Efficient Cellular Delivery of Oligonucleotides

反义寡核苷酸(AON)是特异性调控基因表达的重要工具。它们在基因“敲除”研究的研究实验室中被广泛应用，并被开发为临床治疗药物，用于广泛的适应症。更广泛地实施这项技术的主要挑战是蜂窝递送。虽然许多聚合物和脂类被用来将寡核苷酸输送到细胞，并取得了不同程度的成功，但它们的效率高度不同，取决于ON的化学和结构、所使用的细胞类型和有无血清等因素。此外，对传递效率的生物物理基础了解甚少，因此传递载体的开发在很大程度上仍然是一个经验过程。 在过去的几年里，我们的团队开创了一种定量的、工程化的寡核苷酸输送方法。我们致力于识别和量化细胞递送的主要障碍，并发展聚合物-寡核苷酸相互作用的生物物理与反义活性的程度和动态之间的关系。我们的工作强调了作为限制性细胞因子的内体逃逸的作用，以及作为控制反义活性的主要分子生物物理因子的聚合物-寡核苷酸复合体的竞争性聚电解质解离。我们发现，pH敏感的聚合物，聚丙基丙烯酸，PPAA，能够与阳离子脂类DOTAP一起介导内体逃逸，并改善AONS的递送。最近，我们扩展了这一概念，创造了新型的接枝共聚物，在血清存在的情况下，改善了反义介导的基因沉默。 我们非常有希望的初步结果是基于聚氧亚烷胺接枝到PPAA上的环氧乙烷/环氧丙烷基团的单一组成。在拟议的工作中，我们将合成更多的聚合物，以深入了解接枝密度、聚羟基亚烷胺和丙烯酸化学对接枝共聚物的物理和生物性能的影响。此外，我们将继续开发这种新型的反义递送系统，以应用于癌症。具体地说，我们将：(1)评估我们的接枝共聚物在有血清和无血清的几种细胞类型中的有效性；(3)在肿瘤异种移植模型中评估我们的递送系统。

Antisense oligonucleotides (AONs) are important tools for the specific modulation of gene expression. They are applied widely in the research laboratory for gene “knockdown” studies, and they are being developed clinically as therapeutics for a wide range of indications. The major challenge to more widespread implementation of this technology is cellular delivery. While many polymers and lipids have been used, with varying degrees of success, to deliver oligonucleotides to cells, their efficiency is highly variable depending on factors such as the chemistry and structure of the ON, the cell type used and the presence/absence of serum. Furthermore, the biophysical basis for delivery efficiency is poorly understood, and so development of delivery vectors remains a largely empirical process. Over the past several years, our group has pioneered a quantitative, engineering approach to oligonucleotide delivery. We have devoted considerable effort to identifying and quantifying the major barriers to cellular delivery and to developing relationships between the biophysics of polymer-oligonucleotide interactions and the extent and dynamics of antisense activity. Our work has highlighted the role of endosomal escape as a limiting cellular factor and competitive polyelectrolyte dissociation of polymer-oligonucleotide complexes as a major molecular biophysical factor governing antisense activity. We have found that the pH-sensitive polymer, poly(propylacrylic acid), PPAA, is able to mediate endosomal escape and improve delivery of AONs in conjunction with the cationic lipid, DOTAP. More recently, we have extended this concept to create novel graft copolymers that improve antisense-mediated gene silencing in the presence of serum. Our very promising Preliminary Results were based on a single composition of ethylene oxide/propylene oxide groups in the poly (oxyalkylene amine) grafted onto PPAA. In the proposed work, we will synthesize additional polymers that will provide insight into the effect of grafting density, poly (oxyalkylene amine) and acrylic acid chemistry on the physical and biological properties of the graft copolymers. Furthermore, we will continue to develop this novel antisense delivery system towards applications in cancer. Specifically, we will: (1) evaluate the effectiveness of our graft copolymers in several cell types with and without serum and (3) evaluate our delivery system in a tumor xenograft model.