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. Overall design: 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.

针对大脑多种病理的治疗，可通过开发非侵入性治疗手段得到显著改善——这类手段可在磁共振成像（MR）引导下，于特定靶向脑区域内诱导高效且具有内皮细胞选择性的基因表达。尽管结合充气微泡（gas-filled microbubbles, MBs）的聚焦超声（focused ultrasound, FUS）已成为MR引导下非侵入性脑基因递送的新兴手段，但此前该方法仅被用于暂时性破坏血脑屏障（blood-brain barrier, BBB），这可能引发无菌性炎症反应。在此，我们提出一种全新的MR引导聚焦超声方法，可实现脑血管系统的内皮细胞选择性转染（即“声选择性（sonoselective）”转染），且无需开放血脑屏障。我们首先证实，采用脉冲式低压力（0.1 MPa）1.1 MHz聚焦超声激活携带阳离子质粒的循环微泡，可实现内皮细胞的声选择性基因递送，且不会引发磁共振成像可检测到的血脑屏障破坏。内皮细胞选择性程度与聚焦超声压力呈负相关：更高压力（如0.3 MPa与0.4 MPa聚焦超声）会持续引发血脑屏障开放及血管外转染。批量RNA测序（bulk RNA sequencing）分析显示，声选择性低压力处理方案不会上调炎症或免疫应答。单细胞RNA测序表明，经声选择性转染的脑内皮细胞转录组未受该处理影响。本研究开发的方法可实现血管靶向性基因递送，有望用于促进血管生成、释放内皮细胞分泌因子以刺激神经再生，或募集神经干细胞。整体实验设计：首先采用荧光激活细胞分选术（fluorescence-activated cell sorting, FACS）从混合脑组织中分离mRUBY阳性细胞，这些脑组织来自携带mRUBY质粒的微泡分别经0.1、0.2及0.4 MPa聚焦超声激活的小鼠（每组n=3）。将仅接受mRUBY质粒-微泡注射但未施加聚焦超声的小鼠脑组织，用于构建流式细胞术设门方案。随后，对0.1、0.2、0.4 MPa处理组及未处理组小鼠的mRUBY阳性细胞群开展单细胞RNA测序。