337. Transient Ultrasound-Mediated Microbubble-Assisted Modulation of Blood-Brain Interface in Adult Common Marmoset to Improve rAAV-Mediated Brain Transduction

Background: Non-human primates (NHPs) could provide an appropriate model for neuromuscular diseases because of its cognitive function and physiological resemblance to human. Production of transgenic and knockout NHPs from preimplantation embryos by using genomic modification were reported. However, several decades would be required for establishing homozygous and/or phenotypically stable progenies and for developing the symptoms in case of late onset diseases. In contrast, induction of pathology with recombinant adeno-associated virus (rAAV) has potential to break through this situation, because it is possible to realize with the existing aged animals. In this context, fully maturated blood-brain interface (BBI) significantly limits passive rAAV transport from circulation to the brain. To overcome this issue, we investigated BBI opening as a promising technology to make cerebral capillary open transiently by resonance of i.v. injected microbubble (MB) locally excited with ultrasound irradiation (UI).Methods: Evans blue (EB) and aminoisobutyric acid (AIB) were used for a tracer that has high affinity for serum albumin (ALB). MB and EB were i.v. injected as bolus into the femoral vein of the anesthetized adult marmosets. Transcranial (TC) UI to the brain was performed for 5 minutes, and the brain was examined one day after the UI. To verify the leakage, cryosections were immunostained against ALB and CD31. For live imaging analysis, MB, carbon 11-labeled AIB, and rAAV1 were i.v. injected into the tail vein. Subsequently, TCUI and PET scan were performed. At two days after TCUI, rAAV1 with distinguishable another vector genome was i.v. injected again. At one week after TCUI, the brain was sampled, and pieced into various parts. Then relative rAAV genome copies were measured by qPCR.Results: Macroscopically, ALB leakage was recognized in temporal cortex and hippocampus when TCUI was performed towards the temporal region. When TCUI was performed towards all direction, it was found in whole brain except for white matter. Microscopically, the blood vessels of 10-50 µm in diameter especially in the hippocampus were sensitive to BBI opening. From live image, leakage in the hippocampus, anterior cingulate cortex, basal ganglia, optic tract, amygdala, and superior temporal gyrus started just after TCUI. Lower genome copies of the secondary injected rAAV1 at two days after TCUI revealed that the BBI opening was transient.Conclusion: UI-mediated MB-assisted TC-BBI opening is a promising approach to generate the disease model based on adult common marmoset.