Promoters for Long-term Expression from HSV-1 Vectors

HSV-1 载体长期表达的启动子

• 批准号: 7913100
• 负责人: ALFRED I. GELLER
• 金额: $ 1.15万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-15 至 2010-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7913100
• 关键词: Behavioral; Binding; Biological Assay; Boundary Elements; Chickens; Chromatin; Chromatin Structure; Cloning; Code; Complex; Corpus striatum structure; Cytoplasmic Granules; DNA; DNA Binding; DNA Methylation; Development; Discrimination; Dopamine; Electrophoretic Mobility Shift Assay; Enhancers; Enzymes; Epigenetic Process; Euchromatin; Gene Expression; Gene Transfer; Genes; Genetic; Genome; Globin; Glutamates; Goals; Grant; HSV-1 vector; Helper Viruses; Higher Order Chromatin Structure; Hippocampus (Brain); Human; Hybrids; Knowledge; Learning; Lentivirus Vector; Modeling; Modification; Monkeys; Neurodegenerative Disorders; Neurons; Nucleosomes; Parkinson Disease; Pattern; Physiology; Property; Protamine Kinase; Protein Binding; Protein Kinase C Activation Pathway; Proteins; Rattus; Recombinants; Reporting; Role; Simplexvirus; Subfamily lentivirinae; Support System; System; Testing; Tyrosine 3-Monooxygenase; Upstream Enhancer; Virus; Yeasts; adeno-associated viral vector; aged; chromatin modification; deletion analysis; design; enhancer binding protein; gene therapy; histone acetyltransferase; histone modification; improved; nervous system disorder; neurofilament; novel strategies; overexpression; preproenkephalin; programs; promoter; relating to nervous system; transcription factor; vector; vesicular monoamine transporter; virus development; visual learning

DESCRIPTION (provided by applicant): Long-term expression is essential for virtually all gene therapy treatments of neurodegenerative disorders. Helper virus-free Herpes Simplex Virus (HSV-1) vectors, lentivirus vectors, or adeno- associated virus vectors can support recombinant gene expression for 6 to 14 months. Thus, significant improvements in long-term expression are required for human gene therapy. Our approach is to view the large, ~150 kb, genome of an HSV-1 vector as a minichromosome. We used boundary elements to create a euchromatin-like domain, and enhancers to turn on expression from specific promoters within this domain. During the previous grant period, we identified specific enhancers and promoters that support long- term expression. We isolated an upstream enhancer from the tyrosine hydroxylase promoter that supports long-term expression. We developed HSV-1 vectors that support inducible, long-term expression. We identified promoters that support glutamatergic or GABAergic neuron-specific, long- term expression. We developed targeted gene transfer to specific types of neurons, with long-term expression. We used these advances to support development of specific gene therapies. First, we corrected the rat model of Parkinson's disease by coexpressing three DA biosynthetic enzymes and a vesicular monoamine transporter (4-gene-vector). Expression in GABAergic striatal neurons was observed for 14 months. The 4-gene-vector supported higher levels of behavioral correction, higher levels of DA, and only this vector supported regulated release of DA. Second, we showed that genetic activation of protein kinase C (PKC) pathways in small groups of rat postrhinal cortex neurons enhances learning of visual object discriminations. Moreover, in aged rats, we showed that activation of PKC pathways in small groups of hippocampal dentate granule neurons corrects deficits in spatial learning. The goal of this proposal is to elucidate specific mechanisms that support long-term expression. The first specific aim will isolate and characterize specific enhancers, and their cognate enhancer binding proteins, that support long-term expression. The second specific aim will characterize the chromosomal state of HSV-1 vectors that support long-term expression. The third specific aim will overexpress specific enhancer binding proteins, or chromatin modifying enzymes, to improve long- term expression. Long-term recombinant gene expression is essential for virtually all gene therapy treatments of neurological diseases. During the previous grant period, we identified specific promoters that support long-term expression from Herpes Simplex Virus (HSV-1) vectors. This proposal will elucidate specific mechanisms that support long-term expression.

描述（由申请人提供）：长期表达对于几乎所有神经退行性疾病的基因治疗疗法至关重要。无辅助病毒单纯疱疹病毒（HSV-1）载体，慢病毒载体或腺相关病毒载体可以支持重组基因表达6至14个月。因此，人类基因治疗需要长期表达的显着改善。我们的方法是将HSV-1载体的〜150 Kb基因组视为微小浓度小体。我们使用边界元素来创建一个类似菌膜素的结构域，并增强子来打开该域内特定启动子的表达。在上一个赠款期间，我们确定了支持长期表达的特定增强子和启动子。我们从支持长期表达的酪氨酸羟化酶启动子中分离了上游增强子。我们开发了支持可诱导的长期表达的HSV-1载体。我们确定了支持谷氨酸能或GABA能特异性，长期表达的启动子。我们开发了具有长期表达的特定类型神经元的靶向基因转移。我们使用这些进步来支持特定基因疗法的开发。首先，我们通过共表达三种DA生物合成酶和一个囊泡单胺转运蛋白（4基因载体）来纠正帕金森氏病大鼠模型。观察到GABA能纹状体神经元中的表达14个月。 4-Gene-vector支持较高水平的行为校正，较高的DA水平，并且只有该向量才有支持DA的受调节释放。其次，我们表明蛋白激酶C（PKC）途径的遗传激活在小鼠后皮层神经元中增强了视觉对象区分的学习。此外，在老年大鼠中，我们表明，海马齿状颗粒神经元中PKC途径的激活纠正了空间学习中的缺陷。该提案的目的是阐明支持长期表达的特定机制。第一个特定目标将隔离并表征特定的增强子及其同源增强子结合蛋白，这些蛋白支持长期表达。第二个特定目的将表征支持长期表达的HSV-1载体的染色体状态。第三个特定目的将过表达特定的增强子结合蛋白或染色质修饰酶，以改善长期表达。长期重组基因表达对于几乎所有基因治疗神经疾病的疗法至关重要。在上一个赠款期间，我们确定了支持单纯疱疹病毒（HSV-1）载体长期表达的特定启动子。该建议将阐明支持长期表达的特定机制。