Early detection of infectious agents of human prion diseases such as CJD by using an animal model, etc. is needed in order to rapidly determine prion infections in pharmaceuticals such as blood products, foods, or cosmetics. This invention provides a screening method for infectious agents of human or non-human prion diseases in samples, which employs, as an indication, the deposition of the aberrant prion protein in the follicular dendritic cell (FDC) of a non-human animal.

Description of the Invention

DISCLOSURE OF THE INVENTION

Under the above circumstances, we have conducted concentrated studies in which we prepared novel recombinant prion proteins derived from human and mouse prion proteins and prepared transgenic animals and knock-in animals comprising the aforementioned proteins introduced therein. As a result, we succeeded in developing a very effective screening method which can detect infectious agents of human or non-human prion diseases in samples within an unprecedentedly short period of time.

Specifically, the present invention provides the following (1) to (26).

(1) A screening method for infectious agents of human or non-human prion diseases in a sample, which employs, as an indication, the deposition of an aberrant prion protein in the follicular dendritic cell (FDC) of a non-human animal.

(2) The screening method for according to (1) above, wherein the non-human animal is a transgenic animal that expresses a humanized prion protein gene.

(3) The screening method for according to (1) above, wherein the non-human animal is a knock-in animal that expresses a humanized prion protein gene.

(4) The screening method for according to any one of (1) to (3) above, wherein the sample is administered intraperitoneally, intracerebrally, intravascularly, or orally to the non-human animal.

(5) The screening method for according to any one of (1) to (4) above, wherein the deposition of the aberrant prion protein in the FDC is detected by a histological detection method, electrophoresis, and/or binding assay.

(6) The screening method for according to any one of (1) to (5) above, wherein the deposition of the aberrant prion protein in the FDC is detected 14 to 700 days after the sample administration.

(7) The screening method for according to any one of (1) to (6) above, wherein the humanized prion protein is prepared by substituting a part of the exon 3 of the non-human animal prion protein gene with a part of the exon 3 of the human prion protein.

(8) The screening method for according to any one of (1) to (6) above, wherein the humanized prion protein is prepared by substituting 6 residues on the C-terminal side among human-specific amino acid residues in the human prion protein with corresponding amino acid residues in the non-human animal prion protein that is used in the screening.

(9) The screening method for according to any one of (1) to (6) above, wherein the humanized prion protein comprises an amino acid sequence as shown in SEQ ID NO: 6 or 7.

(10) A recombinant humanized prion protein, which is encoded by a recombinant gene in which a part of the exon 3 of the non-human animal prion protein gene is substituted with a part of the exon 3 of the human prion protein gene.

(11) A recombinant humanized prion protein, wherein 6 residues on the C-terminal side among human-specific amino acid residues in the human prion protein are substituted with corresponding amino acids in non-human animal prion protein.

(12) A recombinant humanized prion protein, which comprises an amino acid sequence as shown in SEQ ID NO: 6 or 7.

(13) A gene or a fragment thereof, which encodes the protein according to any one of (10) to (12) above.

(14) A gene or a fragment thereof, which comprises a nucleotide sequence as described in the following:

(a) the nucleotide sequence as shown in SEQ ID NO: 4;

(b) a nucleotide sequence that is a degenerate sequence of the nucleotide sequence as shown in SEQ ID NO: 4; or

(c) a nucleotide sequence that is hybridizable with the sequence according to (a) or (b) under stringent conditions and encodes the protein according to (10) above.

(15) A vector, which comprises the gene or a fragment thereof according to (13) or (14) above.

(16) A transgenic animal having the gene or a fragment thereof according to (13) or (14) above introduced therein.

(17) A knock-in animal having the gene or a fragment thereof according to (13) or (14) above introduced therein.

(18) A transgenic animal, which expresses a humanized prion protein in the FDC.

(19) A knock-in animal, which expresses a humanized prion protein in the FDC.

(20) A transgenic animal, which expresses the protein according to any one of (10) to (12) above in its brain and/or FDC.

(21) A knock-in animal, which expresses the protein according to any one of (10) to (12) above in its brain and/or FDC.

(22) A method for producing a transgenic animal, which expresses the protein according to any one of (10) to (12) above.

(23) A method for producing a knock-in animal, which expresses the protein according to any one of (10) to (12) above.

(24) The method for producing a knock-in animal according to (23) above comprising the following steps of:

(a) constructing a vector containing a non-human prion protein gene or a fragment thereof;

(b) substituting a part of the exon 3 of the non-human prion protein gene with a part of the exon 3 of a human prion protein;

(c) inserting a loxp-surrounded antibiotic-resistant gene into the 3'-non-translation region;

(d) introducing the resulting modified vector into the non-human ES cell;

(e) producing a chimera animal from a clone having homologous recombination; and

(f) removing the antibiotic-resistant gene by introducing a Cre enzyme-expressing plasmid into a fertilized egg of the F1 animal.

(25) A screening method for a preventive and/or therapeutic agent for human or non-human prion diseases, which utilizes the transgenic animal according to (18) or (20) above or the knock-in animal according to (19) or (21) above.

(26) A method, which utilizes the transgenic animal according to (18) or (20) above or the knock-in animal according to (19) or (21) above, for performing safety tests on various pharmaceuticals such as blood products derived from human or non-human animal organs, foods, or cosmetics.

This description includes part or all of the content as disclosed in the description and/or drawings of Japanese Patent Application No. 2001-24279, which is a priority document of the present application.

EMBODIMENTS FOR CARRYING OUT THE INVENTION

The present invention is hereafter described in more detail.

In this description, the term "non-human animal" refers to: a mammalian animal such as a mouse, rat, hamster, guinea pig, rabbit, pig, cattle, sheep, cat, or dog; bird; or fish. In the present invention, non-human animals are not particularly limited, and a mouse is particularly preferable from the viewpoints of breeding and handling.

In this description, the term "aberrant prion protein" refers to a prion protein, which has a conformation that is different from that of a normal prion protein, is made insoluble by a surfactant, and is not partially digested with protease. In human and animal prion diseases, this aberrant prion protein is always present in addition to a normal prion protein. This presence can be confirmed by, for example, Western blotting after treatment with protease, detection of amyloid fibers constituted by an aberrant prion protein using an electron microscope after the extraction of the protein, or detection by immunostaining utilizing the autoclave method, which we have developed (Shin, R. W. et al., Lab. Invest. 64: 693-702 (1991); Kitamoto, T. et al., J. Virol. 65: 6292-6295 (1991); Kitamoto, T. et al., Am, J. Pathol. 140: 1285-1294 (1992)).

The term "infectious agent" used herein refers to a factor that is capable of developing the prion disease when administered to a human or the transgenic animal or knock-in animal according to the present invention. Specific examples thereof include an aberrant prion protein derived from human or non-human animals or a fragment thereof, and a substance comprising the same. Examples of such samples comprising infectious agents include pharmaceuticals such as blood products derived from human or non-human animals, foods, and cosmetics.

Further, the term "humanized prion protein" used herein refers to a prion protein in which a part thereof, which is originally expressed by its non-human animal host, is substituted with a sequence of a human prion protein. Examples thereof include a recombinant humanized prion protein that is encoded by a recombinant gene in which a part of the encoding region in a non-human animal prion protein gene has been substituted with a corresponding region of a human prion protein gene, and a recombinant humanized prion protein in which a part of the residues in the human-specific amino acid residues of the human prion protein has been substituted with corresponding amino acid residues of the non-human animal prion protein.

An example of a humanized prion protein that is particularly preferably used in the present invention is a recombinant humanized prion protein, which is encoded by a recombinant gene in which a part of the exon 3 in the non-human animal prion protein gene is substituted with a part of the exon 3 in the human prion protein gene. This recombinant humanized prion protein can be obtained in the following manner. A part of the exon 3 in the prion protein gene of the non-human animal such as a mouse is substituted with a part of the exon 3 in the human prion protein gene by recombination to obtain a gene, and the resulting gene is incorporated into, for example, a vector to introduce it into a host to be expressed therein. The phrase "a part of the exon 3" refers to a sequence that essentially comprises the region between the SmaI site and the BstEII site in the prion protein translation region of the exon 3. In the prion protein gene, a protein translation region is present only in the exon 3.

Techniques of genetic engineering that are described in this description such as gene recombination are commonly used in the art, and those skilled in the art can suitably carry out them based on the description given herein.

Specifically, the humanized prion protein according to the present invention is expressed by a knock-in animal that is obtained by, for example, the following steps of:

(a) constructing a vector containing a non-human prion protein gene or a fragment thereof;

(b) substituting a part of the exon 3 of the non-human prion protein gene with a part of the exon 3 of a human prion protein;

(c) inserting a loxp-surrounded antibiotic-resistant gene into the 3'-non-translation region;

(d) introducing the resulting modified vector into the non-human ES cell;

(e) producing a chimera animal from a clone having homologous recombination; and

(f) removing the antibiotic-resistant gene by introducing a Cre enzyme-expressing plasmid into a fertilized egg of the F1 animal.

The humanized prion protein can be obtained by any technique known to those skilled in the art such as site-directed mutagenesis or chemical synthesis as one in which, among amino acid residues that are known to be human-specific, for example, 6 residues on the C-terminal side of the human prion protein are substituted with corresponding amino acid residues of the non-human animal prion protein.

The base sequence of the human prion protein gene is known to be the one as shown in SEQ ID NO: 1 and the amino acid sequence thereof is known to be the total amino acid sequence as shown in SEQ ID NO: 2. It becomes a mature protein through processing with a signal sequence comprising 22 amino acids at the N-terminus and an amino acid sequence as shown in SEQ ID NO: 3 from which 23 amino acids at the C-terminus have been deleted. In SEQ ID NO: 3, residues 33, 34, 50, 58, 75, 87, 90, 116, 121, 123, 133, 144, 146, 193, 197, 198, 205, 206, and 208 are presumed to be human-specific (Kretzschmar, H. A. et al., DNA 1986, 5: 315-24). We have made various studies concerning the substitution of these human-specific amino acids with corresponding amino acids of non-human animals. As a result, we found that a sequence in which 6 residues on the C-terminal side are substituted with corresponding amino acid residues of the non-human animal prion protein is particularly preferably used in the screening method according to the present invention. An embodiment of this sequence is shown in SEQ ID NO: 6 or 7. SEQ ID NO: 6 is methionine at codon 129, and SEQ ID NO: 7 is valine at codon 129. This polymorphism is observed in a normal human prion protein and is also present in aberrant prion proteins that are observed in various human prion diseases.

The present invention also provides a gene or a fragment thereof encoding the humanized prion protein according to the present invention and a vector comprising the gene or a fragment thereof.

The gene encoding the humanized prion protein according to the present invention encodes a recombinant humanized prion protein comprising an amino acid sequence as shown in SEQ ID NO: 6 or 7. Examples of other forms include a gene comprising a nucleotide sequence as shown in SEQ ID NO: 4, a gene comprising a nucleotide sequence that is a degenerate sequence of the nucleotide sequence as shown in SEQ ID NO: 4, and a gene comprising a nucleotide sequence that is hybridizable with these sequences under stringent conditions. The stringent conditions used herein refer to a set of conditions under which a so-called specific hybrid is formed. For example, nucleotide sequences that are highly complementary to each other, i.e., nucleotide sequences that are at least 90%, and preferably at least 95% complementary to each other hybridize with each other, but nucleotide sequences that are less complementary do not hybridize under these conditions. More specifically, sodium concentration is 15 to 300 mM and preferably 15 to 75 mM, and temperature is between 50 and 60.degree. C. and preferably between 55 and 60.degree. C. A gene may be either DNA or RNA, and it may be obtained by synthesis. A "fragment" refers to a part of the prion protein gene and preferably comprises a nucleotide sequence that encodes the region between codon 85 and codon 230 of SEQ ID NO: 2. Alternatively, a fragment may be a defective type of this sequence, which refers to a sequence comprising at least approximately 300 nucleotides.

Any vector comprising the gene or a fragment thereof may be used as long as the gene can be expressed in a host animal cell into which it has been introduced. The vector is not particularly limited as long as it is used in the art. An example thereof is a vector the expression of which can be observed in any cell, which utilizes an actin promoter. A promoter, enhancer, other regulator gene, or the like that can regulate the expression of the gene can be suitably incorporated. Examples of promoters that can be preferably used in the present invention include a promoter that accelerates the expression in the FDC, such as a promoter of the CD21 (Cr2) gene. A reference can be made to literature, for example, Zabel, M. D. et al., J. Immunol. 2000 Oct. 15; 165 (8): 4437-45.

Meanwhile, the preparation of transgenic mice comprising a prion protein gene introduced therein has been already reported (Telling G. C. et al., Cell 1995, Oct. 6: 83 (1) 79-90). One example is a transgenic mouse, which comprises a completely human-type prion protein gene introduced therein as shown in FIG. 1C (see Original Patent). The incubation period therein before the development of CJD was approximately 250 days. Another example is a transgenic mouse, which comprises a protein gene in which the region between the KpnI site and the BstEII site is a sequence derived from a human prion protein, and the N-terminus and the C-terminus are sequences derived from mouse prion proteins introduced therein as shown in FIG. 1A (see Original Patent). The incubation period therein is approximately 200 days. In contrast, the transgenic mouse according to the present invention comprises a protein gene in which the region between the N-terminus and the BstEII site is a sequence derived from a human prion protein and the C-terminus is a sequence derived from a mouse prion protein introduced therein as shown in FIG. 1B (see Original Patent).

We prepared transgenic mice that comprise a gene (SEQ ID NO: 6 or 7) encoding a protein that is schematically shown in FIG. 1B introduced therein, and succeeded in obtaining mice, the incubation period therein before the development of diseases is 147 days on average in the case of homozygous mice.

Accordingly, the present invention provides transgenic animals that express a recombinant humanized prion protein as shown in SEQ ID NO: 6 or 7. In the present invention, transgenic animals may be homozygous or heterozygous, and homozygous animals are more preferable.

However, it is unknown into which position and chromosome of the transgenic animal the gene is incorporated. In order to obtain a transgenic animal having a short incubation period as described above, it has to be mated with a knockout animal of a prion protein gene. We have previously established a method for substituting genes using the ES cell in which a mouse gene is substituted with a human type gene (Kitamoto, T. et al., Biochem. Biophys. Res. Commun. 222: 742-747 (1996)). We improved this method and prepared knock-in mice in which a part of naturally occurring mouse gene was substituted with a human gene having a sequence as shown in SEQ ID NO: 1. These knock-in mice had the incubation period of 151 days on average.

Specifically, the present invention provides knock-in animals that express the recombinant humanized prion protein as shown in SEQ ID NO: 6, 7, or 10. In the present invention, the knock-in animals may be homozygous or heterozygous, and homozygous animals are more preferable. The expression of recombinant humanized prion proteins are observed in their whole bodies, and the expression can be detected particularly in their brains and/or spleens.

An advantage of the method for substituting genes using the ES cell is that it eliminates the need for mating with a knockout animal to remove a mouse endogenous prion protein, which was problematic in the case of the transgenic animals. Also, the distribution and the level of gene expression are similar to those of normal animals, and thus, completely natural expression can be realized.

When human or non-human animal-derived infectious agents are administered to the model animals comprising humanized prion proteins introduced therein, it is presumed that the deposition of the aberrant prion protein is detected in the FDC. Accordingly, human-type transgenic animals can be used for a method for rapidly screening infectious agents that employs the deposition of the aberrant prion protein in the FDC as an indication.

Based on the above presumption, we investigated the FDC after the resulting transgenic and knock-in mice developed the disease. Accordingly, aberrant prion proteins were not substantially detected in the transgenic mice, however, the human-type aberrant prion proteins were found to be deposited in the FDC of the spleen, lymph node, and intestinal lymphoid tissue (Peyer's patch) of the knock-in mice (FIG. 2A (see Original Patent)).

Up to the present, it was impossible to determine whether the deposition of the aberrant prion proteins in the FDC was caused by aggregation of aberrant prion proteins contained in the materials that were administered at the time of infection experiment or caused by conversion of normal prion proteins into aberrant prion proteins in the FDC. The C-terminus of the recombinant prion protein as shown in SEQ ID NO: 6 or 7 according to the present invention is of a mouse-type as described above. Accordingly, we then prepared an antibody to the C-terminus of the human-type prion protein. The FDC was investigated using this antibody, and the FDC could not be stained (FIG. 2B (see Original Patent)). This indicates that the aberrant prion proteins deposited in the FDC were not simple aggregation of human-type aberrant prion proteins contained in the infectious agents.

We further examined the deposition of the aberrant prion proteins in the spleen by Western blotting, which was already confirmed by immunostaining. We confirmed the presence of the aberrant prion protein in the spleen of the knock-in mouse (FIG. 3, see Original Patent). Furthermore, the infectivity in the spleen was confirmed by the direct infection experiment.(Table 2, Inoculation Experiment 3, see Original Patent).

The knock-in mice that we prepared had an incubation period of 151 days on average before the development of the disease. We examined the shortest period before the deposition of the aberrant prion proteins in the FDC could be detected. As a result, it was found out that detection could be made in a very short time period of 14 days in the assay system using the knock-in mice according to the present invention (Table 6, see Original Patent).

In contrast, the Ki-HuM mice that expressed complete human prion proteins (8 amino acids are deleted from SEQ ID NO: 10) required a very long incubation period of 643 days, although all of them developed the disease. In the immunohistostaining of the FDC in the spleen 75 days after the intraperitoneal inoculation, the rate of detection was jut 80% (4 out of 5 mice). As with the case of Ki-ChM, the deposition of the aberrant prion protein in the FDC was confirmed (Table 7, see Original Patent). Specifically, with the use of the knock-in method in which the prion protein is effectively expressed in the FDC, aberration of prion protein was effectively observed in the FDC in the completely human-type animal to some extent. In this type of animal, the aberration of the prion protein is less likely to occur in the brain. Accordingly, with the use of the knock-in animals prepared by the method, which we claimed wherein the prion protein is effectively expressed in the FDC, it is possible to detect the deposition of the aberrant prion protein of non-human animals such as cattle in the FDC of the knock-in mouse, which expresses the prion protein of non-human animals such as cattle, as well as the prion protein gene in which a specific region in the knock-in animal has been substituted.

The knock-in animal according to the present invention was found to be unprecedentedly highly susceptible to prions as described above. Accordingly, the present invention also provides a biological assay, which employs the knock-in animal according to the present invention, and the deposition of the aberrant prion protein in the FDC thereof as an indication, thereby detecting the development of the human or non-human animal prion diseases in any given samples. This assay can be used as a screening method for the infectious agents of the prion diseases in any given samples.

Examples of samples include various pharmaceuticals such as blood or blood products derived from human or non-human animal organs, foods, and cosmetics. The sample is allowed to infect non-human animals, preferably transgenic animals that express humanized prion proteins, and particularly preferably the knock-in animals according to the present invention. The sample may be administered intraperitoneally, intracerebrally, intravascularly, or orally. Intraperitoneal administration is preferable since a relatively large amount of sample can be administered as described below. More specifically, for example, approximately 2 ml of a solution containing blood, organ, or a preparation derived thereform is administered to the knock-in animals intraperitoneally to infect them.

After the infection, the deposition of the aberrant prion protein in the FDC can be detected to determine whether the prion disease was developed or not using the sample. Any detection method may be used as long as it can detect the deposition of the aberrant prion protein in the FDC. Examples of detection methods include histological detection in which the deposition of the aberrant prion protein in the FDC of the Peyer's patch, which is a lymphoid tissue of the spleen, lymph node, or intestine is observed using an electron microscope, etc., electrophoresis, and/or in situ hybridization, Western blotting, or ELISA in which the antibody to the aberrant prion protein is labeled with a radioactive or nonradioactive label and binding assay is conducted. Detection may be carried out with the elapse of time after the infection. Alternatively, the period before the aberrant prion protein is deposited in the FDC in the presence of the infectious agents is previously determined by the control sample, and the deposition of the aberrant prion protein in the FDC may be detected after a determined period has elapsed following sample infection, for example, 75 days. The period of detection is not particularly limited as long as it is between 14 days and 700 days after the administration. The deposition of the aberrant prion protein in the FDC can be employed as an indication to determine the presence of the infectious agent within a significantly shorter period of time than conventional methods.

Up to the present, no cases at all were reported in which the aberrant prion protein has been observed in the FDC of a mouse model of human prion diseases, especially CJD. This is consistent with the fact that CJD is substantially sporadic and is not caused by exogenous infection. nvCJD, which occurred in Britain in 1996, is considered to be associated with the infectious agents derived from bovine spongiform encephalopathy and is classified as a transmissible prion disease. Accordingly, it is very critical to be able to detect the infectious agent.

The present invention also provides a screening method wherein the infectious agent is administered intraperitoneally, intracerebrally, intravascularly, or orally.

In the past, the infection experiments were mainly carried out by intracerebral administration. When a sample containing the infectious agent is administered intracerebrally, the dose is limited. The sample can be administered to the brain of a mouse only in an amount of 20 .mu.l at the maximum, however, 2 ml of the sample can be administered to the abdominal cavity. In addition, the administration can be made several times.

In a biological assay for general organs comprising blood in which the concentration of the infectious agents are considered to be low, this quantitative difference of as much as 100 times is significant and affects the detection sensitivity. In the case of mice infected with CJD, for example, LD50 is 10.sup.-8/g in the brain and 10.sup.-3/g or lower in the blood according to infection experiments by intracerebral administration. These results were obtained when 20 .mu.l of the sample was used for intracerebral administration. In contrast, when 100-fold amount of the sample were intraperitoneally administered, the concentration in the organ (blood) having LD50 of 10.sup.-3/g was brought to the same level as highly sensitive organs (such as the spleen) having LD50 corresponding to 10.sup.-5/g. Accordingly, intraperitoneal administration is a method with much promise as a future screening method for the infectious agents in samples.

Up to the present, no report has been made concerning the development of the disease upon intraperitoneal administration of the infectious agents to a human-type transgenic mouse model. The data on the transgenic mouse that we presented suggests the difficulty of developing the disease from the periphery by intraperitoneal administration without the deposition of the aberrant prion protein in the FDC. In the SCID (deficient of T cell and B cell) mouse, which developed the disease by intracranial administration but does not develop the disease by intraperitoneal administration, the deposition of the aberrant prion protein in the FDC is not observed. This indicates that the aberrant prion protein is not transmitted to the brain without the deposition thereof in the FDC.

In the knock-in mice according to the present invention, the human-type aberrant prion protein was deposited only after the administration of the infectious agents. They were subjected to intraperitoneal administration. After an average of 283 days following the administration, all 11 mice had developed the diseases. Specifically, the infection of a human prion through the periphery was successfully performed for the first time in the present invention (Table 5, see Original Patent).

Further, the present invention provides a screening method for preventive and/or therapeutic agents for human or non-human prion diseases, which utilizes the transgenic animal of the present invention or the knock-in animal of the present invention.

As described above, the deposition of the aberrant prion protein in the FDC can be detected within a short period after administration of infectious agent at high sensitivity with the use of the transgenic animal or knock-in animal according to the present invention. Screening can be performed with this technique in which an agent, which is capable of blocking the deposition of the aberrant prion protein in the FDC or the migration thereof from the FDC to the brain, can be administered to test animals before and after the administration of the infectious agent or simultaneously with the administration of the infectious agent. This enables the development of preventive and/or therapeutic agents for human or non-human prion diseases, which has been and is currently seriously problematic and any effective agent for which has not yet been reported.
 

Claim 1 of 5 Claims

1. A process of diagnosing the presence of infectious agents of human prion diseases in a sample, comprising a) obtaining a knock-in rodent that expresses a humanized prion protein, wherein the humanized prion protein is prepared by substituting the region between the SmaI site and the BstEII site in exon 3 of the non-human animal prion gene with the region between the SmaI site and the BstEII site in exon 3 of the human prion gene; b) administering to the rodent a sample suspected of containing infectious agents of human prion diseases; and c) from 14 to about 75 days after the administration, detecting deposition of an aberrant prion protein in follicular dendritic cell (FDC) of the rodent, wherein the deposition indicates the presence of the infectious agent in the sample.

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