Theranostic nanoparticles to enhance morpholino delivery to the liver for suppres

描述（由申请人提供）：基因疗法为许多疾病的新疗法提供了巨大的希望。尽管取得了巨大的临床前成功，但很少有基因疗法对患者有效。本文提出了一种新型的基因治疗系统，即morpholino-nanoparticles。Morpholinos在抑制基因表达方面非常有效，特别是在抑制microRNA功能方面。所使用的纳米颗粒将具有以下特征：使用二硫醇键连接的morpholinos将在核内体中被切割；一种聚合物涂层，可以破坏核内体，使morpholino释放到细胞质中；PEG链允许较长的循环半衰期；半乳糖靶向肝细胞；用于MRI和TEM检测的铁芯；一个荧光团用于荧光技术。纳米颗粒的氧化铁和荧光团成分允许治疗方法，其中纳米颗粒的性能可以使用成像技术进行评估。由于这些纳米颗粒的循环半衰期长和半乳糖靶向性，纳米颗粒应该在体内肝脏的肝细胞中定位。将尝试两种降低胆固醇产生的方法：1)PCSK9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morpholino- nanopparticle系统将是一种治疗胆固醇水平升高的个体的方法。然而，在这笔资助下开发的morpholino- nanopparticle递送技术随后可以应用于心脏病的其他方面，如肥厚、心肌病和狭窄，或其他疾病，如癌症的治疗。该候选人在合成多功能纳米颗粒方面经验丰富，可作为医学成像的靶向造影剂。该奖项的目的是培训候选人开发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。培训的重点将是开发用于基因治疗的纳米颗粒，以及分析mRNA和microRNA敲低所需的技术，即PCR、Western blotting和Northern blotting。这个指导阶段将为R00独立阶段奠定基础，在这个阶段，纳米颗粒的开发和体外测试将继续进行，体内试验将开始。这项工作的结果将导致建立一个富有成效的调查路线，这将有利于我们对疾病和人类健康的理解。

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.