Noninvasive focal transgene delivery with viral neuronal tracers in the marmoset monkey.
Authors: Parks TV, Szczupak D, Choi SH, Schaeffer DJ
We establish a reliable method for selectively delivering adeno-associated viral vectors (AAVs) across the blood-brain barrier (BBB) in the marmoset without the need for neurosurgical injection. We focally perturbed the BBB (∼1 × 2 mm) in area 8aD of the frontal cortex in four adult marmoset monkeys using low-intensity transcranial focused ultrasound aided by microbubbles. Within an hour of opening the BBB, either AAV2 or AAV9 was delivered systemically via tail-vein injection. In all four marmosets, fluorescence-encoded neurons were observed at the site of BBB perturbation, with AAV2 showing a sparse 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.
Introduction
Purpose
Drug delivery with BBB opening
Study Objective
To establish a focal, noninvasive method using transcranial focused ultrasound to deliver adeno-associated viral vectors across the blood-brain barrier in marmosets for neuronal transduction and anterograde tracing without neurosurgical injection.
Animal model / Human subject
Marmoset monkey (species: marmoset), strain: None, age: adult, sex: None
Targeted brain region(s)
Area 8Ad (Frontal Cortex)
Cargo name and characteristics
Adeno-associated viral vectors (AAV): AAV2 and AAV9 serotypes, systemically delivered gene therapy vectors encoding fluorescent reporter proteins (EGFP or mCherry) to transduce neurons.
Route of administration
Intravenous (tail-vein injection)
Outcomes and Safety
Summary of Outcomes
Microbubble‑aided transcranial focused ultrasound (1.46 MHz single‑element, low‑intensity sonication producing ~1 mm radial × 2 mm axial BBB openings; microbubble dose ~200 μL/kg; single sonication effective in 3/4 animals with one requiring slightly higher pressure and microbubble dose) enabled systemic AAV2 and AAV9 to cross the BBB and selectively transduce neurons at the focal site with long‑range axonal labeling, with AAV9 giving substantially denser transduction than the sparse labeling from AAV2.
Duration of biological effect
>8 h
Safety-related matter
The authors acknowledge safety concerns including potential infection or exposure to circulating toxins during prolonged BBB opening, untoward inflammatory responses from microbubble dosing, peripheral organ effects (notably the liver) and variability from AAV-neutralizing antibodies; however, they report that with titrated microbubble dosage and acoustic parameters BBB opening was achieved without tissue damage and with minimal immune response, while noting openings can last >8 h and dosing may need further optimization to reduce risks.
Brain Region
Ultrasound Parameters
Focal Characteristics
focal depth: None; focal length: None; aperture size: None
Treatment frequency
single
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