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首页> 外文学位 >Enhanced cytosolic delivery of antisense oligonucleotides using listeriolysin O-containingpH-sensitive liposomes.
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Enhanced cytosolic delivery of antisense oligonucleotides using listeriolysin O-containingpH-sensitive liposomes.

机译：使用含有李斯特菌溶血素O的pH敏感脂质体增强反义寡核苷酸的胞质递送。

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摘要

Nucleic acid-based drugs such as antisense oligodeoxynucleotides (ODNs) have great potential to be effective therapeutic agents in the treatment of many disease states. However, their success and widespread utilization are limited primarily by the membrane barriers preventing sufficient concentrations of functionally active drugs from accessing the cytosolic site of action. In order for antisense ODN to deliver on the promise as successful therapeutic agents, an effective delivery system must be formulated to circumvent the membrane barriers, thereby allowing the drug to engender a robust antisense effect.; The work in this thesis focuses on the development and characterization of a liposomal-based cytosolic delivery formulation utilizing listeriolysin O (LLO), the endosomolytic hemolysin from Listeria monocytogenes, to mediate the escape of ODNs from the endocytic compartments into the cytosol. It was demonstrated that LLO-containing liposomes were successful in effectively breaching the endosomal barriers enabling sufficient concentrations of ODNs specific for murine intercellular adhesion molecule-1 (ICAM-1) to be transported to the cytosol. The expression of ICAM-1 was efficiently inhibited in a sequence-specific manner at both the protein and mRNA levels. In addition to demonstrating our delivery strategy, the persistence of antisense activity was estimated for the first time by monitoring the recovery of mRNA levels after bolus administration of phosphorothioate ODNs into the cytosol using LLO-containing liposomes.; Furthermore, preliminary groundwork was performed for investigating the mechanism of LLO-induced hemolysis on cholesterol-containing membranes. More studies are required in order to elucidate the mechanism of LLO-induced hemolysis, which will be important in understanding the endosome lysis mechanism of LLO and designing a new strategy for cytosolic delivery of macromolecules. In addition, an allogenic mixed lymphocyte reaction was utilized in order to demonstrate the biological relevance of ICAM-1 down-regulation in bone marrow-derived macrophages. Due to the inability to establish a robust allogenic response in the proposed allogenic model, the assay was not established.
机译：基于核酸的药物，例如反义寡脱氧核苷酸（ODN），在治疗多种疾病中具有巨大的潜力成为有效的治疗剂。然而，它们的成功和广泛使用主要受到膜屏障的限制，所述膜屏障阻止足够浓度的功能活性药物进入胞质作用位点。为了使反义ODN作为成功的治疗剂发挥作用，必须配制有效的传递系统来规避膜屏障，从而使药物产生强大的反义作用。本文的工作重点是利用利斯特溶血素O（LLO）（来自单核细胞增生李斯特菌的内溶溶血素）介导ODN从内吞区室逸出，从而开发和表征基于脂质体的胞质递送制剂。进入细胞质。已经证明，含有LLO的脂质体成功地有效突破了内体屏障，使得能够将足够浓度的对鼠细胞间粘附分子-1（ICAM-1）特异性的ODN转运至细胞质。在蛋白质和mRNA水平，ICAM-1的表达均以序列特异性方式被有效抑制。除了证明我们的递送策略外，还通过监测使用含LLO脂质体的硫代磷酸酯ODNs推注到细胞质中后mRNA水平的恢复来首次估计反义活性的持久性。此外，进行了初步的基础工作，以研究LLO诱导的含胆固醇膜溶血的机制。为了阐明LLO引起的溶血的机制，还需要进行更多的研究，这对于理解LLO的内体裂解机制和设计新的大分子胞质递送策略非常重要。另外，利用同种异体混合淋巴细胞反应来证明ICAM-1在骨髓源性巨噬细胞中下调的生物学相关性。由于无法在提出的同种异体模型中建立强大的同种异体反应，因此未建立该测定法。

著录项

  • 作者

    Mathew, Elizabeth.;

  • 作者单位

    University of Michigan.;

  • 授予单位 University of Michigan.;
  • 学科 Health Sciences Pharmacy.
  • 学位 Ph.D.
  • 年度 2003
  • 页码 189 p.
  • 总页数 189
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 药剂学;
  • 关键词

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