RNAi (RNA interference) is a phenomenon capable of inducing the selective degradation of target gene mRNA so as to silence the target gene expression by introducing into cells a double-stranded RNA that comprises a sense RNA having the sequence homologous to the target gene mRNA and an antisense RNA having the sequence complementary to the sense RNA. RNAi, because of its capability to selectively silence the target gene expression, has received considerable attention as a simple gene knock-down method that replaces the conventional gene disruption method relying on the tedious, inefficient homologous recombination. The above-mentioned RNAi was originally discovered in nematodes (Nature, 391, 806-811, 1998). Thereafter, it is also observed in various organisms including plants, round worms, Drosophila, and protozoa (Genes Dev. 15, 485-490, 2001). Recently, it was reported that RNAi can be induced also in mammalian cells by transducing the cells with short dsRNAs of 21 or 22 nucleotide long having a single-stranded 2 or 3 nucleotide of 3′-overhang terminus in place of long dsRNAs as those used in other organisms (Nature 411, 494-498, 2001).
An RNAi-inducing entity, siRNA (small interference RNA) is a short, double-helix RNA strand consisting of about 19 to 23 nucleotides, which can suppress expression of a targeted mRNA being related to a disease and having complementary base sequence to the siRNA. However, since siRNA has very low stability and is quickly degraded in vivo, its therapeutic efficiency deteriorates quickly. Even though the dose of expensive siRNA can be increased, the anionic nature of siRNA hinders it from permeating a cell membrane with negative charge, leading to low levels of siRNA transfer into intracellular compartments (Chemical and Engineering News December 22, 32-36, 2003). In addition, the linkage of a ribose sugar in siRNA is chemically very unstable, and thus the majority of siRNA has a half-life of about 30 minutes in vivo and is quickly degraded.
Accordingly, there is a need to develop a technology for the preparation of a novel delivery system that facilitates intracellular transfer of siRNA as a gene-based therapeutic agent. In general, siRNA can be administered to a subject as a recombinant plasmid or a viral vector which expresses the siRNA.
Alternatively, siRNA can be administered to a subject as a naked siRNA in conjunction with a delivery reagent such as Mirus Transit TKO lipophilic reagent, lipofectin, lipofectamine, cellfectin, cationic phospholipid nanoparticles, polycations, liposomes, etc. To improve in vivo stability of siRNA, a biocompatible polymer such as a polyethylene glycol is also conjugated to siRNA to increase the cellular uptake of siRNA. Kataoka et. al. prepared a PEG-siRNA conjugate to improve the low stability against the enzymatic degradation of siRNA and low permeability across the cell membrane, and they developed it as a siRNA delivery system for tumor targeting (J. Am. Chem. Soc. 127, 1624, 2005).
Cationic phospholipid nanoparticles are disclosed in U.S. Pat. No. 5,858,784 and US Patent Publication NO. 20060008910AI, in which cationic lipids are mixed with phospholipids in a predetermined ratio to prepare particles such as cationic liposome, the particles are mixed with nucleic acid to prepare a complex of cationic phospholipids particles and nucleic acid, and the complex is introduced into a cell line to improve gene expression. Cationic polymers have been also studied as a gene delivery vehicle, which are disclosed as follows: DEAE dextran, polylysine having repeating lysine units, and polyethyleneimine having repeating ethyleneimine units, polyamidoamine (U.S. Pat. No. 6,020,457), poly-amino-ester (US Patent Publication NO. 20040071654A1), and a biodegradable cationic copolymer (US patent Publication NO. 20060093674A1).
As a gene delivery vehicle, the synthetic polymers are advantageous in that they are easily prepared, not limited by the size of gene to be introduced, generate fewer side effects that may be induced by immunogenic viral surface protein upon repeated administration, cause no safety problems due to viral genes, and require lower production cost in a commercial process, as compared to viral vectors including lentiviral, adenoviral and retroviral vectors. However, there are drawbacks in that the delivery systems using such cationic polymers have lower transfection efficiency as compared to viral vectors that are effectively transferred via cell surface receptors, and might induce cytoxicity (J. Control. Release 114, 100-109, 2006). Another drawback of the cationic polymer mediated gene transfer is that it does not greatly prolong the half life of the gene in blood (Gene Ther. 8, 1857-1892).
For the purpose of improving the problems, recent studies have been made on a polymer conjugate of polyethylene glycol and chitosan having excellent biocompatibility and industrial availability as a delivery system (U.S. Pat. No. 6,730,742). However, there is a limitation that only the conjugation does not significantly improve the gene transfer efficiency. Accordingly, the present inventors prepared a double conjugate by linking polyamine to chitosan or a triple conjugate by additionally linking polyethylene glycol to the double conjugate, as disclosed in Korean Patent Application NO. 10-2007-0001715. They found that the conjugates had lower cytotoxicity, higher transfer efficiency, and longer retention time in blood than the known gene delivery systems.
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