【作者】 毛成琼；

【导师】 王均；

【作者基本信息】 中国科学技术大学 ， 生物材料， 2013， 博士

【摘要】 癌症是危害人类生命健康的重大疾病之一,放疗和化疗均存在不可忽略的副作用,基于小干扰RNA (siRNA)的核酸类药物为癌症的治疗带来了新的可能。选取合适的靶基因,将针对该基因的siRNA导入肿瘤细胞中,下调其表达,可以安全有效地抑制肿瘤生长。另一方面,由于并不是所有看似“完美”的靶基因都能够通过siRNA直接沉默,当直接干扰其表达存在障碍时,可以绕开目的基因,对其合成致死相关基因进行干扰,在不损伤正常细胞的前提下,达到治疗效果。本论文发展基于纳米胶束的micelleplex载药系统,输运siRNA治疗癌症,探索siRNA药物抗肿瘤治疗方案。在前一部分,论文利用官能化的聚乙二醇单甲醚-聚己内酯-聚磷酸酯三嵌段共聚物(mPEG-b-PCL-b-PPEEA)形成的胶束纳米颗粒,制备micelleplex,携载针对酸性神经酰胺酶的siAC,经系统给药后,将siAC输送至荷瘤小鼠的肿瘤细胞中,显著抑制了乳腺肿瘤的生长,为乳腺癌的治疗提供了新方法。在论文的后一部分,在KRAS突变的非小细胞肺癌肿瘤模型中,绕开了难以直接靶向的KRAS基因,利用KRAS与CDK4基因之间的合成致死效应,使用携载有针对CDK4siRNA (siCDK4)的混合胶束颗粒对荷瘤小鼠进行治疗。结果显示,携载有siCDK4的micelleplex对KRAS突变的肿瘤生长有显著抑制效果,而对正常细胞或KRAS野生型肿瘤的生长影响不明显,从而提供了可用于KRAS突变肿瘤的治疗方案。同时,研究结果提示,基于合成致死效应的RNA干扰治疗手段,不仅可有效杀伤目标肿瘤细胞,而且对正常组织和细胞不造成明显影响。本论文的主要研究内容和结论如下：(1)构建了基于mPEG-b-PCL-b-PPEEA三嵌段两亲性阳离子聚合物的micelleplex,可有效携载siRNA进入乳腺癌细胞BT474中,并在细胞内有效释放siRNA,沉默目的基因表达。当该micelleplex携载针对酸性神经酰胺酶基因的siAC进入BT474细胞后,诱发肿瘤细胞凋亡。在体内研究中,通过系统给药,给原位接种BT474乳腺癌的小鼠注射携载siAC的micelleplex,显著抑制肿瘤生长,研究结果进一步显示上述micelleplex输送siAC,有效抑制了酸性神经酰胺酶在肿瘤的表达。此研究证实了基于RNA干扰的micelleplex用于乳腺癌治疗的有效性和可行性。(2)利用两种嵌段共聚物PCL29-b-PPEEA21和PCL40-b-PEG45制备了混合胶束纳米颗粒,并进一步制备了携载siRNA的micelleplex。研究应用合成致死原理,研究了一种针对KRAS基因突变的肿瘤治疗方案。由于CDK4基因与KRAS基因之间存在合成致死效应,在KRAS基因突变的非小细胞肺癌细胞(NSCLC)中抑制CDK4基因表达可导致肿瘤细胞的凋亡。在体外研究中,我们利用混合胶束纳米颗粒制备的micelleplex携载siCDK4,转染KRAS突变的NSCLC细胞及KRAS野生型的细胞,发现CDK4的表达均被下调。然而,随着CDK4表达的下降,仅KRAS突变的NSCLC细胞增殖受到抑制,KRAS野生型的NSCLC细胞以及正常人肝细胞的生长增殖均影响不明显。同时,构建了小鼠的KRAS突变型以及KRAS野生型的NSCLC细胞皮下植入肿瘤模型,通过尾静脉注射携载有siCDK4的micelleplex进行治疗,发现仅KRAS突变的肿瘤生长被有效抑制,KRAS野生型的肿瘤生长则没有受到显著影响,证实该治疗方案的特异性、高效性和安全性,为开发siRNA药物提供了新的策略。更多还原

【Abstract】 Cancer remains the leading cause of death in the world after heart and infectious diseases. Radiotherapy and chemotherapy are two of the major treatment modalities for cancer, which are accompanied by inevitable side effects. Thus, identifying therapeutics that will kill cancer cells and do not harm patients is the bottleneck, which lies with our inability. Based on the fact that nanoparticulate siRNA delivery system has shown great promise for cancer therapy, the suitable drug target and nanocarrier can be chosen for efficient gene downregulation and tumor growth inhibition. On the other hand, the therapeutics based on RNAi are not omnipotent and a lot of drug targets still undruggable. With a more complete understanding of the complex and extensive network of effectors and regulators, secondary dependencies on genes that are themselves not oncogenes but could lead to vulnerabilities caused by the target genes mutation state can also be developed to provide more efficient and safe therapeutic opportunities.This dissertation aims to exploit safe and efficient treatment against cancer based on siRNA delivery system with micelleplex. The main content and conclusions of this dissertation are summarized as below:1. One of the key challenges in the development of RNA interference-based cancer therapy is the lack of an efficient delivery system for synthetic small interfering RNAs (siRNAs) that would enable efficient uptake by tumor cells and allow for significant knockdown of a target transcriptin vivo. In this part, we have developed a micelleplex system based on an amphiphilic and cationic triblock copolymer, which can systemically deliver siRNA targeting the acid ceramidase (AC) gene for cancer therapy. The triblock copolymer, mPEG-b-PCL-b-PPEEA, can self-assemble into micellar nanoparticles (MNPs) in aqueous solution with an average diameter of60nm and a zeta potential of approximately48mV. The resulting micelleplex, formed by the interaction of MNPs and siRNA, was effectively internalized by BT474breast cancer cells and siRNA was subsequently released, resulting in significant gene knockdown. This effect was demonstrated by significant down-regulation of luciferase expression in BT474-luciferase cells which stably express luciferase, and suppression of AC expression in BT474cells at both the transcriptional and protein level, following delivery of specific siRNAs by the micelleplex. Furthermore, a micelleplex carrying siRNA targeting the AC gene was found to induce remarkable apoptosis and reduce the proliferation of cancer cells. Systemic delivery of micelleplexsiAC significantly inhibited tumor growth in a BT474xenograft murine model, with depressed expression of AC and no positive activation of the innate immune response, suggesting therapeutic promise for micelleplex siRNA delivery in cancer therapy.2. The KRAS mutation is present in approximately20%of lung cancers and has not yet been effectively targeted for therapy. This mutation is associated with a poor prognosis in NSCLC and confers resistance to standard anticancer treatment drugs, including EGFR tyrosine kinase inhibitors. In this part, we have exploited a new therapeutic strategy based on the synthetic lethal interaction between CDK4downregulation and the KRAS mutation to deliver micellar nanoparticles containing siRNA targeting CDK4(MNPsiCDK4) for treatment in NSCLC harboring the oncogenic KRAS mutation. Following MNPSiCDK4administration, CDK4expression was decreased, accompanied by inhibited cell proliferation, specifically in KRAS mutant NSCLC. However, this intervention was harmless to normal KRAS wild-type cells, confirming the proposed mechanism of synthetic lethality. Moreover, systemic delivery of MNPsiCDK4significantly inhibited tumor growth in an A549NSCLC xenograft murine model, with depressed expression of CDK4and mutational KRAS status, suggesting the therapeutic promise of MNPsiCDK4delivery in KRAS mutant NSCLC via a synthetic lethal interaction between KRAS and CDK4. These investigations are promising in terms of exploiting a new therapeutic strategy that is effective and safe in KRAS mutant NSCLC.更多还原