RNA interference (RNAi) is a cellular mechanism for post-transcriptional gene regulation mediated by small interfering RNA (siRNA).1,2 Specific gene silencing by siRNA holds significant promise for providing new treatment strategies in a wide range of diseases, including cancer and viral infections.3 Thus, siRNA-based technology is attractive owing to its
target gene specificity, relatively low siRNA immunogenicity, and simple design.4 However, it has two limitations: (1) As siRNA has no cell-type specificity, its medical application would require high doses resulting in high cost and may cause side effects 4,5 and (2) Because of their hydrophilicity, negative charge, and large molecular weight, siRNA molecules cannot readily cross the cell membrane.6,7, are subject to rapid renal clearance and degradation by endogenous RNases, and can be recognized by the innate immune system.8–10
To overcome these limitations, recent studies have focused on the combination of a target-specific antibody and a cell-penetrating peptide with siRNA.11–16 Antibody is an immune system-related biomolecule that can bind a specific region on an antigen.17 As several kinds of antibody molecules have been developed by genetic engineering for disease treatment,17, 18 it can solve the problem of siRNA specificity. Compared to the IgG type antibodies, small antibodies, such as VHH, scFv, and antibody-like molecules are expected to have stronger penetrating capability and lower immunogenicity19 due to the lack of both the constant region and Fc domain. Cell-penetrating peptides typically contain 5–30 amino acids and are mostly positively charged at physiological pH owing to the presence of several arginine and/or lysine residues.20 These peptides can bind to nucleic acids, such as siRNA, through electrostatic interactions. The arginine-9 (R9) motif, a typical cell-penetrating peptide, is more efficient for cellular uptake than other oligomers containing fewer arginine residues or other cationic amino acids such as histidine, lysine, or ornithine.21 By combining the technologies of cell-penetrating peptides and antibodies, successful cell-specific delivery of siRNA has been reported.15 Moreover, no side effects were reported. However, although several cell-penetrating peptide-fused antibodies are functionally expressed as a soluble fraction,15,16 in many cases their functional expression was found to be very low in the bacterial host, and were functionalized by unfolding the inclusion bodies11–13 or by bio-conjugating.14 To expand this technology, it is necessary to develop a method for stable preparation of cell-penetrating peptide-fused antibodies.
An effective strategy for functional expression is to separate and express each functional domain and then fuse them in vitro.22 This can effectively eliminate the complications related to structure and function, reduce undesirable non-expression, and expression as an insoluble fraction. Reconstruction of each functional unit is generally carried out by bioconjugation using a polymer or particle as support; however, the production of well-defined antibody conjugates is extremely challenging owing to the required selectivity, specificity, and reactivity under physiological conditions.23
Recently, a peptide ligation method using sortase and transglutaminase has attracted attention as a method for specifically ligating proteins under mild conditions.24 Among them, microbial transglutaminase (MTG; EC 22.214.171.124), belonging to the class transferases, is the most widely used because of its high activity, Ca+ independence, and low reverse reaction25 The enzyme catalyzes the formation of an isopeptide bond between the γ-carboxamide group of glutamine residues (donor) and the first-order ε-amine group of other compounds, such as proteins (acceptors of acyl residue). This enzyme can achieve specific ligation by adding peptides, such as K-tag sequence MRHKGS and Q-tag sequence LLQGS, to the protein.26, 27
In this study, Nanobody-R9, which is not expressed as a fusion protein, was subjected to separate domain expression that were enzymatically ligated by MTG in vitro. Finally, the RNAi of a model siRNA created using the resulting Nanobody-R9MTG was evaluated.