The engineered AAV2-HBKO promotes non-invasive gene delivery to large brain regions beyond ultrasound targeted sites.
Authors: Kofoed RH, Noseworthy K, Wu K, Sivadas S, Stanek L, Elmer B, Hynynen K, Shihabuddin LS, Aubert I
Magnetic resonance imaging-guided focused ultrasound combined with microbubbles injected in the bloodstream (MRIgFUS) temporarily increases the permeability of the blood-brain barrier (BBB), which facilitates the entry of intravenously administered adeno-associated viruses (AAVs) from the blood to targeted brain areas. To date, the properties of the AAVs used for MRIgFUS delivery resulted in cell transduction limited to MRIgFUS-targeted sites. Considering future clinical applications, strategies are needed to deliver genes to multiple locations and large brain volumes while creating minimal BBB modulation. Here we combine MRIgFUS with a vector that has enhanced biodistribution following brain entry, AAV2-HBKO, to mediate broad gene delivery to targeted brain regions at levels with potential therapeutic relevance. Expression of a reporter gene was achieved in 13% and 21% of all neurons present in the striatum and thalamus, respectively, while targeting only 28% of the brain regions with MRIgFUS. Compared with AAV9, MRIgFUS-mediated delivery of AAV2-HBKO showed greater diffusion in the brain and a higher percentage of the neurons expressing the transgene. MRIgFUS AAV2-HBKO gene delivery to the brain has the potential to reach levels that are functionally and clinically relevant, and this even when using relatively low intravenous AAV dosages, compared with what is currently used in clinical trials.
Introduction
Purpose
Drug delivery with BBB opening
Study Objective
To evaluate MRIgFUS-mediated delivery of intravenous AAV2-HBKO for achieving broad, neuron-targeted gene transfer to deep brain regions with low immunogenicity and reduced off-target liver transduction.
Disease model
Healthy
MRI or image guidance method
Magnetic resonance imaging (MRI) guidance (MRIgFUS)
Targeted brain region(s)
Striatum
Cargo name and characteristics
Adeno-associated virus vector — engineered AAV2-HBKO (AAV serotype 2 variant with modified capsid for enhanced biodistribution and brain diffusion) carrying a reporter transgene; compared to AAV9 as a reference viral vector
Route of administration
intravenous
Outcomes and Safety
Summary of Outcomes
MRIgFUS combined with intravenous AAV2-HBKO (with microbubbles) produced robust neuronal transduction—achieving ~13% of striatal neurons and ~21% of thalamic neurons while targeting only ~27–29% of each region—and showed greater diffusion and neuronal targeting than AAV9 without causing persistent BBB leakage. Successful conditions were MRIgFUS targeting two 1 mm-diameter, dorsoventral-spanning focal spots per region (covering ~27% striatum / ~29% thalamus) combined with a high intravenous AAV2-HBKO dose (1.7×10^13 GC/kg); lower doses (1.7×10^12 and 3.3×10^12 GC/kg) produced minimal transduction.
Duration of biological effect
3 weeks
Safety-related matter
MRIgFUS-mediated delivery of AAV2-HBKO caused no persistent BBB permeability or microhemorrhage and no extensive T cell infiltration at 3 weeks (no fibrinogen/erythrocytes in parenchyma), although GFAP upregulation indicated astrocyte activation. When larger areas were targeted there was microglial morphological change and a minimal increase in MHCII and T cell infiltration in the thalamus, and the authors caution that AAV2-HBKO transduces dorsal root ganglia (a potential DRG toxicity concern), while peripheral liver biodistribution was substantially lower than AAV9.
Brain Region
Ultrasound Parameters
Focal Characteristics
focal depth: None; focal length: None; aperture size: None
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