Overcoming Temozolomide Resistance using MGMT-targeting Spherical Nucleic Acids

描述(申请人提供)：多形性胶质母细胞瘤(GBM)是最常见的原发中枢神经系统恶性肿瘤。由于这些肿瘤的侵袭性和我们无法对其进行适当的治疗，只有3-5%的患者在确诊后存活超过3年。新诊断的GBM的治疗标准是手术切除，然后辅以放射治疗和替莫唑胺(TMZ)化疗。TMZ的细胞毒性主要是通过鸟嘌呤的O6位甲基化来实现的。在大多数患者中，这种甲基被酶O6-甲基鸟嘌呤-DNA甲基转移酶(MGMT)迅速清除，从而对化疗产生抗药性。然而，在一组GBM患者中，在他们的肿瘤发展过程中的某个时候，MGMT的启动子区域甲基化。这种对MGMT活性的表观遗传沉默使TMZ能够诱导胶质母细胞瘤细胞凋亡，并显著提高GBM患者的存活率。具有甲基化启动子区的患者的两年存活率(49%)远远高于没有MGMT启动子区甲基化的患者(15%)。此外，在罕见的GBM长期幸存者中，74%的患者被发现存在MGMT启动子区甲基化。该项目旨在抑制MGMT在GBM细胞中的表达，并随后应用TMZ来概括在MGMT启动子区甲基化的患者中观察到的改善的生存表型。我们将利用紧密结合在金纳米颗粒(MGMTi-球形核酸)表面的小干扰RNA(SiRNA)双链。这些微粒具有独特的特性，与其他基因转染剂相比具有优势，包括(1)不依赖于辅助转染剂或脂质体的同时转染和基因调控，(2)包括神经元在内的所有类型细胞的快速内化，(3)优越的稳定性，包括对核酸酶降解的抵抗，(4)在动物模型中最小限度地激活固有免疫反应，且在高剂量下没有急性毒性，(5)能够跨越血脑屏障(BBB)和血肿瘤屏障(BTB)，穿透异种移植、颅内肿瘤、GBM癌基因沉默和提高小鼠的存活率。我们将使用患者来源的肿瘤神经球(TNS)进行体外和体内实验；对于体内实验，我们将从TNS培养物中产生颅内异种移植。在进行体内实验之前，我们将在我们的TNS培养中建立MGMT、TMZ和细胞死亡之间的关系。MGMTi-SNAs对MGMT基因敲除的影响将通过RT-qPCR、蛋白质印迹和MGMT修复实验进行评估。细胞凋亡标志物的定量包括：膜联蛋白V阳性、线粒体膜完整性、半胱氨酸天冬氨酸氨基转移酶活性和蛋白水解性切割的评估。用原位GBM小鼠模型进行的活体研究将评估MGMTi-SNAs和TMZ的联合治疗；我们打算(1)促进肿瘤内的凋亡，(2)减少胶质瘤的形成，(3)延长生存时间。

DESCRIPTION (provided by applicant): Glioblastoma multiforme (GBM) is the most prevalent primary central nervous system malignancy. Due to the aggressive nature of these tumors and our inability to adequately treat them, only 3-5% of patients survive longer than 3 years post-diagnosis. The standard of care for newly diagnosed GBM is surgical resection followed by adjuvant radiotherapy and temozolomide (TMZ) chemotherapy. TMZ cytotoxicity is mediated primarily through methylation of the O6-position of guanine. In the majority of patients, this methyl group is rapidly removed by the enzyme O6-methylguanine-DNA methyltransferase (MGMT), conferring resistance to the chemotherapy. However, in a subset of GBM patients, sometime during the course of their tumor development the promoter region for MGMT is methylated. This epigenetic silencing of MGMT activity allows TMZ to induce apoptosis in glioblastoma cells and drastically increases survival in GBM patients. Patients with a methylated promoter region have a much higher two-year survival rate (49%) than patients without a methylated MGMT promoter region (15%). Furthermore, in the rare long-term survivors of GBM, 74% of patients were found to have a methylated MGMT promoter region. This project seeks to knockdown MGMT expression in GBM cells and subsequently administer TMZ to recapitulate the improved survival phenotype observed in patients with a methylated MGMT promoter region. We will utilize small interfering RNA (siRNA) duplexes densely conjugated to the surface of gold nanoparticles (MGMTi-Spherical Nucleic Acids (SNAs)). These particles possess unique characteristics that confer advantages over other gene transfection reagents, including (1) simultaneous transfection and gene regulation independent of auxiliary transfection agents or lipoplexes, (2) rapid internalization by all cell types including neurons, (3) superior stability i physiological environments including resistance to nuclease degradation, (4) minimal activation of the innate immune response and no acute toxicity at high doses in animal models, and (5) capacity to cross the blood-brain barrier (BBB) and blood-tumor barrier (BTB), penetrate xenografted, intracranial tumors, silence GBM oncogenes, and increase survival in mice. We will use patient-derived tumor neurospheres (TNS) for in vitro and in vivo experiments; for in vivo experiments, we will generate intracranial xenografts from TNS cultures. Prior to running in vivo experiments, we will establish a relationship between MGMT, TMZ, and cell death in our TNS cultures. MGMT knockdown by MGMTi-SNAs will be assessed with RT-qPCR, western blot and an MGMT repair assay. Quantification of apoptotic markers will include: annexin V positivity, mitochondrial membrane integrity, caspase activity, and assessment of proteolytic cleavage. In vivo studies with orthotopic GBM mouse models will assess the combination treatment of MGMTi-SNAs and TMZ; we intend to (1) enhance intratumoral apoptosis, (2) reduce glioma formation, and (3) increase survival time.