CPE Peptide-Based Nanoparticles for the Diagnosis and Therapy of Chemotherapy Res

描述（申请人提供）：卵巢癌仍然是妇科肿瘤中死亡率最高的癌症。虽然许多卵巢癌患者对手术和化疗的标准组合完全有反应，但近90%的患者后来发展为复发性化疗耐药癌症，并不可避免地死于疾病。因此，开发针对复发性/化疗耐药性卵巢癌的创新有效疗法仍然是改善公共卫生的高度优先事项。使用高通量技术分析卵巢癌的遗传指纹，我们最近发现了编码蛋白claudin-3和claudin-4的基因的极高表达。因为密蛋白-3和-4是产气荚膜梭菌肠毒素（CPE）的上皮受体，并且足以介导CPE结合，所以我们假设使用可生物降解的纳米颗粒如聚（乳酸-共-乙醇酸，即，包封与CPE结合结构域复合的治疗剂以靶向卵巢癌细胞的PLGA-NP是治疗化疗抗性卵巢癌的新型、潜在高效的治疗方法。与该观点一致，初步的体外数据清楚地显示，荧光素（FITC）标记的-C-CPE有效地结合并快速内化在化疗抗性原代OSPC细胞系中，表明C-CPE能够结合药物并且还能够使药物跨细胞肿瘤膜易位。更重要的是，初步的体内结果表明，CPE-PLGA-NP包封的荧光染料（香豆素）选择性地在化疗耐药的卵巢肿瘤中体内蓄积，在RES器官中具有最小的非特异性蓄积，并且仅在各种其他器官的正常细胞中具有背景结合。1）表征与不同长度的CPE肽复合的荧光PLGA-NP包封模型和治疗药物（即，30 aa、17 aa和9aa），并在针对原发性化疗抗性卵巢癌细胞系的多个体外测定中评估此类NP的结合活性和治疗功效，2）在原发性化疗抗性卵巢癌的临床相关动物模型中检查包封与125 I标记的C-CPE肽复合的香豆素的PLGA-NP的体内分布和药代动力学，以及3）检测PLGA-NP包封的编码白喉毒素A自杀蛋白的质粒DNA在卵巢癌中高度和优先活性的基因的启动子序列控制下的治疗潜力（即，间皮素和HE 4/WFDC 2），在原发性化疗抗性卵巢癌的临床相关动物模型中与CPE肽体内复合。该项目完成后，我们将能够迅速将这项研究转化为针对化疗耐药疾病患者的新型高效治疗方法。 公共卫生相关性：我们的研究小组最近发现，在最致命的妇科恶性肿瘤卵巢癌中，编码蛋白claudin-3和claudin-4的基因表达极高。因为claudin-3和claudin-4是产气荚膜梭菌肠毒素（CPE）的上皮受体，并且足以介导CPE结合，所以在所提出的研究中，我们计划评估可生物降解的聚合物纳米颗粒如聚（乳酸-共-乙醇酸，即，PLGA NP）包封编码白喉毒素A自杀蛋白的质粒DNA，所述白喉毒素A自杀蛋白在卵巢癌中高度和优先活性的基因的启动子序列的控制下（即，间皮素和HE 4/WFDC 2）与CPE的结合结构域复合，作为治疗化疗抗性卵巢癌的新型、潜在高效的治疗方法。该项目完成后，我们将能够迅速将这项研究转化为针对化疗耐药疾病患者的创新和高度靶向治疗。

DESCRIPTION (provided by applicant): Ovarian carcinoma remains the cancer with the highest mortality rate among gynecological tumors. Although many ovarian cancer patients fully respond to the standard combination of surgery and chemotherapy, nearly 90% later develop recurrent chemotherapy-resistant cancer and inevitably succumb to their disease. Thus, development of innovative, effective therapies against recurrent/chemotherapy-resistant ovarian cancer remains a high priority for improving public health. Using high-throughput technologies to analyze genetic fingerprints of ovarian cancer, we have recently discovered extremely high expression of the genes encoding the proteins claudin-3 and claudin-4. Because claudin-3 and -4 are the epithelial receptors for Clostridium perfringens enterotoxin (CPE), and are sufficient to mediate CPE binding, we hypothesize that using biodegradable nanoparticles such as poly (lactic-co-glycolic acid, i.e., PLGA-NP) encapsulating therapeutic agents complexed to the binding domain of CPE, to target ovarian cancer cells, is a novel, potentially highly effective therapeutic approach to treat chemotherapy-resistant ovarian cancer. Consistent with this view, preliminary in vitro data clearly showed that fluorescein (FITC)-labeled-C-CPE effectively binds and rapidly internalizes in chemotherapy-resistant primary OSPC cell lines, suggesting that C- CPE is capable to bind and also of translocating drugs across cell tumor membrane. More importantly, preliminary in vivo results showed that CPE-PLGA-NP encapsulating a fluorescent dye (coumarin) selectively accumulate in vivo in chemotherapy resistant ovarian tumors with minimal nonspecific accumulation in RES organs and only background binding in normal cells of various other organs. Accordingly, this proposal has three related specific aims: 1) Characterize fluorescent PLGA-NP encapsulating model and therapeutic drugs, complexed to CPE peptides of different lengths (i.e., 30aa, 17aa and 9 aa), and evaluate binding activity and therapeutic efficacy of such NP in multiple in vitro assays against primary chemotherapy resistant ovarian carcinoma cell lines, 2) Examine the distribution and pharmacokinetics of PLGA-NP encapsulating coumarin complexed to 125I labeled C-CPE peptide in vivo in clinically relevant animal models of primary chemotherapy resistant ovarian carcinoma and 3) Examine the therapeutic potential of PLGA-NP encapsulating plasmid DNA encoding Diptheria Toxin A suicide protein under the control of promoter sequences of genes highly and preferentially active in ovarian cancer (i.e., mesothelin and HE4/WFDC2), complexed to CPE peptide in vivo in clinically relevant animal models of primary chemotherapy resistant ovarian carcinoma. Upon completion of this project, we will be positioned to quickly translate this research into a novel, highly effective therapeutic treatment for patients with chemotherapy-resistant disease. PUBLIC HEALTH RELEVANCE: Our research group has recently discovered extremely high expression of the genes encoding the proteins claudin-3 and claudin-4 in ovarian cancer, the most lethal gynecologic malignancy. Because claudin-3 and -4 are the epithelial receptors for Clostridium perfringens enterotoxin (CPE), and are sufficient to mediate CPE binding, in the proposed research we plan to evaluate the use of biodegradable polymeric nanoparticles such as poly(lactic-co-glycolic acid, i.e., PLGA NP) encapsulating plasmid DNA encoding Diptheria Toxin A suicide protein under the control of promoter sequences of genes highly and preferentially active in ovarian cancer (i.e., mesothelin and HE4/WFDC2), complexed to the binding domain of CPE, as a novel, potentially highly effective therapeutic approach to treat chemotherapy-resistant ovarian cancer. Upon completion of this project, we will be positioned to quickly translate this research into an innovative and highly targeted therapeutic treatment for patients with chemotherapy-resistant disease.