title: AAV8, 9, Rh10, Rh43 vector gene transfer in the rat brain: effects of serotype, promoter and purification method.
abstract: We compared adeno-associated virus (AAV) serotypes for expression levels of green fluorescent protein (GFP) in the adult rat hippocampus by biophotonic imaging. Preparations of AAV serotypes 8, 9, Rh10, and Rh43 incorporating cytomegalovirus (CMV) promoter-driven GFP were purified by a CsCl method. Neither AAV Rh10 nor AAV Rh43 produced greater levels of GFP than AAV8, which was used as a reference. For AAV9, there was an increase relative to AAV8. The CsCl-purified AAV8 displayed an astroglial transduction pattern in contrast to the expected neuronal expression of other AAVs. After preparing the same CMV-GFP plasmid in AAV8 with an iodixanol purification method, the expected neuronal pattern resulted. The astroglial expression with the CsCl AAV8 was probably due to relatively high levels of protein impurities. We compared the CMV promoter with the CMV/chicken beta-actin (CBA) promoter in the context of AAV8, both prepared by iodixanol, and found the CBA promoter to produce stronger GFP expression. At two doses of vectors optimized for serotype, promoter and purification, we did not observe serotype differences among AAV8, AAV9, or AAV Rh10. The purification method can therefore impact the transduction pattern as well as the results when comparing serotype strengths.
authors.affiliation: Department of Pharmacology, Toxicology, and Neuroscience, Louisiana State University Health Sciences Center, Shreveport, Louisiana, USA.
authors.forename: Ronald L
authors.initials: RL
authors.lastname: Klein
authors.forename: Robert D
authors.initials: RD
authors.lastname: Dayton
authors.forename: Jason B
authors.initials: JB
authors.lastname: Tatom
authors.forename: Karen M
authors.initials: KM
authors.lastname: Henderson
authors.forename: Phillip P
authors.initials: PP
authors.lastname: Henning
mesh_terms: D000818:Animals; D001923:Brain Chemistry / Q000235:genetics*; D002460:Cell Line; D002586:Cesium; D002712:Chlorides; D000229:Dependovirus / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D005822:Genetic Vectors / Q000008:administration & dosage / Q000138:chemical synthesis; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D017919:Hepatitis B Virus, Woodchuck / Q000235:genetics; D006624:Hippocampus / Q000378:metabolism / Q000821:virology; D006801:Humans; D008297:Male; D011401:Promoter Regions, Genetic; D051381:Rats; D017207:Rats, Sprague-Dawley; D012703:Serotyping; D014161:Transduction, Genetic / Q000379:methods; D014283:Triiodobenzoic Acids
chemical_list: D002712:Chlorides; D014283:Triiodobenzoic Acids; D049452:Green Fluorescent Proteins; D002586:Cesium; C028019:cesium chloride; C044834:iodixanol