Targeted Gene Expression in Ischemic Brain by Intravenous Delivery

描述（申请人提供）：缺血性损伤后的先天代偿反应可减轻缺血性损伤程度，促进功能恢复。血管生成因子的外源性递送已被证明可以增强这种先天反应。本研究项目的主要目的是克服将“增强的先天损伤反应”范式转化为临床环境的某些障碍。例如，治疗性蛋白质和基因的全身递送被脑血屏障（BBB）阻止，而脑室内递送导致非特异性血管生成因子分布和基因表达，这可能导致正常组织中不必要的血管生成和不良副作用。此外，向缺血半暗区立体定向注射蛋白质和载体需要侵入性手术，并可能造成额外的损伤。我们已经生成了一个定制设计的腺相关病毒载体（AAV），具有两个主要属性，符合我们的预期目的。首先，载体的缺氧反应元件（HRE）限制了转基因在缺血组织中的表达。其次，AAV血清型9 （AAV9）能有效穿透血脑屏障，使静脉给药成为可能。我们把向量命名为H9。我们建议使用静脉（IV）传递H9载体来实现脑缺血灶的靶向基因表达。新载体的有效载荷将是血管内皮生长（VEGF）和血管生成素-1 （Ang-1）。之所以选择VEGF，是因为它是研究最多的用于缺血性卒中治疗的血管生成因子，具有血管生成和神经生成的作用，并促进神经元的保护和修复。之所以选择Ang-1，是因为它可以减少血管内皮生长因子引起的血管渗漏，并与血管内皮生长因子协同起血管生成和神经保护作用。该项目的目标是开发一种创新的方法来改善缺血性脑损伤的基因治疗结果。我们计划使用小鼠永久性大脑中远端动脉闭塞模型来验证我们的假设，即(1)静脉注射H9载体将导致缺血脑中靶向基因表达；(2)静脉注射H9- vegf和H9- ang -1比立体定向注射效果更好。这项试点研究将为我们的方法提供坚实的原理证明，在随后的应用中（R01），我们将扩展研究以优化载体递送（剂量和递送方案）；更好地了解VEGF和Ang-1的神经保护或神经修复作用以及神经行为恢复的机制；并研究该方法在临床应用的可行性。提出的技术是新颖的，如果成功，将导致发展更具选择性的基因治疗缺血性中风。同样的方法也可以应用于脊髓损伤的治疗。

DESCRIPTION (provided by applicant): Innate compensatory responses after ischemic injury can attenuate the extent of ischemic injury and promote functional recovery. Exogenous delivery of angiogenic factors has been shown to enhance this innate response. This research project is aimed primarily at overcoming certain barriers to the translation of the "enhanced innate response to injury" paradigm to the clinical setting. For example, systemic delivery of therapeutic proteins and genes into the brain is prevented by the brain blood barrier (BBB), and intraventricular delivery results in non-specific angiogenic factor distribution and gene expression, which can cause unwanted angiogenesis in normal tissues and untoward side effects. Moreover, stereotactic injection of proteins and vectors into the ischemic penumbra requires an invasive procedure and can cause additional damage. We have generated a custom-designed adeno-associated viral vector (AAV) with two primary attributes that suit our intended purpose. First, the vector's hypoxia response elements (HRE) restricts transgene expression to ischemic tissue. Second, AAV serotype nine (AAV9) effectively pentrates the BBB, enabling intravenous adminsitration. We have named our vector H9. We propose to use intravenous (IV) delivery of H9 vectors to achieve targeted gene expression in brain ischemic foci. The payload of the novel vector will be both vascular endothelial growth (VEGF) and angiopoietin-1 (Ang-1). VEGF was chosen, because it is the most- studied angiogenic factor for ischemic stroke therapy, has angiogenic and neurogenic effects, and promotes both neuronal protection and restoration. Ang-1 was chosen, because it reduces vessel leakage caused by VEGF and works synergistically with VEGF on angiogenesis and neuroprotection. The goal of this project is to develop an innovative approach to improve the outcomes of gene-based therapies for ischemic brain injury. We plan to use a mouse permanent distal middle cerebral artery occlusion model to test our hypotheses, that (1) IV injection of H9 vector will result in targeted gene expression in the ischemic brain and (2) IV injection of H9-VEGF and H9-Ang-1 results in better outcomes than stereotactic injection. This pilot study will provide solid proof of principle for our approach, and in subsequent applications (R01), we will extend the studies to optimize vector delivery (dosage and delivery schemes); to better understand the mechanisms of the neuroprotective or neurorestorative effects of VEGF and Ang-1, and neurobehavioral recovery; and to study the feasibility of this approach for clinical application. The proposed technology is novel and, if successful, will lead to development of a more selective gene therapy for ischemic stroke. The same approach can also be applied to the treatment of spinal cord injury. PUBLIC HEALTH RELEVANCE: This project will develop a new method to induce new functional small blood vessels in the brain where the blood supply is insufficient. As result, increased blood flow will protect neurons from death.