Topical Delivery of siRNA Nanconjugates: Suppressing Epidermal Hyperplasia

描述(申请人提供)：短干扰核糖核酸(SiRNAs)是经过验证的研究工具，它彻底改变了我们抑制基因的能力。然而，传递仍然是它们在体内使用的主要障碍，包括局部应用后siRNAs穿越高效表皮屏障的能力有限。该项目的目标是优化和评估一种局部应用的siRNA纳米颗粒递送系统，该系统能够穿透皮肤并选择性地抑制基因表达。这样的递送系统将有可能选择性地针对导致遗传疾病的微小突变，其影响范围从抑制癌细胞生长到抑制炎症。我们的实验室已经设计出一种新型的纳米颗粒结合物，它利用紧密堆积在金纳米颗粒(siRNA-Au NPs)表面的siRNA双链，显示出惊人的能力通过小鼠和人类角质层，并抑制两个测试靶点，绿色荧光蛋白和表皮生长因子受体。到目前为止，当siRNA-Au纳米粒局部或静脉注射到小鼠体内的浓度远远超过抑制基因所需的浓度时，还没有发现毒性。在我们提出的工作中，我们将确定siRNA-Au纳米颗粒渗透到人体皮肤的机制(S)，并利用这些信息来优化siRNA-Au纳米颗粒的基因抑制。接下来，我们将评估siRNA-Au纳米颗粒抑制RAS信号和逆转H-RAS皮肤特异、诱导过表达的小鼠模型中表皮增生的能力。在我们的体外和体内小鼠siRNA-Au纳米粒研究的基础上，我们将测试局部应用siRNA-Au纳米粒到RAS过表达的人类移植皮肤模型是否同样会抑制RAS信号的异常，从而导致临床和组织学上可检测到的表皮正常化。这项工作将为siRNA纳米结合物的临床应用奠定基础，并为基因治疗的局部应用建立一个新的范式。

DESCRIPTION (provided by applicant): Short interfering ribonucleic acids (siRNAs) are proven research tools that have revolutionized our ability to suppress genes. However, delivery remains a major hurdle for their use in vivo, including the limited ability of siRNAs to traverse the highly effective epidermal barrier after topical application. The objective of this project is to optimize and evaluate a topically applied nanoparticle delivery system for siRNA that is able to penetrate the skin and selectively suppress gene expression. Such a delivery system would have the potential to selectively target even small mutations that lead to genetic disorders, with implications ranging from inhibiting cancer cell growth to suppressing inflammation. Our laboratories have engineered a novel nanoparticle conjugate utilizing siRNA duplexes that are densely packed on the surface of gold nanoparticles (siRNA-Au NPs) and which demonstrates a surprising ability to transit the mouse and human stratum corneum and suppress two tested targets, green fluorescent protein and the epidermal growth factor receptor. To date, no toxicity has been found when the siRNA-Au NPs are delivered either topically or intravenously into mice at concentrations that far exceed those needed to suppress genes. In our proposed work, we will determine the mechanism(s) by which siRNA-Au NPs penetrate into human skin, and use this information to optimize siRNA-Au NPs for gene suppression. Next, we will assess the ability of siRNA-Au NPs to suppress Ras signaling and reverse epidermal hyperplasia in a mouse model of skin-specific, inducible overexpression of H-Ras. Building on our in vitro and in vivo mouse studies with siRNA-Au NPs, we will test if topical application of siRNA-Au NPs to a human transplanted skin model of Ras overexpression will similarly suppress aberrant Ras signaling to cause clinically and histologically detectable epidermal normalization. This work will lay the foundation for clinical application of the siRNA nanoparticle conjugates, and establish a new paradigm for the topical application of gene therapies.