Sonoselective transfection of cerebral vasculature without blood–brain barrier disruption

Significance Focused ultrasound (FUS) is a targeted and noninvasive technique that can be used to activate gas-filled microbubbles (MBs) to oscillate within the bloodstream. This technique has been used previously to open the blood–brain barrier (BBB) to facilitate the delivery of therapeutics to the surrounding brain tissue. However, disruption of the BBB may be contraindicated in certain disease contexts. Here, we utilize low-pressure FUS to oscillate the MBs just enough to transfect endothelial cells, without opening the BBB. The low-pressure FUS regimen results in enhanced gene delivery to endothelial cells, with none of the inflammatory or immune pathway up-regulation observed at higher FUS pressures.
Treatment of many pathologies of the brain could be improved markedly by the development of noninvasive therapeutic approaches that elicit robust, endothelial cell-selective gene expression in specific brain regions that are targeted under MR image guidance. While focused ultrasound (FUS) in conjunction with gas-filled microbubbles (MBs) has emerged as a noninvasive modality for MR image-guided gene delivery to the brain, it has been used exclusively to transiently disrupt the blood–brain barrier (BBB), which may induce a sterile inflammation response. Here, we introduce an MR image-guided FUS method that elicits endothelial-selective transfection of the cerebral vasculature (i.e., “sonoselective” transfection), without opening the BBB. We first determined that activating circulating, cationic plasmid-bearing MBs with pulsed low-pressure (0.1 MPa) 1.1-MHz FUS facilitates sonoselective gene delivery to the endothelium without MRI-detectable disruption of the BBB. The degree of endothelial selectivity varied inversely with the FUS pressure, with higher pressures (i.e., 0.3-MPa and 0.4-MPa FUS) consistently inducing BBB opening and extravascular transfection. Bulk RNA sequencing analyses revealed that the sonoselective low-pressure regimen does not up-regulate inflammatory or immune responses. Single-cell RNA sequencing indicated that the transcriptome of sonoselectively transfected brain endothelium was unaffected by the treatment. The approach developed here permits targeted gene delivery to blood vessels and could be used to promote angiogenesis, release endothelial cell-secreted factors to stimulate nerve regrowth, or recruit neural stem cells.