Dose and promoter effects of adeno-associated viral vector for green fluorescent protein expression in the rat brain.
Previous studies demonstrated that the rat neuron-specific enolase (NSE) promoter is effective for transgene expression in the brain in a variety of adeno-associated virus-2 vectors. This study evaluated the dose response and longer time course of this promoter and compared it to two cytomegalovirus/chicken beta-actin hybrid (CBA) promoter-based systems. NSE promoter-driven green fluorescent protein (GFP)-expressing neurons were found at doses as low as 10(7) particles, with expression increasing in a dose-dependent manner over a 3.3-log range. Bicistronic expression of GFP via an internal ribosome entry site coupled to the NSE promoter was also dose dependent, although the potency was decreased by 3.4-fold. The number of GFP-expressing neurons was stable for at least 25 months. The CBA promoter increased the numbers of GFP-expressing cells versus the NSE promoter, although the expression pattern remained neuronal and persisted for at least 18 months. The CBA promoter permitted detection of cells distal to the injection site that had retrogradely transported the vector from their terminal areas. Incorporating the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) into a CBA promoter vector induced greater expression levels in the hippocampus, as measured by stereological estimates of cell numbers and by Western blots, which demonstrated an 11-fold increase. Incorporation of the WPRE also improved transgene expression in primary neuronal cultures. The increased efficiency obtained with vector elements such as the CBA promoter and the WPRE may enhance the ability to genetically modify larger portions of the brain while requiring smaller doses and volumes.
Klein
Ronald L
RL
Department of Pharmacology and Therapeutics, University of Florida College of Medicine, Gainesville, Florida 32610, USA. ronklein@ufl.edu
Hamby
Mary E
ME
Gong
Yan
Y
Hirko
Aaron C
AC
Wang
Samuel
S
Hughes
Jeffrey A
JA
King
Michael A
MA
Meyer
Edwin M
EM
D000199:Actins / Q000235:genetics; D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D002452:Cell Count; D002645:Chickens; D003587:Cytomegalovirus / Q000235:genetics; D000229:Dependovirus / Q000235:genetics*; D004305:Dose-Response Relationship, Drug; D018014:Gene Transfer Techniques; D022761:Gene Transfer, Horizontal; D005822:Genetic Vectors / Q000235:genetics* / Q000378:metabolism* / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D017919:Hepatitis B Virus, Woodchuck / Q000235:genetics; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D008297:Male; D011401:Promoter Regions, Genetic / Q000235:genetics*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012045:Regulatory Sequences, Nucleic Acid; D013997:Time Factors; D019076:Transgenes
D000199:Actins; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins