Targeted siRNA nanotechnology for intravesical treatment of urologicaldiseases

靶向 siRNA 纳米技术用于膀胱内治疗泌尿系统疾病

• 批准号: 7832079
• 负责人: ROBERT M WEISS
• 金额: $ 49.86万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7832079
• 关键词: Address; Adherence; Adverse effects; Affect; Angiogenic Factor; Animal Disease Models; Animals; Apoptosis; Apoptosis Inhibitor; Base Pairing; Biomedical Engineering; Bladder; Bladder Neoplasm; Cancer Etiology; Cancer Model; Cancerous; Cell Proliferation; Cells; Collaborations; Complex; Development; Disease; Disease Pathway; Down-Regulation; Drosophila genus; Drug Formulations; Encapsulated; Figs - dietary; Fluorescein; Fluoresceins; Fluorescence; Genes; Goals; Green Fluorescent Proteins; Half-Life; Histone Deacetylase Inhibitor; Histones; Hour; Human; Hyperreflexia; In Vitro; Individual; Inhibitory Concentration 50; Interstitial Cystitis; Intravesical Instillation; Label; Life; Malignant neoplasm of urinary bladder; Mammalian Cell; Materials Testing; Measures; Messenger RNA; Microspheres; Mitomycins; Modeling; Mus; Nanosphere; Nanotechnology; Nitrosamines; Oligonucleotides; Overactive Bladder; Pathogenesis; Peptides; Pharmaceutical Preparations; Play; Polymers; Prevention; Property; Proteins; RNA; RNA Interference; Role; Scanning Electron Microscopy; Site; Small RNA; Specificity; System; Technology; Testing; Therapeutic; Time; Tissues; Toxic effect; Transgenic Mice; Transitional Cell Carcinoma; Treatment Protocols; Tumor Burden; Universities; Urologic Diseases; Urothelial Cell; Urothelium; Vascular Endothelial Growth Factors; Viral; bladder transitional cell carcinoma; cancer cell; common treatment; comparative efficacy; controlled release; cytotoxicity; design; in vivo; inhibitor/antagonist; intravesical; mortality; mouse model; nanoparticle; outcome forecast; overexpression; protein expression; survivin; transcription factor; tumor; tumor growth; tumor xenograft; uptake

DESCRIPTION (provided by applicant): Our Broad Challenge is to develop enabling technologies for the prevention and treatment of diseases affecting the bladder. The Proof of Concept that we are testing is that intravesical instillation of small interference RNA (siRNA) packaged in biodegradable nanospheres provides wide opportunities for the treatment of urologic diseases including transitional cell carcinoma of the bladder (TCC), interstitial cystitis (IC), overactive bladder, and detrusor hyperreflexia. Because of their robust, gene selective silencing of target protein expression, siRNA oligonucleotides are an attractive therapeutic option with high selectivity and specificity and minimal toxicity to neighboring cells. siRNAs, however, have a relatively short half-life and thus we will address the technical challenges of stabilization and intravesical delivery of siRNAs. To achieve this goal, we plan to create and test clinically viable, non-viral nanosphere siRNA complexes that are intravesically instilled for treatment of diseases of the urinary tract. This past year, in collaboration with Drs. W. Mark Saltzman and Kim Woodrow of the Yale University Department of Bioengineering, we have shown that antennapedia (AP), a Drosophila transcription factor that facilitates uptake of peptides and oligonucleotides into mammalian cells, when complexed with PLGA nanoparticles (AP-PLGA) adheres effectively to T-24 bladder cancer cells, and slowly releases complexed siRNA over 10 days in amounts sufficient to downregulate intracellular target mRNA and protein. Conversely, naked survivin and VEGF siRNA are stable for only a few hours. We have complexed two siRNAs with AP-PLGA, survivin siRNA and VEGF siRNA, both with relevance in urological diseases. The Inhibitor of Apoptosis (IAP), survivin, is not detected in normal urothelium and its expression in bladder cancer correlates with poor prognosis. The angiogenic factor, VEGF is overexpressed in TCC and may play an important role in the pathogenesis of TCC and interstitial cystitis. We have shown that these survivin siRNA-AP-PLGA and VEGF siRNA-AP-PLGA are released into the T-24 cells and down-regulate targeted mRNA and protein. Furthermore, VEGF siRNA-AP-PLGA downregulates VEGF levels in normal human urothelium. We now plan to: 1) develop and test a number of nanoparticle controlled release systems containing targeting proteins that will stabilize and deliver siRNAs and drugs, to test their physicochemical properties (materials testing), and the capacity of the nanoparticles to release siRNA efficiently from the nanospheres to an intracellular target site; 2) move from an in vitro to an in vivo system and test in a whole animal the ability of human targeted encapsulated siRNAs to reduce target protein and mRNA and cell/tumor growth; and 3) test whether combinations of encapsulated siRNAs and/or histone deacetylase inhibitors (HDACIs) more effectively reduce tumor burden, the time to onset, rate of occurrence and mortality compared to individual encapsulated siRNAs in a nitrosamine induced bladder cancer model. The ultimate challenge is to design more effective intravesical instillation protocols for treatment of common urological diseases including bladder cancer, overactive bladder and IC using siRNAs encapsulated in microspheres to increase their stability and prolong their efficacy. Standard chemotherapeutic treatment options for urological diseases including bladder cancer, overactive bladder and interstitial cystitis may cause undesirable side effects or may be ineffective. Small interference RNA (siRNA) can specifically and sensitively degrade RNA messages and thus reduce levels of the proteins synthesized from the specific mRNAs that may play a role in disease development. In order to exploit the therapeutic potential of these siRNAs, which are short lived and easily degraded, we have designed strategies to stabilize and test them. We plan to encapsulate the siRNAs in polymer nanoparticles which will release the siRNA over days to weeks. We also will add peptides that will target the nanoparticles to specific cells within the bladder. These siRNA polymers will be instilled into the bladder as a treatment for urological diseases. Because more than one siRNA can be encapsulated and targeted in the same nanoparticle, we can downregulate more than one urologic disease pathway with the same nanoparticles. Our first test of these nanoparticles will use a mouse model of bladder cancer. Thus, we can determine the therapeutic potential of siRNA for treatment of urologic diseases.

描述（由申请人提供）：我们的广泛挑战是开发用于预防和治疗影响膀胱的疾病的支持技术。我们正在测试的概念验证是，将包装在可生物降解的纳米球中的小干扰 RNA (siRNA) 进行膀胱内滴注，为治疗泌尿科疾病提供了广泛的机会，包括膀胱移行细胞癌 (TCC)、间质性膀胱炎 (IC)、膀胱过度活动症和逼尿肌反射亢进。由于其对靶蛋白表达具有强大的基因选择性沉默作用，siRNA 寡核苷酸是一种有吸引力的治疗选择，具有高选择性和特异性，并且对邻近细胞的毒性最小。然而，siRNA 的半衰期相对较短，因此我们将解决 siRNA 的稳定性和膀胱内递送的技术挑战。为了实现这一目标，我们计划创建并测试临床上可行的非病毒纳米球 siRNA 复合物，将其膀胱内滴注用于治疗泌尿道疾病。去年，与博士合作。在耶鲁大学生物工程系的 W. Mark Saltzman 和 Kim Woodrow 的带领下，我们发现，触角足 (AP) 是一种果蝇转录因子，可促进哺乳动物细胞摄取肽和寡核苷酸，当与 PLGA 纳米颗粒 (AP-PLGA) 复合时，它可以有效地粘附在 T-24 膀胱癌细胞上，并在 10 天内缓慢释放复合的 siRNA。 量足以下调细胞内靶 mRNA 和蛋白质。相反，裸露的存活蛋白和 VEGF siRNA 仅稳定几个小时。我们将两种 siRNA 与 AP-PLGA 复合，即生存素 siRNA 和 VEGF siRNA，两者都与泌尿系统疾病相关。细胞凋亡抑制剂 (IAP) 生存素在正常尿路上皮中未检测到，其在膀胱癌中的表达与不良预后相关。血管生成因子VEGF在TCC中过度表达，可能在TCC和间质性膀胱炎的发病机制中发挥重要作用。我们已经证明这些存活蛋白 siRNA-AP-PLGA 和 VEGF siRNA-AP-PLGA 被释放到 T-24 细胞中并下调目标 mRNA 和蛋白质。此外，VEGF siRNA-AP-PLGA 下调正常人尿路上皮中的 VEGF 水平。我们现在计划：1）开发和测试一些含有靶向蛋白的纳米颗粒控释系统，这些靶向蛋白将稳定和递送siRNA和药物，以测试它们的理化性质（材料测试），以及纳米颗粒将siRNA从纳米球有效释放到细胞内靶位点的能力； 2) 从体外系统转向体内系统，并在整个动物中测试人类靶向封装的 siRNA 减少靶蛋白和 mRNA 以及细胞/肿瘤生长的能力； 3) 测试在亚硝胺诱导的膀胱癌模型中，与单独封装的 siRNA 相比，封装的 siRNA 和/或组蛋白脱乙酰酶抑制剂 (HDACIs) 的组合是否更有效地减少肿瘤负荷、发病时间、发生率和死亡率。最终的挑战是设计更有效的膀胱内灌注方案，用于治疗常见泌尿系统疾病，包括膀胱癌、膀胱过度活动症和 IC，使用封装在微球中的 siRNA 来提高其稳定性并延长其疗效。泌尿系统疾病（包括膀胱癌、膀胱过度活动症和间质性膀胱炎）的标准化疗方案可能会导致不良副作用或可能无效。小干扰 RNA (siRNA) 可以特异性、灵敏地降解 RNA 信息，从而降低由可能在疾病发展中发挥作用的特定 mRNA 合成的蛋白质的水平。为了开发这些寿命短且容易降解的 siRNA 的治疗潜力，我们设计了稳定和测试它们的策略。我们计划将 siRNA 封装在聚合物纳米颗粒中，该纳米颗粒将在几天到几周内释放 siRNA。我们还将添加肽，将纳米颗粒靶向膀胱内的特定细胞。这些 siRNA 聚合物将被注入膀胱以治疗泌尿系统疾病。由于可以将不止一种 siRNA 封装并靶向到同一纳米颗粒中，因此我们可以使用相同的纳米颗粒下调不止一种泌尿系统疾病途径。我们对这些纳米颗粒的首次测试将使用膀胱癌小鼠模型。因此，我们可以确定 siRNA 治疗泌尿系统疾病的潜力。