Project 3: Using RNAi-based Spherical Nucleic Acid (SNA) Nanoconjugates Targeting Bcl2L12 to Promote Therapy-Induced Apoptosis in Glioblastoma

项目3：总结 胶质母细胞瘤是恶性胶质瘤中最具侵袭性和最常见的表现，其特点是 抵抗现有的治疗方式，并表现出神经衰弱的过程，最终导致死亡， 通常在确诊后14个月内。在改善治疗结果的关键挑战中， GBM患者正在识别和表征克服臭名昭著的治疗方法的新药靶点 对GBM的耐药性，以及针对不可药物的遗传损害的药物输送平台的开发。 恢复P53活性是治疗GBM的一种有吸引力的治疗策略，因为~65%的 原发GBM患者表达野生型，但功能抑制P53。基因的扩增和过表达 非典型的Bcl2家族蛋白Bcl2L12(Bcl2-like-12)通过阻断P53的功能而损害其功能 P53的转录活性。为了抑制Bcl2L12的功能，我们建议使用新的基于RNAi的 纳米偶联物，称为球形核酸(SNAs)，用于中和已建立的GBM中Bcl2L12的表达。 我们发现靶向Bcl2L12的SNAs(siBcl2L12-SNAs)能够穿过细胞膜 包括血脑屏障。我们确定siBcl2L12-SNAs不需要使用有毒的辅助物质 药物通过血-脑/血-肿瘤屏障后在GBM肿瘤细胞内有效积聚 全身注射SNAs后脑内GBM异种移植。SNAs表现出稳定性 生理环境，激发肿瘤内Bcl2L12 mRNA和蛋白的表达下调，从而导致 在GBM患者来源的异种移植(PDX)模型中，P53重新激活和肿瘤生长缓慢。在这里，我们将进一步 SNA治疗Bcl2L12可增强抑瘤作用、减缓肿瘤生长的假说 GBM进展，并可与传统的遗传毒性疗法以及靶向治疗相结合 改善对肿瘤生长的抑制，并可能导致肿瘤消退的治疗方法。在目标1中， 我们将在体外测定siBcl2L12对患者来源的胶质瘤起始细胞(GICs)和PDX的治疗效果。 体内模型，作为单一治疗和联合放射治疗(RT)。在AIM 2中，使用两个PDX 新诊断和复发肿瘤的模型，以及同基因、免疫活性的小鼠模型， 我们将把siBcl2L12与细胞毒性和P53激活的化疗药物，即DNA烷化剂结合起来 替莫唑胺和MDM2抑制剂RG7388。目标3建议进行0期临床试验 SiBcl2L12-SNAs，以确定SNA的药代动力学、生物分布和下调GBM-1的能力 相关的Bcl2L12基因和蛋白。这项提案的结果将提供对 Bcl2L12癌蛋白作为一种可操作的GBM癌蛋白，将为成功实施 作为一种纳入临床实践的治疗方法，SNA介导的多模式P53重新激活。

PROJECT 3: SUMMARY Glioblastoma (GBM), the most aggressive and prevalent manifestation of malignant glioma, is characterized by resistance to extant therapeutic modalities, and exhibit a neurologically debilitating course culminating in death, often within 14 months after diagnosis. Among the critical challenges for improving treatment outcomes for GBM patients are the identification and characterization of new drug targets to overcome the notorious therapy resistance of GBM, and the development of drug delivery platforms to target undruggable genetic lesions. Restoration of p53 activity represents an attractive therapeutic strategy for the treatment of GBM, as ~65% of primary GBM patients express wildtype but functionally suppressed p53. Amplification and overexpression of the atypical Bcl2 family protein Bcl2L12 (Bcl2-Like-12) compromises p53 function by blocking the transcriptional activity of p53. To inhibit Bcl2L12 function, we propose to use novel RNAi-based nanoconjugates, termed Spherical Nucleic Acids (SNAs) to neutralize Bcl2L12 expression in established GBM. We have found that Bcl2L12-targeting SNAs (siBcl2L12-SNAs) are able to traverse cellular membranes including the blood-brain-barrier. We established that siBcl2L12-SNAs do not require the use of toxic auxiliary reagents and accumulate effectively in GBM tumor cells upon crossing the blood-brain/blood-tumor barrier in intracerebral GBM xenografts following systemic administration of the SNAs. SNAs exhibit stability in physiological environments, provoke robust intratumoral Bcl2L12 mRNA and protein knockdown resulting in p53 reactivation, and slow tumor growth in GBM patient derived xenograft (PDX) models. Here, we will further investigate the hypothesis that Bcl2L12 ablation by SNA treatment increases p53 tumor suppression, slows GBM progression, and can be combined with conventional genotoxic therapies as well as with targeted therapeutics for improved suppression of tumor growth, and possibly for causing tumor regression. In Aim 1, we will determine siBcl2L12 treatment effect in patient-derived glioma-initiating cells (GICs) in vitro, and in PDX models in vivo, as monotherapy and in combination with radiation therapy (RT). In Aim 2, using both PDX models for newly diagnosed and recurrent tumor, together with syngeneic, immunocompetent mouse models, we will combine siBcl2L12 with cytotoxic and p53-activating chemotherapeutic drugs, i.e., the DNA alkylator temozolomide and the MDM2 inhibitor RG7388, respectively. Aim 3 proposes a phase 0 clinical trial of siBcl2L12-SNAs, to determine SNA pharmacokinetics, biodistribution, and ability to downregulate GBM- associated Bcl2L12 mRNA and protein. The results of this proposal will provide an in-depth characterization of the Bcl2L12 oncoprotein as an actionable GBM oncoprotein, and will pave the way to successfully implement SNA-mediated, multi-modal p53 reactivation as a therapeutic approach to incorporate in clinical practice.