Targeted Polymer-Adenovirus Hybrids for Ablation of Bladder Cancer Disease

描述（由申请人提供）：拟议的研究描述了用于晚期膀胱癌疾病靶向消融的基于腺病毒的基因递送载体的开发。膀胱癌是泌尿生殖道第二常见的癌症，2008年有超过68，000例新发病例和14，000例死亡。在美国，大多数膀胱癌是移行细胞癌（TCC），尽管鳞状细胞癌（SCC）和腺癌也会发生。浅表病变可通过经尿道切除术切除，但由于残留病变复发（>80%）和进展（~50%）的风险较高，因此患者需接受额外的膀胱内治疗。长期随访显示，最初有浅表病变的患者中有三分之一死于膀胱癌。根治性膀胱切除术是治疗复发性浅表癌或肌层浸润性膀胱癌的唯一选择。已经侵入肌肉壁之外的疾病，通过淋巴系统扩散，形成肝、肺或骨的转移。晚期肿瘤的多模式治疗导致5年生存率仅为20-40%。因此，需要有效的治疗策略，旨在减少浅表性膀胱癌的复发和进展，以及改善晚期疾病患者的治疗结果。 使用腺病毒的基因治疗是用于递送外源基因的强有力的方法，所述外源基因转录毒性蛋白，导致癌细胞的消融。虽然这是一种有吸引力的方法，但晚期膀胱癌细胞中柯萨奇-腺病毒受体（CAR）的低表达导致癌细胞而不是正常肝细胞的不良转导，这导致全身递送的腺病毒的肝毒性。此外，腺病毒的免疫原性也是令人担忧的原因。因此，研究新的基因治疗方法对膀胱癌的治疗具有重要意义。 我们提出了一种基于聚合物-抗体免疫缀合物或PIC和腺病毒（Ad）的混合系统，用于膀胱癌细胞的靶向消融。新型阳离子聚合物将与抗EGFR抗体缀合，以靶向表皮生长因子受体（EGFR），其在晚期膀胱癌细胞上过表达。PIC将与Ad-GFP和Ad-TRAIL病毒复合，并且将使用膀胱癌细胞系评估所得PIC-腺病毒杂合体（PAH）的体外转导功效。捕获晚期膀胱癌表型的细胞系将用于拟定的研究。基于AdGFP的PAH将用于确定感染性（使用GFP表达）。将研究基于AdTRAIL的多环芳烃在表达TRAIL后的凋亡诱导功效，TRAIL已显示出对诱导癌细胞凋亡的选择性。从这些体外实验中选择的三种PIC将用于膀胱癌的原位动物模型中，以研究PAH的生物分布和抗肿瘤功效。预计所提出的策略将导致识别用于膀胱癌疾病的安全、有效和靶向消融的新型疗法。 公共卫生相关性：该研究的总体目标是开发高效的基于腺病毒的基因递送方法，用于晚期膀胱癌疾病的消融。在美国，晚期膀胱癌疾病每年造成超过68，000例新发病例和14，000例死亡。我们假设，聚合物-免疫缀合物（PIC）可以采用，以选择性地针对恶性膀胱组织，并克服与目前现有的腺病毒癌症基因治疗系统的局限性。

DESCRIPTION (provided by applicant): The proposed research describes the development of adenovirus-based gene delivery vectors for the targeted ablation of advanced bladder cancer disease. Bladder cancer is the second most common cancer of the urogenital tract with over 68,000 new cases and 14,000 deaths in 2008. The majority of bladder cancers in the US are transitional cell carcinomas (TCC), although squamous cell carcinomas (SCC) and adenocarcinomas also occur. Superficial lesions are removed surgically by transurethral resection but since residual lesions have a high risk of recurrence (>80%) and progression (~50%), patients receive additional intravesical therapy. Long-term follow up shows that one-third of the patients who initially had superficial lesions die of bladder cancer. Radical cystectomy is the only therapeutic choice for recurrent superficial cancer or muscle invasive bladder cancer. Disease that has invaded beyond the muscle wall, spreads via the lymphatic system forming metastases in liver, lung or bone. Multimodal therapy of advanced tumors results in only 20-40% survival at 5 years. Thus, effective treatment strategies aimed at reducing the recurrence and progression of superficial bladder cancer, as well as improving therapeutic outcome in patients with advanced disease, are needed. Gene therapy using adenoviruses is a powerful approach for delivering exogenous genes that transcribe toxic proteins leading to the ablation of cancer cells. While this is an attractive approach, low expression of the Coxsackie-Adenovirus receptor (CAR) in advanced bladder cancer cells results in poor transduction of cancer cells but not normal hepatocytes, which results in liver toxicity of systemically delivered adenoviruses. In addition, immunogenicity of adenoviruses is a cause for concern. It is necessary to develop novel approaches for efficacious gene therapy of bladder cancer disease. We propose a hybrid system based on polymer-antibody immunoconjugates or PICs, and adenoviruses (Ad) for the targeted ablation of bladder cancer cells. Novel cationic polymers will be conjugated to anti-EGFR antibodies in order to target the Epidermal Growth Factor Receptor (EGFR), which is over-expressed on advanced bladder cancer cells. PICs will be complexed with Ad-GFP and Ad-TRAIL viruses and the in vitro transduction efficacy of the resulting PIC-adenovirus hybrids (PAHs) will be evaluated using bladder cancer cell lines. Cell lines that capture the advanced bladder cancer phenotype will be employed in the proposed studies. AdGFP-based PAHs will be used to determine infectivity (using GFP expression). AdTRAIL-based PAHs will be investigated for their apoptosis-inducing efficacy following the expression of TRAIL, which has been shown to demonstrate selectivity for inducing apoptosis in cancer cells. Three PICs selected from these in vitro experiments will be employed in an orthotopic animal model of bladder cancer to study the biodistribution and anti-tumor efficacy of the PAH. It is anticipated that the proposed strategy will result in the identification of novel therapeutics for the safe, effective, and targeted ablation of bladder cancer disease. PUBLIC HEALTH RELEVANCE: The overall aim of the proposed research is to develop highly effective adenovirus-based gene delivery methods for the ablation of advanced bladder cancer disease. Advanced bladder cancer disease accounts for over 68,000 new cases and 14,000 deaths every year in the United States. We hypothesize that polymer-immunoconjugates (PICs) can be employed in order to selectively target malignant bladder tissue and overcome the limitations associated with currently existing adenoviral cancer gene therapy systems.