Genome Editing the Blood-Brain Barrier with Sonoselective Focused Ultrasound

利用声选择性聚焦超声对血脑屏障进行基因组编辑

• 批准号: 10554403
• 负责人: Richard J. Price
• 金额: $ 59.35万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-15 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10554403
• 关键词: ABCB1 gene; Acoustics; Amino Acids; Ataxia; Back; Binding; Blood; Blood - brain barrier anatomy; Blood Circulation; Brain; Brain Pathology; CMV promoter; CRISPR/Cas technology; Cells; Central Nervous System; Central Nervous System Diseases; Cerebrum; Coupled; DNA Sequence Alteration; Data; Development; Disease; Drug Delivery Systems; Drug Efflux; Dysarthria; Endonuclease I; Endothelial Cells; Endothelium; Engineering; Epilepsy; Feedback; Focused Ultrasound; Formulation; Functional disorder; Gene Deletion; Gene Delivery; Gene Therapy Agent; Genes; Genome; Genomics; Gliosis; Glucose; Guide RNA; Health; Immunofluorescence Immunologic; Immunohistochemistry; Inflammation; Intellectual functioning disability; Magnetic Resonance Imaging; Microbubbles; Microcephaly; Microcirculation; Molecular Target; Monitor; Neurosciences Research; Nutrient; Pathology; Plasmids; Positron-Emission Tomography; Publications; Recurrence; Role; SLC2A1 gene; Safety; Sorting; Stains; Sterility; Symptoms; Syndrome; System; Technology; Testing; Therapeutic; Tight Junctions; Tissues; Transfection; Treatment Efficacy; Ultrasonic Therapy; United States National Academy of Sciences; Verapamil; Xenobiotics; astrogliosis; blood-brain barrier disruption; brain metabolism; brain tissue; cell type; cerebral capillary; cerebrovascular; fluorodeoxyglucose; genetic manipulation; genome editing; image guided; immune cell infiltrate; improved; minimally invasive; molecular modeling; nanoparticle; nanopolymer; neural; pre-clinical; pressure; promoter; red fluorescent protein; single-cell RNA sequencing; spasticity; targeted treatment; technology platform; tissue preparation; transgene expression; ultrasound

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.

脑微循环的内皮细胞是血脑屏障（BBB）的重要组成部分， 通过内皮细胞和外流之间存在紧密连接来保护神经组织 在制定药物输送策略时，将许多化合物挤出回血液中的转运蛋白。 对于脑部病理学，这些外排转运蛋白 [例如多重耐药 1 (MDR1)] 具有相当大的作用。 对有效药物输送的挑战，因为它们限制了中枢神经系统（CNS）病变的暴露 同时，营养转运蛋白 [例如葡萄糖转运蛋白 1 (GLUT1)] 事实上，GLUT1 的基因突变可能导致复发。 癫痫发作、小头畸形、智力障碍、痉挛、共济失调和构音障碍。 鉴于脑部健康和疾病的核心作用，血脑屏障内皮细胞代表着丰富的 在本提案中，我们将设计一种能够实现治疗性基因组操作的平台技术。 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，具有精确的局部区域 我们将这种方法称为 MR 图像引导提供的定位，它允许非常低的压力聚焦超声。 用于激活质粒包被的微泡，这是因为，而不是“声选择性”基因传递。 使用细胞特异性启动子，超声波（即“sono”）单独“选择”转染哪种细胞类型。 内皮细胞特异性启动子是不必要的，可以采用大量的遗传操作。 在目标 1 中，我们将设计声学激活的递送剂，以声波选择性编辑基因组 这将需要使用不同的向导RNA测试CRISPR-Cas9“切口酶”质粒。 (gRNA) 配对序列，因为它们能够声选择性地从 BBB 内皮细胞中删除 GLUT1 和 MDR1。 还将测试是否可以通过将质粒掺入非病毒聚合物纳米粒子来提高效率 最有前途的组合物是通过非免疫原性接头与 MB 共价偶联的 (NP)。 然后使用正电子发射断层扫描 (PET) [即 18F-FDG 进行脑代谢检查] GLUT1 删除后的变化和 (R)-[11C]维拉帕米 MDR1a 删除后药物流出的变化] 单细胞RNA测序（scRNAseq）将用于评估基因删除的细胞选择性和功效。 在目标 2 中，我们将通过合理操纵来提高声选择性基因组编辑的效率和控制 我们将测试增加 FUS 突发持续时间是否可以改善质粒递送。 以及后续的基因（GLUT1和MDR1）删除效率我们也会测试是否可以控制。 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 图像引导的内皮基因组编辑平台 我们认为这种方法将在临床前神经科学研究中具有多种应用。 作为治疗许多中枢神经系统疾病的治疗方法具有巨大的潜力。