Single-cell mapping of focused ultrasound-transfected brain.
Authors: Mathew AS, Gorick CM, Price RJ
Gene delivery via focused ultrasound (FUS) mediated blood-brain barrier (BBB) opening is a disruptive therapeutic modality. Unlocking its full potential will require an understanding of how FUS parameters (e.g., peak-negative pressure (PNP)) affect transfected cell populations. Following plasmid (mRuby) delivery across the BBB with 1 MHz FUS, we used single-cell RNA-sequencing to ascertain that distributions of transfected cell types were highly dependent on PNP. Cells of the BBB (i.e., endothelial cells, pericytes, and astrocytes) were enriched at 0.2 MPa PNP, while transfection of cells distal to the BBB (i.e., neurons, oligodendrocytes, and microglia) was augmented at 0.4 MPa PNP. PNP-dependent differential gene expression was observed for multiple cell types. Cell stress genes were upregulated proportional to PNP, independent of cell type. Our results underscore how FUS may be tuned to bias transfection toward specific brain cell types in vivo and predict how those cells will respond to transfection.
Introduction
Purpose
Drug delivery with BBB opening
Study Objective
To determine how focused ultrasound peak-negative pressure (PNP) influences which brain cell types are transfected and their transcriptional responses following blood–brain barrier opening.
Animal model / Human subject
Mus musculus (mouse); strain: None; age: None; sex: None
Disease model
Healthy
Cargo name and characteristics
Plasmid DNA encoding mRuby fluorescent protein (plasmid reporter gene)
Outcomes and Safety
Summary of Outcomes
Both 0.2 MPa and 0.4 MPa focused ultrasound successfully enabled plasmid transfection across the BBB: 0.2 MPa preferentially transfected BBB-associated cells (endothelial cells, pericytes, astrocytes), whereas 0.4 MPa produced higher overall transfection and enriched neurons, oligodendrocytes, and microglia; increasing PNP also caused proportional upregulation of cell-stress/inflammatory genes across cell types.
Duration of biological effect
48 h
Safety-related matter
They observed PNP-dependent upregulation of cellular stress and inflammation-associated genes (e.g., CTSD, CTSB, LY86, CD68, LYZ2, TYROBP) consistent with a resolving sterile inflammatory response 48 h post-FUS, though classical markers of sterile neuroinflammation (AIF1, GFAP, ICAM1) were not significantly upregulated. The authors note that prior studies report minimal tissue-level damage but caution that sonoporation can cause cellular harms (irreversible pores, increased ROS, apoptosis) and therefore recommend lower PNPs to reduce cellular stress while higher PNPs increase transfection at the cost of potentially eliciting sterile inflammation.
Brain Region
Ultrasound Parameters
FUS Frequency
1 MHz
FUS Pressure
0 MPa; 0.2 MPa; 0.4 MPa
Focal Characteristics
Focal depth: None; Focal length: None; Aperture size: None
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