Optical imaging in the development of molecularly targeted AAV for cardiac regeneration after myocardial infarction

这项提议的首要目标是设计新型的、多功能的药物和基因输送系统。 它可以针对特定的细胞和细胞内隔间进行治疗，还可以监控药物的传递和 通过整合先进的成像技术来确定治疗效果。为了追求这一目标， 此多PI应用程序建议利用UVA的现有优势，包括：使用专业知识 先进的生物医学成像技术在动物模型和再工程中评估缺血性心脏病 重组腺相关载体(RAAV)(法国)，体内筛选分子文库以鉴定特异性 能够靶向病变组织的多肽(Kelly)，使衣壳表面显示特定的多肽 能够将基因输送到病变组织(DASA)。不仅靶向多肽将在 该项目将用于开发一种新型的基于AAV的心脏基因传递系统，未来的应用包括 用荧光团和/或放射性示踪剂标记亲心多肽，以产生造影剂 缺血性心脏病的分子成像。类似地，相同的心力肽可能在 静脉(Iv)注射后直接将新的治疗药物靶向心脏。在初步研究中， 两个PI已经证明了所需的跨学科技术的健壮性和实用性 实现以下具体目标： 1)通过评估其他针对梗塞边缘区域的多肽的特异性来验证它们 体内光学成像和体外共聚焦显微镜。 2)通过在AAV9中嫁接亲心肽配体来优化心脏基因递送系统 用于由心脏特异性启动子驱动的选择性递送表达盒的衣壳。 3)证明心脏靶向AAV基因递送系统的实用性 心肌梗死后心脏中TTK蛋白激酶和下调Meis1转录因子的作用 然后使用先进的成像技术来量化对左室重构的治疗效果。 这种心脏靶向基因传递系统的发展将为未来的研究奠定基础 努力包括设计和评价心脏靶向纳米粒和脂质体，如 多功能平台，不仅促进缺血性心脏病的分子成像，而且还 靶向递送新型小分子疗法。这项提案采取了多学科的方法，使 生物医学成像的广泛应用，从而跨越了放射学、心脏病学、分子成像和 分子病毒学。联合研究/开发项目将产生：a)新的靶向多肽， 与活体心肌细胞特异性相互作用，b)新的基于AAV的心脏基因传递系统 临床前研究和转化为临床应用的巨大潜力，c)对 一种具有明显转化医学潜力的新型基因治疗的作用机制(S)。

The overarching goal of this proposal is to engineer novel, multifunctional drug and gene delivery systems that can target therapies to particular cells and intracellular compartments and also monitor delivery and determine therapeutic efficacy through the integration of advanced imaging technologies. To pursue this goal, this multi-PI application proposes to leverage existing strengths within UVA including: expertise in using advanced biomedical imaging techniques to assess ischemic heart disease in animal models and re-engineering recombinant adeno-associated vectors (rAAV) (French), screening molecular libraries in vivo to identify specific peptides capable of targeting diseased tissue (Kelly) so that the capsid surface displays specific peptides capable of targeting gene delivery to diseased tissue (Dasa). Not only will the targeting peptides validated during this project be used to develop a novel AAV-based cardiac gene delivery system, future applications include labeling the cardiotropic peptides with fluorophores and/or radiotracers to create contrast agents for the molecular imaging of ischemic heart disease. Similarly, the same cardiotropic peptides may prove valuable in targeting novel therapeutic agents directly to the heart after intravenous (iv) injection. In preliminary studies, the two PIs have already demonstrated the robustness and utility of the interdisciplinary technologies needed to achieve the following specific aims: 1) Validate additional peptides specific for the infarct border zone by evaluating their specificity with in vivo optical imaging and ex vivo confocal microscopy. 2) Optimize the cardiac gene delivery system by grafting cardiotropic peptide ligands into the AAV9 capsid for the selective delivery of expression cassettes driven by a cardiac-specific promoter. 3) Demonstrate the utility of the cardiac-targeted AAV gene delivery system by overexpressing the TTK protein kinase and down-regulating Meis1 transcription factor in the heart after myocardial infarction, then use advanced imaging techniques to quantify the therapeutic effect on LV remodeling. The development of this cardiac-targeted gene delivery system will establish a foundation for future research efforts including the engineering and evaluation of cardiac-targeted nanoparticles and liposomes as multifunctional platforms to facilitate not only the molecular imaging of ischemic heart disease, but also the targeted delivery of novel small molecule therapies. This proposal takes a multidisciplinary approach that makes extensive use of biomedical imaging, thus spanning the fields of radiology, cardiology, molecular imaging and molecular virology. The combined research/development project will result in: a) new targeting peptides that interact specifically with cardiomyocytes in vivo, b) new AAV-based systems for cardiac gene delivery with considerable potential for both pre-clinical research and translation to clinical applications, c) insights into the mechanism(s) of action for a novel gene therapy with clear potential for translational medicine.