Ultrasound Microbubbles Enhance Recombinant Adeno-associated Virus Vector Delivery To Retinal Ganglion Cells In Vivo

BackgroundIt has been reported that ultrasound-mediated microbubbles destruction could enhance obviously transduction efficiency of reporter gene which was transferred to retinal ganglion cells(RGCs) in vitro and in vivo.ObjectiveTo investigate whether ultrasound-mediated microbubbles destruction could enhance transduction efficiency of recombinant adeno-associated virus(rAAV) mediated enhanced green fluorescent protein(EGFP) gene into RGCs of rats and whether it causes relevant adverse effects.Design, time and site of the experimentObservation experiment of gene morphology. It was completed in the laboratory of Institute of Ultrasonic Image in Chongqing Medical University between March and September in 2008.Methods75 adult Sprague-Dawley (SD) rats were divided into five groups randomly. Each group had ten rats. The first group (group A) was used as normal control. Group B and group C received recombinant adeno-associated virus encoding EGFP gene(rAAV2-EGFP) in PBS without or with ultrasound to the retina respectively. Group D and group E received the mixture of microbubbles and rAAV2-EGFP through intravitreal injection without or with ultrasound to the retina respectively. After 21 days, RGCs were labeled retogradely with fluogold. After 28 days, the retinal flatmounts,frozen sections and retinal HE staining were performed with five rats respectively in each group.Major index of observationExpression of EGFP reporter gene was observed on laser scanning confocal microscope and evaluated through average optical intensity (AOD) and transfected RGCs rate. Labeled RGCs were counted with retinal flatmounts and histopathologic slices were observed to evaluate the damage of retinas.ResultsGreen fluorescence can be obeserved exactly in labeled RGCs in B,C,D,E groups. While AOD and transfected rate in group E respectively were (95.02±7.25)and(20.10±0.74)% . They were higher compared with other groups,and no obvious damage to retinas was detected with RGCs counting and pathologic slices.Conclusion In some condition, Ultrasound-mediated microbubbles destruction can effectively and safely enhance rAAV delivery to RGCs in rats, and it may be a novel gene delivery method in gene therapy of glaucomatous optic neuroprotection.