Co-Injection of a Targeted, Reversibly Masked Endosomolytic Polymer Dramatically Improves the Efficacy of Cholesterol-Conjugated Small Interfering RNAsIn Vivo

Abstract

Effective in vivo delivery of small interfering (siRNA) has been a major obstacle in the development of RNA interference therapeutics. One of the first attempts to overcome this obstacle utilized intravenous injection of cholesterol-conjugated siRNA (chol-siRNA). Although studies in mice revealed target gene knockdown in the liver, delivery was relatively inefficient, requiring 3 daily injections of 50 mg/kg of chol-siRNA to obtain measurable reduction in gene expression. Here we present a new delivery approach that increases the efficacy of the chol-siRNA over 500-fold and allows over 90% reduction in target gene expression in mice and, for the first time, high levels of gene knockdown in non-human primates. This improved efficacy is achieved by the co-injection of a hepatocyte-targeted and reversibly masked endosomolytic polymer. We show that knockdown is absolutely dependent on the presence of hepatocyte-targeting ligand on the polymer, the cognate hepatocyte receptor, and the cholesterol moiety of the siRNA. Importantly, we provide evidence that this increase in efficacy is not dependent on interactions between the chol-siRNA with the polymer prior to injection or in the bloodstream. The simplicity of the formulation and efficacy of this mode of siRNA delivery should prove beneficial in the use of siRNA as a therapeutic.

Going beyond the liver: Progress and challenges of targeted delivery of siRNA therapeutics

“…induction of influx of hydrogen ions followed by water and swelling of the endosomes, finally causing their disruption [43]. While initially presented data on polyconjugates relied on a covalent, but labile attachment of the siRNA cargo to the polymer via a disulfide linkage, more recent evolution of the systems uses simple co-application of siRNA and the carrier [44]. Because little interaction of the uncharged carrier with siRNA can be expected, it is not fully clear to what extent the polymer influences direct siRNA uptake or is simply an adjuvant for increasing endosomal escape.…”

Synthetic RNAi triggers and their use in chronic hepatitis B therapies with curative intent

“…The masking agent is bifunctional and is also used as a linker to attach the hepatocyte targeting ligand N-acetylgalactosamine (GalNAc or NAG) to the peptide. Once inside the endosome, the low pH environment triggers unmasking, allowing the peptide to interact with and disrupt the endosomal membrane which allows release of the co-injected RNAi trigger (Wong et al, 2012). The mechanism of endosomal release of RNAi trigger conjugates, when conjugated to GalNAc for hepatocyte targeting, is not known.…”

“…These include substitution of the 2 0 OH of the sugar with 2 0 OMe or 2 0 F groups and a phosphorothioate linkage between the terminal 3 0 nucleotides of the guide strand. A cholesterol moiety was also conjugated to the RNAi triggers to enhance liver uptake (Soutschek et al, 2004;Wong et al, 2012). Two mouse models of CHB were used in these studies.…”

The Role of micro RNAs in Breast Cancer Metastasis: Preclinical Validation and Potential Therapeutic Targets

“…Upon entry into the endosome, the acidic conditions make the PEG molecules dissociate and unleash the endosomal escape capacity of the lipid polymers. Thereby, the nucleic acid payload is released into the cytosol in a prodrug-like manner (192,193).…”