Targeted theranostic microbubble vectors for transcription factor decoy delivery

描述(由申请人提供)：对参与肿瘤发生的途径和蛋白质的扩展知识导致了靶向分子疗法的发展，包括使用设计用作转录因子诱饵的小核酸结构。尽管有太多基于核酸的小分子疗法具有潜在的临床价值，但没有一种可用的方法能够靶向、安全、微创和重复地将这些药物输送到癌症中。因此，以小核酸为基础的疗法的临床翻译一直很慢。因此，在这项提案中，一个独特的多学科团队，包括化学和生物医学工程、声学物理、基础和临床肿瘤学、分子生物学和成像方面的专业知识，将开发一种新的方法，用于定向递送转录因子诱饵，利用超声诱导的微泡振动(MBS)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通透性，如转录因子诱骗，可将其加载到MBS上，并在靶点通过指向靶点的超声束诱导MB断裂时单独释放。由于超声波还可以提供成像能力，项目团队将创新一种声学超声-MB传递系统，该系统将超声成像与核酸携带能力相结合，共同可能克服目前治疗转录因子诱骗传递的障碍，同时允许实时可视化 肿瘤和诱饵的分布情况。我们将测试总体假设，即MB可以装载治疗性核酸(转录因子诱饵)，超声波介导的治疗性递送将导致癌基因沉默并减少肿瘤生长，以及使用肿瘤特异性靶向部分将增强治疗效果，同时允许特异性肿瘤检测。我们将利用STAT3诱饵作为测试治疗，因为这种小核酸在动物癌症模型中具有治疗潜力和安全性。首先，我们将在不同的超声条件下将培养的癌细胞暴露于携带STAT3诱骗的MBS，分析所产生的下游靶基因的表达，并确定对成功传递该特定制剂至关重要的超声和MB特征。然后，为了确定超声-MB递送平台是否在体内诱导STAT3信号沉默和肿瘤生长抑制，我们将静脉递送STAT3诱饵负载的MBS并对携带鳞状细胞癌的小鼠施加超声波，检测STAT反应基因的表达，并通过超声跟踪肿瘤随时间的生长。这些研究将最终形成一种有效的、非侵入性的、靶向的转录因子诱饵传递策略，应有助于临床实施。重要的是，虽然我们建议的递送策略针对该项目中的特定癌基因，但这项工作将为图像引导的超声-MB治疗性递送平台建立一般原则，该平台可以扩展到其他小核酸递送代表治疗方法的疾病。 与公共卫生相关：临床采用分子疗法来定向沉默与癌症等疾病有关的异常基因，包括使用旨在充当转录因子诱饵的小核酸结构，但由于缺乏定向、安全、微创和重复传递这些结构的策略，因此受到限制。在这项提案中，一个独特的多学科团队将开发一种床边超声-微泡诊断和传递系统，该系统将实时超声成像与加载转录因子诱饵的微泡相结合，用于专门针对肿瘤的超声诱导诱骗传递。在这个项目中开发的传递平台应该促进使用转录因子诱骗治疗癌症的方法的临床翻译，最终导致患有癌症的患者获得更好的结果。该平台还可以应用于靶向基因沉默是治疗选择的其他疾病的分子治疗。

DESCRIPTION (provided by applicant): Expanding knowledge of pathways and proteins involved in oncogenesis has led to the development of targeted molecular therapeutics, including the use of small nucleic acid constructs designed to act as transcription factor decoys. Despite the plethora of small nucleic acid-based therapeutics of potential clinical value, no available method is capable of targeted, safe, minimally invasive, and repeated delivery of therapeutic quantities of these agents to cancers. As a result, clinical translation of small nuclec acid-based therapeutics has been slow. Accordingly, in this proposal, a unique multi-disciplinary team embodying expertise in chemical and biomedical engineering, acoustic physics, basic and clinical oncology, molecular biology, and imaging, will develop a new approach for targeted delivery of transcription factor decoys that capitalizes on unique bioeffects ensuing from ultrasound-induced vibrations of microbubbles (MBs). These bioeffects include enhanced cell membrane permeability to macromolecules, such as transcription factor decoys, which can be loaded on MBs and released solely at the target site upon induction of MB rupture by an ultrasound beam directed at the site. Because ultrasound also confers imaging capability, the project team will innovate a theranostic ultrasound- MB delivery system that combines ultrasound imaging with nucleic acid carrying capacity, which together may overcome current barriers to therapeutic transcription factor decoy delivery, while allowing real time visualization of the tumor and distribution of the decoy. We will test the overall hypothesis that MB can be loaded with a therapeutic nucleic acid (transcription factor decoy), that ultrasound-mediated delivery of the therapeutic will cause oncogene silencing and reduce tumor growth, and that the use of tumor-specific targeting moieties will enhance the therapeutic effect and simultaneously allow specific tumor detection. We will utilize the STAT3 decoy as a test therapeutic, as this small nucleic acid is known to have therapeutic potential and safety in animal cancer models. First, we will expose cultured carcinoma cells to STAT3 decoy- loaded MBs under varying ultrasound conditions, assay the resulting expression of downstream target genes, and determine the ultrasound and MB features which are critical for successful delivery of this particular agent. Then, to determine if the ultrasound-MB delivery platform induces STAT3 signal silencing and tumor growth suppression in vivo, we will intravenously deliver STAT3 decoy-loaded MBs and administer ultrasound to mice bearing squamous cell carcinomas, assay expression of STAT responsive genes, and ultrasonically track tumor growth over time. These studies will culminate in an efficient, non-invasive, targeted transcription factor decoy delivery strategy that should facilitate clinical implementation. Importantly, while our proposed delivery strategy targets a specific oncogene in this project, this work will establish general principles fr an image- guided ultrasound-MB therapeutic delivery platform that can be extended to other diseases for which small nucleic acid delivery represents a therapeutic approach. PUBLIC HEALTH RELEVANCE: Clinical adoption of molecular therapeutics for targeted silencing of abnormal genes involved in diseases such as cancer, including the use of small nucleic acid constructs designed to act as transcription factor decoys, is limited by the lack of strategies for targeted, safe, minimally invasive, and repeated delivery of these constructs. In this proposal, a unique multi-disciplinary team will develop a bedside ultrasound-microbubble diagnostic and delivery system that combines real time ultrasound imaging with transcription factor decoy-loaded microbubbles for ultrasound-induced decoy delivery specifically to tumors. The delivery platform developed in this project should facilitate the clinical translation of approaches using transcription factor decoys for treatment of cancer, ultimately leading to better outcome in patients afflicted with cancer. This platform can also be applied to molecular therapy of other diseases for which targeted gene silencing represents a treatment option.