리간드 부착 올리고-아르기닌 siRNA 전달체의 매커니즘적 이해에 대한 연구

Abstract

Since the discovery of RNA interference, there were many attempts to harness RNAi-based therapeutics in gene therapy. RNAi provide sequence specific degradation of mRNA transcript and can be expected to apply most of the genes that organism has. However, its translation to therapeutic application is hindered greatly by two major obstacles. First, it is not easy to deliver siRNA, negative charged macromolecule, into the cells by passing through the negative charge on the cell surface. Secondly, efficient delivery systems that can deliver siRNA to its site of action-cytosol, crossing the ultimate endosomal barrier are another problem. Thus solving these two obstacles have become the rate limiting steps in success towards RNAi therapeutics and efficient cellular uptake carriers to deliver siRNA to cytosol where the RNAi machinery resides is required. RNAi can be elicited by two major siRNA delivery system. One is viral vector and the other is non-viral vector. Viral vector system is promising carrier for efficient siRNA delivery but safety concerns exist based on their intrinsic property that brings severe side effect in gene therapy. Non-viral vectors are less cytotoxic but it needs to improve delivery efficiency for applying in vivo. One of the well-studied strategies is using cell-penetrating peptides (CPPs), such as nona-arginine (R9). It has been very well established that CPPs alone poorly translocate siRNA into cells. In this work, we have explored the possibility of enhancing cargo delivery efficiency of R9 by attaching it to peptide or antibody based ligands that bind cell to surface receptors and increase siRNA uptake. Further, we have elucidated the mechanism by which these targeting ligands enter the cell and enhance the delivery of functional siRNA. The mechanism involved accumulation of ligand-R9:siRNA microparticles on the cell membrane, which induced transient membrane inversion at the site of ligand-R9 binding and rapid siRNA translocation into the cytoplasm. siRNA release also occurred late after endocytosis when the ligand was attached to the L-isoform of R9, but not the protease-resistant D-isomer of R9, prolonging mRNA knockdown (Both peptide and antibody ligands). This critically depended on endosomal proteolytic activity, implying that partial CPP degradation is required for endosome to cytosol translocation. In conclusion, attaching target ligands to CPPs are not only improve delivery of functional siRNA but also produce therapeutic outcomes with lesser doses of siRNA. Next, we tested the application of ligand-R9 in 2 different therapeutic settings. We used a peptide ligand composed of 29 amino acid derived from rabies virus glycoprotein (RVG) known to bind to nicotinic acetylcholine receptor (nAchR) which attached to L-R9 (RVG-(L)R9). We test whether targeted siRNA delivery to lung epithelia reduces airway inflammation in a mouse model for allergic asthma. Allergic pulmonary inflammation and airways hyper-reactivity (AHR) occurs through the binding of T helper cell type II cytokines IL-4 and IL-13 to its cognate receptor present on lung epithelial cells. IL-13 induces inflammation through a multi-subunit receptor that includes the alpha chain of the IL-4 receptor (IL-4Rα), which also functions in IL-4 signaling. We used a RVG-(L)R9 that specifically binds to lung epithelial cells expressed nAchR and tested the therapeutic efficacy of siRNA targeting IL-4Rα in vivo. Intra-trachea inoculation of RVG-(L)R9:siRNA efficiently delivers siRNA to the lungs. Our targeting method allows superior penetration of lung epithelia with minimal toxicity and extended silencing of target gene expression compared to the naked siRNA. Silencing IL-4Rα expression in an OVA-challenged mouse model of airway inflammation showed prolonged relief from asthma symptoms compared to non-carrier mediated siRNA treatment. Our data suggest that RVG-(L)R9: siRNA microparticle can specifically deliver siRNA to the lung epithelial cells with a high efficacy and silencing IL-4Rα can be a novel RNAi-based approach to reducing respiratory inflammation. In second therapeutic setting, we tested whether intra-articular (I.A) injection of RVG-R9:siRNA targeting tumor necrosis factor-alpha (TNF-α) reduces rheumatoid arthritis in a mouse model. Tumor necrosis factor-alpha (TNF-α) secreted from macrophage plays a central role in development of rheumatoid arthritis (RA). In this experiment, we evaluate intra-articular (I.A) administration of RVG-(L)R9 binds to CD11b+ macrophages located in joint area and also known to be involved in production of enhanced TNF-α under inflammatory conditions resulting in arthritis. Intra-articular injection of RVG-(L)R9 delivers siRNA to nicotinic acetylcholine receptor expressing macrophage in synovium. RVG-(L)R9:siTNF-α decrease clinical score of paw swelling significantly as well as joint destruction by reducing TNF-α level in a collagen antibody-induced arthritis (CAIA) RA mouse model. These results indicate that macrophage specific suppression of TNF-α through intra-articular administration of RVG-(L)R9:siTNF-α can be used as a novel therapeutic intervention in rheumatoid arthritis. In this dissertation, we evaluated the molecular mechanism of ligand-R9 mediated siRNA delivery and its therapeutic effects in two different disease settings which are intra-trachea administration in asthma model or intra-articular injection in RA model. Our delivery of siRNA by ligand-(L)R9 used herein topical application where disease is onset is a novel method with minimizing unwanted side effects.