Optimizing a Novel AAV Vector to Selectively Influence Seizure Networks In Vivo

优化新型 AAV 载体以选择性影响体内癫痫网络

基本信息

批准号：
10740434
负责人：
Thomas J. McCown
金额：
$ 42.76万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-06-15 至 2025-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10740434
关键词：
Acute Affinity Alanine American Amino Acids Animal Model Anticonvulsants Appearance Area Attenuated Blood - brain barrier anatomy Body Weight Brain Capsid Cell Count Chimera organism Chronic Development Diagnosis Dose Drug resistance Elements Ensure Epilepsy Exhibits Funding Gene Expression Goals Hour Human Immunohistochemistry Incidence Individual Injections Intravenous Kainic Acid Mediating Monitor Neurons Organ Outcome Perfusion Peripheral Pharmacotherapy Population Rattus Recombinant adeno-associated virus (rAAV)Reporting Risk Saline Seizures Serotyping Site Therapeutic Therapeutic Effect Treatment Efficacy Validation Variant Virus adeno-associated viral vector attenuation experience gene therapy high reward high risk in vivo in vivo evaluation intravenous administration neuron loss novel novel therapeutics prevent promoter recombinant virus therapeutic gene transgene expression vector vector genome

项目摘要

Abstract Approximately 3 million Americans have a diagnosis of epilepsy (Hirtz et al., 2007) where approximately one third of this population experience inadequate seizure control (Cascino, 2008; Kwan and Brodie, 2000; Braakman et al., 2011). Based upon the fact that chronic seizures compromise the blood-brain barrier (BBB) in areas of seizure activity, we discovered a novel chimeric AAV vector, clone 83, that selectively crossed the seizure compromised BBB after intravenous administration (Gray et al., 2010). Unfortunately, the amount of therapeutic gene expression was not sufficient to alter chronic spontaneous seizures in rats (unpublished findings). However, we recently discovered that specific elements of the AAV9 capsid interact with promoters to a degree that significantly alters in vivo gene expression (Powell et al., 2020). For example, in the context of the artificial Jeti promoter the insertion of 6 alanines into aa138 of AAV9 VP1/2 resulted in a 50% increase in neuronal gene expression (Bohlen et al., 2020). Therefore, we propose that a substantial amount of the clone 83 vector actually crosses the seizure compromised BBB, but transgene expression is attenuated by an interaction of the capsid with the promoter. By inserting amino acids into the analogous clone 83 capsid region, we hypothesize that the increase in in vivo gene expression will prove adequate to exert a therapeutic effect. First the clone 83 AAV capsid will be modified with 6 amino acid alanine insertions into VP1, VP2 or VP1 and 2 at the site analogous to aa138 of the AAV 9 capsid. Subsequently, these AAV clone 83 capsid variants will be packaged into recombinant virus that expresses and constitutively secrets NPY[13-36], or appropriate GFP controls. Twenty- four hours after induction of acute kainic acid seizures, the AAV viruses will be injected intravenously. One week later, the appearance of limbic seizure activity will be monitored daily for one month. Given that AAV mediated expression and constitutive secretion of NPY[13-36] significantly blocks kainic acid induced seizures in vivo, we predict that the increased clone 83 expression will prove sufficient to prevent the development of chronic seizure activity. Such findings will not only advance clone 83 as an intravenous seizure therapeutic but have significant impact on the understanding and application of AAV vectors to CNS gene therapies.

抽象的大约 300 万美国人被诊断为癫痫症（Hirtz 等人，2007 年），其中大约该人群中有三分之一的癫痫发作控制不充分（Cascino，2008 年；Kwan 和 Brodie，2000 年；布拉克曼等人，2011）。基于慢性癫痫发作会损害血脑屏障 (BBB) 的事实癫痫发作活动区域，我们发现了一种新型嵌合 AAV 载体，克隆 83，它选择性地穿过静脉注射后癫痫发作会损害 BBB（Gray 等，2010）。不幸的是，金额治疗基因表达不足以改变大鼠的慢性自发性癫痫发作（未发表发现）。然而，我们最近发现 AAV9 衣壳的特定元件与启动子相互作用显着改变体内基因表达的程度（Powell et al., 2020）。例如，在以下背景下人工Jeti启动子将6个丙氨酸插入AAV9 VP1/2的aa138导致神经元增加50% 基因表达（Bohlen 等人，2020）。因此，我们建议大量的克隆83载体实际上穿过了癫痫发作受损的 BBB，但转基因表达因衣壳与启动子。通过将氨基酸插入类似的克隆 83 衣壳区域，我们假设体内基因表达的增加将证明足以发挥治疗效果。首先是克隆83 AAV 衣壳将被修饰，在 VP1、VP2 或 VP1 和 2 的类似位点插入 6 个氨基酸丙氨酸至 AAV 9 衣壳的 aa138。随后，这些AAV克隆83衣壳变体将被包装成表达并组成型分泌 NPY[13-36] 的重组病毒，或适当的 GFP 对照。二十- 诱导急性红藻氨酸癫痫发作后四小时，将静脉注射 AAV 病毒。一周之后，将在一个月内每天监测边缘系统癫痫发作活动的出现。鉴于 AAV 介导 NPY[13-36]的表达和组成性分泌显着阻断红藻氨酸诱导的体内癫痫发作，我们预测克隆 83 表达增加将足以预防慢性癫痫发作活动。这些发现不仅将推动克隆 83 作为静脉注射癫痫治疗药物，而且具有重要意义影响 AAV 载体在 CNS 基因治疗中的理解和应用。