Targeted Nanoparticle DNA Therapy for Ovarian Cancer

描述（由申请人提供）：对于晚期卵巢癌的更有效的治疗有明确和立即的需求。为了满足这一需求，我们的长期目标是开发一种用于晚期转移性卵巢癌的纳米疗法，有效地控制癌症，从而延长健康寿命。所提出的实验的目的是引入一种有前途的新型阳离子、可生物降解的聚（2-氨基酯）聚合物的改性，其导致DNA向卵巢肿瘤细胞的有效的、靶向的纳米颗粒递送。所提出的实验测试了这样的假设，即腹膜内施用旨在将编码所谓自杀基因的DNA靶向递送到卵巢癌细胞的聚合物纳米颗粒将有效地杀死细胞，导致卵巢肿瘤消退。我们将以三种方式修饰聚合物，旨在增强其将DNA靶向递送至卵巢癌细胞的能力，并提高其对血清灭活的抵抗力。一种方法将是聚乙二醇化聚合物并将其缀合至两个单链可变抗体片段（scFv）之一，所述单链可变抗体片段（scFv）对卵巢肿瘤细胞表面上发现的蛋白质或叶酸具有反应性。第二种方法是将DNA聚（2-氨基酯）纳米颗粒与抗坏血酸抗体或叶酸缀合的聚（丙烯酸）复合。第三种方法是对聚（2-氨基酯）的结构进行组合末端修饰。我们将使用基于细胞的高通量转染测定来鉴定赋予最高程度的血清抗性和DNA递送的改性聚合物。然后，我们将在三种体内小鼠模型中测试所选聚合物制剂的有效性：卵巢癌的转基因模型、同基因小鼠/肿瘤细胞模型和人肿瘤细胞异种移植模型。除了靶向DNA递送到卵巢癌细胞，我们的目标是使用卵巢特异性启动子序列靶向基因表达。我们将使用非侵入性成像技术（生物发光和microCT）来纵向评估肿瘤对纳米颗粒递送DT-A治疗的反应。此外，我们将确定这种疗法对寿命的影响，并确定这种疗法是否会导致非特异性毒性。目前还没有有效的治疗晚期卵巢癌患者。我们的期望是，创新性地使用纳米颗粒将自杀基因靶向递送到卵巢癌细胞，结合靶向这些细胞的基因表达策略，将导致这种致命疾病的有效治疗。虽然我们在这里专注于卵巢癌新疗法的开发，但这项研究将有助于建立纳米颗粒在基因治疗中的效用，并为其在治疗其他类型的癌症和其他疾病方面的更广泛应用铺平道路。 项目叙述：聚（2-氨基酯）是阳离子的、可生物降解的聚合物，其作为非病毒载体用于将治疗性DNA递送至癌细胞显示出巨大的前景。目前，对于对初始治疗无反应或复发性疾病的晚期卵巢癌患者没有有效的治疗方法。本研究的目的是引入对聚（2-氨基酯）的修饰，其导致在腹膜内施用后将编码自杀基因的DNA高效、靶向纳米颗粒递送至卵巢肿瘤细胞。靶向纳米治疗，单独使用或作为辅助治疗，应该会为转移性卵巢癌患者带来更有效的治疗。

DESCRIPTION (provided by applicant): There is a clear and immediate need for a more effective therapy for advanced ovarian cancer. To address this need, our long-term goal is to develop a nanotherapy for advanced-stage metastatic ovarian cancer that effectively manages the cancer, resulting in a longer, healthy life. The objective of the proposed experiments is to introduce modifications to a promising new class of cationic, biodegradable poly(2-amino ester) polymers that result in efficient, targeted nanoparticle-delivery of DNA to ovarian tumor cells. The proposed experiments test the hypothesis that intraperitoneal administration of polymeric nanoparticles designed to target the delivery of DNA encoding so-called suicide genes to ovarian cancer cells will effectively kill the cells, resulting in ovarian tumor regression. We will modify polymers in three ways, aiming to enhance their ability to target DNA delivery to ovarian cancer cells and to improve their resistance to inactivation by serum. One approach will be to pegylate polymers and conjugate them to one of two single chain variable antibody fragments (scFvs) having reactivity to proteins found on the surface of ovarian tumor cells, or to folic acid. The second approach will be to complex DNA poly(2-amino ester)-nanoparticles with scFv- or folic acid-conjugated poly(acrylic acid). The third approach will be to make combinatorial end-modifications to the structure of poly(2-amino ester)s. We will use a high-throughput cell-based transfection assay to identify the modified polymers imparting the highest degree of serum resistance and DNA delivery. We will then test the effectiveness of selected polymer formulations in three in vivo mouse models: a transgenic model for ovarian cancer, a sygeneic mouse/tumor cell model, and a human tumor cell xenograft model. In addition to targeting DNA delivery to ovarian cancer cells, we aim to target gene expression using ovarian-specific promoter sequences. We will use non-invasive imaging technologies (bioluminescence and microCT) to assess longitudinally the response of tumors to the nanoparticle-delivers DT-A therapy. In addition, we will determine the effect of this therapy on life span and determine whether the therapy results in non-specific toxicity. There are currently no effective therapies for advanced-stage ovarian cancer patients. Our expectation is that the innovative use of nanoparticles to target delivery of suicide genes to ovarian cancer cells, combined with a strategy for targeting gene expression to these cells, will lead to an effective treatment for this deadly disease. While we focus here on the development of a new therapy for ovarian cancer, this study will help establish the utility of nanoparticles for gene therapy and pave the way for their broader application for treating additional types of cancer and other diseases. Project Narrative: Poly(2-amino ester)s is cationic, biodegradable polymers that show great promise as non-viral vectors for the delivery of therapeutic DNA to cancer cells. There are currently no effective therapies for advanced-stage ovarian cancer patients that either do not respond to initial therapy or those with recurrent disease. The purpose of this study is to introduce modifications to poly(2-amino ester)s that result in efficient, targeted nanoparticle-delivery of DNA encoding suicide genes to ovarian tumor cells following intraperitoneal administration. Targeted nanotherapy, used alone or as an adjuvant therapy, should lead to a more effective treatment for patients with metastatic ovarian cancer.