Efficient Cellular Delivery of Oligonucleotides

寡核苷酸的高效细胞递送

• 批准号: 7654447
• 负责人: CHARLES M. ROTH
• 金额: $ 34.26万
• 依托单位: RUTGERS, THE STATE UNIV OF N.J.
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7654447
• 关键词: Acids; Advanced Development; Adverse effects; Amines; Animals; Antisense Oligonucleotides; Area; Biodistribution; Biological; Biological Assay; Biophysics; Breast Cancer Cell; Cell Communication; Cell Culture Techniques; Cells; Chemistry; Complex; Development; Disease model; Dissociation; Effectiveness; Engineering; Equipment; Ethylene Oxide; Evaluation; Family; Gene Expression; Gene Silencing; Genes; Image; Injection of therapeutic agent; Investigation; Label; Laboratories; Laboratory Research; Lead; Ligands; Lipids; Malignant Neoplasms; Mediating; Molecular; Molecular Weight; Mus; Oligonucleotides; Peripheral; Polymers; Process; Property; RGD (sequence); Resources; Role; STAT3 gene; Screening procedure; Serum; Structure; System; Tail; Technology; Therapeutic; Time; Titrations; Variant; Veins; Vertebral column; Work; Xenograft Model; acrylic acid; base; cell type; copolymer; density; design; gene function; human disease; improved; in vivo; insight; neoplastic cell; next generation; novel; physical property; polyterephthalic acid anhydride; prevent; propylene oxide; success; targeted delivery; tool; tumor; tumor xenograft; vector

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.

反义寡核苷酸是特异性调控基因表达的重要工具。它们被广泛应用于基因“敲除”研究的研究实验室中，并且它们正在临床上被开发为用于广泛适应症的治疗剂。更广泛地实施这项技术的主要挑战是蜂窝传送。虽然许多聚合物和脂质已被用于将寡核苷酸递送至细胞，并具有不同程度的成功，但它们的效率取决于诸如ON的化学和结构、所用的细胞类型和血清的存在/不存在等因素而高度可变。此外，传递效率的生物物理基础知之甚少，因此传递载体的开发仍然是一个主要的经验过程。 在过去的几年里，我们的团队开创了一种定量的寡核苷酸递送工程方法。我们已经投入了相当大的努力，以确定和量化的主要障碍，细胞交付和发展之间的关系的聚合物-寡核苷酸相互作用的生物物理学和反义活性的程度和动态。我们的工作突出了内体逃逸作为限制性细胞因子和聚合物-寡核苷酸复合物的竞争性解离作为控制反义活性的主要分子生物物理因子的作用。我们已经发现，pH敏感性聚合物，聚（丙基丙烯酸），PPAA，能够介导内体逃逸，并与阳离子脂质，DOTAP一起改善AON的递送。最近，我们已经扩展了这一概念，以创建新的接枝共聚物，提高反义介导的基因沉默在血清的存在下。 我们非常有希望的初步结果是基于接枝到PPAA上的聚（氧化烯胺）中的环氧乙烷/环氧丙烷基团的单一组合物。在拟议的工作中，我们将合成额外的聚合物，这将提供深入了解接枝密度，聚（氧化烯胺）和丙烯酸化学的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传递系统，以应用于癌症。具体而言，我们将：（1）评价我们的移植共聚物在有血清和无血清的几种细胞类型中的有效性和（3）评价我们的递送系统在肿瘤异种移植模型中的有效性。