title: Quantitative comparison of expression with adeno-associated virus (AAV-2) brain-specific gene cassettes.
abstract: This study compared a range of mammalian CNS expression cassettes in recombinant adeno-associated virus (AAV-2) vectors using strong endogenous promoter sequences, with or without a strong post-regulatory element and polyadenylation signal. Changes in these elements led to transgene expression varying by over three orders of magnitude. In experiments conducted in primary cell culture and in >100 stereotactically injected rats, we observed highly efficient and stable (>15 months) gene expression in neurons and limited expression in glia; the highest expression occurred with endogenous, nonviral promoters such as neuron-specific enolase and beta-actin. The packaging size of AAV-2 was maximized at 5.7 kb without impairing gene expression, as judged by direct comparison with a number of smaller AAV-2 constructs. The genomic insert size and titer were confirmed by Southern blot and quantitative PCR, and infectivity was tested by particle titer using ELISA with a conformation-dependent epitope that requires the full intact capsid. A packaging and purification protocol we describe allows for high-titer, high-capacity AAV-2 vectors that can transduce over 2 x 10(5) neurons in vivo per microliter of vector, using the strongest expression cassette.
authors.affiliation: University of Auckland Medical School, Department of Molecular Medicine, Auckland, New Zealand.
authors.forename: R
authors.initials: R
authors.lastname: Xu
authors.forename: C G
authors.initials: CG
authors.lastname: Janson
authors.forename: M
authors.initials: M
authors.lastname: Mastakov
authors.forename: P
authors.initials: P
authors.lastname: Lawlor
authors.forename: D
authors.initials: D
authors.lastname: Young
authors.forename: A
authors.initials: A
authors.lastname: Mouravlev
authors.forename: H
authors.initials: H
authors.lastname: Fitzsimons
authors.forename: K L
authors.initials: KL
authors.lastname: Choi
authors.forename: H
authors.initials: H
authors.lastname: Ma
authors.forename: M
authors.initials: M
authors.lastname: Dragunow
authors.forename: P
authors.initials: P
authors.lastname: Leone
authors.forename: Q
authors.initials: Q
authors.lastname: Chen
authors.forename: B
authors.initials: B
authors.lastname: Dicker
authors.forename: M J
authors.initials: MJ
authors.lastname: During
mesh_terms: D000818:Animals; D015139:Blotting, Southern / Q000379:methods; D015153:Blotting, Western / Q000379:methods; D001921:Brain / Q000201:enzymology*; D002478:Cells, Cultured; D000229:Dependovirus / Q000235:genetics*; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000008:administration & dosage / Q000235:genetics*; D007267:Injections; D008156:Luciferases / Q000235:genetics*; D051381:Rats; D017208:Rats, Wistar
chemical_list: D008156:Luciferases