Noninvasive focal transgene delivery with viral neuronal tracers in the marmoset monkey

Abstract

Although preclinical neuroscientific modeling species permit invasive intracranial delivery of targeted neurotropic agents, direct intracranial injections are not readily translatable to clinical therapeutics. Transcranial focused ultrasound (tFUS) has been identified as a technique to circumvent surgical injections altogether by transiently opening the blood-brain barrier (BBB) with selective focus. We have recently characterized the ability to focally deliver substances across the BBB in the marmoset, a non-human primate model with similar husbandry requirements to rodents but with cortical topologies more similar to humans. Here, we establish a reliable method for selectively delivering adeno-associated viral vectors (AAVs) across the BBB in marmoset frontal cortex with tFUS and demonstrate long-range anterograde neuronal tracing. Using a single-element 1.46 MHz transducer, we focally perturbed the BBB ([~]1 x 2 mm) in area 8aD of frontal cortex in four adult marmoset monkeys using low-intensity focused ultrasound aided by microbubbles. We confirmed BBB opening via a gadolinium-enhanced MRI at 9.4 T prior to AAV delivery. Within an hour of opening the BBB, either AAV2 or AAV9 was delivered systemically via tail-vein injection. Four to six weeks later, animals were sacrificed, and microscopy was performed to confirm the presence of neurons transduced as indicated by EGFP or mCherry fluorescence. In all four marmosets, neurons were observed at the site of BBB perturbation, with AAV2 showing an exiguous distribution of transduced neurons when compared to AAV9. The results are compared to direct intracortical injections of anterograde tracers into area 8aD and similar (albeit sparser) long-range connectivity was observed. With evidence of transduced neurons specific to the region of BBB opening as well as long-distance tracing, we establish a framework for focal noninvasive transgene delivery to the marmoset brain. This technique will be of utility for the burgeoning marmoset model, with applications for noninvasive delivery of therapeutics, genetic delivery of precursors for techniques like two-photon imaging, or neuronal tracing across the lifespan.