Novel Oligonucleotide Delivery Vehicles for Gene Tharapy

描述（由申请人提供）：本项目的总体目标是阐明多胺类似物在促进反义寡核苷酸（ODN）摄取和功能方面的作用机制。假设多胺类似物在DNA缩合和/或RNA稳定中的选择性，DNA杂合体可用于促进细胞摄取和功能反义ODNs用于乳腺癌治疗。针对c-myc和HER-2基因的反义ODNs将被研究。为了评价多胺类似物对RNA、DNA杂交稳定性的结构特异性影响，将使用分子信标技术测定缔合常数。ODN纳米颗粒在促进细胞摄取ODN中的作用将通过动态光散射以及电子、扫描力和偏振光谱来量化。将使用MCF-7和SK-BR-3乳腺癌细胞测量ODN的细胞摄取和功能。将使用放射性和荧光ODN测定ODN摄取。将检查多胺类似物/ODN组合对靶基因表达、细胞生长停滞和凋亡的影响，以证明ODN的增强功能。随后，HER-2基因靶向的ODN和选择的多胺类似物将使用乳腺癌的转基因小鼠模型检查体内ODN递送和功能。多胺类似物和ODN对天然多胺的影响将通过HPLC定量。这些研究将促进我们对ODNs细胞摄取机制的认识，并有助于开发乳腺癌治疗的新方法。

DESCRIPTION (provided by applicant): The overall goal of this project is to elucidate the mechanism of action of polyamine analogues in facilitating the uptake and function of antisense oligonucleotides (ODNs). The hypothesis is that the selectivity of polyamine analogues in DNA condensation and/or stabilization of RNA.DNA hybrids can be utilized to facilitate the cellular uptake and function antisense ODNs for breast cancer therapy. Antisense ODNs targeted to c-myc and HER-2 genes will be studied. To evaluate the structural specificity effects of polyamine analogues on RNA.DNA hybrid stabilization, association constants will be determined using a molecular beacon technique. The role of ODN nanoparticles in facilitating cellular uptake of ODNs will be quantified by dynamic light scattering as well as electron, scanning force and polarizing spectroscopy. Cellular uptake and function of ODNs will be measured using MCF-7 and SK-BR-3 breast cancer cells. ODN uptake will be determined using radioactive and fluorescent ODNs. Effects of polyamine analogue/ODN combinations on target gene expression, cell growth arrest, and apoptosis will be examined to demonstrate enhanced function of ODNs. Subsequently, HER-2 gene targeted ODN and selected polyamine analogues will be examined for in vivo ODN delivery and function using a transgenic mouse model of breast cancer. The effects of polyamine analogues and ODNs on natural polyamines will be quantified by HPLC. These studies will advance our knowledge of the mechanism of cellular uptake of ODNs, and help to develop novel approaches for breast cancer therapy.