Systemic RNA interference to reactivate p53 tumor suppression

多形性胶质母细胞瘤（GBM）是恶性胶质瘤中最具侵袭性和最常见的表现， 以抵抗现存的治疗方式为特征，并表现出神经衰弱过程 通常在确诊后14个月内死亡。预后如此糟糕，几乎100%失败 随着GBM药物开发速度的加快，胶质瘤领域面临的关键挑战是识别和表征 新的药物靶点，以克服臭名昭著的治疗耐药性GBM，并开发药物输送平台 来治疗不可治愈的基因损伤p53活性的恢复代表了一种有吸引力的治疗策略 对于GBM的治疗，约65%的原发性GBM患者表达功能缺陷的野生型p53。 非典型Bcl 2家族蛋白Bcl 2L 12（Bcl 2-Like-12）的扩增和过表达损害p53 通过阻断p53的转录活性发挥作用。为了抑制Bcl 2L 12功能，我们建议使用新的 基于RNAi的纳米缀合物，称为球形核酸（SNA），以中和Bcl 2L 12在细胞中的表达。 建立了神经胶质瘤我们已经发现，Bcl 2L 12靶向SNA（siBcl 2L 12-SNA）能够穿过细胞， 膜，包括血脑屏障。我们确定siBcl 2L 12-SNA不需要使用 有毒的辅助试剂，并在细胞中和血脑/血肿瘤交叉时有效积累 在脑内胶质瘤中的屏障。它们在生理环境中表现出稳定性， 引起强有力的肿瘤内Bcl 2L 12 mRNA和蛋白敲低以及p53再活化，并减少肿瘤 GBM PDX模型中的负荷。将SNA平台确立为适用的p53激活治疗方式 对于其他高度恶性和致命的实体癌的治疗，我们选择皮肤黑色素瘤作为治疗的一种方法。 第二种癌症类型，用于评估siBcl 2L 12-SNA。与GBM相似，皮肤黑色素瘤是 其特征是罕见的p53突变和Bcl 2L 12表达升高，这与肿瘤的程度有关。 黑色素瘤的抗药性和进展。在这里，我们将测试Bcl 2L 12消融的假设， 高活性SNA缀合物增加p53肿瘤抑制，减少GBM和黑素瘤进展， 因此代表了一种新的、广泛适用的治疗策略，用于激活固体中的野生型p53， 癌的在目标1中，我们将通过以下方法确定p53再激活的机制并鉴定其替代标记物： siBcl2L12-SNA。在目标2中，我们将优化SNA表面化学以优化siBcl 2L 12的递送。 针对GBM和黑素瘤肿瘤的寡核苷酸。目的3将在基因工程中评估siBcl 2L 12-SNA 黑色素瘤（Aim 3a）和GBM小鼠模型（Aim 3b），作为单一疗法，以及与DNA 烷化剂替莫唑胺（Aim 3c）。这项建议的结果将提供一个深入的特点 Bcl 2L 12癌蛋白在细胞和生物水平，并将铺平道路，成功地实现多- 模式p53再激活作为治疗进入临床实践。

Glioblastoma multiforme (GBM), the most aggressive and prevalent manifestation of malignant glioma, are characterized by resistance to extant therapeutic modalities, and exhibit a neurologically debilitating course culminating in death often within 14 months after diagnosis. With this dismal prognosis and a near 100% failure rate of GBM drug development, the critical challenges facing the glioma field are to identify and characterize new drug targets to overcome the notorious therapy resistance of GBM, and to develop 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 functionally defective wildtype 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 glioma. 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 cells and upon crossing of the blood-brain/blood-tumor barrier in intracerebral gliomas upon systemic delivery. They exhibit stability in physiological environments, provoke robust intratumoral Bcl2L12 mRNA and protein knockdown and p53 reactivation, and reduce tumor burden in GBM PDX models. To establish the SNA platform as a p53 activating therapeutic modality applicable to the treatment of other highly malignant and lethal solid cancers, we elected cutaneous melanoma as a second cancer type for the evaluation of siBcl2L12-SNAs. Similar to GBM, cutaneous melanoma are characterized by infrequent p53 mutation and elevated Bcl2L12 expression, which correlates with the degree of melanoma drug resistance and progression. Here, we will test the hypothesis that Bcl2L12 ablation by a high activity SNA conjugate increases p53 tumor suppression, reduces GBM and melanoma progression, and thus represent a novel, broadly applicable therapeutic strategy for the activation of wild-type p53 in solid cancers. In Aim 1, we will determine the mechanism and identify surrogate markers of p53 reactivation by siBcl2L12-SNAs. In Aim 2, we will optimize SNA surface chemistry for optimized delivery of siBcl2L12 oligonucleotides to GBM and melanoma tumors. Aim 3 will evaluate siBcl2L12-SNAs in genetically engineered melanoma (Aim 3a) and GBM mouse models (Aim 3b), as monotherapies, and in combination with the DNA alkylator temozolomide (Aim 3c). The results of this proposal will provide an in-depth characterization of the Bcl2L12 oncoprotein at cellular and biological levels, and will pave the way to successfully implement multi- modal p53 reactivation as therapy into clinical practice.