Protease-triggered siRNA delivery vehicles

Abstract

The safe and efficacious delivery of membrane impermeable therapeutics requires cytoplasmic access without the toxicity of nonspecific cytoplasmic membrane lysis. We have developed a mechanism for control of cytoplasmic release which utilizes endogenous proteases as a trigger and results in functional delivery of small interfering RNA (siRNA). The delivery approach is based on reversible inhibition of membrane disruptive polymers with protease-sensitive substrates. Proteolytic hydrolysis upon endocytosis restores the membrane destabilizing activity of the polymers thereby allowing cytoplasmic access of the co-delivered siRNA. Protease-sensitive polymer masking reagents derived from polyethylene glycol (PEG), which inhibit membrane interactions, and N-acetylgalactosamine, which targets asialoglycoprotein receptors on hepatocytes, were synthesized and used to formulate masked polymer-siRNA delivery vehicles. The size, charge and stability of the vehicles enable functional delivery of siRNA after subcutaneous administration and, with modification of the targeting ligand, have the potential for extrahepatic targeting.

HIF2α-Targeted RNAi Therapeutic Inhibits Clear Cell Renal Cell Carcinoma

“…Protease masking reagent PEG 12 -L-analine-L-citrulline-p-aminobenzyl alcohol p-nitrophenyl carbonate (PEG 12 -ACit-PABC-PNP, PEG 12 refers to a discrete 12-mer of PEG) was synthesized as described previously (28). RGD-or RGE mimetic-conjugated-PEG masking reagents, RGD-PEG 20 -FCitFP-TFP or RGE-PEG 20 -FCitFP-TFP (PEG 20 is a discrete 20-mer PEG, FCitFP refers to the amino acid sequence phenylalanine-citrulline-phenylalanineproline, TFP (tetrafluorophenyl ester), were synthesized using standard amide coupling reagents as described previously (34,35).…”

“…Polymer N-Boc-ethoxyethylamine acrylate and propyl acrylate (EAP) synthesis is similar as described for N-Boc-ethoxyethylamine acrylate and sec-butyl acrylate (EAB) (28). For polymer EAP, propyl acrylate was used instead of sec-butyl acrylate.…”

“…Previously, we described an efficient and selective liver targeting siRNA delivery system termed dynamic polyconjugates (DPC; refs. 28,29). This delivery approach included the use of a membrane active polymer that is reversibly modified with shielding and targeting moieties to provide highly selective target tissue delivery of siRNA (28,29).…”

“…28,29). This delivery approach included the use of a membrane active polymer that is reversibly modified with shielding and targeting moieties to provide highly selective target tissue delivery of siRNA (28,29). For liver targeting, DPC uses N-acetylgalactosamine as the ligand to target asialoglycoprotein receptors (ASGPr) that are abundantly expressed by hepatocytes (30).…”

“…For liver targeting, DPC uses N-acetylgalactosamine as the ligand to target asialoglycoprotein receptors (ASGPr) that are abundantly expressed by hepatocytes (30). The incorporation of a targeting ligand and the reversible masking of a membrane active polymer allowed efficient target cell uptake and subsequent RNAi trigger escape from endosomes (28,29). The modular nature of DPCs allows for exchange of targeting moiety and RNAi trigger for a specific tissue and therapeutic gene of interest.…”