In vivo targeted gene silencing, a novel method

描述（由申请人提供）：最近有报道称，每150名美国儿童中就有1名患有自闭症，每年给美国造成的损失估计为900亿美元。自闭症是一种具有广泛社会缺陷的谱系障碍，很难在动物研究中建立模型。在最近一项用大鼠进行的动物研究中，我们描述了一种调节对社交线索的情绪反应的神经回路，这种神经回路可能与谱系障碍患者观察到的社交加工缺陷有关。该神经回路的一个关键组成部分是位于杏仁核内含有物质P1受体（也称为速激肽受体1；Tacr1）的离散中间神经元群。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。选择性地用靶向毒素ssp -皂苷损伤这些中间神经元，这种化合物选择性地结合并（通过受体内化）将毒素仅传递给具有Tacr1的细胞，导致焦虑增加，而焦虑性社会线索（如社会熟悉度）无法减轻焦虑。这些Tacr1中间神经元代表了含有生长抑素（Sst）和胆囊收缩素（Cck）神经肽的bla中间神经元亚群，尽管这些细胞似乎在调节焦虑样反应中起关键作用，但这些神经肽对这些反应的贡献尚不清楚。此外，由于tacr1 -中间神经元只是含有Sst-和Cck-的BLA中间神经元的一个亚群，拮抗剂或基因抑制等传统方法无法阐明这些神经肽在tacr1 -神经元间回路中的具体作用。提出的研究目标是开发一种针对特定细胞的体内基因沉默技术，并证明在体内使用的可行性。本研究的目的是将反义PNAs的基因沉默特性与靶向毒素的细胞靶向剂结合起来，创造一种能够靶向基因沉默的化合物。具体来说，我们将把已证实的Tacr1靶向剂SSP与阻断Sst mRNA翻译的反义PNA （antiPNASst）结合起来，产生SSP- antiPNASst。该研究的中心假设是，SSP-antiPNAsst仅在质膜上有Tacr1的细胞中抑制Sst的表达。一旦这项技术建立起来，就可以用来阐明神经肽在BLA中间神经元中的焦虑调节作用。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PNA在体内传递的可行性，可能影响生物医学研究的多个领域。这将为靶向药物和基于PNAs的诊断和治疗药物的几乎无限组合打开大门，迄今为止，由于PNAs的膜渗透性差，这些药物在体内的使用受到限制。

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.