Tumor-targeted silencing of Bcl-2/Bcl-xL by the self-assembled siRNA-nanovectors

描述（由申请人提供）：抗凋亡蛋白Bcl-2和Bcl-xL在许多癌症中过表达，并有助于肿瘤的发生、进展和对治疗的抵抗。Bcl-2/Bcl-xL的分子调控是克服当前癌症治疗诱导的细胞凋亡抗性的一种有希望的策略。小干扰RNA （small interfering RNA, siRNA）有效的序列特异性基因沉默已成为癌症研究的有力工具，并具有作为新型癌症分子治疗的巨大潜力。然而，将基于sirna的治疗方法有效且特异性地传递给肿瘤及其转移仍然是一个巨大的挑战。我们已经开发了一种肿瘤特异性、配体靶向、自组装dna纳米载体系统，该系统在将各种基因和反义寡核苷酸靶向递送到人类癌症方面显示出良好的效率和特异性，对正常组织的影响有限（美国专利号：6,749,863）。我们还设计了针对人类Bcl-2和Bcl-xL的sirna，可以有效地敲除Bcl-2/Bcl-xL，导致广泛的癌细胞死亡（美国专利正在申请中）。我们建议使用我们的专利纳米载体系统开发基于sirna的肿瘤靶向沉默Bcl-2/Bcl-xL的治疗方法。我们将验证两个相互关联的假设：(1)肿瘤靶向递送siRNA将有效地沉默Bcl-2/Bcl- xl，并诱导依赖Bcl-2/Bcl- xl生存的人类癌细胞凋亡；(2)抗凋亡基因Bcl-2/Bcl-xL的敲低反过来会克服耐药，恢复癌细胞对化疗/放疗的敏感性。我们的长期目标是开发靶向肿瘤的sirna纳米载体，作为靶向Bcl-2/Bcl- xl的新型分子疗法，用于Bcl-2/Bcl- xl过表达的人类癌症。为了验证我们的假设，我们提出了三个特定的目标：目标1：制备和优化siRNA纳米载体，使siRNA在体外和体内有效地递送到人类肿瘤；目的2：探讨sirna -纳米载体联合化疗/放疗的体外抗肿瘤活性及作用机制；目的3：探讨Bcl-2/Bcl-xL sirna纳米载体在高水平Bcl-2/Bcl-xL人类肿瘤裸鼠异种移植模型中的体内治疗潜力。基于sirna的Bcl-2/Bcl-xL分子治疗与常规治疗相结合，可以提高疗效，克服当前癌症治疗的耐药性，特别是对于Bcl-2/Bcl-xL蛋白过表达、常规治疗效果不佳的肿瘤转移。如果成功实施，我们的研究将提供概念证明，siRNA可以通过自组装的纳米载体传递，用于肿瘤靶向沉默对癌症进展和耐药性至关重要的基因。

DESCRIPTION (provided by applicant): Anti-apoptotic proteins Bcl-2 and Bcl-xL are overexpressed in many cancers and contribute to tumor initiation, progression and resistance to therapy. Molecular modulation of Bcl-2/Bcl-xL represents a promising strategy for overcoming the resistance to apoptosis induced by current cancer therapy. The potent, sequence-specific gene silencing by small interfering RNA (siRNA) has become a powerful tool in cancer research and holds significant potential as novel molecular therapy for cancer. However, delivering the siRNA-based therapeutics efficiently and specifically to tumor and its metastases remains a great challenge. We have developed a tumor-specific, ligand-targeting, self-assembled DNA-nanovector system which shows promising efficiency and specificity in targeted delivery of various genes and anti-sense oligonucleotides to human cancer, with limited effect on normal tissues (US Patent No. 6,749,863). We have also designed siRNAs for human Bcl-2 and Bcl-xL that can potently knock-down Bcl-2/Bcl-xL leading to extensive cancer cell death (US Patent pending). We propose to use our patented nanovector system to develop siRNA-based therapeutics for tumor- targeted silencing of Bcl-2/Bcl-xL. We will test two inter-related hypotheses: (1) Tumor-targeted delivery of siRNA will efficiently silence Bcl-2/Bcl-xL, and induce apoptosis in human cancer cells that depend on Bcl- 2/Bcl-xL for survival; (2) Knock-down of the anti-apoptotic Bcl-2/Bcl-xL in turn will overcome resistance and restore sensitivity of cancer cells to chemo/radiotherapy. Our long-term goal is to develop the tumor- targeting siRNA-nanovectors as novel molecular therapy targeting Bcl-2/Bcl-xL for human cancers with Bcl- 2/Bcl-xL over-expression. To test our hypothesis, we propose to carry out three SPECIFIC AIMS: AIM 1: To prepare and optimize the siRNA-nanovectors for efficient siRNA delivery to human tumors in vitro and in vivo; AIM 2: To investigate in vitro anti-tumor activities and the mechanism of action of siRNA-nanovectors in combination with chemo/radiotherapy; AIM 3: To investigate the in vivo therapeutic potential of Bcl-2/Bcl-xL siRNA-nanovectors in nude mouse xenograft models of human cancers with high levels of Bcl-2/Bcl-xL Combining siRNA-based Bcl-2/Bcl-xL molecular therapy with conventional therapy would improve the efficacy and overcome the resistance to current cancer treatment, especially for tumor metastasis, in which Bcl-2/Bcl-xL protein is overexpressed and for which conventional therapy is not very effective. Successfully carried out, our studies will provide proof-of-concept that siRNA can be delivered by the self-assembled nanovectors for tumor-targeted silencing of the genes critical for cancer progression and resistance.