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，并导致肿瘤发生、进展和对治疗的抗性。Bcl-2/Bcl-xL的分子调控代表了克服当前癌症治疗诱导的细胞凋亡抗性的有希望的策略。小分子干扰RNA（small interfering RNA，siRNA）是一种有效的序列特异性基因沉默技术，已成为肿瘤研究的有力工具，并有望成为一种新型的肿瘤分子治疗手段。然而，将基于siRNA的治疗剂有效且特异性地递送至肿瘤及其转移灶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、自组装的DNA-纳米载体系统，其在将各种基因和反义寡核苷酸靶向递送至人类癌症中显示出有希望的效率和特异性，对正常组织的影响有限（美国专利号6，749，863）。我们还设计了用于人Bcl-2和Bcl-xL的siRNA，其可以有效地敲低Bcl-2/Bcl-xL，导致广泛的癌细胞死亡（美国专利申请中）。我们建议使用我们的专利纳米载体系统来开发基于siRNA的治疗剂，用于Bcl-2/Bcl-xL的肿瘤靶向沉默。我们将测试两个相互关联的假设：（1）siRNA的肿瘤靶向递送将有效地沉默Bcl-2/Bcl-xL，并诱导依赖Bcl- 2/Bcl-xL存活的人类癌细胞的凋亡;（2）抗凋亡Bcl-2/Bcl-xL的敲低反过来将克服癌细胞对化疗/放疗的抗性并恢复癌细胞对化疗/放疗的敏感性。我们的长期目标是开发肿瘤靶向siRNA纳米载体，作为针对Bcl-2/Bcl-xL过表达的人类癌症的新型分子疗法。为了验证我们的假设，我们提出了三个具体的目标：目的1：制备和优化siRNA纳米载体，用于体外和体内有效地将siRNA递送到人类肿瘤;目的2：研究siRNA纳米载体与化疗/放疗组合的体外抗肿瘤活性和作用机制;目的3：为了研究Bcl-2/Bcl-xL siRNA纳米载体在具有高水平Bcl-2/Bcl-xL的人癌症的裸鼠异种移植模型中的体内治疗潜力，xL分子疗法与常规疗法将提高疗效并克服对当前癌症治疗的抗性，特别是对于其中Bcl-2/Bcl-xL蛋白过表达且常规疗法不是非常有效的肿瘤转移。成功进行，我们的研究将提供概念验证，即siRNA可以通过自组装纳米载体递送，用于肿瘤靶向沉默对癌症进展和抗性至关重要的基因。

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.