Targeted siRNA nanotechnology for intravesical treatment of urologicaldiseases

描述(由申请人提供)：我们的广泛挑战是开发使能技术来预防和治疗影响膀胱的疾病。我们正在测试的概念证据是，将包装在可生物降解纳米球中的小干扰RNA(SiRNA)注入膀胱内，为治疗包括膀胱移行细胞癌(TCC)、间质性膀胱炎(IC)、膀胱过度活动和逼尿肌反射亢进在内的泌尿系统疾病提供了广泛的机会。由于siRNA寡核苷酸对靶蛋白表达的基因选择性沉默，具有高选择性和特异性，对邻近细胞的毒性最小，是一种有吸引力的治疗选择。然而，siRNAs的半衰期相对较短，因此我们将解决siRNAs的稳定和膀胱内递送的技术挑战。为了实现这一目标，我们计划创造和测试临床上可行的、非病毒纳米球siRNA复合体，这些复合体可以通过膀胱内注入治疗尿路疾病。在过去的一年里，我们与耶鲁大学生物工程系的W.Mark Saltzman博士和Kim Woodrow博士合作，证明了触角蛋白(AP)是一种促进多肽和寡核苷酸进入哺乳动物细胞的转录因子，当与PLGA纳米颗粒(AP-PLGA)复合时，它有效地附着在T-24膀胱癌细胞上，并在10天内缓慢释放复杂的siRNA，数量足以下调细胞内的靶mRNA和蛋白质。相反，裸露的Survivin和VEGFsiRNA只能稳定几个小时。我们已经将两个siRNA与AP-PLGA、Survivin siRNA和VEGFsiRNA复合，两者都与泌尿系统疾病相关。凋亡抑制因子Survivin在正常尿路上皮细胞中不表达，其在膀胱癌中的表达与预后不良有关。血管生成因子血管内皮生长因子在TCC中高表达，可能在TCC和间质性膀胱炎的发病机制中起重要作用。我们已经证明，这些Survivin siRNA-AP-PLGA和VEGFsiRNA-AP-PLGA可以释放到T-24细胞中，并下调靶基因和蛋白的表达。此外，VEGFsiRNA-AP-PLGA下调正常人尿路上皮中的VEGF值。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制释放系统，以稳定和传递siRNA和药物，测试它们的物理化学性质(材料测试)，以及纳米颗粒将siRNA有效地从纳米球释放到细胞内靶点的能力；2)从体外系统转移到体内系统，并在整个动物中测试人类靶向包裹的siRNA减少靶蛋白和mRNA以及细胞/肿瘤生长的能力；3)在亚硝胺诱导的膀胱癌模型中，测试微囊化siRNAs和/或组蛋白脱乙酰酶抑制剂(HDACIs)的组合是否比单独微囊化siRNAs更有效地降低肿瘤负担、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，利用包裹在微球中的siRNA来治疗常见的泌尿系统疾病，包括膀胱癌、膀胱过度活动症和IC，以增加其稳定性和延长其疗效。对膀胱癌、膀胱过度活跃和间质性膀胱炎等泌尿系统疾病的标准化疗方案可能会导致不良副作用或无效。小干扰RNA(SiRNA)可以特异而敏感地降解RNA信息，从而降低由特定mRNAs合成的蛋白质水平，这些蛋白质可能在疾病的发展中发挥作用。为了挖掘这些短命且容易降解的siRNAs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包裹在聚合物纳米颗粒中，这将在几天到几周内释放siRNA。我们还将添加多肽，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中，作为治疗泌尿系统疾病的药物。由于同一纳米颗粒中可以包裹和靶向不止一种siRNA，所以我们可以用相同的纳米颗粒下调不止一种泌尿系统疾病的通路。我们对这些纳米颗粒的第一次测试将使用膀胱癌的小鼠模型。因此，我们可以确定siRNA在治疗泌尿系统疾病方面的治疗潜力。

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.