Quantitative comparison of expression with adeno-associated virus (AAV-2) brain-specific gene cassettes.
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.
Xu
R
R
University of Auckland Medical School, Department of Molecular Medicine, Auckland, New Zealand.
Janson
C G
CG
Mastakov
M
M
Lawlor
P
P
Young
D
D
Mouravlev
A
A
Fitzsimons
H
H
Choi
K L
KL
Ma
H
H
Dragunow
M
M
Leone
P
P
Chen
Q
Q
Dicker
B
B
During
M J
MJ
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
D008156:Luciferases