Targeted Gene Expression in Ischemic Brain by Intravenous Delivery

描述(申请人提供)：缺血损伤后的先天代偿反应可以减轻缺血损伤的程度，促进功能恢复。外源性血管生成因子的传递已被证明能增强这种先天反应。这项研究项目的主要目的是克服将“增强的先天损伤反应”范例转化到临床环境中的某些障碍。例如，治疗性蛋白质和基因的系统输送被脑血屏障(BBB)阻止，脑室内输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，立体定向将蛋白质和载体注射到缺血半暗带需要一种侵入性的程序，可能会造成额外的损害。我们已经产生了一种定制的腺相关病毒载体(AAV)，它具有两个主要属性，适合我们的预期目的。首先，载体的缺氧反应元件(HRE)将转基因表达限制在缺血组织中。其次，AAV血清型9(AAV9)有效地使血脑屏障五价，使静脉给药成为可能。我们已将我们的矢量命名为H9。我们建议使用静脉(IV)递送H9载体来实现脑缺血灶中的靶向基因表达。新载体的有效载荷将是血管内皮生长因子(VEGF)和血管生成素-1(Ang-1)。之所以选择血管内皮生长因子，是因为它是治疗缺血性卒中研究最多的血管生成因子，具有血管生成和神经生成作用，并促进神经元保护和修复。选择Ang-1是因为它减少了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在血管生成和神经保护方面具有协同作用。该项目的目标是开发一种创新的方法，以改善基于基因的治疗缺血性脑损伤的结果。我们计划使用一种永久性的小鼠大脑中动脉远端闭塞模型来验证我们的假设，即(1)静脉注射H9载体将导致缺血脑内靶向基因的表达，(2)静脉注射H9-VEGF和H9-Ang-1比立体定向注射产生更好的结果。这项初步研究将为我们的方法提供坚实的原则证据，在后续的应用(R01)中，我们将扩展研究以优化载体传递(剂量和传递方案)；更好地了解VEGF和Ang-1的神经保护或神经修复作用的机制，以及神经行为恢复；并研究该方法临床应用的可行性。这项拟议的技术是新的，如果成功，将导致开发一种更具选择性的缺血性中风基因疗法。同样的方法也可以应用于脊髓损伤的治疗。 公共卫生意义：该项目将开发一种新的方法，在血液供应不足的大脑中诱导新的功能性小血管。因此，增加的血流量将保护神经元免受死亡。

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.