Theranostic nanoparticles to enhance morpholino delivery to the liver for suppres

描述（由申请人提供）：基因治疗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，但很少有基因治疗对患者有效。在该资助申请中，提出了一种新型的基因治疗系统，即吗啉代纳米颗粒。吗啉代对于抑制基因表达，特别是对于抑制microRNA功能非常有效。所使用的纳米颗粒将具有以下特征：使用将在内体中裂解的二硫醇键连接的吗啉代;可以破坏内体以将吗啉代释放到细胞质中的聚合物涂层;允许长循环半衰期的PEG链;靶向肝细胞的半乳糖;用于MRI和TEM检测的铁芯;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治疗诊断方法，其中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，纳米颗粒应定位于体内肝细胞中。将尝试两种减少胆固醇产生的方法：1）PCSK 9敲低和2）miR-122抑制。胆固醇水平与心脏病的风险相关，因此这种吗啉代纳米颗粒系统将是胆固醇水平升高的个体的治疗方法。然而，在该资助下开发的吗啉代纳米颗粒递送技术随后可用于治疗心脏病的其他方面，如肥大，心肌病和狭窄，或其他疾病，如癌症。候选人在多功能纳米颗粒的合成方面具有丰富的经验，这些纳米颗粒可作为医学成像的靶向造影剂。该奖项的目的是培养候选人开发和应用新型纳米颗粒用于基因治疗目的。该奖项的K99指导阶段将在西奈山医学院的Roger Hajjar教授和Zahi Fayad教授的指导下进行。培训的重点将是用于基因治疗的纳米颗粒的开发以及mRNA和microRNA敲低分析所需的技术，即PCR，Western印迹和北方印迹。这个指导阶段将为R 00独立阶段奠定基础，在R 00独立阶段，纳米颗粒开发和体外测试将继续进行，体内试验将开始。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，将收获我们对疾病和人类健康的理解的好处。 公共卫生相关性：心脏病是美国的头号杀手，也导致患有心脏病的人生活质量低下。高胆固醇水平导致心脏病发作的可能性更高。虽然被称为他汀类药物的药物已经成功地降低了许多患者的胆固醇水平，但仍有大量人群的胆固醇水平过高。在这项应用中，提出了降低胆固醇水平的新方法，其中纳米颗粒提供称为吗啉代的药物，将关闭胆固醇的产生。我们将专注于开发这些吗啉纳米颗粒系统，并评估其降低胆固醇水平的潜力。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: Heart disease is the number one killer in the USA, and also leads to poor quality of life for those who suffer from it. High cholesterol levels lead to a higher likelihood a heart attack. While the class of drugs known as statins has been successful for lowering the cholesterol levels of many patients, there are still large populations for whom cholesterol levels are too high. In this application are proposed new methods for reducing cholesterol levels where nanoparticles deliver drugs called morpholinos that will shut down cholesterol production. We will focus on developing these morpholino-nanoparticle systems and evaluating their potential to reduce cholesterol levels.