Développement de procédés efficaces pour la construction et la production de vecteurs adénoviraux

The adenovirus has numerous interesting characteristics making this particular virus an ideal candidate for the construction of vector for conducting studies in functional genomics.  The vast majority of those applications rely on a so-called “first-generation vector” in which the E1 region is replaced by a transgene.  Despite all their advantages, there are 2 weak links associated with first-generation vector:  the efficient construction of the actual vector and its production.  Therefore, the development of alternative methods for construction and production is necessary to ensure their usefulness.  The development will involve 2 axes: the reengineering of the transfer vector for the construction of recombinant adenovirus and the reengineering of the cell line capable of producing the vector.  Using a transfer vector co-expressing the adenoviral protease (PS) gene and GFP by using an IRES under the control of a tetracycline-regulated promoter, our laboratory previously established the proof of concept that positive selection of recombinant adenovirus through ectopic expression of the PS gene was an efficient approach to generate adenoviral libraries. However, the diversity achieved was quite low, around 1 recombinant adenovirus per 1,000 cells. The goal of this thesis was to design a new transfer vector in which the PS expression was independent from the expression of the transgene in order to be able to optimize its expression independently.  We also improved library diversity by lowering the amount of PS in order to reduce the the trans-complementation from the transfer vector.  Using this method, at least 1 recombinant adenovirus per 75 cells was generated with 100% of the plaques being recombinant. This system was successfully used to generate an antisense library targeting GFP. The diversity of the library was high enough to allow the selection of an antisense that inhibited 75% of GFP expression.  Amplification of those first-generation recombinant adenoviruses must take place in an E1-expressing cell such as 293 cells. However, when replicating in 293 cells, the recombinant adenovirus can exchange their transgene with the E1 region of the cell by homologous recombination, which results in the generation of a fully replicative adenovirus (RCA), a situation that compromises the purity of the viral preparation. Our laboratory has previously patented an A549 cell line expressing the E1 region and producing RCA-free recombinant adenovirus (BMAdE1). However, the replication of E1-deleted adenovirus in BMAdE1 cells was sub-optimal, in the range of 15-30% the level obtained in 293 cells.  The work done in this thesis establishes that the low level of E1B-55K could be responsible for the lower productivity of BMAdE1 cells. Thus, we have derived new clones following lentiviral transduction in order to increase E1B-55K expression. Western blot confirmed that some clones expressed more E1B-55K than BMAdE1, and this correlated with a more robust replication of a recombinant adenovirus in those clones. This newly optimized BMAdE1 cell line was adapted to serum-free suspension culture.

Keyphrases: Adenovirus, RCA, E1B-55K, Viral vector, Adenovirus, Positive selection, Library, Protease, Cell line