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The use of an adeno-associated viral vector for efficient bicistronic expression of two genes in the central nervous system

Research output: Chapter in Book/Report/Conference proceedingChapter

Thomas Haynes Hutson, Claudia Kathe, Sean Christopher Menezes, Marie-Claire Rooney, Hansruedi Bueler, Lawrence David Falcon Moon

Original languageEnglish
Title of host publicationAxon Growth and Regeneration
Subtitle of host publicationMethods and Protocols
EditorsAndrew J. Murray
Place of PublicationN/A
PublisherSpringer New York
Pages189-207
Number of pages19
VolumeN/A
EditionN/A
ISBN (Print)9781493907762
DOIs
Published2014

Publication series

NameMethods in Molecular Biology
PublisherSpringer New York
Volume1162
ISSN (Print)1064-3745

King's Authors

Abstract

Recombinant adeno-associated viral (AAV) vectors are one of the most promising therapeutic delivery systems for gene therapy to the central nervous system (CNS). Preclinical testing of novel gene therapies requires the careful design and production of AAV vectors and their successful application in a model of CNS injury. One major limitation of AAV vectors is their limited packaging capacity (<5 kb) making the co-expression of two genes (e.g., from two promoters) difficult. An internal ribosomal entry site has been used to express two genes: However, the second transgene is often expressed at lower levels than the first. In addition to this, achieving high levels of transduction in the CNS can be challenging. In this chapter we describe the cloning of a bicistronic AAV vector that uses the foot-and-mouth disease virus 2A sequence to efficiently express two genes from a single promoter. Bicistronic expression of a therapeutic gene and a reporter gene is desirable so that the axons from transduced neurons can be tracked and, after CNS injury, the amount of axonal sprouting or regeneration quantified. We go on to describe how to perform a pyramidotomy model of CNS injury and the injection of AAV vectors into the sensorimotor cortex to provide efficient transduction and bicistronic gene expression in cortical neurons such that transduced axons are detectable in the dorsal columns of the spinal cord.

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