Sonoselective Transfection of Cerebral Vasculature without Blood-Brain Barrier Disruption - Singel Cell RNA seq

Treatment of many pathologies of the brain could be improved markedly by the development of non-invasive 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 non-invasive 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 a new 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 upregulate 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. FACS was first used to isolate mRUBY+ cells from pooled brain tissue wherein mRUBY plasmid-bearing MBs were activated with FUS at 0.1, 0.2, and 0.4 MPa (n = 3 per group). Brain tissue from mice that received mRUBY plasmid-MB injection, but without FUS application, were used to generate the flow cytometry gating scheme. Single cell RNA sequencing was then performed on mRUBY+ cell populations from 0.1, 0.2, and 0.4 MPa treated mice, as well as from untreated mice.