Bioengineering of highly effective AAV vectors for noninvasive gene delivery to the nervous system

高效 AAV 载体的生物工程，用于将基因非侵入性传递至神经系统

• 批准号: 10597682
• 负责人: SHUXIN LI
• 金额: $ 39.63万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-01 至 2027-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10597682
• 关键词: Adult; Age; Alzheimer' s Disease; Astrocytes; Axon; Biomedical Engineering; Blood - brain barrier anatomy; Capsid; Capsid Proteins; Cd68; Cells; Central Nervous System Diseases; Chimeric Proteins; Chondroitin Sulfate Proteoglycan; Cicatrix; Clinical; Effectiveness; Engineering; Evaluation; Failure; Gene Delivery; Gene Targeting; Genes; Glial Fibrillary Acidic Protein; Growth; Growth Inhibitors; Injections; Intravenous; Invaded; Locomotor Recovery; Mammals; Mediating; MicroRNAs; Microglia; Molecular Target; Motor; Mouse Strains; Mus; Mutagenesis; Natural regeneration; Nervous System; Nervous System Trauma; Neurons; Neurosciences; Neurosciences Research; Oligodendroglia; PTEN gene; Pathway interactions; Penetration; Peptides; Pericytes; Population; Probability; Protein Tyrosine Phosphatase; Recovery; Recovery of Function; Research Personnel; Rodent; SOX11 gene; Signal Transduction; Spinal cord injury; Synapsins; Technology; Transduction Gene; Transgenic Mice; Translating; Tropism; Up-Regulation; Viral Vector; Virus; Wild Type Mouse; adeno-associated viral vector; age related; axon growth; axon regeneration; blood-brain barrier crossing; c-myc Genes; cellular transduction; central nervous system injury; design; effective therapy; improved; in vivo; inhibitor; intravenous injection; nervous system disorder; neural; neuronal growth; novel; promoter; receptor; receptor function; regenerative; regenerative approach; regenerative therapy; repaired; technology validation; tool; transduction efficiency; transgene expression; vector

Abstract: This project is designed to identify a noninvasive and highly effective gene delivery strategy to target specific neural cells in the CNS and to validate this technology by delivering regenerative molecules to mammals with CNS injury. We will determine whether systemic delivery of our newly engineered AAV9 vectors can transduce most target CNS cells and whether noninvasive delivery of the genes that target neuronal intrinsic and extrinsic factors can promote robust axon regeneration and functional recovery in rodents with spinal cord injury (SCI). A major challenge in neuroscience research is to deliver target genes to specific types of neural cells widely distributed in CNS. Engineered AAV9 vectors usually show limited efficacy after intravenous (IV) injection by transducing cells only in some CNS regions. We thus created new AAV9 vectors that include multiple features of engineered AAV9 capsids, aiming to develop highly efficient BBB-crossing AAV9 vectors (HEBC-AAV9) that can transduce most target CNS cells after IV injection. In Aim 1, we will study efficiency of our novel HEBC-AAV9-GFP vectors for selectively transducing each type of neural cells (neurons, astrocytes, oligodendrocytes, and microglia) in several strains of adult mice. To solve a crucial issue in neuroscience research with this technology, in Aim 2 we will develop a regenerative therapy for SCI by systemic delivery of genes to target neuronal let-7 miRNA. After SCI, severed axons fail to regenerate partly because of reduced intrinsic growth capacity of mature neurons. Many genes are known to control the growth ability of mature neurons, but none have been translated to clinical use. The best targets are probably those with potential to impact multiple genes. Among them, let-7 is important for regulating age-dependent decline in axon regeneration in worms. In Aim 2, we propose to use unique HEBC-AAV9-synapsin vectors to target neurons selectively for inducing expression of let-7 inhibitor, lin28, and lin41, aiming to promote robust regeneration of multiple axon tracts by enhancing growth capacity of mature neurons in SCI rodents. Chondroitin sulfate proteoglycans (CSPGs) generated by glial scars strongly suppress axon extension and are major extrinsic molecular targets for treating CNS injury. Our lab designed small peptides to block functions of CSPG receptor LAR, PTPσ, and PTPδ by targeting their critical activity domains and demonstrated their high efficiency for promoting axon regrowth. In Aim 3, we will induce astrocytic expression of secreted 3 peptides for each of LAR, PTPσ, and PTPδ with HEBC- AAV9-GFAP vectors, aiming to promote robust axon regeneration after SCI by targeting extrinsic CSPGs alone or combined with intrinsic let-7 signals. Our new viral vectors should provide a powerful tool for gene delivery in CNS and for developing effective regenerative therapies for SCI and other neurological disorders.

摘要： 该项目旨在确定一种非侵入性和高效的基因递送策略， 在中枢神经系统中的特定神经细胞，并通过提供再生分子， 中枢神经系统损伤的哺乳动物。我们将确定我们新设计的AAV 9 载体可以最大限度地靶向CNS细胞，并且是否可以无创递送靶向CNS细胞的基因， 神经元内源性和外源性因子可以促进轴突再生和功能恢复 脊髓损伤（SCI）的啮齿动物。神经科学研究中的一个主要挑战是将靶点传递给 基因的特定类型的神经细胞广泛分布在中枢神经系统。工程化的AAV 9载体通常 通过仅在某些CNS区域转导细胞，在静脉内（IV）注射后显示出有限的功效。 因此，我们创建了新的AAV 9载体，其包括工程化的AAV 9衣壳的多种特征， 开发高效BBB交叉AAV 9载体（HEBC-AAV 9）， IV注射后的CNS细胞。在目标1中，我们将研究新型HEBC-AAV 9-GFP载体的效率 用于选择性地转导每种类型的神经细胞（神经元、星形胶质细胞、少突胶质细胞和 小胶质细胞）。为了解决神经科学研究中的一个关键问题， 技术，在目标2中，我们将开发一种再生疗法，通过全身性基因传递， 靶向神经元let-7 miRNA。脊髓损伤后，切断的轴突不能再生，部分原因是由于减少了 成熟神经元的内在生长能力。已知许多基因控制植物的生长能力。 成熟的神经元，但没有一个被转化为临床应用。最好的目标可能是那些 有可能影响多个基因其中let-7对调节年龄依赖性 蠕虫的轴突再生能力下降。在目标2中，我们提出使用独特的HEBC-AAV 9-突触蛋白， 选择性靶向神经元的载体，用于诱导let-7抑制剂、lin 28和lin 41的表达， 通过增强成熟神经元的生长能力来促进多个轴突束的稳健再生 在脊髓损伤的啮齿动物中。神经胶质瘢痕产生的硫酸软骨素蛋白聚糖（CSPG）强烈抑制了 轴突延伸，是治疗CNS损伤的主要外源性分子靶点。我们的实验室设计 通过靶向CSPG受体LAR、PTPσ和PTPδ的关键功能， 活性结构域，并证明其促进轴突再生的高效率。在目标3中，我们 用HEBC诱导星形胶质细胞表达LAR、PTPσ和PTPδ中每一种分泌的3种肽， AAV 9-GFAP载体，旨在通过靶向外源性神经元来促进SCI后稳健的轴突再生。 CSPG单独或与内在let-7信号组合。我们新的病毒载体应该能提供一个强大的 用于CNS中的基因递送和用于开发SCI和其他疾病的有效再生疗法的工具 神经系统疾病