Ultrasound-activated microbubbles for targeted siRNA delivery to tumor

用于将 siRNA 靶向递送到肿瘤的超声激活微泡

基本信息

批准号：
8501449
负责人：
Flordeliza S Villanueva
金额：
$ 53.4万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-07-01 至 2016-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8501449
关键词：
Acoustics Address Autoimmune Diseases Binding Biodistribution Biological Assay Cell Culture Techniques Cell Membrane Permeability Cells Charge Chemicals Chemistry Clinical Clinical Medicine Contrast Media Cytoplasm Cytosol Diffusion Disease Dose Drug Carriers Drug Formulations Emerging Technologies Endocytosis Epidermal Growth Factor Receptor Future Gene Expression Gene Silencing Gene Targeting Genes Growth Image In Vitro Label Learning Lipids Liposomes Malignant Epithelial Cell Malignant Neoplasms Mediating Medicine Membrane Membrane Lipids Messenger RNA Methods Microbubbles Modeling Modification Molecular Target Mus Nature Neurodegenerative Disorders Oncogenes Pathogenesis Pathway interactions Polymers Proteins RNA RNA Interference RNA-Induced Silencing Complex Regulation Research Design Role Serum Small Interfering RNA Squamous cell carcinoma System Testing Therapeutic Therapeutic Effect Tissues Toxic effect Transduction Gene Translations Tumor Markers Ultrasonography Virus Diseases Work base c-erbB-1 Proto-Oncogenes cancer cell cancer therapy cellular targeting cytotoxicity design immune activation in vitro Model in vivo innovation insight macromolecule multidisciplinary novel novel strategies novel therapeutics nuclease plasmid DNA prevent receptor expression submicron targeted delivery theories theranostics therapeutic gene trafficking tumor tumor growth uptake vector vibration

项目摘要

DESCRIPTION (provided by applicant): The use of RNA interference (RNAi) to silence the expression of specific genes associated with disease is one of the most promising new therapeutic paradigms in medicine. Although RNAi, and specifically small interfering RNA (siRNA), has shown much promise for the treatment of cancer and a broad spectrum of other diseases, delivery of RNAi is a challenge due to the vulnerability of RNA to serum nucleases, the propensity to cause off- target effects, and the requirement for delivery into the cytosol of target cells. Thus, new methods and vectors are needed to effectively deliver RNAi to target tissue. The objective of this proposal is to develop a new approach for targeted delivery of siRNA that capitalizes on the unique bioeffects that result from ultrasound (US)-induced vibrations of microbubles (MB) carrying siRNA. These bioeffects include enhanced cell membrane permeability to macromolecules, which is thought to facilitate siRNA internalization. We have developed an acoustically active cationic lipid MB carrier of EGFR siRNA that retards growth of murine squamous cell carcinomas. We have also recently designed a novel submicron polymer MB carrying EGFR siRNA-loaded liposomes that silences EGFR expression in vitro, and may have unique potential to facilitate extravascular siRNA transfer. Accordingly, we will test the overall hypothesis that siRNA delivery, gene silencing, and therapeutic effects can be achieved by optimal combinations of these 2 MB formulations and US parameters, using in vivo and in vitro models of squamous cell carcinoma as the test system. Four Aims are proposed: (1) To test the hypothesis that the loading efficiency of siRNA on MB and target cellular uptake can be increased by manipulation of MB chemistry and US parameters, we will experimentally determine the loading and cellular internalization of siRNA using a matrix of MB/US combinations ("platforms") in cell culture. (2) To test the hypothesis that our US-MB siRNA delivery platform induces specific gene silencing, we will determine levels of EGFR silencing in vitro (and assess toxicity) using platforms from Aim 1. (3) To test the hypothesis tha our US-MB siRNA theranostics approach induces therapeutic gene silencing in vivo and that the RNAi will suppress tumor growth with favorable toxicity and biodistribution, we will determine EGFR silencing at a variety of doses in tumor bearing mice, assay tumor growth inhibition upon EGFR siRNA treatment, and determine biodistribution and toxicity profiles. (4) To investigate the mechanisms of US and MB mediated siRNA delivery, we will microscopically observe the trafficking of labeled siRNA, focusing on endocytotic and endocytosis-independent mechanisms. These studies will culminate in a non-invasive, targeted siRNA delivery strategy that will facilitate the clinical implementation of RNAi. Importantly, while our proposed siRNA delivery platform targets mRNA (and subsequent protein levels) of a specific oncogene, our work will establish general principles that can be extended to US-MB siRNA platforms for image-guided targeted gene silencing in other diseases for which specific gene silencing represents a therapeutic approach.

描述（由申请人提供）：使用RNA干扰（RNAi）来沉默与疾病相关的特定基因的表达是医学上最有前途的新治疗范例之一。尽管 RNAi，特别是小干扰 RNA (siRNA)，在治疗癌症和广泛的其他疾病方面显示出很大的希望，但由于 RNA 对血清核酸酶的脆弱性，RNAi 的传递是一个挑战，容易导致- 目标效应，以及递送到目标细胞的细胞质中的要求。因此，需要新的方法和载体来有效地将RNAi递送至靶组织。该提案的目的是开发一种新的 siRNA 靶向递送方法，该方法利用超声波 (US) 引起的携带 siRNA 的微泡 (MB) 振动产生的独特生物效应。这些生物效应包括增强细胞膜对大分子的渗透性，这被认为有助于 siRNA 内化。我们开发了一种 EGFR siRNA 的声学活性阳离子脂质 MB 载体，可延缓小鼠鳞状细胞癌的生长。我们最近还设计了一种新型亚微米聚合物 MB，其携带 EGFR siRNA 负载脂质体，可在体外沉默 EGFR 表达，并且可能具有促进血管外 siRNA 转移的独特潜力。因此，我们将使用鳞状细胞癌的体内和体外模型作为测试系统，通过这些 2 MB 制剂和 US 参数的最佳组合来测试 siRNA 递送、基因沉默和治疗效果的总体假设。提出了四个目标：（1）为了测试通过操作 MB 化学和 US 参数可以增加 siRNA 在 MB 上的负载效率和靶细胞摄取的假设，我们将使用基质通过实验确定 siRNA 的负载和细胞内化细胞培养中的 MB/US 组合（“平台”）。 (2) 为了检验我们的 US-MB siRNA 递送平台诱导特定基因沉默的假设，我们将使用目标 1 的平台确定体外 EGFR 沉默的水平（并评估毒性）。 (3) 为了检验我们的 US-MB siRNA 传递平台诱导特定基因沉默的假设。 -MB siRNA治疗诊断方法在体内诱导治疗性基因沉默，RNAi将抑制肿瘤生长，并具有良好的毒性和生物分布，我们将在荷瘤中确定各种剂量的EGFR沉默小鼠，测定 EGFR siRNA 治疗后的肿瘤生长抑制，并确定生物分布和毒性特征。 (4) 为了研究US和MB介导的siRNA递送机制，我们将在显微镜下观察标记的siRNA的运输，重点关注内吞作用和内吞作用无关的机制。这些研究最终将形成一种非侵入性、靶向 siRNA 递送策略，从而促进 RNAi 的临床实施。重要的是，虽然我们提出的 siRNA 递送平台针对特定癌基因的 mRNA（以及随后的蛋白质水平），但我们的工作将建立可扩展到 US-MB siRNA 平台的一般原则，用于在其他特定疾病中进行图像引导的靶向基因沉默。基因沉默代表了一种治疗方法。