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

摘要： 该项目旨在确定一种非侵入性和高效的靶向基因传递策略。 并通过将再生分子输送到 有中枢神经系统损伤的哺乳动物。我们将确定是否系统交付我们新设计的AAV9 载体可以转导大多数靶向中枢神经系统细胞，以及靶向基因的非侵入性传递 神经元内在和外在因素可促进强健的轴突再生和功能恢复 在有脊髓损伤(SCI)的啮齿类动物中。神经科学研究中的一个主要挑战是交付目标 基因到特定类型的神经细胞广泛分布于中枢神经系统。工程化的AAV9载体通常 静脉注射(IV)后，仅在某些中枢神经系统区域进行细胞转导，显示出有限的疗效。 因此，我们创建了新的AAV9载体，其中包括工程AAV9衣壳的多种特征，瞄准 构建高效的BBB交叉AAV9载体(HEBC-AAV9)，可转导大部分靶点 静脉注射后的中枢神经系统细胞。在目标1中，我们将研究我们的新型HEBC-AAV9-GFP载体的效率 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 在几个品系的成年小鼠中)。用它来解决神经科学研究中的一个关键问题 技术，在目标2，我们将开发一种再生疗法，通过系统地将基因输送到 靶向神经元let-7miRNA。脊髓损伤后，切断的轴突不能再生，部分原因是 成熟神经元的内在生长能力。已知有许多基因控制植物的生长能力。 成熟的神经元，但没有一个转化为临床使用。最好的目标可能是那些 有可能影响多个基因。其中，let-7在调节年龄依赖性方面起重要作用 蠕虫的轴突再生能力下降。在目标2中，我们建议使用独特的HEBC-AAV9-突触素 选择性靶向神经元诱导let-7抑制物LIN28和lin41表达的载体，目的 通过增强成熟神经元的生长能力促进多轴索的强健再生 在脊髓损伤啮齿类动物中。胶质瘢痕产生的硫酸软骨素蛋白多糖(CSPGs)受到强烈抑制 轴突延伸，是治疗中枢神经系统损伤的主要外在分子靶点。我们的实验室设计了 靶向CSPG受体LAR、PtPσ和PtPδ功能的小肽 并证明了它们在促进轴突再生方面的高效率。在《目标3》中，我们将 用HEBC-1诱导LAR、PtPσ和PtPδ各自分泌的3个多肽的星形胶质细胞表达 AAV9-GFAP载体，旨在通过靶向外源性促进脊髓损伤后强大的轴突再生 CSPG单独或与固有的LET-7信号结合。我们的新病毒载体应该会提供一种强大的 中枢神经系统基因传递工具和开发脊髓损伤等有效再生疗法的工具 神经紊乱。

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.