Abstract
Ultrasound-targeted microbubble destruction has been utilized to deliver a drug/gene into cells in both in vitro and in vivo studies. This work was performed to investigate the feasibility of gene transfer to human retinal pigment epithelium cell line(ARPE-19) and rat retinal pigment epithelium cell line(RPE-J) by a combinatorial use of recombinant adeno-associated virus (rAAV) and ultrasound (US) or/and microbubbles (MBs) and compare the difference between them. Different doses of serotype 2 rAAV encoding a enhanced green fluorescent protein (rAAV2-EGFP) gene and MBs was administered to ARPE-19 and RPE-J cells under different US conditions. Transfection efficiency and cell viability were assessed by fluorescence microscopy, flow cytometry (FCM) analysis, trypan blue staining. The results indicated that US and MBs could respectively improve rAAV2-mediated gene transfer to RPE-J cells, but neither US nor MBs could do so in ARPE-19 cells. US plus MBs could significantly enhance rAAV2-mediated gene transfer to ARPE-19 cells, however, the same effects were not seen in RPE-J cells. These findings demonstrated it is not always coincident that US, MBs and US plus MBs exert the similar effects on gene transfer in vitro RPE cells. So, it is necessary to choose appropriate RPE cell line for the study of US or/and MBs-mediated rAAV gene transfer in retinal gene therapy.
Highlights
The retinal pigment epithelium (RPE) cell plays many vital functions, this single layer of polarized cells between the photoreceptors and vascular choroid phagocytizes shed photoreceptor outer segments, transports nutrients, growth factors to the rods and cones, helps remove waste products and absorbs scattered light ( )
The AAV infectious process includes multiple-step intracellular events that are approximately divided into seven stages: adhesion to the surface of the target cell, receptor-mediated endocytosis, vesicular trafficking, endosomal escape, nuclear transport, viral uncoating and genome conversion of the single-stranded recombinant adeno-associated virus (rAAV) genome to double stranded DNA intermediates capable of expressing transgenes (, )
In case of rAAV-mediated gene delivery, US or US plus MBs were presumed to overcome most of the rate-limiting steps, from stage to stage and accelerate rAAV -mediated gene expression
Summary
The retinal pigment epithelium (RPE) cell plays many vital functions, this single layer of polarized cells between the photoreceptors and vascular choroid phagocytizes shed photoreceptor outer segments, transports nutrients, growth factors to the rods and cones, helps remove waste products and absorbs scattered light ( ). US and MBs could respectively improve rAAV -mediated gene transfer to RPE-J cells, but neither US nor MBs could do so in ARPE cells. US plus MBs could significantly enhance rAAV -mediated gene transfer to ARPE- cells, the same effects were not seen in RPE-J cells.
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