In vivo targeted gene silencing, a novel method

体内靶向基因沉默，一种新方法

• 批准号: 8030280
• 负责人: WILLIAM Anthony TRUITT
• 金额: $ 21.85万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 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/8030280
• 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; receptor; receptor internalization; relating to nervous system; response; social; targeted delivery

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）：最近有报道称，自闭症影响150名美国儿童中的1名，估计每年给美国造成900亿美元的损失。自闭症是一种具有广泛社会缺陷水平的谱系障碍，并且难以在动物研究中建模。在最近的一项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，可能与频谱障碍患者观察到的社会处理缺陷有关。这种神经回路的一个关键组成部分是位于杏仁核内的含有P1物质受体（也称为速激肽受体1; Tacr 1）的中间神经元的离散群体。这些Tacr 1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。用靶向毒素SSP-皂草素选择性地损伤这些中间神经元，SSP-皂草素是一种选择性地结合毒素并仅将毒素传递（通过受体内化）到具有Tacr 1的细胞的化合物，导致焦虑措施增加，而这种焦虑措施并没有通过抗焦虑的社交线索（如社交熟悉度）缓解。这些Tacr 1中间神经元代表了含有神经肽生长抑素（Sst）和胆囊收缩素（Cck）的BLA-interneurons亚群，尽管这些细胞似乎在焦虑样反应的调节中起关键作用，但这些神经肽对这些反应的贡献仍然未知。此外，由于Tacr 1-interneurons只是一个亚群的Sst-和Cck-含有BLA interneurons，传统的方法，如拮抗剂或基因抑制不能阐明的具体贡献，这些神经肽内的Tacr 1-interneurons电路。 这项研究的目的是开发一种体内基因沉默技术，该技术将靶向特定细胞并证明在体内使用的可行性。该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联合收割机结合，以产生能够靶向基因沉默的化合物。具体而言，我们将联合收割机结合已证实的Tacr 1靶向剂SSP和阻断Sst mRNA翻译的反义PNA（antiPNASst），产生SSP-antiPNASst。这项研究的中心假设是，SSP-antiPNASst仅在质膜上具有Tacr 1的细胞中抑制Sst表达。一旦建立了这种技术，然后可以用来阐明BLA中间神经元内的神经肽的焦虑调节作用。这项研究的结果将通过提供一种新的方法来确定社会行为背后的关键神经基质，从而影响自闭症领域。此外，这些研究通过证明靶向体内PNA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，其在体内使用受到限制。 公共卫生关系：肽核酸是用于遗传操作的强大分子，并且代表了对当前基因治疗方法的潜在改进，包括增加的特异性、调节基因表达的多种策略以及在没有病毒转染的情况下对基因表达的持久作用。然而，PNA对膜是相对不可渗透的，从而将体内使用保持在最低限度。为了克服这个问题，我们计划开发一种新的方法，在体内有针对性地传递PNA，并证明在动物模型中使用这些化合物的可行性。