Spontaneously Biotinylated Lentiviral Vectors For Envelope Independent Targeting Of Infection

As a result of a BBSRC funded project we have generated a new 293T based cell line specifically for the production of metabolically biotin labelled lentiviral vectors. These bio-lentiviral vectors have been demonstrated to efficiently complex with streptavidin paramagnetic particles. This combination has resulted in the most efficient purification and concentration method yet described for lentiviral vectors. We wish to use the platform of these new biotin-lentiviral packaging cells to exemplify their value to the gene therapy community. By virtue of their biotinylated surface these viruses can be attached to specific cell binding proteins so that they can be targeted to specific sites of disease. Their unique properties also allow us to increase our knowledge of the cell derived accessory proteins that are also resident on the lentiviral surface. Information on the influence of these proteins on the process of lentiviral infection can then be applied to more efficient targeting of gene therapy vectors to specific sites of infection.

For lentiviral vectors we have succeeded in engineering an entirely new producer cell type in which the extracellular domain of LNGFR is fused to a biotin acceptor peptide (BAPref). Coordinate expression with the bacterial Bir A gene in human 293T cells results in metabolic biotinylation of a specific lysine residue in the BAP region. LNGFR-BAP-BIOTIN appears on the surface of lentiviral vector making them susceptible to Streptavidin capture. We now propose to use the platform bio-lentivector technology to increase the efficiency of targeting and to test the hypothesis that 'infection attenuated, and non-infectious lentiviral vectors can be manipulated to generate an exclusively ligand dependent, tissues specific infection of target cells'. To do this we will pursue two strategies: 1) Incorporation of cell/tissue specific ligands onto lentivirus particles, thus enabling them to regain infectivity only for the targeted cells. 2) Ligand dependent targeting of non-infectious vectors to specific cells, followed by a second stage targeting delivery of infection promoting factors, thus restricting infection to cells that have been successfully targeted by both components (the non-infectious virus, and the infection agent).