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）：最近据报道，自闭症影响着每 150 名美国儿童中就有 1 名，估计每年给美国造成 900 亿美元的损失。自闭症是一种谱系障碍，具有广泛的社交缺陷，很难在动物研究中建立模型。在最近一项使用大鼠的动物研究中，我们描述了一种调节对社交线索的情绪反应的神经回路，并且可能与谱系障碍中观察到的社交处理缺陷有关。该神经回路的一个关键组成部分是位于杏仁核内的一组离散的中间神经元，其中含有 P1 物质受体（也称为速激肽受体 1；Tacr1）。这些 Tacr1 中间神经元接收与社会识别相关的皮质输入，并抑制 BLA 投射神经元的焦虑样输出。使用靶向毒素 SSP-皂草素（一种选择性结合并仅向具有 Tacr1 的细胞传递毒素（通过受体内化）的化合物）选择性损害这些中间神经元，会导致焦虑感增加，但这种焦虑感并不能通过社交熟悉等抗焦虑社交线索得到缓解。。这些 Tacr1 中间神经元代表 BLA 中间神经元的一个亚群，其中含有神经肽生长抑素 (Sst) 和胆囊收缩素 (Cck)，尽管这些细胞似乎在调节焦虑样反应中至关重要，但这些神经肽对这些反应的贡献仍然存在未知。此外，由于 Tacr1 中间神经元只是包含 Sst 和 Cck 的 BLA 中间神经元的一个亚群，因此拮抗剂或基因抑制等传统方法无法阐明这些神经肽在 Tacr1 中间神经元回路中的具体贡献。拟议研究的目标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体内使用的可行性。拟议研究的目的是将反义 PNA 的基因沉默特性与靶向毒素的细胞靶向剂结合起来，创建一种能够靶向基因沉默的化合物。具体来说，我们将经过验证的 Tacr1 靶向剂 SSP 与反义 PNA 结合起来，该反义 PNA 阻断 Sst mRNA (antiPNASst) 的翻译，从而产生 SSP-antiPNASst。本研究的中心假设是 SSP-antiPNASst 只会抑制质膜上有 Tacr1 的细胞中的 Sst 表达。一旦确定，这项技术就可以用来阐明 BLA 中间神经元内神经肽的焦虑调节作用。该研究产生的结果将通过提供一种确定社会行为背后的关键神经基质的新方法来影响自闭症领域。此外，这些研究通过证明体内 PNA 靶向递送的可行性，可能会影响生物医学研究的多个领域。这将为靶向剂和基于 PNA 的诊断和治疗剂的近乎无限的组合打开大门，迄今为止，由于 PNA 的膜渗透性差，这些组合在体内的使用受到限制。公共卫生相关性：肽核酸是用于基因操作的强大分子，代表着对当前基因治疗方法的潜在改进，包括增加特异性、多种调节基因表达的策略以及无需病毒转染即可对基因表达产生持久影响。然而，PNA 相对不渗透膜，因此可将体内使用量降至最低。为了克服这个问题，我们计划开发一种 PNA 体内靶向递送的新方法，并证明在动物模型中使用这些化合物的可行性。