Overcoming Temozolomide Resistance using MGMT-targeting Spherical Nucleic Acids

描述（由申请方提供）：多形性胶质母细胞瘤（GBM）是最常见的原发性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充分治疗它们，只有3-5%的患者在诊断后存活超过3年。新诊断GBM的标准治疗是手术切除，然后进行辅助放疗和替莫唑胺（TMZ）化疗。TMZ的细胞毒性主要通过鸟嘌呤O 6位的甲基化介导。在大多数患者中，这种甲基基团被酶O 6-甲基鸟嘌呤-DNA甲基转移酶（MGMT）迅速去除，从而对化疗产生耐药性。然而，在GBM患者的子集中，在其肿瘤发展过程中的某个时候，MGMT的启动子区域被甲基化。这种MGMT活性的表观遗传沉默允许TMZ诱导胶质母细胞瘤细胞的凋亡并显著增加GBM患者的存活率。具有甲基化启动子区的患者的两年生存率（49%）比没有甲基化MGMT启动子区的患者（15%）高得多。此外，在罕见的GBM长期存活者中，发现74%的患者具有甲基化的MGMT启动子区域。 该项目旨在敲低GBM细胞中的MGMT表达，随后给予TMZ以重现在具有甲基化MGMT启动子区的患者中观察到的改善的存活表型。我们将利用紧密结合到金纳米颗粒（MGMTi-球形核酸（SNA））表面的小干扰RNA（siRNA）双链体。这些颗粒具有独特的特性，赋予其优于其它基因转染试剂的优点，包括（1）不依赖于辅助转染剂或脂质复合物的同时转染和基因调节，（2）被包括神经元在内的所有细胞类型快速内化，（3）在生理环境中的上级稳定性，包括对核酸酶降解的抗性，（4）在动物模型中先天免疫应答的最小激活和在高剂量下无急性毒性，和（5）穿过血脑屏障（BBB）和血肿瘤屏障（BTB）、穿透异种移植的颅内肿瘤、沉默GBM癌基因和增加小鼠存活的能力。我们将使用患者源性肿瘤神经球（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物。在进行体内实验之前，我们将在我们的TNS培养物中建立MGMT、TMZ和细胞死亡之间的关系。将使用RT-qPCR、蛋白质印迹和MGMT修复测定来评估MGMTi-SNA的MGMT敲低。凋亡标志物的定量将包括：膜联蛋白V阳性、线粒体膜完整性、半胱天冬酶活性和蛋白水解裂解的评估。原位GBM小鼠模型的体内研究将评估MGMTi-SNA和TMZ的联合治疗;我们打算（1）增强肿瘤内细胞凋亡，（2）减少胶质瘤形成，和（3）增加存活时间。

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.