Phage with nuclear localization signal

Details Phage with nuclear localization signal Phage with nuclear localization signal

2001-04-27

2004-07-06

Leffers, Jr., Gerald G. (Department: 1636)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Bacteria or actinomycetales; media therefor

C435S235100, C435S320100, C435S252300, C435S325000, C435S440000, C435S456000, C435S471000, C536S023100, C536S024100

Reexamination Certificate

active

06759231

ABSTRACT:

TECHNICAL FIELD
The present invention relates to the field of genetic engineering, especially to the transportation of exogenous materials by means of virus particles.
BACKGROUND ART
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 Japan No. 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.
DISCLOSURE OF THE INVENTION
An objective of the present invention is to provide a system that enables delivering genes introduced into cells to the nucleus. More specifically, the objective of the invention is to provide a &lgr; phage with a nuclear localization signal exposed on the outer surface of its head, and capable of packaging long chain DNA.
In order to translocate long chain DNA into the nucleus, it is necessary to condense the DNA to nearly 40 nm, the size of nuclear membrane pore. The present inventors paid attention to the head of a &lgr; phage, which is able to compactly package desired long chain DNA in vitro and to protect the DNA from the attack by external DNases, and used it as a carrier of the DNA. Furthermore, we paid attention to the phenomena that the viruses that infect animals can invade the nucleus in the form of virion in virtue of nuclear localization signals in their capsid proteins, and attempted to actively transport DNA into the nucleus by preparing and using the &lgr; phage head to which a nuclear localization signal has been attached. More specifically, we used the following steps.
First, we constructed a vector that expresses a fusion protein between the gpD protein, which is one of the proteins to constitute the &lgr; phage head, and the nuclear localization signal sequence, transformed
Escherichia coli
with this vector, then infected the transformants with a mutant &lgr; phage incapable of expressing gpD in the
E. coli
cells (hereinafter designated as “D amber phage”). By plaque formation analysis and western blot analysis using an anti-gpD antibody, we have confirmed that the mutant phage was complemented by the fusion protein between the gpD protein and the nuclear localization signal sequence expressed by the vector and, a &lgr; phage having the nuclear localization signal attached to its head was obtained. That is, we have found that the fusion protein expressed in
E. coli
has been complementarily integrated into the phage head which does not express the protein.
Next, we have obtained a similar result by introducing the vector that expresses the fusion protein between the gpD protein and the nuclear localization signal sequence into the
E. coli
lysogenized by the mutant &lgr; phage, and by heat-inducing the lysogenic phage. More specifically, we introduced the vector that expresses the above fusion protein into the
E. coli
lysogenized by the D amber phage, and heat-induced the transformants. As the result, the phage whose head has not incorporated the fusion protein and consists of the gpE protein became sensitive to EDTA, while the phage which has incorporated the fusion protein exhibited resistance to EDTA. Next, we treated the resulting phage with EDTA and measured the titer. As a result, it was revealed that the phage packaged with 80% genome size DNA was constructed, and that the fusion protein was incorporated in the phage head. In addition, we have confirmed that the phage had the nuclear localization signal exposed on the outer surface of the head. We also confirmed that the phage incorporating the fusion protein was formed in the same manner even when we used 100% genome size DNA. Furthermore, we introduced the phage having the nuclear localization sign

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