Lentiviral vectors for gene delivery to the nervous system

Abstract

The effi cient management and development of therapeutic strategies for disorders of the nervous system still remains a major medical challenge. Gene therapy for the nervous system diseases is particularly challenging due to the post-mitotic nature of neuronal cells and the restricted accessibility of the brain itself. Viral vectors based on lentiviruses are particularly attractive vehicles, routinely used in developing genebased therapies to treat neurological diseases. Due to their unique properties, which allow them to transducer most nervous system cell types, maintaining strong, and long-term transgene expression, they present a versatile and powerful tool for many research and gene therapy applications. Lentiviral vectors pseudotyped with envelope glycoproteins derived from various viruses, such as VSV and rabies have been shown to be able to genetically modify cells with good effi ciency and broad tropism. This chapter discusses lentiviral vectors properties and applications in gene therapy for neurodegenerative diseases, presenting some of the recent progress in this fi eld. We also present the materials and methods necessary to generate hightiter lentiviral vectors. Methods and applications involving lentiviral production are frequently changing. Here we describe the current protocols used and optimized in our laboratories that allow us to produce high-titer lentiviral vector preparations for both in vitro and in vivo applications. Full detailed protocols describe here step-by-step the lentiviral vector production from DNA preparation, culturing of 293T producer cells to transfection for viral production, and titration of the lentiviral vector preparations. We further present standard in vitro transduction experiments and the in vivo applications for which these vectors are used in our research. In the in vivo application section surgical details are presented.