RESUMO
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.
Assuntos
Oligonucleotídeos Antissenso , Polissacarídeos , Técnicas de Silenciamento de Genes , Ligantes , Polissacarídeos/metabolismo , RNA Interferente Pequeno/genéticaRESUMO
Affinity purification is one of the most prevalent methods for the target identification of small molecules. Preparation of an appropriate chemical for immobilization, however, is a tedious and time-consuming process. A decade ago, a photoreaction method for generating affinity beads was reported, where compounds are mixed with agarose beads carrying a photoreactive group (aryldiazirine) and then irradiated with ultraviolet light under dry conditions to form covalent attachment. Although the method has proven useful for identifying drug targets, the beads suffer from inefficient ligand incorporation and tend to shrink and aggregate, which can cause nonspecific binding and low reproducibility. We therefore decided to craft affinity beads free from these shortcomings without compromising the ease of preparation. We herein report a modified method; first, a compound of interest is mixed with a crosslinker having an activated ester and a photoreactive moiety on each end. This mixture is then dried in a glass tube and irradiated with ultraviolet light. Finally, the conjugates are dissolved and reacted with agarose beads with a primary amine. This protocol enabled us to immobilize compounds more efficiently (approximately 500-fold per bead compared to the original method) and generated beads without physical deterioration. We herein demonstrated that the new FK506-immobilized beads specifically isolated more FKBP12 than the original beads, thereby proving our method to be applicable to target identification experiments.