Tumor targeted RNAi by novel nanovectors for molecular therapy of prostate cancer

描述(申请人提供)：通过小干扰RNA(SiRNA)有效的、序列特异性的基因沉默已成为癌症研究的强大工具，并具有作为癌症新的分子治疗方法的巨大潜力。然而，将基于siRNA的治疗有效且特异地用于前列腺癌及其转移仍然是一个巨大的挑战。我们开发了一种肿瘤特异的、配体靶向的、自组装的DNA纳米载体系统，用于前列腺癌基因的传递。纳米载体在靶向运送各种基因和反义寡核苷酸方面表现出良好的效率和特异性，而对正常组织的作用有限(美国专利号6,749,863)。这种纳米载体系统目前正处于非病毒p53基因治疗的第一阶段临床试验。这一试点项目提案的目的是探索使用我们的专利纳米导向器系统用于肿瘤靶向递送基于siRNA的治疗人类前列腺癌的可行性。在我们的初步研究中，我们设计了针对人Bcl2和Bclxl的siRNAs，它可以有效地击倒Bcl2/Bclxl高达95%，导致广泛的癌细胞死亡(美国专利申请中)。我们建议利用Bcl2/Bclxl siRNA来检验两个相互关联的假说：(1)自组装的纳米载体可以选择性地将siRNA输送到前列腺癌，并有效地沉默靶基因Bcl2/Bclxl；(2)敲除抗凋亡的Bcl2/Bclxl将诱导依赖Bcl2/Bclxl生存的人前列腺癌细胞发生凋亡，从而克服耐药性，恢复前列腺癌细胞对化疗/放疗的敏感性。我们的长期目标是开发肿瘤靶向的siRNA纳米载体，作为前列腺癌的新型分子治疗方法。为了验证我们的假设，我们提出了两个特定的目标：目的1：制备和优化高效的体内外RNA干扰人前列腺癌的纳米载体；目的2：研究Bcl2/BclxL siRNA纳米载体在高表达Bcl2/BclxL的人前列腺癌裸鼠移植瘤模型中的抗肿瘤活性和靶向性。抗凋亡蛋白Bcl2和Bclxl在大多数前列腺癌中过表达，与前列腺癌的发生、发展和耐药有关。Bcl2/Bclxl的分子调控是克服当前肿瘤治疗诱导的细胞凋亡抵抗的一种有前途的策略。将siRNA为基础的分子治疗与常规治疗相结合，可提高疗效并克服目前肿瘤治疗方法的耐药性，特别是对肿瘤转移瘤，即Bcl2/Bclxl蛋白过度表达且常规治疗效果不佳的肿瘤。成功开展后，我们的研究将提供概念证明，即siRNA可以通过自组装的纳米载体传递，用于肿瘤靶向的RNA干扰前列腺癌进展和耐药的关键基因。将肿瘤靶向的Bcl2/Bclxl RNA干扰与常规治疗相结合，可提高疗效并克服目前肿瘤治疗方法的耐药性，特别是对Bcl2/Bclxl蛋白过度表达且常规治疗效果不佳的转移瘤。成功开展后，我们的研究将提供概念证明，即siRNA可以通过自组装的纳米载体传递，用于肿瘤靶向沉默前列腺癌进展和耐药的关键基因。纳米载体体内肿瘤靶向RNA干扰的成功将对基于siRNA的前列腺癌各种分子靶点的新疗法的发展产生重大影响。

DESCRIPTION (provided by applicant): 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 prostate cancer and its metastases remains a great challenge. We have developed a tumor-specific, ligand-targeting, self-assembled DNA-nanovector system for prostate cancer gene delivery. The nanovectors show promising efficiency and specificity in targeted delivery of various genes and anti-sense oligonucleotides to human prostate cancer, with limited effect on normal tissues (US Patent No. 6,749,863). This nanovector system is now in Phase I clinical trial for non-viral p53 gene therapy. The objective of this pilot project proposal is to explore the feasibility of using our patented nanovector system for tumor-targeted delivery of siRNA-based therapeutics for human prostate cancer. In our preliminary studies, we have designed siRNAs for human Bcl-2 and Bcl-xL that can potently knock-down Bcl-2/Bcl-xL up to 95%, leading to extensive cancer cell death (US Patent pending). We propose to use Bcl-2/Bcl-xL siRNA to test two inter-related hypotheses: (1) the self-assembled nanovectors can selectively deliver siRNA to prostate cancer and efficiently silence the target Bcl-2/Bcl-xL; (2); Knock-down of the anti-apoptotic Bcl-2/Bcl-xL will induce apoptosis in human prostate cancer cells that depend on Bcl-2/Bcl-xL for survival, thus overcome resistance and restore sensitivity of prostate cancer cells to chemo/radiotherapy. Our long-term goal is to develop the tumor-targeting siRNA-nanovectors as novel molecular therapy for prostate cancer. To test our hypothesis, we propose to carry out two SPECIFIC AIMS: AIM 1: To prepare and optimize the nanovectors for efficient RNA interference for human prostate cancer in vitro and in vivo; AIM 2: To investigate anti-tumor activities and target validation of Bcl-2/Bcl-xL siRNA-nanovectors in nude mouse xenograft models of human prostate cancer with high levels of Bcl-2/Bcl-xL. Anti-apoptotic proteins Bcl-2 and Bcl-xL are overexpressed in most of prostate cancer and contribute to prostate 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. Combining siRNA-based 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 study will provide proof-of-concept that siRNA can be delivered by the self-assembled nanovectors for tumor-targeted RNA interference of the genes critical for prostate cancer progression and resistance. Combining tumor-targeted RNA interference of Bcl-2/Bcl-xL with conventional therapy would improve the efficacy and overcome the drug resistance to current cancer treatment, especially for 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 prostate tumor progression and resistance. The success of tumor-targeted RNA interference by nanovectors in vivo will have a major impact on the development of siRNA-based novel therapeutics for various molecular targets of human prostate cancer.