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

摘要 大约300万美国人被诊断为癫痫（Hirtz等人，2007年），约 三分之一的这一人群的癫痫控制不足（Cascino，2008年; Kwan和Brodie，2000年; Braakman等人，2011年）。基于慢性癫痫发作损害脑内血脑屏障（BBB）的事实， 我们发现了一种新的嵌合AAV载体，克隆83，它选择性地穿过癫痫活动的区域， 静脉内给药后癫痫损害BBB（Gray等人，2010年）。不幸的是， 治疗性基因表达不足以改变大鼠的慢性自发性癫痫发作（未发表 调查结果）。然而，我们最近发现AAV 9衣壳的特定元件与启动子相互作用， 显著改变体内基因表达的程度（Powell等，2020年）。例如，在 在人工Jeti启动子中，将6个丙氨酸插入AAV 9 VP 1/2的aa 138中导致神经元细胞凋亡增加50%。 基因表达（Bohlen等，2020年）。因此，我们建议将大量的克隆83载体 实际上穿过癫痫受损的血脑屏障，但转基因的表达被减弱的相互作用， 衣壳与启动子。通过将氨基酸插入类似的克隆83衣壳区，我们假设 体内基因表达的增加将证明足以发挥治疗效果。第83章克隆人 AAV衣壳将用6个氨基酸丙氨酸插入VP 1、VP 2或VP 1和2的类似位点进行修饰 至AAV 9衣壳的aa 138。随后，这些AAV克隆83衣壳变体将被包装到 表达和组成型分泌NPY的重组病毒[13-36]，或适当的GFP对照。二十- 在诱导急性红藻氨酸发作后4小时，静脉内注射AAV病毒。一周 之后，将每天监测边缘癫痫活动的出现，持续一个月。考虑到AAV介导的 NPY的表达和组成性分泌[13-36]在体内显著阻断红藻氨酸诱导的癫痫发作，我们 预测增加的克隆83表达将被证明足以防止慢性癫痫发作的发展 活动这些发现不仅将促进克隆83作为静脉内癫痫治疗， 对AAV载体在中枢神经系统基因治疗中的理解和应用的影响。

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the amount of therapeutic gene expression was not sufficient to alter chronic spontaneous seizures in rats (unpublished findings).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.