Selective gene transfer to the retina using intravitreal ultrasound irradiation.

Shozo Sonoda,Makoto Shirasawa,Katsuro Tachibana,Ryo Suzuki,Kazuo Maruyama,Taiji Sakamoto,Toshifumi Yamashita,Hiroto Terasaki,Eisuke Uchino

doi:10.1155/2012/412752

Shozo Sonoda, Makoto Shirasawa + Show 7 more

Open Access

https://doi.org/10.1155/2012/412752

Copy DOI

Abstract

This paper aims to evaluate the efficacy of intravitreal ultrasound (US) irradiation for green fluorescent protein (GFP) plasmid transfer into the rabbit retina using a miniature US transducer. Intravitreal US irradiation was performed by a slight modification of the transconjunctival sutureless vitrectomy system utilizing a small probe. After vitrectomy, the US probe was inserted through a scleral incision. A mixture of GFP plasmid (50 μL) and bubble liposomes (BLs; 50 μL) was injected into the vitreous cavity, and US was generated to the retina using a SonoPore 4000. The control group was not exposed to US. After 72 h, the gene-transfer efficiency was quantified by counting the number of GFP-positive cells. The retinas that received plasmid, BL, and US showed a significant increase in the number (average ± SEM) of GFP-positive cells (32 ± 4.9; n = 7; P < 0.01 ). No GFP-positive cells were observed in the control eyes (n = 7). Intravitreal retinal US irradiation can transfer the GFP plasmid into the retina without causing any apparent damage. This procedure could be used to transfer genes and drugs directly to the retina and therefore has potential therapeutic value.

Highlights

Ultrasound (US) increases the permeability of the plasma membrane and reduces the thickness of the unstirred layer at the cell surface, thereby facilitating the entry of DNA into cells
The retina deeper part of ocular tissue was more hard to deliver DNA because of difficulties of US exposure, we demonstrated a possibility of transcorneal US irradiation with MB transfer of DNA plasmids into the retina (Sonoda S, et al IOVS 2006;47:ARVO E-Abstract 828)
No green fluorescent protein (GFP)-positive cells were observed in the control eyes (n = 7; Figure 2(a)); the retinas that received plasmid and US concomitantly with or without bubble liposomes (BLs) showed GFPpositive cells (Figure 2(b))

Summary

Introduction

Ultrasound (US) increases the permeability of the plasma membrane and reduces the thickness of the unstirred layer at the cell surface, thereby facilitating the entry of DNA into cells. A combination of low-intensity US and microbubble (MB) echocontrast agents allows direct DNA transfer into the cytosol through small pores in the cells caused by cavitation effects, and dramatically enhances gene transfection both in vitro and in vivo [1,2,3,4]. Our group reported that combination of US and MB increases the induction efficiency of plasmid DNA in the surface of ocular tissues such as cornea, conjunctiva, and eyelid [1, 5, 6]. The retina deeper part of ocular tissue was more hard to deliver DNA because of difficulties of US exposure, we demonstrated a possibility of transcorneal US irradiation with MB transfer of DNA plasmids into the retina (Sonoda S, et al IOVS 2006;47:ARVO E-Abstract 828). Selective retinal transfection would be advantageous to improve induction efficiency and avoid unexpected US exposure

Objectives

Methods

Results

Conclusion