Overcoming Temozolomide Resistance using MGMT-targeting Spherical Nucleic Acids

使用 MGMT 靶向球形核酸克服替莫唑胺耐药性

• 批准号: 8788811
• 负责人: Timothy L Sita
• 金额: $ 4.81万
• 依托单位: NORTHWESTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-01-01 至 2015-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8788811
• 关键词: Actins; Acute; Adjuvant Radiotherapy; Alkylating Agents; Alkylating Antineoplastic Agents; Animal Model; Apoptosis; Apoptotic; Biological Assay; Blood; Blood - brain barrier anatomy; Brain Neoplasms; Caspase; Cell Death; Cell Survival; Cell model; Cells; Cessation of life; Characteristics; DNA Damage; DNA Repair; Development; Diagnosis; Dose; Enrollment; Environment; Enzymes; Epigenetic Process; Excision; Gene Expression Regulation; Gene Silencing; Genes; Glioblastoma; Glioma; Goals; Gold; Guanine; Health; Hour; Immune response; In Vitro; Intracranial Neoplasms; Lamins; Life; Long-Term Survivors; MGMT gene; Malignant - descriptor; Malignant Neoplasms; Mediating; Methylation; Mus; Nature; Neuraxis; Neurons; Newly Diagnosed; Oligonucleotides; Oncogenes; Operative Surgical Procedures; Oral; Patients; Phenotype; Physiological; Poly(ADP-ribose) Polymerases; Polymerase Chain Reaction; Positioning Attribute; Predisposition; Preparation; Prognostic Factor; Promoter Regions; Proteins; Radiation therapy; Reagent; Regimen; Resistance; Running; Small Interfering RNA; Spherical Nucleic Acids; Surface; Survival Rate; Testing; Time; Tissues; Toxic effect; Transfection; Tumor-Derived; Western Blotting; Xenograft procedure; annexin A5; base; cell type; chemotherapeutic agent; chemotherapy; cytotoxicity; experience; improved; in vivo; killings; mRNA Expression; methyl group; mitochondrial membrane; mouse model; nanoparticle; neoplastic cell; nuclease; particle; pre-clinical; prevent; promoter; repaired; research study; response; standard of care; temozolomide; therapy resistant; tumor; tumor xenograft; validation studies

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.

描述（由申请人提供）：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以及我们无法充分治疗它们，只有 3-5% 的患者在诊断后存活时间超过 3 年。新诊断 GBM 的护理标准是手术切除，然后进行辅助放疗和替莫唑胺 (TMZ) 化疗。 TMZ 细胞毒性主要通过鸟嘌呤 O6 位甲基化介导。在大多数患者中，该甲基会被 O6-甲基鸟嘌呤-DNA 甲基转移酶 (MGMT) 快速去除，从而产生对化疗的耐药性。然而，在部分 GBM 患者中，在肿瘤发展过程中的某个时候，MGMT 启动子区域会被甲基化。这种 MGMT 活性的表观遗传沉默使得 TMZ 能够诱导胶质母细胞瘤细胞凋亡，并大大提高 GBM 患者的生存率。具有甲基化启动子区域的患者的两年生存率 (49%) 比没有甲基化 MGMT 启动子区域的患者 (15%) 高得多。此外，在罕见的 GBM 长期幸存者中，74% 的患者被发现具有甲基化的 MGMT 启动子区域。 该项目旨在敲低 GBM 细胞中的 MGMT 表达，然后施用 TMZ 以重现在具有甲基化 MGMT 启动子区域的患者中观察到的生存表型改善。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，与其他基因转染试剂相比具有优势，包括（1）同时转染和基因调控，不依赖于辅助转染剂或脂质复合物，（2）被包括神经元在内的所有细胞类型快速内化，（3）在生理学中具有优异的稳定性环境，包括对核酸酶降解的抵抗力，（4）先天免疫反应的最小激活，并且在动物模型中高剂量时没有急性毒性，以及（5）穿过血脑屏障（BBB）和血肿瘤屏障的能力(BTB)，穿透异种移植的颅内肿瘤，沉默 GBM 癌基因，并提高小鼠的存活率。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物。在进行体内实验之前，我们将在 TNS 培养物中建立 MGMT、TMZ 和细胞死亡之间的关系。 MGMTi-SNA 造成的 MGMT 敲低将通过 RT-qPCR、蛋白质印迹和 MGMT 修复测定进行评估。细胞凋亡标记物的定量包括：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切割的评估。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）增强瘤内细胞凋亡，（2）减少神经胶质瘤形成，（3）增加生存时间。