Targeted In Vivo Delivery of siRNA and an Endosome-Releasing Agent to Hepatocytes

The discoveries of RNA interference (RNAi) and short interfering RNAs (siRNAs) have provided the opportunity to treat diseases in a fundamentally new way: by co-opting a natural process to inhibit gene expression at the mRNA level. Given that siRNAs must interact with the cells' natural RNAi machinery in order to exert their silencing effect, one of the most fundamental requirements for their use is efficient delivery to the desired cell type and, specifically, into the cytoplasm of those cells. Numerous research efforts involving the testing of a large number of delivery approaches using various carrier molecules and inventing several distinct formulation technologies during the past decade illustrate the difficulty and complexity of this task. We have developed synthetic polymer formulations for in vivo siRNA delivery named Dynamic PolyConjugates™ (DPCs) that are designed to mimic the features viruses possess for efficient delivery of their nucleic acids. These include small size, long half-life in circulation, capability of displaying distinct host cell tropism, efficient receptor binding and cell entry, disassembly in the endosome and subsequent release of the nucleic acid cargo to the cytoplasm. Here we present an example of this delivery platform composed of a hepatocyte-targeted endosome-releasing agent and a cholesterol-conjugated siRNA (chol-siRNA). This delivery platform forms the basis of ARC-520, an siRNA-based therapeutic for the treatment of chronic hepatitis B virus (HBV) infection. In this chapter, we provide a general overview of the steps in developing ARC-520 and detailed protocols for two critical stages of the discovery process: (1) verifying targeted in vivo delivery to hepatocytes and (2) evaluating in vivo drug efficacy using a mouse model of chronic HBV infection.