Cancer cell-specific RNA interference in vivo

小干扰RNA （sirna）引起的RNA干扰（RNAi）是体外和体内细胞中基因表达序列特异性控制的常用方法。为每个感兴趣的基因设计sirna的问题可以通过生物信息学在计算机上解决，并在有限数量的体外和体内实验中得到验证。因此，开发基于sirna的药物可能比传统药物更节省时间和成本，并可以进一步促进个性化医疗的发展。最近sirna在体内传递方面的突破使得一些基于sirna的药物（治疗高脂血症、年龄相关性黄斑变性等）的临床试验开始，其中一些已进入晚期（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，sirna的癌细胞特异性传递/激活仍然是一个问题，目前还没有一个简单而通用的解决方案。因此，siRNA药物的靶标数量仅限于在癌细胞中强烈过表达或只存在的基因。癌症特异性前药的应用不仅有望扩大siRNA药物的可能靶点数量，而且有望减少治疗的副作用。在这个项目中，我们将致力于设计一种简单的解决方案，用于设计在活性氧（ROS）存在下被激活的癌症特异性sirna前药。细胞特异性将基于癌细胞过量产生ROS的实验事实，而其在正常细胞中的浓度可以忽略不计。ROS诱导激活是设计癌症特异性sirna的一种潜在的通用方法，因为升高的ROS被认为是癌症表型的关键。因此，建议的sirna前药应该对许多不同类型的癌症有效。作为一项原理证明，针对Ubr连接酶的ros应答sirna前药物将在小鼠模型中用于肝细胞癌（HCC）的治疗和测试。选择这种模式是因为目前还没有令人满意的HCC治疗方法。此外，肝脏特异性递送sirna的方法也很发达。在这个项目中制定的原则，将潜在地适用于设计用于治疗肝脏以外器官癌症的sirna前药物，前提是器官特异性递送将在未来成为可能。本研究利用了(a) Mokhir团队在“笼状”siRNA的合成和细胞应用方面的丰富经验，以及(b) Zatsepin团队在siRNA设计和配方优化以及siRNA偶联物的合成和应用方面的丰富经验，以抑制小鼠模型体内基因表达。

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.