Insertional mutagenesis at positions 520 and 584 of adeno-associated virus type 2 (AAV2) capsid gene and generation of AAV2 vectors with eliminated heparin- binding ability and introduced novel tropism.
Recombinant adeno-associated virus (AAV) vectors are promising in the context of gene therapy because of their ability to mediate efficient gene transfer and stable gene expression. AAV2 uses heparin sulfate as its primary receptor, which is widely expressed on the various tissues and organs. This limits the application of AAV2 in targeting specific tissues. To make an AAV2 vector with modified tropism, we constructed various AAV2 capsid mutants by inserting RGD-4C peptide at position 520 and/or at position 584. Eight mutants were generated, identified, and characterized. Heparin-binding ability was completely abrogated in five mutants, and partially reduced in three mutants. Solid-phase ELISA and gene transduction assays confirmed that the novel tropism is determined by the introduced RGD epitope, which binds to cellular integrin receptor. Our observations suggest that simultaneous modification at both sites, tentatively involved in heparin binding, results in altered tropism and improved transduction efficiency in vitro.
Shi
Xiangqun
X
Department of Neurology, Lanzhou Military District General Hospital, Lanzhou 730050, People's Republic of China.
Fang
Guangguang
G
Shi
Wenfang
W
Bartlett
Jeffrey S
JS
D000818:Animals; D001665:Binding Sites; D036022:Capsid Proteins / Q000235:genetics* / Q000378:metabolism; D002478:Cells, Cultured / Q000378:metabolism / Q000821:virology; D000229:Dependovirus / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D005822:Genetic Vectors; D006367:HeLa Cells / Q000378:metabolism / Q000821:virology; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000378:metabolism / Q000821:virology; D051379:Mice; D016254:Mutagenesis, Insertional; D009154:Mutation / Q000235:genetics; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism; D014161:Transduction, Genetic; D018523:Tropism
D036022:Capsid Proteins; D009842:Oligopeptides; C047981:arginyl-glycyl-aspartic acid; D006493:Heparin