| Objective: The achievement of organ-specific delivery of gene/drug represents a major problem limiting the clinical application of retroviral vectors for gene therapy, whilst non-viral techniques have the disadvantage of lack of efficiency and longevity of gene expression. Ultrasound-targeted microbubble destruction (UTMD) has been newly developed for destructing the bubbles carrying drugs or genes, and thus for achieving the local release of these target molecules. The aim of this study was to evaluate the potential for achieving to the effective, local delivery of recombinant adeno-associated virus serotypes 2 (rAAV2) transgene into rat myocardium by UTMD. This study involves three parts. The aim of the first part is to study the best titer of rAAV2 delivered to myocardium by targeted ultrasound microbubbles and being infused into tail vein. The aim of the second part is to study the changes of left ventricular function before and after rAAV2-GFP targeted to myocardium by the ultrasound mediated microbubbles. The aim of the third part is to evaluate the potential for achieving to the effective, local delivery of recombinant adeno-associated virus serotype-2 (rAAV2) transgene into rat myocardium by UTMD. Methods: The methods of the fist part: Twenty one adult SD rats were divided into seven groups. SonoVue attached with different titers (1.5×109vg/mL ;3.0×109vg/mL;1.5×1010 vg/mL;3.0×1010vg/mL; 1.5×1011vg/mL; 3.0×1011vg/mL) of rAAV2-GFP was infused into the tail vein of rats, following ultrasound mediated microbubbles destruction, as experiment groups. Normal saline was infused into the tail vein of rats as the control group (without rAAV2). Rats were killed after 14 days and hearts were harvested. GFP protein expression which showed rAAV2 transfer was observed under fluorescence microscope in frozen section. The methods of second part: The rats were divided into 2 groups of 10 rats. Control group: Echocardiographic destruction of microbubbles without rAAV2-GFP. Experimental group: Echocardiographic destruction of microbubbles containing rAAV2-GFP. The mean value of Left ventricular rotation of rats was obtain in apical and base plane using speckle tracking imaging (STI). LV twist was defined as apical rotation relative to the base. Circumferential strain; systolic and diastolic circumferential strain rate E and A; torsion and time to peak were measured using STI, before and after 14 days of experiments. Rats were killed after 14 days and hearts were harvested. GFP protein expression which showed rAAV2 transfer was observed under fluorescence microscope in frozen section. The methods of the third part: Recombinant adenovirus vector marked with Green Fluorescent Protein (GFP) was attached to the surface of the SonoVue, sulphur hexafluoride-filled microbubbles. These bubbles were infused into the tail vein of rats with or without simultaneous echocardiography. Rats were divided into six groups of 10: Group 1 echocardiographic destruction of microbubbles without rAAV2-GFP; Group 2 infusion of rAAV2-GFP (no microbubbles) without echocardiography; Group 3 echocardiography during infusion of rAAV2-GFP (no microbubbles); Group 4: echocardiographic destruction of microbubbles followed by rAAV2-GFP infusion; Group 5: microbubbles containing rAAV2-GFP without echocardiography; Group 6: echocardiographic destruction of microbubbles containing rAAV2-GFP. Group 6 was the experimental target; the remaining groups were controls. We administered infusions (1.2 mL) over 5～8 minutes because of the risk of volume overload. Results: The results of the first part: When the titer of rAAV2 was 1.5×1011 vg/ml infused into the tail vein of rats there was much more GFP expression in myocardium than lower titers (P<0.01) However the 3.0×1011 vg/ml group also showed a large quantity of GFP expression, the GFP expression in the 1.5×1011 vg/ml group is clear and enough to observe the rAAV2 transfection rate. The results of the second part:There is no significant difference of left ventricular myocardial function of rats between before and after rAAV2-GFP targeted to myocardium by the ultrasound mediated microbubbles. The results of the third part: The hearts of all 10 rats in the experimental groups showed luciferase activity, indicating rAAV2-GFP expression. All of the treatment groups that received the virus showed less luciferase activity in the myocardium than the group of echocardiographic destruction of microbubbles containing rAAV2-GFP. This confirmed that destruction of the microbubbles containing the virus was responsible for the observed GFP expression in the myocardium of rats. The livers of all rats that received the virus showed extensive GFP activity, whereas none of the brains showed GFP activity. GFP expression was 2.2-fold higher in the group treated with echocardiographic destruction of microbubbles (group 6) containing rAAV2-GFP than in the controls (P, 0.001). The group that underwent microbubble destruction followed by an infusion of rAAV-GFP had a 1.6-fold increase in GFP expression, suggesting that disruption of the endothelial barrier is an important factor in viral transduction. There is significant difference of GFP activity only in group of echocardiographic destruction of microbubbles containing rAAV2-GFP, between liver and heart (P<0.01), however other groups showed no significant difference (P>0.05). Conclusion: Ultrasound-mediated destruction of SonoVue is a promising method for the delivery of rAAV2 to the heart in vivo. And it is safe invasive and convenient and efficient way for gene transfection. 1) The first part of conclusion is that 1.5×1011 vg/ml is the best titer of rAAV2-GFP delivered to myocardium by being infused into tail vein and ultrasound mediated microbubbles destruction. The second part of conclusion is that the left ventricular function between before and after the experiment had not significant difference. It means the method of enhancement of rAAV2 delivered to the myocardium by ultrasound mediated microbubbles destruction is safe and efficient. The third part of conclusion is that ultrasound-mediated destruction of microbubbles is a promising method for the delivery of rAAV2 to the heart in vivo.
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