Focused Ultrasound And Recombinant Adeno-associated Virus Non-invasively Promote Neuron Specific Gene Therapy

[Background]The recombinant adeno-associated virus(rAAV)has shown great potential in the treatment of neurodegenerative disease.However,the existence of BBB hinders the effective transmission of virus vectors.The most common method to achieve rAAV transfection is to open the head for targeted injection,but it increases the risk of intracranial infection and other complications and makes its clinical application limited.At present,it is shown that the combination of low-frequency focused ultrasound(FUS)and microbubble technology can open the blood-brain barrier non-invasively and transport drugs into brain tissue in a targeted way.On this basis,to further explore the main factors affecting the biological activity and selectivity of rAAV in the brain.To clarify the mechanism of the multiple opening BBB and the combination of drug regimens on brain tissue,we intend to use the focused ultrasound to burst drug-loaded microbubbles and open the blood-brain barrier in the cortex movement area of rats.The changes of ultrastructure and brain tissue morphology of nerve cells were observed by comparing the intensity time mechanical index of ultrasound irradiation in each group.The GFP protein after rAAV transfection was detected in the brain and outside the brain,to evaluate the feasibility and safety of the opening of blood-brain barrier.Our study will provide the theoretical basis for non-invasive and targeted gene transfer therapy for brain diseases.[Objectives]1.To explore the feasibility of using different acoustic parameters(sound pressure intensity irradiation time mechanical index)to open the blood-brain barrier.To preliminarily determine the key parameters of the noninvasive and effective opening of the blood-brain barrier.2.Targeted delivery rAAV in vivo can be achieved by using focused ultrasound as a technology which can accurately locate,temporarily,and refolded the blood-brain barrier.The molecular imaging method is used to explore the transmission mechanism of gene therapy.[Methods]It mainly includes:1.Safety and renaturation evaluation of focused ultrasound combined with microbubbles to temporarily open the blood-brain barrie.2.Evans blue(EB)tracer experiment.3.To study the mechanism of focused ultrasound combined with microbubbles to increase the permeability of blood-brain barrier.4.The GDPA enhanced T1W-MR imaging was used to monitor the BBB permeability.5,Using the focused ultrasound combined with microbubble,rAAV6-eGFP can cross the blood-brain barrier and transinfected the neuron.6.Behavioral tests of rats to assess the safety of BBB opening and rAAV transfection.[Results]1.Under the different ultrasound power and irradiation time,rats were injected with the same dose of microbubbles.The average extravasation of gadolinium contrast agent/EB per unit mass of brain tissue increased with the increase of ultrasound power and irradiation time.2.Light microscope and transmission electron microscope showed that there was no obvious damage or morphological change on the surface of cells in each experimental group,after MBs mediated focused ultrasound irradiation.The leakage was observed at 1 h and 2 h after the ultrasound treatment,but 4 h in a few vessels.These changes were parallel to the obvious disintegration of the TJ complex,which showed the redistribution and loss of immune signals of Occludin,Claudin-5,and ZO-1.3.In the MWM experiment,the crossing times of rats in the ultrasound irradiation group were(2.98±0.31)second and dwell time in platform area(1.21±0.16)second compared with the control group(3.37±0.7 sec;1.44±0.32 sec;p>0.05).There was no significant difference in the time spent in the target quadrant and the speed of movement among the groups.These results suggested that the memory and cognitive function of the rats did not change significantly after the opening of FUS.4.The expression of GFP was directly detected by the confocal microscope,which was enhanced after immunostaining with an anti-GFP antibody.In all the rats treated with FUS,GFP was highly expressed in the target cortex.However,GFP expression was almost undetectable in non-FUS treated areas.[Conclusions]1.The combination of focused ultrasound and microbubbles will lead to the disintegration of TJ molecular structure,resulting in the loss of brain microvascular connection and barrier function.Optimization of ultrasound parameters can induce BBB to open effectively,relatively and safely,while minimizing the damage to normal brain tissue.2.Intravenous rAAV combined with FUS caused BBB opening can provide effective gene delivery,followed with the gene expression in the central nervous system.This technique has a great potential of non-invasive and targeted gene delivery in the treatment of central nervous system diseases.