Theranostic Nanoparticles to enhance morpholino delivery to the liver for suppres

摘要 基因疗法为许多疾病的新疗法带来了巨大的希望。尽管临床前取得了巨大的成功， 很少有基因疗法对患者有效。在本次拨款申请中，提出了一种新的类型 基因治疗系统的最新进展，即吗啉纳米粒。Morpholinos对抑制 基因表达，尤其是抑制microRNA的功能。所使用的纳米颗粒将具有 以下特征：使用二硫醇键连接的吗啉，该键将在内涵体中被裂解；聚合物 一种涂层，可以破坏内体，使吗啡释放到细胞质中；聚乙二醇链，以允许长 循环半衰期；靶向肝细胞的半乳糖；用于核磁共振和透射电子显微镜检测的铁核；以及 用于荧光技术的荧光团。纳米颗粒的氧化铁和荧光团成分允许 用于治疗方法，其中纳米颗粒的性能可以使用成像技术进行评估。 由于这些纳米粒子的循环半衰期很长，并且具有半乳糖靶向性，因此纳米粒子应该 在体内定位于肝细胞。减少胆固醇产生的两种方法是 尝试：1)PCSK9基因敲除和2)miR-122抑制。胆固醇水平与患糖尿病的风险相关 心脏病，因此这种吗啉-纳米颗粒系统将是一种治疗 胆固醇水平升高。然而，在此基础上开发的吗啉纳米粒递送技术 格兰特随后可能被应用于心脏病的其他方面的治疗，如肥厚， 心肌病和狭窄，或其他疾病，如癌症。 应聘者在合成用作靶向的多功能纳米颗粒方面经验丰富 用于医学成像的造影剂。该奖项的目的是培养候选人的开发和应用能力。 用于基因治疗目的的新型纳米粒子。 该奖项的K99指导阶段将在Roger Hajjar教授和Zahi教授的指导下进行 西奈山医学院的法亚德。培训的重点将是纳米粒子的开发 用于基因治疗以及分析mRNA和microRNA敲除所需的技术，即聚合酶链式反应， Western印迹和Northern印迹。此指导阶段将为R00独立阶段奠定基础 在那里，纳米颗粒的开发和体外测试将继续进行，并将启动体内试验。这个 这项工作的结果应导致建立对候选人的富有成效的调查路线， 对我们对疾病和人类健康的了解大有裨益。

ABSTRACT Gene therapy holds great promise for new treatments for many diseases. Despite great pre-clinical successes, few gene therapy treatments have been effective in patients. In this grant application, is proposed a new type of gene therapy system, namely morpholino-nanoparticles. Morpholinos are highly effective for suppressing gene expression and, notably, for suppressing microRNA function. The nanoparticles used will possess the following features: morpholinos attached using a dithiol bond that will be cleaved in endosomes; a polymer coating that can disrupt endosomes for morpholino release into the cytoplasm; PEG chains to allow a long circulation half-life; galactose targeting to hepatocytes; an iron core for MRI and TEM detection; and a fluorophore for fluorescence techniques. The iron oxide and fluorophore components of the nanoparticle allow for a ¿theranostic¿ approach, where the nanoparticle performance can be evaluated using imaging techniques. Due to the long circulation half-life of these nanoparticles and the galactose targeting, the nanoparticles should localize in the hepatocytes of the liver in vivo. Two approaches for reducing cholesterol production will be attempted: 1) PCSK9 knockdown and 2) miR-122 suppression. Cholesterol levels are correlated with the risk of heart disease and therefore this morpholino-nanoparticle system would be a treatment for individuals with elevated levels of cholesterol. However, the morpholino-nanoparticle delivery technology developed under this grant could subsequently be applied for therapy of other aspects of heart disease such as hypertrophy, cardiomyopathy and stenosis, or to other diseases such as cancer. The candidate is highly experienced in the synthesis of multifunctional nanoparticles that act as targeted contrast agents for medical imaging. The purpose of this award is to train the candidate to develop and apply novel nanoparticles for gene therapy purposes. The K99 mentored phase of the award will take place under the guidance of Prof. Roger Hajjar and Prof. Zahi Fayad of Mount Sinai School of Medicine. The focus of the training will be on the development of nanoparticles for gene therapy and the techniques required for analysis of mRNA and microRNA knockdown, i.e. PCR, Western blotting and Northern blotting. This mentored phase will set the stage for the R00 independent phase where the nanoparticle development and in vitro testing will continue and in vivo trials will be initiated. The results of this work should lead to the establishment of a fruitful line of investigation for the candidate that will reap benefits for our understanding of disease and human health.