ABSTRACT
The major impediment in development of macromolecular drug therapeutics, including peptides, proteins, and siRNAs, is delivery of the drug into the cytoplasm or nucleus of cells where it will exert its biological effect. Macromolecular siRNAs are too large, too charged, and too hydrophilic to cross the hydrophobic plasma membrane, which serves as a protective barrier to protect keep foreign molecules and pathogens out of the cell. Thus, cellular delivery of siRNAs remains a significant barrier and rate-limiting step for development of siRNA therapeutics. Current, siRNA delivery approaches rely on condensing and packaging siRNAs into ~100 nm liposome or polymer nanoparticles. However, lipid nanoparticles are 100 megaDaltons in size, roughly 5,000-fold larger than the 14 kDaltons siRNA being delivered, which severely limits the diffusion coefficient, pharmacokinetics, and potential of lipid nanoparticle therapeutics. An alternative siRNA delivery approach is to deliver one siRNA into a cell at a time using Peptide Transduction Domains (PTDs) delivery
peptides, also called Cell-Penetrating Peptides (CPPs). However, due to the negative charge of the siRNA, conjugation of positively charged PTDs results in aggregation, cytotoxicity, and poor delivery into cells. This chapter describes a new delivery strategy that utilizes a PTD fusion protein with a double-stranded RNA-binding domain (DRBD) that binds to and masks the siRNA negative charge, allowing for PTDmediated delivery into cells and induction of RNAi responses.
