Patent Application
20050053583
This Nonprovisional application claims priority under 35 U.S.C. §119(a) on Patent Application No. 2003/307666 filed in Japan on Aug. 29, 2003, the entire contents of which are hereby incorporated by reference.
The present invention relates to a method of inhibiting the prion protein expression and use of the same.
“Prion disease” is a generic name of fatal and transmissible spongiform encephalophaties, which is characterized by pathological changes such as astrocyte hyperplasia, microgria hyperplasia, amyloid plaque, and neuron death associated with spongiform degeneration. A typical prion disease in animals is scrapie, which occurs in sheep and goats. Human prion diseases include Creutzfeldt-Jakob disease, Gerstmann-Sträussler-Scheinker syndrome, Kuru, fatal familial insomnia. Those diseases are infectious.
It is postulated that a pathogenic factor of the prion disease is a “prion” that is constituted of only protein but does not have a nucleic acid. All mammals have one prion protein gene. It is considered that prion protein has important roles in the nervous system. The prion protein is of two types: normal prion protein (PrPc: cellular prion protein) and abnormal prion protein (PrPsc: scrapie prion protein), which are same in terms of amino acid sequence but different in protein conformation. The normal prion protein is expressed in normal tissues and has no pathogenic activity, whereas the abnormal prion protein has photogenic activity. Moreover, the normal prion protein is sensitive to protease and soluble in detergents, while the abnormal prion protein is resistance to protease and insoluble in the detergents.
The following model is suggested as a hypothesis for mechanism of the pathogenesis of prion disease: an abnormal prion protein taken into a normal individual enters the brain thereof, and makes a contact with a normal prion protein already existing inside the brain. As a result, the normal prion protein is converted into an abnormal prion protein by some kind of post-translational modification. The abnormal prion protein thus generated by the conversion converts normal prion protein nearby. As a result, the abnormal prion proteins are accumulated inside the brain, thereby causing the prion disease. Today, this model is widely accepted.
Intensive researches have been carried out for development of effective methods of treating prion diseases. For example, researches have been carried out for a method of inhibiting the conversion from the normal prion protein to the abnormal prion protein, a method of converting the abnormal prion protein back to the normal prion protein, development of vaccines by using anti-prion protein antibodies or attenuated prion strains, and the like attempts.
On the other hand, RAN interference (RNAi) is a phenomenon that double-stranded RNA entering cells causes degradation of mRNA that is homologous to a sequence of the RNA and thereby inhibits gene expression. The researches have showed that siRNA (small interfering RNA), which is short double-stranded RNA, specifically inhibits expression of the gene that is homologous to the siRNA.
The inventors have already developed vectors for producing siRNA in mammalian cells by using a U6 promoter, which is a RNA polymerase III type promoter (Miyagishi M, Taira K. U6 promoter-driven siRNAs with four uridine 3′ overhangs efficiently suppress targeted gene expression in mammalian cells. Nat Biotechnol. 2002 May; 20(5): 497-500.)
Recently, Tilly et al. reported that expression of the normal prion protein is inhibited in prion non-infected cells by using an siRNA expression vector having the U6 promoter. (Tilly G, Chapuis J, Vilette D, Laude H, Vilotte J L. Efficient and specific down-regulation of prion protein expression by RNAi. Biochem Biophys Res Commun. 2003 Jun. 6;305(3):548-51.)
Even though intensive studies have been carried out for developing effective methods of treating prion diseases as described above, no effective treating method has been successfully developed. There is a desperate desire for effective methods of treating prion diseases, while there are a large number of reports on a novel type Creutzfeldt-Jakob disease caused by infection from the mad-cow disease (bovine spongiform encephalopathy) which is a prion disease for cattle, and on an iatrogenic prion disease caused as a result of transplantation of dura mater of an infected brain. Therefore, there is a need of a novel knowledge that is applicable in prevention and treatment of the prion disease.
According to the hypothesis of the mechanism of the pathogenesis of prion disease, it is considered that the abnormal prion protein is produced by structural conversion of the normal prion protein. Therefore, it is expected that inhibition of expression of the normal prion protein inhibits the accumulation of the abnormal prion protein.
Tilly et al. who reported the inhibition of the normal prion protein expression in the prion non-infected cells by using an siRNA expression vector having the U6 promoter. However, Tilly et al. did not report that the abnormal prion protein accumulation is inhibited by inhibiting the expression of the normal prion protein in the prion-infected cells.
In view of the aforementioned problem, an object of the present invention is to provide a method of inhibiting accumulation of an abnormal prion protein, the method being applicable in prevention and treatment of the prion diseases, and to provide a use of the method.
The inventors of the present invention accomplished the present invention by finding out that a prion protein specific siRNA inhibits expression of a normal prion protein in prion-infected cells, and also inhibits the accumulation of the abnormal prion protein. An abnormal prion protein inhibition method of the present invention inhibits the accumulation an abnormal prion protein by inhibiting expression of a normal prion protein in prion-infected cells. As a method of inhibiting the expression of the normal prion protein, it is preferable that the expression of the normal prion protein be inhibited by using a prion protein specific siRNA. Especially, it is preferable that an siRNA expression vector (prion protein specific siRNA expression vector) be introduced into the prion-infected cells, the siRNA expression vector having a prion protein specific sequence. It is preferable that the siRNA expression vector be a virus vector. It is especially preferable that the virus vector (that is, the siRNA expression vector) be an adenovirus vector, a herpesvirus vector, or a retrovirus vector.
As to a prion protein targeted for the inhibition of the expression, it is preferable that the prion proteins be human prion proteins, domestic animal prion proteins, or laboratory animal prion proteins.
Moreover, the present invention is a method of preventing and/or treating a prion disease by using the abnormal prion protein inhibition method.
Moreover, the present invention includes an siRNA expression vector having a prion protein specific sequence. The siRNA expression vector is preferably a virus vector, especially preferably an adenovirus vector, a herpesvirus vector, or a retrovirus vector.
Further, the present invention includes a medicament for preventing and/or treating a prion disease, the medicament containing a prion protein specific siRNA or an siRNA expression vector having a prion protein specific sequence. Further, the present invention includes a kit for use in prion research, containing the siRNA expression vector.
For a fuller understanding of the nature and advantages of the invention, reference should be made to the ensuing detailed description taken in conjunction with the accompanying drawings.
(1) Method of Inhibiting Abnormal Prion Protein
A method of the present invention for inhibiting abnormal prion protein (hereinafter, may be referred to as the present inhibition method) is a method of inhibiting accumulation of an abnormal prion protein by inhibiting expression of a normal prion protein in prion-infected cells. As a result of the inhibition of the normal prion protein expression in the prion-infected cells, accumulation of the abnormal prion protein is inhibited. There is no particular limitation in terms of a method of inhibiting the expression of the normal prion protein, provided that the method can inhibit the expression of the normal prion protein in the prion-infected cells. Here, the “prion” means an infectious protein, which is considered as a pathogenic factor for human Creutzfeldt-Jakob disease or the like diseases, that is, the “prion” means the abnormal prion protein. The “prion-infected cells” are persistently producing the abnormal prion protein. Moreover, the “normal prion protein” is a prion protein being expressed in a normal tissue and having no pathogenic activity. The “abnormal prion protein” is a prion protein having the same amino acid sequence as the normal prion protein but having a different protein conformation from the normal prion protein, and having a pathogenic activity.
[siRNA (small interfering RNA)]
One method of inhibiting the expression of the normal prion protein is use of siRNA (No.1) expression vector having a prion protein specific sequence, that is, an RNA interfering method. “RNA interference” is a phenomenon that double-stranded RNA entering cells causes degradation of mRNA that is homologous to a sequence of the RNA and thereby inhibits gene expression. The “siRNA” is short double-stranded RNA that can cause the RNA interference. Many studies have demonstrated that the siRNA inhibits the gene expression specifically.
A nucleotide sequence of siRNA may be designed based on a sequence of a prion protein gene of species to which the method of the present invention for inhibiting the abnormal prion protein accumulation is applied. Manuals for designing siRNA have been published in many literatures and books. The prion protein specific siRNA can be designed in accordance with the manuals. In Examples descried later, 5 kinds of siRNAs were designed based on a nucleotide sequence of a mouse prion protein gene (GenBank accession No. M13685), in order to inhibit the expression of the normal prion protein in a mouse neuroblast cell line. Targeted nucleotides were 83rd to 103rd nucleotides in No. 1, 180th to 200th nucleotides in No. 2, 370th to 390th nucleotides in No. 3, 651st to 671st nucleotides in No. 4, and 785th to 805th nucleotides in No.5.
The siRNA may be directly introduced into cells by lipofection method or the like. Moreover, a vector which is so constructed as to express siRNA in the cells (siRNA expression vector), may be introduced into cells thereby expressing the siRNA in the cells. In case the siRNA expression vector is used, the siRNA can exist in a longer time in the cells than in case where the siRNA is directly introduced into the cells. Thus, the use of the siRNA expression vector is advantageous over the direct introduction of siRNA in that the use of the siRNA expression vector gives higher efficiency in the inhibition of the gene expression. Therefore, it is preferable to inhibit the expression of the normal prion protein by introducing the siRNA expression vector into the cells.
[siRNA Expression Vector]
The siRNA expression vector is not particularly limited provided that the vector can express the siRNA in the cells when introduced in the cells. An example of such a vector is shown in
While the siRNA expression vector may be plasmid vector, a virus vector is preferable in case the vector is to introduced into a mammal. The use of the virus vector attains efficient expression of the siRNA in the cells. In case of the virus vector, a promoter for the expression of the siRNA, and a siRNA sequence are inserted in part of a virus gene. For example, an siRNA expression virus vector may be constructed by inserting, in part of the virus gene, the U6 promoter, the sense sequence, the loop sequence, the antisense sequence, and ttttt shown in
There is no particular limitation in terms of the virus vector. However, it is preferable to use adenovirus vector, herpesvirus vector, or retrovirus vector. The adenovirus vector is a vector for gene therapy, and has been already used for humans. The herpesvirus vector is known for high selectivity for nerve cells, the nerve cells being target cells for the prion. The retrovirus vector has such an advantage that a gene of the retrovirus is integrated into chromosomal DNA of host cells so that the siRNA can be permanently expressed. Therefore, adenovirus vector, herpesvirus vector, or retrovirus vector is suitable as the prion protein specific siRNA vector for use in mammals including humans.
Note that the present invention includes an siRNA expression vector having a prion protein specific sequence, this siRNA expression vector being formed by inserting the prion protein specific sequence into the siRNA expression vector.
[Target Organism]
Organisms to which the method of the present invention for inhibiting the abnormal prion protein are not particularly limited as long as the organism has the prion protein gene. Among the organisms, humans, domestic animals, and laboratory animals are organisms to which the present inhibition method is preferably applicable. For humans, the present inhibition method has a possibility to be used for prevention and treatment of the prion diseases, such as Creutzfeldt-Jakob disease. The domestic animals include cattle, sheep, goats, and the like animals. As the prion diseases infecting the domestic animals, the mad-cow disease for cattle, the scrapie for sheep and goats are known. The present inhibition method has a possibility to be used as prevention and treatment of the prion diseases for the domestic animals. Moreover, the use of the present inhibition method to the laboratory animals can contribute to progress of basic study of the prion diseases. The laboratory animals include: mice, rats, hamsters, guinea pigs, rabbits, dogs, monkeys, chimpanzees, and the like.
[Method of Preventing and/or Treating Prion Diseases]
The present invention includes a method of preventing and/or treating the prion disease by using the method of inhibiting the abnormal prion protein. If the accumulation of the abnormal prion protein was inhibited by the present inhibition method, it would be possible to prevent and treat the prion disease.
(2) Medicament for Preventing and/or Treating Prion Disease
By introducing, into prion-infected cells, (a) the prion protein specific siRNA or (b) the siRNA expression vector having the prion protein specific sequence, it is possible to inhibit the expression of the normal prion protein in the cells, and consequently to inhibit the accumulation of the abnormal prion protein. Therefore, for a medicament for preventing and/or treating the prion disease, it is possible to use (a) the prion protein specific siRNA or (b) the siRNA expression vector having the prion protein specific sequence. In other words, the present invention includes a medicament for preventing and/or treating the prion disease, the medicament containing the prion protein specific siRNA, and a medicament for preventing and/or treating the prion disease, the medicament containing the siRNA expression vector, of the present invention, having the prion proteins specific sequence.
The medicament contains (a) the prion protein specific siRNA or (b) the siRNA expression vector having the prion protein specific sequence, and may contain other component. For example, the medicament may contain a buffer liquid for dissolving or suspending the siRNA or the vector.
Moreover, the present invention may be put in practice as a kit including an siRNA expression vector having a prion protein specific sequence; and a member for administering the vector. The kit may have any arrangement, provided that the kit includes the siRNA expression vector having the prion protein specific sequence; and a member for administering the vector.
The member (means) for administrating the vector is not particularly limited, and may be selected arbitrarily from well-known administrating members. Specific examples of the administrating member include a syringe, a tube, a catheter, and the like.
(3) Prion Research Kit
The siRNA expression vector, of the present invention, having the prion protein specific sequence is very useful as a tool for functional analysis of the normal prion protein or analysis of accumulation mechanism of the abnormal prion protein, because the siRNA expression vector can inhibit the expression of the normal prion protein. Therefore, a prion research kit including the siRNA expression vector having the prion protein specific sequence is applicable for promoting the prion researches. That is, the present invention includes the prion research kit (kit for use in prion research) including the siRNA expression vector having the prion protein specific sequence.
The present kit contains may have any composition, as long as the present kit contains at least the siRNA expression vector having the prion protein specific sequence. Specifically, for examples, a control vector, protenase K, a gel for electrophoresis, an agent for electrophoresis, an agent for western blotting, an antibody for prion protein detection, and/or the like may be contained.
The following describes the present invention in further details, but not limit the present invention. First, siRNAs, cells, prion stains, experiment methods used in respective Examples will be explained.
[Target Regions of siRNAs]
5 kinds of siRNAs were designed based on the nucleotide sequence of a mouse prion protein gene (GenBank accession No. M13685).
[Prion protein specific siRNA expression vector]
[Cell Line Used]
N2a-58 cells that overexpressed a mouse normal prion protein was used, the N2a cells transformed by transfecting, into mouse neuroblastoma cells (N2a, ATCC CCL131), a plasmid having cDNA of a wild type mouse prion protein.
[Prion Strain Used]
Three kinds of prion strains were used in Examples, namely, Chandler prion strain, Fukukoka-1 prion strain, and 22L prion strain.
[Method of preparation of Prion-infected cells]
The prion-infected cells can be prepared by adding 1% emulsion of prion infected mouse brain to a culture medium.
[Experiment Method]
The prion protein specific siRNA expression vector or a control vector was transfected into the prion-infected cells by lipofection method. As the control vector, siRNA expression vector having a luciferase protein sequence that is irrelevant to the prion protein. After incubated for 48 hours or 96 hours, cell extracts were collected from the respective cultures. In case all the prion protein (normal prion protein and abnormal prion protein) were to be detected, the cell extracts were subjected to polyacrylamide gel electrophoresis without treating the cell extracts with protenase K. In case where only the abnormal prion protein was to be detected, the cell extracts were treated with the protenase K at 37° C. for 30 minutes, and then subjected to polyacrylamide gel electrophoresis. After that, detection of the prion protein was carried out by the western blotting method. An anti-prion protein antibody was used in the detection.
Into Chandler prion stain infected cells, the 5 kinds of prion protein specific siRNA expression vectors were transfected. After 96 hour incubation, detection of the prion proteins was carried out by the western blotting method. Results are shown in
1 μg, 2 μg, 4 μg, and 8 μg of the prion protein specific siRNA (No. 1) expression vector were transfected into Chandler prion strain infected cells. An amount of the control vector was 4 μg. After 96 hours incubation, detection of the prion protein was carried out by the western blotting method. Results are shown in
On the other hand, in PK(+) the accumulation of abnormal prion protein was inhibited in dependence on the DNA amount of the siRNA expression vector until 4 μg. However, there was almost no difference between 4 μg and 8 μg. This indicated that in the present experiment system, the DNA amount of 4 μg was enough for the siRNA expression vector to cause the inhibition of the abnormal prion protein accumulation.
The prion protein specific siRNA (No.1) expression vector was transfected into Chandler prion strain infected cells. After 48-hour incubation and 96-hour incubation, detection of the prion protein was carried out by the western blotting method. Results are shown in
In
The prion protein specific siRNA (No.1) expression vector was transfected into Fukuoka-1 prion strain infected cells. After 96-hour incubation, detection of the prion protein was carried out by the western blotting method. Results are shown in
The prion protein specific siRNA (No.1) expression vector was transfected into a 22L prion strain infected cells. After 96-hour incubation, detection of the prion protein was carried out by the western blotting method.
Results are shown in
From those results, it was proved that the abnormal prion protein inhibition method of the present invention is effective in inhibiting the abnormal prion protein accumulation in plural kinds of prion strains, even though different prion strains show different sensitivities. Hence, the abnormal prion protein inhibition method of the present invention is quite useful in clinical application.
The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2003-307666 | Aug 2003 | JP | national |
