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the amount of therapeutic gene expression was not sufficient to alter chronic spontaneous seizures in rats (unpublished findings).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

摘要