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。尽管在临床前取得了巨大的成功， 很少有基因治疗对患者有效。在这项拨款申请中，提出了一种新的类型， 基因治疗系统，即吗啉纳米颗粒。吗啉代对抑制 基因表达，特别是抑制microRNA功能。所使用的纳米颗粒将具有 以下特征：使用将在内涵体中裂解的二硫醇键连接的吗啉代;聚合物 可以破坏内体以将吗啉释放到细胞质中的涂层; PEG链， 循环半衰期;靶向肝细胞的半乳糖;用于MRI和TEM检测的铁核心;以及 用于荧光技术的荧光团。纳米颗粒的氧化铁和荧光团组分允许 对于治疗诊断学方法，可以使用成像技术评估纳米颗粒的性能。 由于这些纳米颗粒的长循环半衰期和半乳糖靶向，纳米颗粒应该 定位于体内肝脏的肝细胞中。减少胆固醇产生的两种方法是 尝试：1）PCSK 9敲除和2）miR-122抑制。胆固醇水平与以下风险相关： 因此，这种吗啉代纳米颗粒系统将是治疗患有心脏病的个体的一种方法。 胆固醇水平升高然而，在此基础上开发的吗啉代纳米颗粒递送技术 资助其后可用于治疗心脏病的其他方面，例如心脏肥大， 心肌病和狭窄，或其他疾病如癌症。 该候选人在合成具有靶向作用的多功能纳米颗粒方面经验丰富 用于医学成像的造影剂。这个奖项的目的是培养候选人的发展和应用 用于基因治疗目的的新型纳米颗粒。 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 西奈山医学院的法耶德。培训的重点将是纳米粒子的发展 用于基因治疗和分析mRNA和microRNA敲低所需的技术，即PCR， Western印迹和北方印迹。该指导阶段将为R 00独立阶段奠定基础 其中纳米颗粒的开发和体外试验将继续进行，体内试验将开始。的 这项工作的结果应导致建立一个富有成效的调查路线的候选人， 为我们理解疾病和人类健康带来益处。

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.