Pitt Shield

Strategy to enhance transgene expression in proximity of amyloid plaques in a mouse model of Alzheimer's disease.

Authors: Weber-Adrian D, Kofoed RH, Chan JWY, Silburt J, Noroozian Z, Kügler S, Hynynen K, Aubert I

Gene therapy can be designed to efficiently counter pathological features characteristic of neurodegenerative disorders. Here, we took advantage of the glial fibrillary acidic protein (GFAP) promoter to preferentially enhance transgene expression near plaques composed of amyloid-beta peptides (Aβ), a hallmark of Alzheimer's disease (AD), in the TgCRND8 mouse model of amyloidosis. <b>Methods:</b> The delivery of intravenously injected recombinant adeno-associated virus mosaic serotype 1/2 (rAAV1/2) to the cortex and hippocampus of TgCRND8 mice was facilitated using transcranial MRI-guided focused ultrasound in combination with microbubbles (MRIgFUS), which transiently and locally increases the permeability of the blood-brain barrier (BBB). rAAV1/2 expression of the reporter green fluorescent protein (GFP) under a GFAP promoter was compared to GFP expression driven by the constitutive human beta actin (HBA) promoter. <b>Results:</b> MRIgFUS targeting the cortex and hippocampus facilitated the entry of rAAV1/2 and GFP expression under the GFAP promoter was localized to GFAP-positive astrocytes. Adjacent to Aβ plaques where GFAP is upregulated, the volume, surface area, and fluorescence intensity of the transgene GFP were greater in rAAV1/2-GFAP-GFP compared to rAAV1/2-HBA-GFP treated animals. In peripheral organs, GFP expression was particularly strong in the liver, irrespective of the promoter. <b>Conclusion:</b> The GFAP promoter enhanced transgene expression in proximity of Aβ plaques in the brain of TgCRND8 mice, and it also resulted in significant expression in the liver. Future gene therapies for neurological disorders could benefit from using a GFAP promoter to regulate transgene expression in response to disease-induced astrocytic reactivity.

Introduction

Purpose Drug delivery with BBB opening
Study Objective To determine whether a GFAP promoter drives increased rAAV1/2-mediated transgene (GFP) expression in astrocytes adjacent to amyloid-β plaques after MRI-guided focused ultrasound delivery to the TgCRND8 mouse brain.
Animal model / Human subject Mouse (Mus musculus), TgCRND8, 15 weeks old, sex None
Disease model Alzheimer's disease
MRI or image guidance method MRI-guided focused ultrasound (MRIgFUS) using a 7T MRI with a radiofrequency surface coil to image and target the cortex and hippocampus; contrast-enhanced T1-weighted MRI was used to visualize BBB permeability/confirm targeting.
Targeted brain region(s) Cortex And Hippocampus
Cargo name and characteristics Recombinant adeno-associated virus mosaic serotype 1/2 (rAAV1/2) vector expressing enhanced green fluorescent protein (GFP). Features: mosaic rAAV1/2 packaging (50:50 rAAV1/rAAV2), GFP reporter under either a 2,210 bp human GFAP promoter or a constitutive human beta actin (HBA) promoter with a CMV enhancer; contains woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) and bovine growth hormone polyA signal. Administered IV at 3 × 10^9 vector genomes per gram.
Route of administration intravenous (tail vein)

Outcomes and Safety

Summary of Outcomes MRI-guided focused ultrasound (MRIgFUS) enabled systemic rAAV1/2 delivery to the cortex and hippocampus producing astrocyte-specific GFP expression from a GFAP promoter that was ~37% higher in intensity and had increased volume/surface area in astrocytes adjacent to Aβ plaques in TgCRND8 mice, with notable off-target GFP expression in the liver.
Duration of biological effect 14 days
Safety-related matter The paper reports off-target transgene expression particularly in the liver (and to a limited extent the kidney) despite absence of detectable endogenous GFAP, raising concerns about peripheral/transient hepatic expression after systemic rAAV delivery and suggesting organ-specific microRNA inhibition as a possible mitigation. It also notes that MRIgFUS can increase GFAP expression (astrogliosis) for days and that anti-AAV antibody responses after initial administration may limit safe re-administration (immunogenicity).

Brain Region

Ultrasound Parameters

Ultrasound instrument 1.68 MHz spherically focused transducer (aperture: 7 cm, F-number: 0.8); manufacturer: None reported
FUS Frequency 1.68 MHz; 840 kHz
FUS Mode pulsed
Pulse duration 10 ms
Duration of a single FUS session 120 seconds
Focal Characteristics Focal depth: None; Focal length: None; Aperture size: 7 cm
Treatment frequency Single session

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