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知识不断扩展，导致了靶向分子治疗的发展，包括使用设计用作转录因子诱饵的小核酸构建体。尽管存在过多的具有潜在临床价值的基于小核酸的治疗剂，但没有可用的方法能够靶向、安全、微创和重复递送治疗量的这些药剂至癌症。因此，基于小核酸的治疗剂的临床转化一直很缓慢。因此，在这项提案中，一个独特的多学科团队体现了化学和生物医学工程，声学物理学，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，用于靶向递送转录因子诱饵，利用超声诱导的微泡振动（MB）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，如转录因子诱饵，其可以加载在MB上，并在通过指向靶位点的超声波束诱导MB破裂后仅在靶位点释放。由于超声波也赋予成像能力，项目团队将创新一种治疗诊断超声- MB输送系统，该系统将超声成像与核酸携带能力相结合，这两者可以共同克服目前治疗性转录因子诱饵输送的障碍，同时允许真实的时间可视化 和诱饵的分布我们将检验以下总体假设：MB可以装载治疗性核酸（转录因子诱饵），超声介导的治疗剂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并同时允许特异性肿瘤检测。我们将利用STAT 3诱饵作为测试治疗剂，因为已知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装载STAT 3诱饵的MB，测定下游靶基因的所得表达，并确定对于成功递送该特定试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STAT 3信号沉默和肿瘤生长抑制，我们将静脉内递送装载STAT 3诱饵的MB并向携带鳞状细胞癌的小鼠施用超声，测定STAT应答基因的表达，并超声跟踪肿瘤随时间的生长。这些研究将最终在一个有效的，非侵入性的，有针对性的转录因子诱饵交付策略，应促进临床实施。重要的是，虽然我们提出的递送策略在该项目中靶向特定的致癌基因，但这项工作将建立图像引导的超声MB治疗递送平台的一般原理，该平台可以扩展到小核酸递送代表治疗方法的其他疾病。 公共卫生相关性：用于靶向沉默涉及疾病如癌症的异常基因的分子治疗的临床采用，包括设计用作转录因子诱饵的小核酸构建体的使用，受到缺乏用于靶向、安全、微创和重复递送这些构建体的策略的限制。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输送系统，该系统将真实的超声成像与装载转录因子诱饵的微泡结合起来，用于超声诱导的诱饵输送，特别是肿瘤。该项目中开发的递送平台应促进使用转录因子诱饵治疗癌症的方法的临床转化，最终导致癌症患者的更好结果。该平台还可以应用于靶向基因沉默代表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.