title: Ultrasound-targeted microbubble destruction enhances AAV-mediated gene transfection in human RPE cells in vitro and rat retina in vivo.
abstract: This study was conducted to investigate the efficacy and safety of ultrasound (US)-targeted microbubble (MB) destruction (UTMD)-mediated rAAV2-CMV-EGFP transfection to cultured human retinal pigment epithelium (RPE) cells in vitro and to the rat retina in vivo. In the in vitro study, cultured human RPE cells were exposed to US under different conditions with or without MBs. Furthermore, the effect of UTMD on rAAV2-CMV-EGFP itself and on cells was evaluated. In the in vivo study, gene transfer was examined by injecting rAAV2-CMV-EGFP into the subretinal space of rats with or without MBs and then exposed to US. We investigated enhanced green fluorescent protein (EGFP) expression in vivo by stereomicroscopy and performed quantitative analysis using Axiovision 3.1 software. Hematoxylin and eosin staining and frozen sections were used to observe tissue damage and location of the EGFP gene expression. In the in vitro study, the transfection efficiency of rAAV2-CMV-EGFP under optimal UTMD was significantly higher than that of the control group (P=0.000). Furthermore, there was almost no cytotoxicity to the cells and to rAAV2-CMV-EGFP itself. In the in vivo study, UTMD could be used safely to enhance and accelerate the transgene expression of the retina. Fluorescence expression was mainly located in the retinal layer. UTMD is a promising method for gene delivery to the retina.
authors: Department of Ultrasound, Shanghai Jiaotong University Affiliated First People's Hospital, Shanghai, China.
authors: H L
authors: HL
authors: Li
authors: X Z
authors: XZ
authors: Zheng
authors: H P
authors: HP
authors: Wang
authors: F
authors: F
authors: Li
authors: Y
authors: Y
authors: Wu
authors: L F
authors: LF
authors: Du
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D000229:Dependovirus / Q000235:genetics*; D005822:Genetic Vectors / Q000008:administration & dosage*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D045423:Microbubbles; D051381:Rats; D017208:Rats, Wistar; D012160:Retina / Q000378:metabolism*; D055213:Retinal Pigment Epithelium / Q000378:metabolism; D013010:Sonication / Q000379:methods*; D014162:Transfection / Q000379:methods*; D014465:Ultrasonics
chemical_list: C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins