Dengue virus is a virus belonging to the family Flaviviridae, which has a single strand RNA and a single envelope having a diameter of 30 nm and shows positive polarity. It is one of disease that most commonly affect people in the world. Dengue fever is transmitted by mosquito bites or infected people, causes a high fever and rash with muscle and joint pains, and also causes hemorrhage in some cases. People with dengue hemorrhagic fever are high risk of losing their life. In addition, people have permanent immunity against a dengue viral type that infected them, but are not protected from other viral types. For this reason, in the case people who live in endemic area, infections with all four types of dengue virus may occur throughout their life.
The World Health Organization estimates that over 50 million dengue infected people and over one million dengue infected patients each year in worldwide occur. Thus, dengue is considered as one of key public health issues, but a dengue virus preventive vaccine has not yet been reported. Southeastern Asia is the largest endemic area for dengue fever, in which reinfections with several serotypes of dengue virus easily occur. Dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) in secondary dengue infections are the major causes of death (Qiu F X et al., Bull World Health Organ, 71:349-359, 1993; Tassniyom S et al., Pediatrics, 92:111-115, 1993). In Korea, an outbreak of dengue fever has not yet occurred, but it is expected that the number of Korean people infected with dengue virus will increase due to the expansion of trade with dengue endemic areas, trip abroad, overseas residence and the like. In addition, an increase in infection opportunities such as off-road traveling and repeated visits to dengue endemic areas can increase the possibility of reinfection with dengue virus and can also increase the risk of development of DHF or DSS by secondary infection. In fact, in the Korea National Institute of Health, serological tests for 99 persons, who returned from abroad and showed symptoms suspected as dengue fever, were performed during a period from 2001 to June, 2003, and 33 people positive for dengue virus antibodies were found.
Dengue virus has four serotypes and a genome size of about 11 kb. The dengue virus genome is translated into a single polyprotein, and then cleaved into functional proteins by host or viral proteases into functional proteins. In a previous vaccine strategy, there was great difficulty in inhibiting dengue virus replication, because effective protection against four similar serotypes with a polyvalent vaccine was not induced. However, in a genetic engineering approach for the viral genome, studies on a vaccine effective against all the four serotypes are in progress, and final clinical trials are in progress by a certain multi-national pharmaceutical company. Generally, primary infection is not fatal, but when secondary infection with different serotypes occurs, effective protection will not be made due to a previously formed antibody, and the viral infection will be increased by ADCC (antibody dependent cell cytotoxicity) to pose great threat to life. To date, only a primary approach has been performed in which cytokine is administered to such patients. Meanwhile, technologies of inhibiting the expression of genes are an important tool in developing a therapeutic agent for treating diseases and validating a target. Since the roles of RNA interference (hereinafter referred to as ‘RNAi’) among these technologies was found, it was found that the RNAi acts on sequence-specific mRNA in various kinds of mammalian cells (Silence of the Transcripts: RNA Interference in Medicine. J Mol Med (2005) 83: 764-773). When long-chain double-stranded RNA is delivered into cells, the delivered double stranded RNA is converted into a small interfering RNA (hereinafter referred to as ‘siRNA’) processed into 21 to 23 base pairs (bp) by the endonuclease Dicer, wherein the siRNA inhibits the expression of the target gene in a sequence-specific manner by a process in which the antisense strand recognizes and degrades the target mRNA (NUCLEIC-ACID THERAPEUTICS: BASIC PRINCIPLES AND RECENT APPLICATIONS. Nature Reviews Drug Discovery. 2002. 1, 503-514).
Bertrand et al. have reported that siRNA has an excellent inhibitory effect on the expression of mRNA in vitro and in vivo compared to an antisense oligonucleotide (ASO) for the same target gene and that the effect is long lasting (Comparison of antisense oligonucleotides and siRNAs in cell culture and in vivo. Biochem. Biophys. Res. Commun. 2002. 296: 1000-1004). Also, because siRNA complementarily binds to the target mRNA to regulate the expression of the target gene in a sequence-specific manner, it can be advantageously used in a wider range of applications compared to conventional antibody-based drugs or chemicals (small molecule drugs) (Progress Towards in Vivo Use of siRNAs. MOLECMLAR THERAPY. 2006 13(4):664-670).
siRNA has excellent effects and can be used in a wide range of applications, but in order for siRNA to be developed into therapeutic agents, it is required to improve the in vivo stability and intracellular delivery efficiency of siRNA so as to effectively deliver siRNA into its target cells (Harnessing in vivo siRNA delivery for drug discovery and therapeutic development. Drug Discov Today. 2006 January; 11(1-2):67-73).
In order to solve the above-mentioned problem, studies on a technology of either modifying some nucleotides of siRNA or the backbone of siRNA to improve the in vivo stability so as to have resistance against nuclease or using carriers such as a viral vector, liposome or nanoparticles have been actively conducted.
Delivery systems comprising a viral vector such as adenovirus or retrovirus have high transfection efficiency, carry the risks of immunogenicity and oncogenicity. However, non-viral carriers including nanoparticles are evaluated to have low intracellular delivery efficiency compared to viral carriers, but have advantages, including high safety in vivo, target-specific delivery, efficient uptake and internalization of RNAi oligonucleotides into cells or tissues, and low cytotoxicity and immune stimulation. Thus, these non-viral carriers are considered as a promising delivery method compared to the vial delivery system (Nonviral delivery of synthetic siRNA s in vivo. J Clin Invest. 2007 December 3; 117(12): 3623-3632).
In a method of using nanocarriers among the non-viral delivery systems, nanoparticles are formed using various polymers such as liposome, a cationic polymer complex and the like, and iRNA is supported on these nanoparticles that are nanocarriers, and is delivered into the cell. In the method of using nanocarriers, a polymeric nanoparticle, polymer micelle, lipoplex and the like are mainly used. Among them, the lipoplex is composed of cationic lipid and interacts with anionic lipid of endosome in the cell to destabilize the endosome, thereby serving to deliver the iRNA into the cell (Proc. Natl. Acad. Sci. 15; 93(21):11493-8, 1996).
In addition, it is known that the efficiency of siRNA in vivo can be increased by conjugating a chemical compound or the like to the end region of the passenger (sense) strand of the siRNA so as to have improved pharmacokinetic characteristics (Nature 11; 432(7014):173-8, 2004). In this case, the stability of the siRNA changes depending on the property of the chemical compound conjugated to the end of the sense (passenger) or antisense (guide) strand of the siRNA. For example, siRNA conjugated with a polymer compound such as polyethylene glycol (PEG) interacts with the anionic phosphoric acid group of siRNA in the presence of a cationic compound to form a complex, thereby providing a carrier having improved siRNA stability (J Control Release 129(2):107-16, 2008). Particularly, micelles made of a polymer complex have a very small size and a very uniform size distribution compared to other drug delivery systems such as microspheres or nanoparticles, and are spontaneously formed. Thus, these micelles have advantages in that the quality of the formulation is easily managed and the reproducibility thereof is easily secured.
Further, in order to improve the intracellular delivery efficiency of siRNA, a technology for securing the stability of the siRNA and increasing the cell membrane permeability of the siRNA using a siRNA conjugate obtained by conjugating a hydrophilic compound (for example, polyethylene glycol (PEG)), which is a biocompatible polymer, to the siRNA via a simple covalent bond or a linker-mediated covalent bond, has been developed (Korean Patent Registration No. 883471). However, even when the siRNA is chemically modified and conjugated to polyethylene glycol (PEG) (PEGylation), it still has low stability in vivo and a disadvantage in that it is not easily delivered into a target organ. In order to solve these disadvantages, double-stranded oligo RNA structures comprising hydrophilic and hydrophobic compounds bound to an oligonucleotide, particularly double-stranded oligo RNA such as siRNA have been developed. These structures form self-assembled nanoparticles, named SAMiRNA™ (Self Assembled Micelle Inhibitory RNA), by hydrophobic interaction of the hydrophobic compound (see Korean Patent Registration No. 1224828). The SAMiRNA™ technology has advantages over conventional delivery technologies in that homogenous nanoparticles having a very small size can be obtained.
Specifically, in the SAMiRNA™ technology, PEG (polyethylene glycol) is used as the hydrophilic compound. PEG is a synthetic polymer and is generally used to increase the solubility of medical drugs, particularly proteins, and control the pharmacokinetics of drugs. PEG is a polydisperse material, and a one-batch polymer is made up of different numbers of monomers, and thus shows a molecular weight in the form of a gauss curve. Also, the homogeneity of a material is expressed as the polydisperse index (Mw/Mn). In other words, when PEG has a low molecular weight (3-5 kDa), it shows a polydisperse index of about 1.01, and when it has a high molecular weight (20 kDa), it shows a high a polydisperse index of about 1.2, indicating that the homogeneity of PEG decreases as the molecular weight of PEG increases (F. M. Veronese. Peptide and protein PEGylation: a review of problems and solutions. Biomaterials (2001) 22:405-417). Thus, when PEG is bound to a medical drug, there is a disadvantage in that the polydisperse properties of PEG are reflected to the conjugate so that the verification of a single material is not easy. Due to this disadvantage, processes for the synthesis and purification of PEG have been improved in order to produce materials having a low polydisperse index. However, when PEG is bound to a compound having a low molecular weight, there are problems associated with the polydisperse properties of the compound, causing a problem in that it is not easy to confirm whether the binding was easily achieved (Francesco M. Veronese and Gianfranco Pasut. PEGylation, successful approach to drug delivery. DRUG DISCOVERY TODAY (2005) 10(21):1451-1458).
Accordingly, in recent years, the SAMiRNA™ technology (that is self-assembled nanoparticles) has been improved by forming the hydrophilic compound of the double-stranded RNA structure (constituting SAMiRNA™) into basic unit blocks, each comprising 1-15 monomers having a uniform molecular weight, and if necessary, a linker, so that a suitable number of the blocks will be used, if required. Thus, new types of delivery system technologies, which have small sizes and significantly improved polydisperse properties, compared to conventional SAMiRNA™, have been developed.
As described above, a therapeutic agent having an excellent therapeutic effect against most serotypes of dengue virus is not currently present, and conventional vaccination methods have limitations. Thus, there is an urgent need for the treatment of a new type of therapeutic agent. A dengue virus infection therapeutic agent based on RNAi technology has a potential to be used as an alternative thereto, but an effective therapeutic agent based on RNAi technology, particularly a therapeutic agent having a therapeutic effect against all dengue virus serotypes, has not yet been developed.