Reversible Phosphorothioate Backbone Modification for Oligonucleotide Delivery and Control

背景：寡核苷酸疗法在基因水平上治疗多种疾病显示出巨大的前景。目前寡核苷酸疗法面临的一个主要问题是递送能力差。寡核苷酸是一种大的、高负电荷的生物分子，生物利用度低，细胞递送效率低。由于它们的高度阴离子性质，这些药物很难与血浆蛋白结合，从而减少了组织分布。此外，由于它们不会自发地穿过细胞膜，内化到细胞内的过程会受到阻碍。众所周知，由于第一代寡核苷酸治疗药物周转快，无法达到足够的细胞内浓度，因此对寡核苷酸的修饰对于改善其药理特性是必不可少的。为了提高寡核苷酸治疗药物的稳定性和递送性，人们采用了大量的修饰，其中最常见的是硫代磷酸骨架。这些修饰允许更好的分布，同时仍然保持所需的活性；然而，组织水平仍然较低，并且经常观察到偏离目标的效果。项目摘要：该项目旨在利用硫代磷酸盐的内在反应性进行与刺激响应的化学基团的化学修饰。然后，可以通过施加刺激来选择性地去除这些组，从而允许对寡核苷酸释放的空间和时间进行控制。认为通过化学修饰掩蔽电荷和提高亲脂性，可以改善药物的分布和细胞穿透的药理性质。此外，通过未修饰的寡核苷酸的刺激响应再生，脱靶效应将被减少。已有研究表明，硫代磷酸对化学基团具有反应性，修饰增加了其亲脂性。然而，目前还没有关于硫代磷的可逆掩蔽及其在生命系统中的应用的报道。为了研究这一点，将合成各种反应性刺激响应性化学基团，并研究它们对硫代磷的反应性。在对这些修饰进行化学评价后，将如上所述对已知的治疗序列进行修饰，并将研究它们进入活细胞的情况和它们的生物活性。这些生物学研究将有助于进一步修改的设计。这些修饰的寡核苷酸随后将被用于细胞疾病模型，以衡量它们对当前寡核苷酸疗法的有效性。该项目是由牛津大学生物和医学合成中心博士培训中心(SBM CDT)的Michael Booth博士团队进行的。该项目属于EPSRC化学生物学和生物化学研究领域。

Background:Oligonucleotide therapeutics show great promise to treat a variety of diseases on a genetic level. One major problem current oligonucleotide therapeutics face is poor delivery. Oligonucleotides are large, highly negatively charged biomolecules with poor bioavailability and ineffective cell delivery. Due to their highly anionic nature, these drugs poorly bind to plasma proteins, which reduces tissue distribution. Additionally, internalisation into cells is hindered, as they do not spontaneously cross cellular membranes. It is well known that modification of oligonucleotides is essential to improve their pharmacological properties, as first generation oligonucleotide therapeutics suffered from fast turnover and the inability to achieve sufficient intracellular concentrations. To improve stability and delivery of oligonucleotide therapeutics, a large number of modifications are employed, the most common of which is the phosphorothioate backbone. These modifications allow for better distribution, while still retaining the desired activity; however, tissue levels are still low and off-target effects are often observed.Project Summary:This project aims at exploiting the inherent reactivity of the phosphorothioate for chemical modification with stimuli-responsive chemical groups. These groups can then be selectively removed by application of the stimulus, allowing for spatial and temporal control of oligonucleotide release. It is believed that through the masking of the charge and the increase in lipophilicity with chemical modification, the pharmacological properties of distribution and cell penetration should be improved. Additionally, through the stimuli-responsive regeneration of the unmodified oligonucleotide, off-target effects will be reduced.It has been shown that the phosphorothioate is reactive towards chemical groups and that modification increases its lipophilicity. However, there are no accounts yet on the reversible masking of phosphorothioates and their applications in living systems. To study this, a variety of reactive stimuli-responsive chemical groups will be synthesised and their reactivity towards phosphorothioates studied. Following evaluation of the chemistry of these modifications, known therapeutic sequences will be modified as above and their delivery into living cells and their biological activity will be investigated. These biological studies will aid in the design of further modifications. These modified oligonucleotides will then be used on cellular disease models to measure their efficacy against current oligonucleotide therapies.This project is undertaken in the group of Dr. Michael Booth within the Synthesis for Biology and Medicine Centre for Doctoral Training (SBM CDT) at the University of Oxford. The project falls within the EPSRC chemical biology and biological chemistry research area.