Genome Editing the Blood-Brain Barrier with Sonoselective Focused Ultrasound

脑微循环内皮是血脑屏障（BBB）的重要组成部分， 其通过内皮细胞之间的紧密连接和外排保护神经组织 将许多化合物排回血液的转运蛋白。当开发药物输送策略时， 对于脑病理，这些外排转运蛋白[例如多药耐药1（MDR 1）]呈现出相当大的 有效药物递送的挑战，因为它们限制了中枢神经系统（CNS）病变暴露于 许多全身施用的药剂。同时，营养转运蛋白[例如葡萄糖转运蛋白1（GLUT 1）] 对维持正常的大脑功能至关重要事实上，GLUT 1的基因突变可以导致复发性 癫痫发作、小头畸形、智力残疾、痉挛状态、共济失调和构音障碍。 考虑到在大脑健康和疾病中的这种中心作用，BBB的内皮代表了丰富的脑血管内皮细胞。 用于治疗性基因组操作靶点。在本提案中，我们将设计一种平台技术， 以安全、内皮细胞选择性和非侵入性的方式对BBB进行基因组编辑， 通过MR图像引导提供靶向。我们称之为超低压聚焦超声 用于激活质粒包被的微泡，“声选择性”基因递送。这是因为， 使用细胞特异性启动子，单独的超声波（即“Sono”）“选择”转染的细胞类型。以来 虽然内皮细胞特异性启动子是不必要的，但是可以采用大量的遗传操作。 在Aim 1中，我们将设计声激活递送剂，其声选择性地编辑 血脑屏障内皮这将需要用不同的指导RNA测试CRISPR-Cas9“切口酶”质粒 针对它们从BBB内皮中声选择性地缺失GLUT 1和MDR 1的能力，对gRNA配对序列进行了研究。我们 还将测试是否可以通过将质粒并入非病毒聚合物纳米颗粒来提高效率 (NPs)其用非免疫原性接头共价偶联至MB。最有前途的组合物将 然后使用正电子发射断层扫描（PET）进行功能检查[即用于大脑代谢的18 F-FDG GLUT 1缺失后的变化和（R）-[11 C]维拉帕米用于MDR 1a缺失后的药物外排变化]。此外，本发明还 将使用单细胞RNA测序（scRNAseq）来评估细胞选择性和基因缺失的功效。 在目标2中，我们将通过合理地操纵基因组，提高声选择性基因组编辑的效率和控制。 聚焦超声参数。我们将测试增加FUS爆发持续时间是否改善质粒递送 以及随后的基因（GLUT 1和MDR 1）缺失效率。我们还将测试我们是否能够控制 使用基于声发射的反馈控制系统进行声选择性基因组编辑。一旦完成， 我们将建立一个安全、非侵入性、MR图像引导的内皮细胞基因组编辑平台， 的BBB。我们认为，这种方法将在临床前神经科学研究中有多种应用 并且作为治疗许多CNS疾病的治疗方法具有相当大的潜力。

The endothelium of the cerebral microcirculation is a critical component of the blood-brain barrier (BBB), which protects neural tissue through the presence of tight junctions between endothelial cells and efflux transporters that extrude many compounds back into the bloodstream. When developing drug delivery strategies for brain pathologies, these efflux transporters [e.g. multi drug resistance 1 (MDR1)] present a considerable challenge to effective drug delivery, as they limit the exposure of central nervous system (CNS) pathologies to many systemically administered agents. Meanwhile, nutrient transporters [e.g. glucose transporter 1 (GLUT1)] are critical for maintaining normal brain function. Indeed, genetic mutation(s) of GLUT1 can cause recurrent epileptic seizures, microcephaly, intellectual disability, spasticity, ataxia, and dysarthria. Given this central role in health and disease in the brain, the endothelium of the BBB represents a rich target for therapeutic genomic manipulation. In this proposal, we will engineer a platform technology capable of genome editing the BBB in a safe, endothelial cell-selective, and non-invasive manner, with precise loco-regional targeting provided by MR image-guidance. We call this approach, wherein very low pressure focused ultrasound is used to activate plasmid-coated microbubbles, “sonoselective” gene delivery. This is because, instead of employing a cell-specific promoter, ultrasound (i.e. “sono”) alone “selects” which cell type is transfected. Since endothelial cell-specific promoters are unnecessary, a vast array of genetic manipulations may be employed. In Aim 1, we will engineer acoustically-activated delivery agents that sonoselectively edit the genome of blood-brain barrier endothelium. This will entail testing CRISPR-Cas9 “nickase” plasmids with varying guide RNA (gRNA) pair sequences for their ability to sonoselectively delete GLUT1 and MDR1 from BBB endothelium. We will also test whether efficiency can be improved by incorporating plasmids into non-viral polymer nanoparticles (NPs) that are coupled covalently to MBs with non-immunogenic linkers. The most promising compositions will then be examined functionally using positron emission tomography (PET) [i.e. 18F-FDG for brain metabolism changes after GLUT1 deletion and (R)-[11C]verapamil for drug efflux changes after MDR1a deletion]. Further, single cell RNA sequencing (scRNAseq) will be used to assess the cell selectivity and efficacy of gene deletion. In Aim 2, we will augment the efficiency and control of sonoselective genome editing by rationally manipulating focused ultrasound parameters. We will test whether increasing FUS burst duration improves plasmid delivery and subsequent gene (GLUT1 and MDR1) deletion efficiency. We will also test whether we can control sonoselective genome editing using a feedback control system based on acoustic emissions. Once completed, we will have established a safe, non-invasive, MR image-guided, platform for genome editing of endothelium in the BBB. We submit that such an approach will have multiple applications in pre-clinical neuroscience research and considerable potential as a therapeutic approach to treating many diseases of the CNS.