Focused ultrasound-mediated APOE4 knockdown in mouse brain.

The apolipoprotein E (APOE) ε4 allele is widely recognized as the strongest genetic risk factor for late-onset Alzheimer's disease. Therapeutic strategies to reduce apoE4 expression in APOE ε4 carriers hold promise to mitigate neuroinflammatory and neurodegenerative processes driving disease progression. Focused ultrasound (FUS) was employed to transiently open the blood-brain barrier (BBB) for efficient knockdown of humanized APOE ε4 in the mouse brain via gene editing. The all-in-one clustered regularly interspaced short palindromic repeats (CRISPR)-based adeno-associated virus (AAV) vectors were administered intravenously at a dose of 1.5×10<sup>12</sup> vg per mouse to determine the gene-editing efficacy within the hippocampus. FUS-enhanced delivery of AAV resulted in a 12.6% knockdown of APOE ε4 gene expression in the targeted hippocampus, accompanied by an over 20% decrease in apoE4 protein levels and significant reductions in astrocyte and microglia levels. Our findings demonstrate a noninvasive, targeted approach for APOE ε4 knockdown, highlighting FUS-mediated brain-directed interventions as a promising therapeutic strategy for Alzheimer's disease. Focused ultrasound (FUS) enables noninvasive, transient blood-brain barrier (BBB) opening for enhanced adeno-associated virus (AAV) delivery. FUS-mediated gene editing achieves a 12.6% knockdown in APOE ε4 expression within the hippocampus of mouse brain. APOE ε4 knockdown significantly reduces apoE4 protein levels and astrocyte and microglia levels. No detectable gross toxicity was observed following the FUS-mediated gene editing.

Introduction

Purpose Drug delivery with BBB opening

Study Objective Evaluate focused ultrasound-mediated blood-brain barrier opening for enhanced systemic delivery of CRISPR-based AAV vectors targeting humanized APOE ε4 knockdown in mouse brain

Animal model / Human subject humanized APOE4KI mouse (B6(SJL)-ApoEtm1.1(APOE+4)Adiuj/J), 8 weeks old, both sexes

Disease model Alzheimer's disease (humanized APOE ε4 carrier)

MRI or image guidance method Yes (B-mode ultrasound imaging for targeting; 9.4T MRI for BBB opening confirmation)

Targeted brain region(s) Hippocampus ( Hemisphere)

Target coordinates 3 mm anterior, 2 mm lateral relative to lambda

Cargo name and characteristics AAV9 vector carrying all-in-one CRISPR-SaCas9 and sgRNA targeting APOE ε4, dose 1.5×10^12 vg/mouse

Route of administration intravenous (tail vein)

Outcomes and Safety

Summary of Outcomes FUS-mediated BBB opening (1.5 MHz, 1.5 MPa, 2 min) enhanced AAV delivery to hippocampus, achieving 12.6% APOE ε4 gene editing (sgRNA2) in targeted hemisphere vs 2.2% in non-targeted hemisphere. APOE4 mRNA reduced by 21%, apoE4 protein reduced by >20%. Significant reductions in astrocyte (GFAP/S100β) and microglia (Iba1/CD68) levels. No gross toxicity or off-target effects.

Safety-related matter No apparent edema or hemorrhage on T2-weighted MRI; minimal red blood cell extravasation at day 0 resolved by day 2; no off-target effects detected; no significant toxicity.

Brain Region

Ultrasound Parameters

Ultrasound instrument Verasonics Vantage 256 system with P4-1 phased array (ATL, Philips), 96 elements, 2.5 MHz center frequency, 1.5-3.5 MHz bandwidth

FUS Frequency 1.5 MHz

FUS Pressure 1.5 MPa (free-field peak negative pressure)

FUS Mode pulsed

Pulse duration ~1 µs (≈3 half cycles at 1.5 MHz, calculated)

Duration of a single FUS session 2 min (60 bursts)

Focal Characteristics focal depth 35 mm from transducer face

Treatment frequency single sessions

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