The use of an adeno-associated viral vector for efficient bicistronic expression of two genes in the central nervous system.
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.
Neurorestoration Group, Wolfson Centre for Age-Related Diseases, King's College London, Guy's Campus, London, SE1 1UL, UK, thomas.hutson@kcl.ac.uk.
Thomas Haynes
TH
Hutson
Claudia
C
Kathe
Sean Christopher
SC
Menezes
Marie-Claire
MC
Rooney
Hansruedi
H
Bueler
Lawrence David Falcon
LD
Moon
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002490:Central Nervous System / Q000293:injuries* / Q000378:metabolism / Q000502:physiology; D003001:Cloning, Molecular / Q000379:methods; D000229:Dependovirus / Q000235:genetics*; D018274:Electroporation / Q000379:methods; D005260:Female; D015870:Gene Expression; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors / Q000008:administration & dosage / Q000235:genetics / Q000627:therapeutic use*; D006651:Histocytochemistry / Q000379:methods; D007267:Injections; D008853:Microscopy / Q000379:methods; D008969:Molecular Sequence Data; D009416:Nerve Regeneration; D016133:Polymerase Chain Reaction / Q000379:methods