Extrahepatic delivery of small interfering RNAs (siRNAs) may have applications in the development of novel therapeutic approaches. However, reports on such approaches are limited, and the scarcity of reports concerning the systemically targeted delivery of siRNAs with effective gene silencing activity presents a challenge. We herein report for the first time the targeted delivery of CD206-targetable chemically modified mannose–siRNA (CMM–siRNA) conjugates to macrophages and dendritic cells (DCs). CMM–siRNA exhibited a strong binding ability to CD206 and selectively delivered contents to CD206-expressing macrophages and DCs. Furthermore, the conjugates demonstrated strong gene silencing ability with long-lasting effects and protein downregulation in CD206-expressing cells in vivo. These findings could broaden the use of siRNA technology, provide additional therapeutic opportunities, and establish a basis for further innovative approaches for the targeted delivery of siRNAs to not only macrophages and DCs but also other cell types.

Selection of antibody library in vitro is almost always performed on a certain solid-phase with immobilized antigen. However, for the selection of small molecule binders, conjugation of the antigen to a carrier molecule is indispensable, which often leads to the selection of unwanted binders such as conjugate-binders or those with insufficient specificity. Here we describe a rapid and efficient way to improve the affinity of an anti-small molecule antibody without antigen derivatization. The method is based on the open-sandwich (OS) principle, which utilizes the antigen-dependent stabilization of antibody variable domain Fv. We used an anti-osteocalcin C-terminal peptide Fv that showed a good response but with moderate sensitivity in OS ELISA as a model. By selecting PCR-randomized V(H)-displaying phages for superior binders to the immobilized V(L) fragment in the presence of limited amount of antigen peptide, V(H) mutants that show superior detection sensitivity in OS ELISA were obtained, and were characterized to retain improved antigen-binding affinity. Furthermore, saturation mutagenesis of a mutant resulted in further improvement in sensitivity. This 'OS-selection' will be the first to select anti-small molecule antibodies without using conjugated antigens, and especially useful in the affinity maturation of antibodies whose Fv has limited stability in the absence of antigen.

Extrahepatic targeted delivery of oligonucleotides, such as small interfering RNA (siRNA) and antisense oligonucleotides (ASOs), is an attractive technology for the development of nucleic acid-based medicines. To target CD22-expressing B cells, several drug platforms have shown promise, including antibodies, antibody–drug conjugates, and nanoparticles, but to date CD22-targeted delivery of oligonucleotide therapeutics has not been reported. Here we report the uptake and enhancement of siRNA gene expression knockdown in CD22-expressing B cells using a chemically stabilized and modified CD22 glycan ligand-conjugated siRNA. This finding has the potential to broaden the use of siRNA technology, opening up novel therapeutic opportunities, and presents an innovative approach for targeted delivery of siRNAs to B cell lymphomas.

We report a potent cationic lipid, SST-02 ((3-hydroxylpropyl)dilinoleylamine), which possesses a simple chemical structure and is synthesized just in one step. Cationic lipids are key components of siRNA-lipid nanoparticles (LNP), which may serve as potential therapeutic agents for various diseases. For a decade, chemists have given enhanced potency and new functions to cationic lipids along with structural complexity. In this study, we conducted a medicinal chemistry campaign pursuing chemical simplicity and found that even dilinoleylmethylamine (SST-01) and methylpalmitoleylamine could be used for the in vitro and in vivo siRNA delivery. Further optimization revealed that a hydroxyl group boosted potency, and SST-02 showed an ID 50 of 0.02 mg/kg in the factor VII (FVII) model. Rats administered with 3 mg/kg of SST-02 LNP did not show changes in body weight, blood chemistry, or hematological parameters, while the AST level decreased at a dose of 5 mg/kg. The use of SST-02 avoids a lengthy synthetic route and may thus decrease the future cost of nucleic acid therapeutics.

Regulation of enzyme activity either by its substrates or by effectors is generally known as allostery. However, it has been considered hard to alter its effector specificity, despite its potential utility as a sensitive molecular sensor. To this end, we made fusion proteins consisting of an antibody variable region Fv and a circularly permutated TEM-1 β-lactamase cpBLA. Two expression vectors encoding Fv-cpBLA with different antigen specificities were made, in which cpBLA was inserted into the linker region of the single chain Fv that specifically binds either bone-related disease marker osteocalcin (BGP) C-terminal peptide or neonicotinoid insecticide imidacloprid (ICP). The cpBLA having new termini near the active site was activated upon binding with its cognate antigen, owing to the stabilization of tethered Fv by bound antigen. As a result, both Fv-cpBLA showed specific antigen binding as well as antigen-induced enhancement in catalytic activity. Moreover, E. coli cells expressing Fv-cpBLA for ICP showed ICP concentration dependent growth in the medium containing ampicillin. The system was also applied to select for Fv-cpBLA linker mutants that confer faster growth. This will be the first of an antibody-based small molecule indicator enzyme.