Oligonucleotide therapeutics based on antisense and RNA interference methods are attracting attention as effective drugs for diseases that were difficult to treat with conventional drugs. However, the major problem is that there is no effective delivery method that efficiently introduces pharmacologically active molecules such as antisense oligonucleotides and siRNA into the cytoplasm. As existing methods, two major methods are available; a liposome method in which an oligonucleotide is complexed with a cationic polymer, and a ligand conjugate method in which a ligand for a cell membrane receptor is introduced into the oligonucleotide. The critical problem of these methods is that the efficiency of oligonucleotide release from endosomes to the cytoplasm is extremely low. Furthermore, the former methods has properties that are unsuitable for pharmaceutical applications, such as cytotoxicity derived from cationic substrates and the undefined and non-uniform molecular composition. In the pharmaceutical industry, there is a strong demand for the development of a new cytoplasmic delivery method that replaces the liposome method.
In this research, we will develop a delivery method called “disulfide method” that we have independently developed, and will establish a method for cytoplasmic delivery of oligonucleotide drugs. In this method, an oligonucleotide with a disulfide group at the end is used as the active molecule(referred to as MPON; Membrane Permeable OligoNucleotide). As a possible mechanism, it is presumed that the disulfide structure forms a bond with proteins on the cell membrane, and the oligonucleotide is directly taken into the cytoplasm. In cell experiments, MPON was efficiently distributed in the cytoplasm in a very short time of 10 minutes, and suppressed gene expression more strongly than t the conventional lipofection method. Furthermore, on the on in vivo distribution analysis of MPON in mice, the uptake of MPON was predominantly increased in specific organs and tissues such as lung, brain, and muscle compared to the normal oligonucleotide. Based on these results, we will perform the analysis of the detailed molecular mechanism of cellular uptake, the optimization of the disulfide unit structure and the tuning of lipophilicity, and the optimization of the molecular structure such as the introduction of a target-directing ligand. Through these investigations, we will establish a delivery method that combines high cytoplasmic efficiency and high target selectivity. We will also develop siRNA-MPON and antisense-MPON that can treat mesothelioma and other diseases, and aim to establish effective treatments for diseases for which no effective molecular targeted drug is available. Through the series of studies described above, we will demonstrate that the disulfide method is a revolutionary method for oligonucleotide delivery that overcomes the bottleneck in the development of oligonucleotide therapeutics.