The gene transfer technology to artificially introduce an exogenous gene into cells is an important technology not only as a fundamental technology to analyze a variety of biological phenomena but also as one which leads to useful applications such as gene therapy and production of beneficial animals. Generally, two methods have been used for gene transfer. One is a biological method using a virus having an exogenous gene, and the other is a physical method in which an exogenous gene is physically introduced into cells.
The method using a virus is based on the principle that a cell is infected with a recombinant virus in which the gene of interest is incorporated, and the entire recombinant virus genome integrates into the genome of the host cell. This method is currently attracting much attention as a technological basis for gene therapy for such diseases as Lesch-Nyhan syndrome and adenosine deaminase (ADA) deficiency. However, it has been pointed out that the method has various problems such as the pathogenicity of the virus since it utilizes the biological properties of the virus itself. For this reason, modified retroviral vectors without the regions associated with the viral pathogenicity and replication have currently being developed. However, these modified vectors have yet many problems that they might still cause some undesirable effects on cells, and they can infect only dividing cells.
Therefore, physical methods to introduce non-viral vectors are now used as well as the above-mentioned methods using viruses. In one of the established physical methods, non-viral vectors are introduced into cells in combination with chemicals such as calcium phosphate, DEAE-dextran, polycations, or liposomes. However, these physical methods have such problems that the transfection efficiency of genes into cells is low, and that the exogenous gene on a non-viral vector thus transfected does not reach the cell nucleus in many cases. Therefore, the methods have many difficulties to be overcome for being applied to gene therapy.
Recently, it was reported that the proteins which are transported into the nucleus of eukaryotic cells and function there have a specific amino acid sequence that functions as a signal (NLS: nuclear localization signal) for their transportation into the nucleus (G. Garcia-Bustos et al., Biochem. Biophys. Acta 1071: 83-101 (1991)). Moreover, it was also reported that attaching the nuclear localization signal to a protein that normally does not translocate to the nucleus will confer the nuclear translocation activity on this protein (R. E. Lanford et al., Cell 46: 575-582 (1986), Y. Yoneda et al., Exp. Cell. Res. 170: 439-452 (1987), D. Chelsky et al., Mol. Cell. Biol. 9: 2487-2492 (1989)). Based on this knowledge, researches have been made using the nuclear localization signal so that the gene introduced by physical methods can reach the nucleus with a high probability. That is, the techniques are studied to condense DNA as close as possible to 40 nm, the size of the nuclear membrane pore, attach the nuclear localization signal to this condensate, and thereby actively transport the DNA to the nucleus. For example, efforts have been made to make DNA more compact by using proteins such as HMG-1 and histones, as well as poly-L-lysines (Jose C. Perales et al., E. J. B. 266: 255-266 (1994)), and cationic liposomes (J. Zabner et al., J. B. C. 270: 18997-19007 (1995)).
However, the synthetic chemical approach had problems with solubility and homogeneity of the complex with DNA, and with the varying degrees of condensation of DNA dependent on the salt concentration. Moreover, construction of the complex is possible only under highly alkaline conditions and impossible under physiological conditions, which has been one of the problems to be solved for practical use.
It has been suggested that, on the viruses that infect animals such as adenovirus and Sv40, the nuclear localization signals exist in their capsid proteins, and they function to actively translocate their DNA at the early stage of infection (Urs. F. Greber and Harumi Kasamatsu, Trends in Cell Biology 6: 189-195 (1996)). It has been also suggested that the SV40 particle with its diameter of 45 nm invade the nucleus in the form of virion (K. Hummeler et al., J. Virol. 6: 87-93 (1970)). Furthermore, MS-2 phage is reported to have a transport system in which exogenous substances are enveloped by the capsid (International Application published in JapanNo. Hei-508168). However, any transport system using virus particles, which is capable of using long chain DNA and translocating the DNA into the nucleus, has not been reported.
USPTO Backgrounds
USPTO Backgrounds