Cancer cell-specific RNA interference in vivo

由小干扰RNA(SiRNAs)引起的RNA干扰(RNAi)是一种在体外和体内对细胞中基因表达进行序列特异性控制的通用方法。针对每个感兴趣的基因的siRNAs设计问题可以通过生物信息学在电子计算机中解决，并在有限的体外和体内实验中得到验证。因此，基于siRNA的药物的开发可能比经典药物耗时更少、成本更高，并可以进一步促进个性化药物的发展。最近在体内传递siRNAs的突破使得以siRNA为基础的药物开始了一些临床试验(治疗高脂血症、老年性黄斑变性等)，其中一些达到了晚期(如TTR淀粉样变性)。然而，尽管在该领域取得了这些重要的成功，但癌细胞特异性传递/激活siRNAs仍然是一个问题，对于这个问题，还没有简单和通用的解决方案。因此，siRNA药物的靶点数量仅限于基因，这些基因要么强烈过度表达，要么仅存在于癌细胞中。癌症特异性前体药物的应用不仅有望扩大siRNA药物的可能靶点数量，而且还将减少治疗的副作用。在这个项目中，我们将致力于设计一种简单的解决方案，用于设计癌症特异的siRNA前体药物，这些药物在活性氧物种(ROS)存在的情况下被激活。细胞特异性将基于这样一个实验事实，即癌细胞过度产生ROS，而正常细胞中的ROS浓度可以忽略不计。ROS诱导的激活是设计癌症特异性siRNAs的一种潜在的通用方法，因为ROS的升高被认为对癌症表型至关重要。因此，建议的siRNA前体药物应该对许多不同的癌症类型具有活性。作为一项原则证明，针对UBR连接酶的ROS响应性siRNA前药将被开发出来，并在小鼠模型中用于肝细胞癌的治疗。之所以选择这种模式，是因为目前尚无令人满意的肝癌治疗方法。此外，肝脏特异性递送siRNA的方法也得到了很好的发展。该项目制定的原则可能适用于siRNA前药的设计，用于治疗肝脏以外的器官癌症，前提是将来可以提供器官特异性给药。这项研究利用了(A)Mokhir小组在“笼式”siRNA合成和细胞应用方面的丰富经验，以及(B)Zatsepin小组在siRNA设计和配方优化以及基于siRNA的偶联物的合成和应用以抑制小鼠体内基因表达方面的丰富经验。

RNA interference (RNAi) caused by small interfering RNAs (siRNAs) is a general method of sequence-specific control of gene expression in cells, both in vitro and in vivo. A problem of design of siRNAs for every gene of interest can be solved in silico by bioinformatics and validated in a limited number of experiments in vitro and in vivo. Therefore, development of siRNA-based drugs can potentially be less time-consuming and costly than that of classical drugs and can further promote development of personalized medicine. Recent breakthrough in delivery of siRNAs in vivo enabled the initiation of a number of clinical trials with siRNA-based drugs (treatment of hyperlipidemia, age related macular degeneration, etc.), some of which reached advanced stages (e.g. TTR amyloidosis). However, despite these important successes in the field, cancer-cell specific delivery/activation of siRNAs still remains a problem, for which no simple and general solution is known. Therefore, the number of targets for siRNA drugs is limited to genes, which are either strongly overexpressed or present exclusively in cancer cells. Application of cancer-specific prodrugs is expected to not only extend the number of possible targets for siRNA drug, but also reduce side effects of the treatment. In this project we will work on a simple solution for designing cancer specific siRNA-prodrugs, which are activated in the presence of reactive oxygen species (ROS). The cell specificity will be based on the experimental fact that cancer cells overproduce ROS, whereas their concentration in normal cells is negligible. ROS-induced activation is a potential general approach for the design of cancer specific siRNAs, since elevated ROS is believed to be crucial for cancer phenotypes. Therefore, the proposed siRNA-prodrugs should be active against many different cancer types. As a proof-of-principle, ROS-responsive siRNA-prodrugs targeting Ubr ligases will be developed and tested in the treatment of hepatocellular carcinoma (HCC) in mice models. This model was selected since no satisfactory treatment for HCC is currently known. Moreover, methods of liver specific delivery of siRNAs are well developed. The principles, worked out in this project, will be potentially applicable towards the design of siRNA-prodrugs for the treatment of cancers in organs other than the liver, providing that organ-specific delivery will become available in the future. This study capitalizes on the extensive experience of (a) the group of Mokhir in synthesis and cellular applications of “caged” siRNAs and (b) the group of Zatsepin on optimization of siRNA design and formulation as well synthesis and application of siRNA-based conjugates for inhibition of gene expression in vivo in mice models.