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Ultrasound Microbubbles Enhance Recombinant Adeno-Associated Virus Vector Delivery to Retinal Ganglion Cells In Vivo

Posted Sep 30, 2010 Updated Apr 7, 2023
By Wenyue Xie MS , Su Liu MS , Hong Su MS , Zhigang Wang MS , Yuanyi Zheng PhD , Yi Fu MS
11 min read

Rationale and Objectives

The aim of this study was to investigate whether ultrasound-mediated microbubble destruction enhances the transduction efficiency of recombinant adeno-associated virus (rAAV)–mediated enhanced green fluorescent protein (EGFP) gene into retinal ganglion cells (RGCs) of rats and whether it causes relevant adverse effects.

Materials and Methods

Thirty-two adult Sprague-Dawley rats were divided into four groups with different ultrasound powers, and retinal flat mounts and hematoxylin and eosin staining sections were made for optimization of parameters. A further 70 adult Sprague-Dawley rats were divided into four groups randomly. The first group (group A) was used as a normal control with 10 rats, and the remaining rats were evenly divided into groups B, C, and D. Each group included 20 rats. Groups B and C received rAAV-encoding EGFP gene (rAAV 2 -EGFP) in phosphate-buffered saline without and with ultrasound to the retina, respectively. Group D received microbubbles and rAAV 2 -EGFP mixture and ultrasound to the retina. The injection approach was intravitreal injection for all eyes. After 21 days, RGCs were labeled retrogradely with Fluoro-Gold. After 28 days, retinal flat mounts, frozen sections, and pathologic sections were assessed in each group. Expression of EGFP reporter gene was observed on laser confocal microscopy and evaluated according to average optical density and transfected RGC rate. To evaluate adverse effects with retinal flat mounts, labeled RGCs were counted, and retinal pathologic sections were examined.

Results

When ultrasound parameters (frequency, 0.3 MHz; power, 0.5 W/cm 2 ; total time, 60 seconds [irradiation time, 5 seconds; interval time, 10 seconds; four times]) were selected, EGFP expression was stronger, and retinas were not damaged. In the second part of the experiment, RGCs were labeled with Fluoro-Gold successfully. Green fluorescence can be observed in labeled RGCs in groups B to D. While average optical density and transfected RGC rate in group D were the highest compared to the other groups, no significant reduction in RGC number was detected with retrograde labeling. No obvious damage was observed with pathologic sections.

Conclusions

Ultrasound-mediated microbubble destruction can effectively and safely enhance rAAV delivery to RGCs in rats, and it may represent a novel gene delivery method in gene therapy for glaucomatous optic neuroprotection.

Retinal ganglion cell (RGC) apoptosis results in optic nerve damage in glaucoma. It has been proven that RGC apoptosis can be adjusted with apoptosis-related genes , and the progress of apoptosis can be prevented. Thus, gene therapy will probably become a potential treatment for glaucomatous optic neuroprotection . However, how to make foreign gene expression safe and efficient in target cells has been the key issue of gene therapy. Recombinant adeno-associated virus (rAAV) vectors have become promising gene delivery tools for optic neuroprotection treatment. These vectors possess a number of features that render them suited for this purpose, including a lack of pathogenicity and the ability to transfect retinal cells in a stable and long-term manner. But the application of viral vectors has been limited because of difficult production in sufficient quantities with viral particles, so that gene transfection efficiency is not easy to enhance . Unger et al showed that ultrasound contrast agent microbubbles can be a targeted gene therapy vector by using ultrasound cavitation erosion. As the cavitation nuclei in the cavitation progress, the gene released after microbubble destruction can enter into the vessel wall or even the tissue, depending on the energy of the ultrasound radiation . Microbubbles, as a novel gene vector, could enhance target gene transfection and expression safely and efficiently . In this study, we investigated whether ultrasound-mediated microbubble destruction enhances rAAV delivery of enhanced green fluorescent protein (EGFP) reporter gene into RGCs of rats and whether it causes relevant adverse effects.

Materials and methods

rAAV and Microbubble Mixture Preparation

According to a previously described method , lipid microbubbles were prepared at the Institute of Ultrasound Imaging at Chongqing University of Medical Sciences. The lipid shells of the microbubbles were composed of 90 mol% 1,2-distearoyl-sn-glycero-3-phosphocholine, 5 mol% 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, and 1 mol% 1,2-distearoyl-sn-glycero-phosphoacid (Avanti Polar Lipids, Inc, Alabaster, AL). The lipids were mixed in chloroform and dried for 20 minutes under flowing nitrogen gas. To further dry the lipids, they were heated under vacuum for 45 minutes at −50°C. Glycerol (Sigma-Aldrich Corporation, St Louis, MO) and phosphate-buffered saline (PBS) (pH 7.0; Sigma-Aldrich Corporation) were added to the lipids. The solution was placed into a sonicating bath for 45 minutes and then aliquoted into gas-tight vials into which perfluoropropane gas (SynQuest Labs, Alachua, FL) was added. Microbubbles were formed by the shaking method using a VialMix mixing machine (DuPont Pharmaceuticals, Billerica, MA) for 45 seconds.

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Part 1 of the Experiment

Selection of Ultrasound Parameters

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Retinal Flat-mount Preparation

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Retinal Pathologic Section Preparation

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Part 2 of the Experiment: Comparison of Gene Transfection Efficiency

Intravitreal Injection and Gene Transfer

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Retrograde Labeling of RGCs

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Retinal Flat-mount preparation

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Retinal Frozen Section Preparation

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Retinal Pathologic Section Preparation

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EGFP Expression in RGCs and RGC Counting

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Statistical Methods

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Results

Ultrasound Parameter Selection

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Figure 1, Comparison of average optical density (AOD) in groups A and B for the optimization of ultrasound parameters. In group A, the AOD was 92.03 ± 2.59, and in group B, the AOD was 78.96 ± 1.12%. Thus, enhanced green fluorescent protein expression in group A was stronger than in group B ( P < .05).

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EGFP Expression in Retina

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Figure 2, Enhanced green fluorescent protein expression (EGFP) expression in retinal ganglion cells (RGCs) with retinal frozen sections in groups B, C, and D. All retinas were examined 28 days after intravitreal injections. No EGFP-positive cells were observed in group A. Some EGFP-positive RGCs were observed in the ganglion cell layer (GCL) in group B. EGFP-positive cells in groups C and D were observed clearly in the GCL, inner nuclear layer, and pigment epithelial layer. The green fluorescence intensity was strongest compared to each layer of retinas in groups B and C.

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Figure 3, Enhanced green fluorescent protein (EGFP) expression in retinal ganglion cells (RGCs) with retinal flat mounts in groups B, C, and D. All retinas were examined 28 days after intravitreal injections. (B1–D1) Labeled RGCs in groups B to D. (B2–D2) EGFP-positive cells in groups B to D. (B3–D3) Merged images from (B1,B2), (C1,C2), and (D1,D2): yellow-light cells represent EGFP expression in labeled RGCs. Green fluorescence in all groups was distributed extensively all over the retinal flat-mounts in groups B, C, and D. The green fluorescence intensity was strongest and the transfected rate of RGC highest in group D compared to groups B and C. In the merged images (B3–D3), occasional green-light cells were observed, and they may have represented amacrine cells or unlabeled RGCs.

Table 1

AODs for EGFP Expression in Retinal Ganglion Cells on Retinal Flat Mounts

Group AOD 1 (%) AOD 2 (%) AOD 3 (%) AOD 4 (%) AOD 5 (%) Group B (rAAV 2 -EGFP) 45.55 53.67 49.20 64.97 49.08 Group C (rAAV 2 -EGFP plus ultrasound irradiation) 73.72 87.84 58.68 57.53 65.48 Group D (ultrasound irradiation plus rAAV 2 -EGFP plus microbubbles) 104.92 88.93 95.23 98.81 87.23

AOD, average optical density; EGFP, enhanced green fluorescent protein; rAAV, recombinant adeno-associated virus.

P < .05, group D versus groups B and C.

Figure 4, Retinal ganglion cell (RGC) transfection rate (mean ± standard deviation) comparison in each group with retinal flat mounts (%). All retinas were examined 28 days after intravitreal injections. Compared to groups B and C, the RGC transfection rate was highest in group D ( P < .05).

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RGC Counting

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Histologic Analyses of the Retinas

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Discussion

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Academic Radiology, Volume 17, Issue 10
Journals General Radiology
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