Cancer-specific nanoparticle-mediated gene therapy to treat hepatocellular carcinoma

 描述(申请人提供)：建议工作的目标是创建一个新的基因传递平台来成像和杀死人类肝癌，特别是肝细胞癌(HCC)。目前的治疗标准采用化疗，化疗通常通过动脉途径进行，以加强对肿瘤组织的治疗定位。然而，经动脉化疗仅被证明对姑息治疗有效，而且经常会导致周围健康的肝组织受损，从而导致肝功能衰竭和死亡。我们建议通过设计一种新的药物输送系统来解决这一问题，该系统可以在增强肿瘤杀伤力的同时减少对健康细胞的损害。我们将运送编码单纯疱疹病毒胸苷激酶(HSVtk)的DNA，并利用HSVtk将前药更昔洛韦(GCV)转化为细胞杀伤药物。HSVtk DNA将使用聚合物纳米颗粒输送，我们将对其进行工程设计，以选择性地将DNA输送到肝癌，从而实现癌症特异性细胞杀伤。我们将通过设计新的质粒启动子来限制HSVtk在肝癌细胞中的表达，从而进一步提高肝癌的选择性。我们将使用人肝癌的小鼠原位模型，通过动脉途径给药这些纳米颗粒，以模拟目前在肝癌治疗中采用的微创外科技术。HSVtk还将核苷类似物(放射性示踪剂)磷酸化，使它们通过PET成像可见，这将使我们能够在体内对肿瘤进行成像和监测。在前期工作的基础上，我们假设我们将能够设计出纳米颗粒和质粒，从而限制对人肝癌的细胞杀伤，同时避免对人肝细胞的非靶向影响。我们假设，这种传递系统将能够延长在原位人类肝癌小鼠模型中的存活时间，同时能够在体内进行肿瘤成像。在特定的目标1中，我们将展示通过纳米颗粒向9个人肝癌细胞系和3个人肝细胞系的体外比较的癌症特异性基因传递，并进一步在小鼠原位人类肝细胞癌模型中检验这种选择性。此外，我们将研究这些纳米颗粒显示癌症选择性的机制，这可能有助于阐明未来如何进一步改进癌症靶向纳米颗粒。在特定的目标2中，我们将设计一种转录启动子，使肝癌特异性基因能够表达，并使用纳米颗粒递送该质粒，以在体外和体内证明增强的癌症特异性。在具体目标3中，我们将展示HSVtk/GCV细胞杀伤系统在体内外的有效性和肿瘤选择性，此外，我们还将使用HSVtk和放射性示踪剂在小鼠原位肿瘤模型中对人肝癌进行成像。这项研究的最终目标是创造一种针对癌症的、无创的、微创的治疗方法 治疗人类肝细胞癌。

 DESCRIPTION (provided by applicant): The goal of the proposed work is to create a novel gene delivery platform to image and kill human liver cancer, specifically hepatocellular carcinoma (HCC). Current standard of care treatments employ chemotherapy, which is often delivered transarterially to enhance localization of treatment to the tumor tissue. However, transarterial chemotherapy has only proven effective for palliative care, and often results in damage to surrounding healthy liver tissue which can lead to liver failure and death. We propose to address this concern by designing a novel drug delivery system that could enhance tumor killing while reducing damage to healthy cells. We will deliver DNA encoding herpes simplex virus thymidine kinase (HSVtk), and use HSVtk to convert the prodrug ganciclovir (GCV) into a cell-killing drug. HSVtk DNA will be delivered using polymeric nanoparticles which we will engineer to selectively deliver DNA to HCC, thus enabling cancer specific cell killing. We will further promote HCC selectivity by designing novel plasmid promoters that restrict HSVtk expression to HCC cells. We will administer these nanoparticles transarterially using a mouse orthotopic model of human HCC to mimic the minimally invasive surgical technique currently employed in HCC therapy. HSVtk also phosphorylates nucleoside analogs (radiotracers), making them visible via PET imaging, which will enable us to image and monitor the tumor in vivo. Based on preliminary work, we hypothesize that we will be able to engineer both nanoparticles and plasmids that restrict cell killing to human HCC while avoiding off-target effects to human hepatocytes. We hypothesize that this delivery system will enable prolonged survival in an orthotopic human HCC mouse model while simultaneously enabling tumor imaging in vivo. In Specific Aim 1 we will demonstrate cancer- specific gene delivery via nanoparticles to nine human HCC lines as compared to three human hepatocyte cell lines in vitro, and further examine this selectivity in a mouse orthotopic human HCC model. Additionally, we will examine the mechanism by which these nanoparticles exhibit cancer selectivity, which may help to elucidate how cancer-targeting nanoparticles can be improved even further in the future. In Specific Aim 2, we will design a transcriptional promoter that enables HCC-specific gene expression, and deliver this plasmid using nanoparticles to demonstrate enhanced cancer specificity both in vitro and in vivo. In Specific Aim 3, we will demonstrate the efficacy and cancer selectivity of the HSVtk/GCV cell killing system in vitro and in vivo, in addition to using HSVtk and a radiotracer to image human HCC in a mouse orthotopic tumor model. The ultimate goal of this research is to create a cancer-specific, theranostic, and minimally invasive treatment for human HCC.