Delivery of Oligonucleotides to Hepatocytes In Vivo

描述(申请人提供)：反义疗法在治疗各种各样的人类传染病和遗传病方面有着巨大的前景。这些疗法的基础是特定序列对特定基因表达的抑制。由于反义药物的高选择性，与传统药物相比，反义药物有可能引起更少的副作用和更少的毒性。此外，由于反义药物通过与靶RNA分子中的互补序列结合来发挥作用，因此设计反义疗法来特异性地抑制特定基因是非常简单的。 阻碍反义药物有效使用的一个主要因素是这些分子在体内以低效率的功能输送到细胞。Mirus公司的研究人员已经开发出新的非病毒颗粒技术，已被证明在体内将质粒DNA输送到肝细胞方面非常有效。我们在第一阶段研究中提出的研究的一个主要目标是确定这种颗粒技术是否可以被用来将一类新的、高效的反义药物--吗啉寡核苷酸(PMO)--运送到体内的肝细胞。作为这一目标的一部分，我们表征了含有PMO的颗粒的配方，并研究了它们在体内的循环和摄取。通过这些研究，我们成功地制备并鉴定了体内肝细胞显著摄取的颗粒的特征。我们第一阶段研究的另一个完成目标是开发一种检测方法，以评估反义吗啉寡核苷酸在体内抑制内源性肝基因表达的能力。除了在我们的第一阶段研究计划中特别提到的努力外，我们还开发了一种新的方法来实现内溶，即释放细胞质中的内体内容物。这一突破意义重大，因为内化后内化的PMO被包裹在内吞体内是成功传递寡核苷酸的主要障碍。作为我们方法的内溶活性的演示，我们已经展示了生物活性PMO的功能性递送到组织培养中的细胞，这些PMO被复合在一个颗粒中。拟议的第二阶段研究将结合我们在低聚物颗粒配方、肝细胞递送颗粒和使用内溶剂方面的专业知识，开发能够以生物活性形式将PMO有效地体内递送到肝细胞的颗粒。

DESCRIPTION (provided by applicant): Antisense therapies hold tremendous promise for treating a wide variety of human infectious and genetic diseases. These therapies are based on the sequence-specific inhibition of expression of specific genes. Because of their high selectivity, antisense agents have the potential to elicit fewer side effects and display less toxicity compared to traditional drugs. In addition, because antisense agents exert their effects by binding to a complementary sequence in a target RNA molecule, designing antisense therapeutics to specifically inhibit a particular gene is extremely straightforward. A major factor hindering the effective use of antisense agents is the low efficiency with which these molecules are functionally delivered to cells in vivo. Researchers at Mirus Corporation have developed novel, non-viral particle technologies that have been shown to be highly effective at delivering plasmid DNA to hepatocytes in vivo. A major goal of the research proposed in our Phase I study was to determine whether this particle technology could be utilized to deliver a new, highly effective class of antisense agents, morpholino oligonucleotides (PMO), to hepatocytes in vivo. As part of this goal, we characterized the formulation of PMO-containing particles and studied their circulation and uptake in vivo. From these studies, we successfully prepared and identified characteristics of particles with significant uptake by hepatocytes in vivo. Another completed Aim of our Phase I research was the development of an assay to assess the ability of antisense morpholino oligonucleotides to inhibit the expression of an endogenous hepatic gene in vivo. In addition to the efforts specifically mentioned in our Phase I research plan, we have developed a new approach to achieve endosomolysis, i.e., release of endosomal contents in the cell cytoplasm. This breakthrough is significant since entrapment of internalized PMOs in endosomes following internalization is a major barrier to successful oligonucleotide delivery. As a demonstration of our method's endosomolytic activity, we have shown functional delivery of biologically active PMO's, which were complexed in a particle, to cells in tissue culture. The proposed phase II research will combine our expertise in the formulation of oligo-containing particles, hepatocyte delivery of particles, and the use of endosomolytic agents, to develop particles for efficient in vivo delivery of PMO's to hepatocytes in a biologically active form.