HIV-1-derived lentiviral vectors pseudotyped with envelope glycoproteins derived from Ross River virus and Semliki Forest virus.

摘要

To improve gene transfer, HIV-1-derived lentiviral vectors can be "re-targeted" to infect different ranges of target cells by incorporating glycoproteins originating from foreign viruses in their envelope shell---a process named pseudotyping. The commonly used vesicular stomatitis virus glycoprotein (VSV-G) confers a broad host range upon the lentiviral vector. However, VSV-G suffers from a number of drawbacks, such as cell toxicity, that poses limitations on its use in gene therapy protocols. This dissertation wanted to investigate the potential of less toxic glycoproteins derived from Ross River virus (RRV) and Semliki Forest virus (SFV) for pseudotyping lentiviral vectors. RRV and SFV glycoproteins incorporated into lentiviral vectors, and formed infectious pseudotypes that could be efficiently concentrated by ultracentrifugation and were able to infect a wide range of target cells, similar to VSV-G pseudotypes. A systematic analysis revealed that lentiviral vectors incorporate RRV glycoproteins with a comparable efficiency to VSV-G. Both pseudotypes have comparable physical titers, but infectious titers with the RRV pseudotype are lower than with VSV-G. Incorporation of SFV glycoproteins into lentiviral vector is less efficient, leading to decreased physical and infectious titers. Transduction with VSV-G, RRV, and SFV pseudotypes in adherent cell lines can be significantly increased by using recombinant fibronectin and polybrene in combination, Together, the findings suggest that RRV glycoproteins may be an attractive alternative to VSV-G for pseudotyping lentiviral vectors. Further experiments showed a particular resistance of most non-adherent hematopoietic cells to infection with the RRV pseudotype, but not to the VSV-G-pseudotype. Exceptions were erythroleukemia-derived cells, and differentiated macrophage-like cells that were moderately permissive for infection with RRV pseudotype. Enzymatic digestion of cell surface proteins, but not other molecules, had an inhibitory effect on infection with RRV pseudotypes. The data suggest that efficient transduction with RRV-pseudotyped lentiviral vectors is mainly dependent on the cell type and the degree of cellular differentiation. In addition, cell surface proteins may play an important role in the viral entry pathway of RRV. These findings will be important in elucidating the mechanism of RRV uptake and infection and may prove useful in designing strategies for selective transduction of target cell populations.