Using Directed Evolution to Improve Lentiviral Vector Targeting

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We have developed a novel SIV-based lentiviral vector that directs highly efficient in vivo gene transfer to the lungs (PMID: 27852956). The vector incorporates the fusogenic F protein and sialic-acid (SA) binding HN protein from murine parainfluenza (Sendai) virus. Other members of the Paramyxoviridae family, of which Sendai virus is a member, have distinct F and HN-like proteins with alternate fusion activities and SA binding properties allowing them to infect alternate respiratory targets. The aim of this project is to generate improved lentiviral vector envelope proteins for enhanced in vivo gene transfer via the directed evolution of F and HN sequences.The project involves the generation of error-prone and/or DNA-shuffled libraries of Paramyxoviridae envelope protein genes, the creation of viral vectors that encode their own envelope sequences and the screening/selection of enhanced lentiviral pseuodtypes. The project will involve extensive viral vector molecular biology, vector production, and ex vivo and in vivo gene transfer. The evolution of the libraries will be monitored by conventional and next generation DNA sequencing. Overall, this project is an opportunity to both understandand manipulate the biological interaction of lentiviral vector envelopes with cell surface receptors resulting in improved viral vector targeting and tropism.