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the amount of therapeutic gene expression was not sufficient to alter chronic spontaneous seizures in rats (unpublished findings).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300万美国人诊断出癫痫病（Hirtz等，2007）这种人口经验的三分之一不足（Cascino，2008; Kwan and Brodie，2000; 2000; Braakman等，2011）。基于慢性癫痫发作损害血脑屏障（BBB）的事实癫痫发作活动的区域，我们发现了一个新型的嵌合AAV矢量克隆83，它有选择地越过静脉给药后癫痫发作损害了BBB（Gray等，2010）。不幸的是，数量治疗基因表达不足以改变大鼠的慢性自发癫痫发作（未发表发现）。但是，我们最近发现AAV9 CAPSID的特定元素与启动子相互作用在体内基因表达中显着改变的程度（Powell等，2020）。例如，在人工码头启动子将6种丙氨酸插入AAV9 VP1/2的AA138中，导致神经元增加了50％基因表达（Bohlen等，2020）。因此，我们建议大量克隆83矢量实际上越过癫痫发作损害的BBB，但是转基因表达会通过带有发起人的衣壳。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被证明足以发挥治疗作用。首先是克隆83 AAV CAPSID将用6个氨基酸丙氨酸插入在现场类似的VP1，VP2或VP1和2 到AAV 9 CAPSID的AA138。随后，这些AAV克隆83 Capsid变体将被包装到重组病毒表达和组成性秘密NPY [13-36]或适当的GFP对照。二十- 诱导急性海藻酸癫痫发作后四个小时，将静脉注射AAV病毒。一周后来，边缘癫痫发作活动的出现将每天监视一个月。鉴于AAV介导 NPY的表达和本构分泌[13-36]显着阻塞了迦尼酸在体内诱发癫痫发作，我们预测升高的克隆83表达将被证明足以防止慢性癫痫发作的发展活动。这样的发现不仅将克隆83作为静脉癫痫发作治疗，而且具有显着影响AAV矢量对中枢神经系统基因疗法的理解和应用。