS1, but randomly. Therefore, there is a risk of insertional mutagenesis and activation of oncogenes that have important implications for the gene therapy applications of AAV vectors. Since the molecular mechanisms responsible for AAV random integration are largely unknown, we would explore the possibility to study this process in yeast. Taken together these results will have a great impact for AAV therapeutic applications.