title: Directed evolution of adeno-associated virus for glioma cell transduction.
abstract: Glioblastoma multiforme (GBM) is a serious form of brain cancer for which there is currently no effective treatment. Alternative strategies such as adeno-associated virus (AAV) vector mediated-genetic modification of brain tumor cells with genes encoding anti-tumor proteins have shown promising results in preclinical models of GBM, although the transduction efficiency of these tumors is often low. As higher transduction efficiency of tumor cells should lead to enhanced therapeutic efficacy, a means to rapidly engineer AAV vectors with improved transduction efficiency for individual tumors is an attractive strategy. Here we tested the possibility of identifying high-efficiency AAV vectors for human U87 glioma cells by selection in culture of a newly constructed chimeric AAV capsid library generated by DNA shuffling of six different AAV cap genes (AAV1, AAV2, AAV5, AAVrh.8, AAV9, AAVrh.10). After seven rounds of selection, we obtained a chimeric AAV capsid that transduces U87 cells at high efficiency (97% at a dose of 10(4) genome copies/cell), and at low doses it was 1.45-1.6-fold better than AAV2, which proved to be the most efficient parental capsid. Interestingly, the new AAV capsid displayed robust gene delivery properties to all glioma cells tested (including primary glioma cells) with relative fluorescence indices ranging from 1- to 14-fold higher than AAV2. The selected vector should be useful for in vitro glioma research when efficient transduction of several cell lines is required, and provides proof-of-concept that an AAV library can be used to generate AAV vectors with enhanced transduction efficiency of glioma cells.
authors.affiliation: Department of Neurology, Massachusetts General Hospital, and Neuroscience Program, Harvard Medical School, Boston, MA, USA.
authors.forename: Casey A
authors.initials: CA
authors.lastname: Maguire
authors.forename: Davide
authors.initials: D
authors.lastname: Gianni
authors.forename: Dimphna H
authors.initials: DH
authors.lastname: Meijer
authors.forename: Lev A
authors.initials: LA
authors.lastname: Shaket
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Wakimoto
authors.forename: Samuel D
authors.initials: SD
authors.lastname: Rabkin
authors.forename: Guangping
authors.initials: G
authors.lastname: Gao
authors.forename: Miguel
authors.initials: M
authors.lastname: Sena-Esteves
mesh_terms: D036022:Capsid Proteins / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D032621:DNA Shuffling / Q000379:methods; D000229:Dependovirus / Q000235:genetics*; D005434:Flow Cytometry / Q000379:methods; D015723:Gene Library; D005822:Genetic Vectors / Q000502:physiology; D005910:Glioma / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D009447:Neuroblastoma / Q000473:pathology; D013997:Time Factors; D014161:Transduction, Genetic / Q000379:methods*
chemical_list: D036022:Capsid Proteins; D049452:Green Fluorescent Proteins; D006493:Heparin