Dicer-Labile PEG Conjugates for siRNA Delivery

Abstract

Poly(ethylene glycol) (PEG) conjugates of Dicer-substrate small interfering RNA (DsiRNA) have been prepared to investigate a new siRNA release strategy. 3′-sense or 5′-antisense thiol-modified, blunt-ended DsiRNAs, inhibiting enhanced green fluorescent protein (eGFP) expression, were covalently conjugated to PEG with varying molecular weights (2, 10, and 20 kg/mol) through a stable thioether bond using a Michael addition reaction. The DsiRNA conjugates with 2 kg/mol PEG (both 3′-sense or 5′-antisense strand conjugated) and the 10 kg/mol PEG conjugated to the 3′-sense strand of DsiRNA were efficiently cleaved by recombinant human Dicer to 21-mer siRNA, as determined by gel electrophoresis. Importantly, 2 and 10 kg/mol PEG conjugated to the 3′-sense strand of DsiRNA showed potent gene silencing activity in human neuroblastoma (SH-EP) cells, stably expressing eGFP, at both the mRNA and protein levels. Moreover, the 10 kg/mol PEG conjugates of the 3′-sense strand of DsiRNA were less immunogenic when compared with the unmodified DsiRNA, determined via an immune stimulation assay on human peripheral blood mononuclear cells.

Polymer–Peptide Delivery Platforms: Effect of Oligopeptide Orientation on Polymer-Based DNA Delivery

“…Cationic polymers can be designed as various architectures, such as linear, graft (comb), branched, and dendritic, which influence complexation and delivery capabilities of the polymer vehicle. 7–12 Novel synthetic approaches allow integration of many features needed for gene therapy into one macromolecular delivery system, including functionality to prevent immunogenicity, 13 recognize cell surfaces, 14,15 and localize to the nucleus. 16–18 …”

In Vivo RNAi Efficacy of Palmitic Acid‐Conjugated Dicer‐Substrate siRNA in a Subcutaneous Tumor Mouse Model

“…The development of functional siRNAs that can solve these problems is essential for application in the clinic. To enhance the nuclease stability or the cell membrane permeability of siRNAs, direct conjugation of siRNAs to functional molecules, including polymers, peptides, and lipids, has been developed . Although direct conjugations could solve some of the problems associated with RNAi, additional points would need to be addressed prior to clinical use, such as insufficient delivery to target cells, concerns regarding the immune response, and the uncontrolled release of siRNAs into cells.…”

Polymeric siRNA delivery vectors: knocking down cancers with polymeric‐based gene delivery systems

“…Kow et al . demonstrated the formation of physiologically‐stable thioether linkages connecting thiol‐modified DsiRNA (27‐nt) and a PEG‐acrylate vector via the Michael‐addition reaction (Scheme ) . The conjugates were found to be stable in the endosomal pH range (4.5–5.5) while effectively cleaved by recombinant human Dicer to 21‐nt siRNA.…”