In vivo targeted gene silencing, a novel method

体内靶向基因沉默，一种新方法

基本信息

批准号：
8204595
负责人：
WILLIAM Anthony TRUITT
金额：
$ 19.25万
依托单位：
INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-12-10 至 2012-10-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8204595
关键词：
Affect Affinity American Amygdaloid structure Animal Model Animals Anti-Anxiety Agents Anxiety Autistic Disorder Binding Biomedical Research Cell Membrane Permeability Cell membrane Cells Child Cholecystokinin Cues Data Diagnostic Disease Emotional Enzyme Immunoassay Familiarity Gene Expression Gene Silencing Gene Targeting Genes Goals Immunofluorescence Immunologic In Vitro Interneurons Lesion Measures Membrane Messenger RNA Methods Modeling Neurons Neuropeptides Oligonucleotides Output Peptide Nucleic Acids Peptides Population Procedures Process Property Rattus Regulation Reporting Research Resistance Role Social Behavior Somatostatin Specificity Substance P Receptor System Targeted Toxins Techniques Technology Testing Therapeutic Agents Toxin Transfection Translations Viral Work antigene autism spectrum disorder base biological systems cellular targeting cost design gamma-Aminobutyric Acid gene therapy genetic manipulation in vivo knock-down neural circuit novel public health relevance receptor receptor internalization relating to nervous system response social targeted delivery

项目摘要

DESCRIPTION (provided by applicant): Autism has recently been reported to affect 1 in 150 American children with an estimated cost of 90 billion dollars per year to the US. Autism is a spectrum disorder with wide ranging levels of social deficits and has been difficult to model in animal studies. In a recent animal study using rats, we described a neural circuit that regulates emotional responses to social cues and may be related to deficits in social processing observed with spectrum disorders. A key component of this neural circuit is a discrete population of interneurons containing substance P1 receptors (also known as tachykinin receptor 1; Tacr1) located within the amygdala. These Tacr1 interneurons receive cortical inputs related to social recognition and suppress anxiety-like outputs of BLA projection neurons. Selectively lesioning these interneurons with a targeted toxin, SSP-saporin, a compound that selectively binds and delivers (via receptor internalization) a toxin only to cells with Tacr1, resulted in increased anxiety measures that were not alleviated with anxiolytic social cues such as social familiarity. These Tacr1 interneurons represent a subpopulation of BLA-interneurons that contain the neuropeptides somatostatin (Sst) and cholecystokinin (Cck), and although these cells appear to be pivotal in the regulation of anxiety-like responses, the contribution of these neuropeptides to these responses remains unknown. Furthermore, since the Tacr1-interneurons are only a subpopulation of the Sst- and Cck- containing BLA interneurons, traditional methods like antagonists or gene suppression cannot elucidate the specific contributions of these neuropeptides within the Tacr1-interneuronal circuit. The goal of the proposed research is to develop an in vivo gene silencing technique that will target specific cells and demonstrate the feasibility of use in vivo. The objective of the proposed research is to combine the gene-silencing properties of antisense PNAs with cellular targeting agents of targeted toxins to create a compound capable of targeted gene silencing. Specifically, we will combine the proven Tacr1 targeting agent, SSP with an antisense PNA that blocks translation of Sst mRNA (antiPNASst) resulting in SSP- antiPNAsst. The central hypothesis for the proposed research is that SSP-antiPNAsst will inhibit Sst expression only in cells that have Tacr1 on their plasma membranes. Once this is established this technology can then be used to elucidate the anxiety-modulating role of neuropeptides within the BLA interneurons. Results generated from the study will impact the field of autism by providing a novel method to determine key neural substrates underlying social behaviors. Additionally, these studies potential impact multiple fields of biomedical research by demonstrating the feasibility of targeted, in vivo PNA delivery. This will open the doors to near limitless combinations of targeting agents and PNA-based diagnostic and therapeutic agents, which to date have been limited for in vivo use due to poor membrane permeability of PNAs. PUBLIC HEALTH RELEVANCE: Peptide nucleic acids are powerful molecules for genetic manipulations and represent potential improvement to current methods of gene therapies, including increased specificity, multitude of strategies to regulate gene expression and long-lasting effects on gene expression without viral transfection. However, PNAs are relatively impermeable to membranes, keeping in vivo uses to a minimum. To overcome this problem we plan to develop a novel method of in vivo targeted delivery of PNAs and demonstrate the feasibility of using these compounds in an animal model.

描述（由申请人提供）：据报道，自闭症会影响150名美国儿童中的1人，估计每年对美国的成本为900亿美元。自闭症是一种具有广泛社会缺陷水平的频谱障碍，在动物研究中很难建模。在最近使用大鼠的动物研究中，我们描述了一种调节对社会线索的情绪反应的神经回路，可能与频谱障碍观察到的社会处理缺陷有关。该神经回路的关键成分是位于杏仁核内的含有P1受体的中间神经元的离散群体（也称为Tachykinin受体1; TACR1）。这些TACR1中间神经元会接受与社会识别有关的皮质输入，并抑制BLA投射神经元的焦虑样产量。选择性地将这些神经元用靶向毒素SSP-肉饼蛋白（一种选择性结合并通过受体内在化）A毒素与带有TACR1细胞的细胞递送（通过受体内在化）A的化合物，导致焦虑措施增加，从而增加了焦虑措施的增加，而焦虑措施并未通过诸如社会熟悉之类的抗焦虑社交线索来缓解。这些TACR1中间神经元代表了含有神经肽生长抑素（SST）和胆囊基蛋白（CCK）的BLA互肽的亚群，尽管这些细胞在调节焦虑症反应中似乎是关键的，但这些神经肽对这些反应的贡献仍然是未知的。此外，由于tACR1-互操神经元只是含有SST和CCK-含有BLA的神经元的亚群，因此诸如拮抗剂或基因抑制之类的传统方法无法阐明这些神经肽在TACR1-INTRENERENARANARAL NERENARAL回路中的特定贡献。拟议的研究的目的是开发一种体内基因沉默技术，该技术将针对特定细胞并证明在体内使用的可行性。拟议的研究的目的是将反义PNA的基因沉淀特性与靶向毒素的细胞靶向剂相结合，以创建能够靶向基因沉默的化合物。具体而言，我们将将验证的TACR1靶向剂，SSP与反义PNA相结合，该反义PNA阻断了SST mRNA（antipnAsst）的翻译，从而导致SSP-抗精性。拟议的研究的中心假设是SSP抗逆转肌症将仅在其质膜上具有TACR1的细胞中抑制SST表达。一旦建立了这项技术，就可以使用该技术来阐明神经肽在BLA中间神经元中的焦虑调节作用。从研究产生的结果将通过提供一种新的方法来确定社会行为的关键神经底物，从而影响自闭症领域。此外，这些研究的潜在影响生物医学研究的多个领域，证明了靶向的体内PNA递送的可行性。这将为靶向剂和基于PNA的诊断和治疗剂的几乎无限组合打开大门，迄今为止，由于PNA的膜渗透性差，因此在体内使用的限制。公共卫生相关性：肽核酸是用于遗传操纵的强大分子，代表了对基因疗法当前方法的潜在改善，包括提高特异性，调节基因表达的众多策略和对基因表达而无需病毒转染的持久作用。但是，PNA对膜相对不渗透，使体内使用最少。为了克服这个问题，我们计划开发一种新型的在体内靶向PNA的方法，并证明在动物模型中使用这些化合物的可行性。