Comparative analysis of gene transfer to human and rat retinal pigment epithelium cell line by a combinatorial use of recombinant adeno- associated virus and ultrasound or/and microbubbles

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.

FIGURE 1

The transfectíon efficiency of r AAV in ARPE-19 and RPE-J cells under MBs, US and US plus MBs conditions. (A) EGFP expression in ARPE-19 and RPE-J cells observed 48h after infection with rAAV2-EGFP.(400xmagnification) (B) EGFP positive rate of ARPE-19 and RPE-J cells analyzed by FCM 48h after infection with rAAV2-EGFP (**p <0,01, *p<0,05). The ARPE-19 cells were infected with rAAV2-EGFP at a dose of lxlO3 vg/cell and RPE-J cells at a dose of 1x104 vg/cell in all the above experiments. All data were presented as means±SD of three independent experiments.

FIGURE 2

Gene transfer by US plus MBs under different conditions. EGFP-positive ratios of ARPE-19 cells at different concentrations of MBs(A), US intensities(C), US exposure times (E) and duty cycles(G) were examined by FCM analysis 48h after infection with rAAV2-EGFP (*p <0.05). Likewise, EGFP-positive ratios of RPE-J cells at different concentrations of MBs(B), US intensities(D), US exposure times (F) and duty cycles(H) were also examined by FCM analysis 48h after infection with rAAV2-EGFP (*p <0.05). All data were presented as means±SD of three independent experiments.

FIGURE 3

Cell viability assessed by trypan blue exclusion test. Cell viability of APRE-19 cells at different (A) MBs concentrations, (B) US intensities, (C) exposure time and (D)duty cycles, and cell viability of PRE-J cells at different (E) MBs concentrations, (F) US intensities, (G) exposure time and (H) duty cycles were assessed immediately after transfection. (’p <0,05). All data were presented as means±SD of three independent experiments.

FIGURE 4

Phase-contrast photograph of ARPE-19 cells immediately after US plus MBs. No apparent cell damage was observed in (A) ARPE-19(1,2W/cm²; 60,120 seconds; 10%, 20%, 50% duty cycle; 20%, 33.3% MBs) and (C) RPE-J cells(0.5, lW/cm²; 30,60 seconds; 10%, 20%, 50% duty cycle; 6.7%,13.3%MBs). Apparent cell damage was found in (B) ARPE-19 ((3W/cm²,60 seconds, 50% duty cycle, 100%MBs) and (D) RPE-J cells (2W/cm²,90 seconds, 50% duty cycle, 26.7%MBs). (A,C,D, E: lOOxmagnification).