Using Directed Evolution to Improve Lentiviral Vector Targeting

我们开发了一种新型的基于SIV的慢病毒载体，它可以将基因高效地转移到肺部(PMID：27852956)。该载体融合了融合基因F蛋白和结合唾液酸(SA)的小鼠副流感病毒(Sendai)HN蛋白。副粘病毒科的其他成员，仙台病毒是其中的一员，具有不同的F和HN样蛋白，具有交替的融合活性和SA结合特性，使它们能够感染交替的呼吸目标。这个项目的目的是通过F和HN序列的定向进化来产生用于增强体内基因转移的改进的慢病毒载体包膜蛋白，该项目包括产生容易出错和/或DNA改组的副粘病毒科包膜蛋白基因文库，创建编码其包膜序列的病毒载体，以及筛选/选择增强型慢病毒伪型。该项目将涉及广泛的病毒载体分子生物学、载体生产以及体内和体外基因转移。文库的进化将通过传统和下一代DNA测序进行监测。总体而言，该项目是一个了解和操纵慢病毒载体包膜与细胞表面受体的生物相互作用的机会，从而改善病毒载体的靶向性和趋向性。

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.