Delivery of Oligonucleotides to Hepatocytes In Vivo

描述（由申请人提供）：反义疗法在治疗各种人类传染病和遗传疾病方面具有巨大的前景。这些疗法是基于对特定基因表达的序列特异性抑制。由于其高选择性，与传统药物相比，反义药物具有较少副作用和毒性的潜力。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抑制特定基因是非常简单的。

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.