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

• 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
• 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

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中最有前途的新治疗范例之一。尽管RNAi，特别是小干扰RNA（siRNA），已经显示出治疗癌症和广谱其他疾病的很大希望，但是由于RNA对血清核酸酶的脆弱性、引起脱靶效应的倾向以及需要递送到靶细胞的胞质溶胶中，RNAi的递送是一个挑战。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靶向递送siRNA的方法，该方法利用了由携带siRNA的微泡（MB）的超声（US）诱导的振动产生的独特生物效应。这些生物效应包括增强的细胞膜对大分子的渗透性，这被认为有助于siRNA内化。我们已经开发了一种声活性阳离子脂质MB载体EGFR siRNA，延缓小鼠鳞状细胞癌的生长。我们最近还设计了一种新的亚微米聚合物MB携带EGFR siRNA负载的脂质体，在体外沉默EGFR表达，并可能具有独特的潜力，以促进血管外siRNA转移。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，测试siRNA递送、基因沉默和治疗效果可以通过这2种MB制剂和US参数的最佳组合来实现的总体假设。提出了四个目标：（1）为了检验siRNA对MB和靶细胞摄取的负载效率可以通过MB化学和US参数的操作来增加的假设，我们将在细胞培养物中使用MB/US组合（“平台”）的基质实验性地确定siRNA的负载和细胞内化。(2)为了检验我们的US-MB siRNA递送平台诱导特异性基因沉默的假设，我们将使用来自目标1的平台在体外测定EGFR沉默的水平（并评估毒性）。(3)为了检验我们的US-MB siRNA治疗诊断方法在体内诱导治疗性基因沉默以及RNAi将以有利的毒性和生物分布抑制肿瘤生长的假设，我们将在荷瘤小鼠中测定各种剂量的EGFR沉默，测定EGFR siRNA治疗后的肿瘤生长抑制，并测定生物分布和毒性特征。(4)为了研究US和MB介导的siRNA递送的机制，我们将在显微镜下观察标记的siRNA的运输，重点是内吞和内吞非依赖性机制。这些研究将最终形成一种非侵入性的靶向siRNA递送策略，这将促进RNAi的临床实施。重要的是，虽然我们提出的siRNA递送平台靶向特定癌基因的mRNA（和随后的蛋白质水平），但我们的工作将建立可以扩展到US-MB siRNA平台的一般原则，用于在其他疾病中进行图像引导的靶向基因沉默，其中特定基因沉默代表治疗方法。