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人患有自闭症，估计每年给美国造成的损失为900亿美元。自闭症是一种谱系障碍，具有广泛的社会缺陷水平，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，我们描述了一种神经回路，它调节对社交线索的情绪反应，可能与观察到的谱系障碍的社交处理缺陷有关。这个神经回路的一个关键组成部分是一个离散的中间神经元群体，其中含有位于杏仁核内的P1物质受体(也称为速激肽受体1；Tacr1)。这些Tacr1中间神经元接受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。用一种靶向毒素SSP-Saporin选择性地损害这些中间神经元，SSP-Saporin是一种选择性地结合并将毒素传递(通过受体内化)只有Tacr1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社交熟悉度等缓解焦虑的社交线索来缓解。这些Tacr1中间神经元代表了含有神经肽生长抑素(SST)和胆囊收缩素(CCK)的BLA中间神经元的一个亚群，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，传统的拮抗剂或基因抑制方法不能阐明这些神经肽在Tacr1中间神经元环路中的具体作用。这项拟议的研究的目标是开发一种体内基因沉默技术，该技术将针对特定的细胞，并证明在体内使用的可行性。这项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。具体地说，我们将把经过验证的Tacr1靶向剂SSP与反义PNA结合起来，后者可以阻止SST mRNA(AntiPNASst)的翻译，从而产生SSP-antiPNAsst。这项研究的中心假设是，SSP-antiPNAsst只会在质膜上有Tacr1的细胞中抑制SST的表达。一旦确定了这一点，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。这项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背后的关键神经基础，从而影响自闭症领域。此外，这些研究通过证明靶向、体内PNA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PNA的诊断和治疗药物的近乎无限的组合打开大门，到目前为止，由于PNA的膜通透性较差，这些药物在体内的使用一直受到限制。 与公共健康相关：肽核酸是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，包括提高特异性，多种调控基因表达的策略，以及在没有病毒转基因的情况下对基因表达的长期影响。然而，PNA相对不透膜，将体内使用量保持在最低水平。为了克服这个问题，我们计划开发一种新的体内靶向递送PNA的方法，并在动物模型中证明使用这些化合物的可行性。

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.