JPH07231784A - Rna-breaking agent - Google Patents

Abstract

PURPOSE:To obtain a new RNA-breaking agent capable of entering specifically into a cell containing a virus causing an intractable infectious disease and specifically breaking the virus RNA and useful as an antiviral agent or an anticancer agent. CONSTITUTION:This RNA-breaking agent is a complex material composed of a ribozyme of the formula, 3'-X1-Y-X2-X2-5' (X1 and X2 are each independently a base sequence of a nucleic acid complemental to the base sequence of the target RNA; Y is an active base sequence for RNA breakage) and a (glyco) protein-or polypeptide-polycation conjugate of the formula, E-L-F [E is a (glyco) protein or a polypeptide the receptor of which exists on the cell membrane surface; F is a polycation; L is a linker capable of forming a covalent bond to each amino group end of E and F]. The complex material composed of the ribozyme of the formula and an ASOR-poly-L-lysin.conjugate can break RNA of hepatitis C virus. Ribozyme is bonded to the (glyco)protein- or polypeptide-polycation conjugate in a saline to form a complex material.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a specific R in a specific cell.
An RNA cleaving agent that specifically and selectively cleaves NA, and more specifically, specifically cleaves a viral RNA causing a refractory infectious disease and specifically cleaves the viral RNA, or a specific cell R that specifically enters into cancer cells and cancer cells and specifically cleaves messenger RNA (hereinafter referred to as mRNA) transcribed from a specific oncogene in the cells.
NA cleaving agent.

[0002]

BACKGROUND OF THE INVENTION 1984 T.S. of the University of Colorado. Cech
An RNA molecule having enzymatic activity, that is, a ribozyme was discovered by professors. This is because it has been proved that the ribosomal RNA (hereinafter referred to as rRNA) precursor of the protozoa Tetrahymena removes an intron unnecessary for genetic signal transmission by self-splicing without involvement of protein (Nature Vol 308,820-). 8
25, 1984).

Subsequently, similar RNA molecules having a function of self-splicing the phosphodiester bond of RNA without the involvement of proteins were discovered one after another, and a ribozyme that cleaves other RNA was also discovered. In addition, recently, an artificial ribozyme having RNA-cleaving activity has been successfully produced in a 24-base RNA molecule by using a nucleotide sequence commonly conserved among ribozymes (Nature Vo).
1 334, 585-591, 1988).

At present, the most commonly known and most well-studied ribozyme is called the hammerhead ribozyme. Since it was revealed using hammerhead ribozymes that sequence-specific cleavage can be used not only within RNA molecules of its own but also between molecules (Nature Vol 32
8, 596-600, 1987: Nature Vol.
334, 585-591, 1988), and has been attracting attention as a means for inhibiting a specific gene including medical applications.

This hammerhead ribozyme has three structural regions. First, it is the part of the base sequence that complementarily joins with the highly conserved part of the nucleotide sequence located 5'to the cleavage site of the substrate RNA. This sequence of substrate RNA is usually 5'-GUC-3 ', which is cleaved immediately after C by the ribozyme, but 5'-N ₁ UN _2-.
In the 3'sequence, N ₁ may have a cleavage activity at any nucleotide in A, G, C, U, and N ₂ may have a cleavage activity at any nucleotide in A, C, U (Nucleic
Acids Res. Vol 17, 7059-707
1, 1989). Secondly, a part consisting of a highly conserved base sequence part that imparts a ribozyme with a cleavage activity and a part of a variable base sequence. Third, there is a base sequence site that recognizes the base sequence of the RNA molecule that serves as a cleavage substrate and forms base pairs on the 5'side and 3'side. A ribozyme can be produced based on this basic structure, and it has already been proved that it has an activity of cleaving the substrate RNA in a sequence-specific manner (Nature Vol 334, 585-591, 1).
988).

Conventionally, when a ribozyme is used to cleave RNA in a sequence-specific manner in a cell, the ribozyme is transcribed as an RNA molecule from the corresponding DNA sequence operably linked to the promoter of RNA polymerase. A method of incorporating a viral vector designed to be incorporated into cells (Clin. Biotech. Vol.
2, 23-31, 1990) have been used. However, when a DNA vector encoding a ribozyme is introduced into a cell, there are some serious problems, such as mutation of the chromosomal gene of the cell by recombination and recombination with a viral gene.

On the other hand, several methods have been used as methods for introducing nucleic acids into cells. First, there is a method of incorporating nucleic acid into cells by utilizing the phagocytosis of cells, and a method of using a coprecipitation product of nucleic acid and calcium phosphate (V
irology Vol 52,456-467,19
73) or a method using DEAE dextran having a positive charge. Also, by electroporation,
There is also a method of introducing a nucleic acid using a hole temporarily formed in a cell membrane (Nucleic Acids Res.
Vol 15, 1311-1326, 1987: Nuc.
leic Acids Res. Vol 16,612
7-6145, 1988). There are also methods for directly injecting nucleic acid into cells by microinjection, but these methods are not suitable for in vivo administration.

Recently, as a method suitable for introducing a nucleic acid into a cell into a living body, a method of introducing a nucleic acid into a cell using a liposome has been used. In this method, the nucleic acid in the liposome or the nucleic acid complexed with the liposome is introduced into the cell by fusing the liposome with the cell membrane (Proc. Natl. Acad. Sci. U.
SA Vol 84,7413-7417,198
7). In addition, recently, a retrovirus vector (Science Vol 230, 10
57-1062, 1985; Mol. Cell. Bio
l. Vol 7, 3459-3465, 1987; Pr.
oc. Natl. Acad. Sci. USAVol 8
7,439-443,1990; Clin. Inv
est. Vol 88, 1043-1047, 199
1; Science Vol 256, 1550-15
52, 1992; Cancer Res. Vol 5
3, 4129-4133, 1993) and various viral vectors have been developed. However, all of these methods have the drawback that nucleic acids cannot be introduced in a tissue-specific or cell-specific manner.

Another method of introducing a nucleic acid to solve this drawback is a receptor-mediated method of incorporating a nucleic acid into a cell. That is, a complex of (glyco) protein-polycation conjugate and DNA in which a polycation is covalently bound to a protein or glycoprotein having a receptor on the cell membrane surface of a target cell [hereinafter referred to as (glyco) protein] It has been reported that those that have been formed are incorporated into the cell by a receptor present on the cell membrane of the target cell (Proc. Natl. Ac.
ad. Sci. USA Vol 87, 3655-36.
59, 1990: J. Biol. Chem. Vol 2
62,4429-4432,1987).

[0010]

[Problems to be Solved by the Invention] Conventionally, as described above,
Has a DNA sequence encoding the base sequence of ribozyme,
A DNA fragment from which a ribozyme can be obtained as a transcription product, or a DNA vector containing this DNA fragment was used, and this was introduced into cells by various means to study an RNA fragment by a ribozyme as a transcription product. As one of the cell introduction methods, the above-mentioned method of preparing a complex with a protein-polycation conjugate and introducing it into cells was also used. However, the method of producing a ribozyme as a transcription product from DNA in a cell has problems with respect to genetic safety and measures against a mutation of a target gene. The problem of genetic safety is that DNA may cause recombination with a chromosomal gene of a cell or a viral gene existing in the cell to cause a mutation in the gene. In addition, the problem regarding the response to the gene mutation is that when a target RNA of a ribozyme is a viral RNA that is apt to mutate a gene such as AIDS, a mutation occurs in the target RNA and a change in the base sequence causes The ribozyme-encoding DNA introduced into Escherichia coli becomes completely useless for therapy, and rather, only the adverse effects such as the gene mutations described above and the inhibition of various mRNAs related to cell function appear, which causes serious genotoxicity and There is a point that it causes side effects.

Therefore, the present invention directly administers a sequence-specific RNA-cleaving agent that is specifically taken up by cells into a living body, whereby the above-mentioned genetic safety and the target gene It eliminates the possibility of causing problems in response to mutations, and furthermore, using a receptor-mediated method of nucleic acid uptake into cells, selective transcription of viral genes RNA or oncogenes is performed only in the target cells. By cleaving mRNA or the like at a specific base sequence site, an attempt is made to develop a drug for regulating gene expression that acts cell-specifically and gene-specifically.

[0012]

Therefore, as a result of earnest studies, the present inventor has found that by forming a complex between a ribozyme capable of cleaving RNA and a (glyco) protein-polycation conjugate. Succeeded in solving the problem.

[0013] Namely, the present invention has the general formula _{(1) 3'-X 1 -Y} -X 2 -5 '(1) ( wherein, nucleic acids complementary X ₁ and X ₂ are the nucleotide sequence of the target RNA Of the general formula (2) A-LB (2) (in the formula, A has a receptor on the cell membrane surface of an animal). (Glyco) protein or polypeptide-polycation conjugate represented by (glyco) protein or polypeptide, B a polycation, and L a linker forming a covalent bond with the amino group terminals of A and B). An RNA cleaving agent comprising a complex with

X ₁ and X _{2 in} the general formula (1) are target RNs.
A base sequence of a nucleic acid complementary to the base sequence of A, and the target R
As for the base sequence of NA, if the cleavage site is present in it, the site and length are not a problem, but X ₁
The total number of bases of X and X ₂ is preferably 10 to 20.
As the RNA-cleaving active base sequence represented by Y, known ones can be used. Such ribozymes are RNA
It can be produced by a method known in the technical field relating to the synthesis of a molecule, a DNA molecule or an RNA-DNA chimeric molecule.

Examples of the ribozyme of the present invention for cleaving RNA of hepatitis C virus (HCV) include those represented by the following formula (3).

[0016]

[Chemical 1]

As the (glyco) protein represented by A in the general formula (2), any protein having a receptor on the membrane surface of the target cell may be used. When the target cell is a hepatocyte, acylorosomucoid (ASOR) Etc. This (glyco) protein may contain a fluorescent substance, biotin, horseradish peroxidase, carbonic anhydrase, colloidal gold or silver, and the like.

The polycation represented by B may be any one that serves as a medium for binding the ribozyme with the (glyco) protein of A, for example, for binding the ribozyme of formula (3) with ASOR. Is poly-L-lysine.

ASOR-poly-L represented by the formula (2)
-The lysine conjugate is (i) ASOR is reacted with N-succinimidyl-3- (2-pyridylthio) propionate (SPDP) to give ASOR-pyridylthiopropionate, and (b) poly-L-lysine is added. S
Reacting PDP and then dithiothreitol to give poly-L-lysine mercaptopropionate,
Then, (c) it is prepared by reacting ASOR-pyridylthiopropionate with poly-L-lysine mercaptopropionate.

The ribozyme of the formula (1) thus prepared has a negative charge, and ASOR-poly-L
Since the lysine conjugate carries a positive charge, a complex can be obtained by contacting both in saline.

[0021]

The object of the present invention is to directly administer a ribozyme, which has been given an activity by containing an RNA molecule, as a component of a sequence-specific RNA cleaving agent that is taken up cell-specifically in vivo. However, it is further advantageous in the following points as compared with the method of incorporating the above-mentioned DNA vector into cells. First, it is a small nucleic acid having a molecular size of 100 bases or less, and can be easily introduced into cells. Secondly,
Since the ribozyme itself does not have the self-replicating ability or the function of producing RNA by transcription, it does not affect the intracellular genetic information or protein synthesis. Thirdly, it is a small nucleic acid of 100 bases or less, and by including an RNA molecule at the site that imparts the cleavage activity, recombination with the chromosomal gene of the cell or the viral gene existing in the cell is caused, resulting in the gene. Does not cause mutation. Fourthly, the inclusion of RNA in the molecule is safe because after cleavage of the substrate RNA, it is easily decomposed and metabolized and disappears from the cell.

Furthermore, since the RNA cleaving agent of the present invention is specifically taken up by cells for the purpose of treatment, it has the advantage that it does not affect other cells and that the dosage is small. . After being taken up into the cell, it cleaves only a part of the specific nucleotide sequence in the RNA existing in the cell and inhibits only the processes involved in protein synthesis and growth replication of that RNA. Has the advantage that it does not affect the other RNAs present in. Therefore, the RN of the present invention
The A-cleaving agent can be widely used as an antiviral agent or an anticancer agent.

[0023]

The RNA cleaving agent of the present invention has a wide range of therapeutic and biological applicability. One of them is a human pathogenic virus infection caused by hepatitis C virus (HCV), AIDS virus (HIV), etc.
Alternatively, it can hybridize with RNA as a transcription product of viral DNA and cleave it to inactivate it. For this purpose, it can be incorporated into target cells in vivo by intravenous or inhalation or other administration methods.

Furthermore, it can be inactivated by cleaving it by hybridizing with mRNA of an oncogene or a mutated gene that causes abnormal expression, and thus it can be applied to the treatment at the gene level for cancer. In this case, although it is ideal that the present invention is taken up by the cancer cell through a receptor that is specifically present on the surface of the cancer cell, the present invention is genetically safe and is easily metabolized and decomposed, so that the occurrence of cancer occurs. By incorporating ribozymes through receptors that are tissue-specifically present on the cell membrane surface in both cancerous and non-cancerous cells of the tissue,
It is possible to inhibit the mRNA of an oncogene or a mutant gene that is abnormally expressed in cancer cells. In vivo administration in this case can be performed by intravenous or inhalation or other administration methods as described above.

As another application, it can be used to inhibit the expression of a gene which is not desired for biological functions. For example, inhibiting the production of a mutated amyloid precursor protein produced in Alzheimer's disease for which there is no effective treatment at present at the mRNA level can also contribute to the development of therapeutic agents.

[0026]

EXAMPLES Next, examples will be described. a) Preparation of ribozyme: The two oligonucleotides shown in FIG. 1 were synthesized using a DNA synthesizer according to a standard method. Reaction solution containing 50 pmol of each of these oligonucleotides (10 mM Tris-HCl buffer, pH 8.4; 50 mM
KCl; 1.5 mM MgCl ₂ ; 400 μM dAT
P; 400 μM dATP; 400 μM dCTP; 4
Polymerase chain reaction (PCR) was performed by setting a tube obtained by adding 2.5 units of Taq DNA polymerase to 50 μl of 00 μM dGTP to a gene amplification device (thermal reactor of High Bay Co.). PCR reaction conditions are 94 ° C, 1 minute → 55 ° C, 1 minute →
The reaction temperature change was repeated 35 times at 72 ° C. for 1 minute as one cycle, and after the reaction at 72 ° C. for 7 minutes, the temperature of 4 ° C. was maintained. As a result of this reaction, a double-stranded DNA as shown in FIG. 1 was obtained as a PCR product. This double-stranded D
As shown in the figure, NA is obtained by joining a nucleotide sequence of DNA corresponding to the nucleotide sequence of ribozyme downstream of the promoter sequence of T7 RNA polymerase. This T7 R
T7 RNA as a promoter sequence of NA polymerase
Ribozyme RN as a transcript by the action of polymerase
A is obtained. However, in this example, in order to enhance the transcriptional activity, the promoter sequence was taken longer (Nucleic
Acids Res. Vol 15,8783-87
98, 1987), the transcription initiation site is indicated by the arrow G in FIG.

The tube containing the PCR reaction solution was placed in boiling water for 10 minutes and then left at room temperature to gradually lower the temperature of the reaction solution, thereby inactivating the enzymatic activity of the polymerase in the reaction solution. Annealed complete double-stranded DNA is obtained. Using this, the following in vitro transcription reaction (Proc. Natl. Acad. Sci. USA V
Nu 83, 2330-2334, 1986; Nucl.
eic Acid Res. Vol 15,8783-
8798, 1987; Cell Vol 50, 104.
7-1055, 1987), ribozyme RNA is obtained. 0.5 μl of PCR product and 10 μl of T7 RNA polymerase (50 units / μl) were added to a reaction solution for transcription (10 × transcription buffer (400 mM Tris-HCl buffer, pH 8.0; 80 mM MgCl ₂ ; 20 mM spermidine) 5 μl, 50 mM DTT 5 μl, NTP mixture (100 each for UTP, ATP, CTP, GTP
mM) 1 μl, ribonuclease inhibitor (120 units / μl) 0.5 μl, water 28 μl), and add 1 at 37 ° C.
RNA was transcribed by reacting for a time. After transcription reaction, deoxyribonuclease 1 (10 units / μl) 5μ
1 was added to the reaction solution and kept at 37 ° C. for 10 minutes to decompose the DNA serving as the template for ribozyme RNA. Then, the tube containing the reaction solution was kept in boiling water for 10 minutes and then immediately placed on ice to deactivate the activity of the enzyme in the reaction solution, and then extracted once with phenol / chloroform and precipitated with ethanol, Ribozyme RNA (hereinafter, this ribozyme RNA is referred to as RZ) was obtained as a precipitate.

B) Preparation of substrate RNA: The two oligonucleotides shown in FIG. 2 were synthesized using a DNA synthesizer according to the standard method. Using this oligonucleotide, a substrate RNA (hereinafter, this substrate RNA is referred to as THCV) was obtained in the same manner according to the above method a). T7 RNA
The base sequence connected to the downstream of the promoter sequence of the polymerase is the base sequence of DNA corresponding to RNA encoding a part of the HCV genotype II core protein. Figure 3
Like RN, which encodes a part of this HCV core protein
The substrate RNA molecule (THCV), which is the A fragment, has a target base sequence for ribozyme RNA (RZ).
The cleavage site of the substrate RNA is indicated by an arrow.

C) Demonstration of ribozyme activity: FIG. 4 shows the in vitro activity of ribozyme RZ on substrate RNA (THCV) by ribozyme RZ. Reaction solution containing 58 μg of ribozyme and 86 μg of substrate RNA (50 mM
Tris-HCl buffer, pH 7.5; 10 mM MgC
15 μl of 1 ₂ ) was reacted at 37 ° C. for 3 hours, then 15 μl of a reaction stop solution (7M urea; 50 mM EDTA) was added to stop the reaction, and electrophoresis was performed using 20% polyacrylamide / 7M urea / TBE gel. RNA molecules were detected by silver staining. RNA when reacted only with RZ (lane 1) or when reacted only with THCV (lane 3)
Although no cleavage product of RZ and THC was detected, when THCV was reacted with RZ (lane 2), as shown in FIG.
In addition to the V band, RNA bands of 27 bases and 15 bases after cleavage of THCV targeted by ribozyme were observed. As a result, it was confirmed that the substrate RNA (THCV) was cleaved in the presence of ribozyme RNA (RZ).

D) Demonstration of ribozyme activity against full-length HCV-RNA obtained from HCV antibody-positive patient serum: (1) Extraction of serum HCV-RNA Acid guanidine / phenol from HCV antibody-positive patient serum / Chloroform method (Anal. Biochem.
Vol 162, 156-159, 1987) in RN.
A was extracted. Acidic guanidine solution containing 100 μl of patient serum (4M guanidine thiocyanate; 25 mM sodium citrate, pH 7.0; 0.5% sarcoziel;
The protein component was denatured in 1000 μl of 0.1 M mercaptoethanol), extracted with phenol / chloroform, and RNA was obtained by the ethanol precipitation method.

(2) Determination of HCV-RNA Genotype Determination of HCV-RNA genotype is performed by a method known in the literature (J. Gen. Virol. Vol 73, 673-6).
79, 1992). First, HCV-R
In the core region of NA, genotypes I, II, III
Design antisense primer for PCR by shifting the position to the part showing the nucleotide sequence specific to each type, IV type, and the sense primer (A) in the genotype common base sequence part upstream from that part. design. When PCR is carried out with a mixture of the above-mentioned four types of gene-specific antisense primers and a common sequence sense primer, PCR products are obtained as DNAs having different base lengths depending on the genotypes. It can be judged.

Using the genotype common antisense primer (B) located downstream of the four genotype-specific antisense primers, R obtained as described above was used.
CDNA was synthesized from NA by a reverse transcription reaction. That is, 10 μl containing 100 pmol of this antisense primer (B) and 100 μl of RNA obtained from patient serum
Reverse transcription reaction solution (10 mM Tris-HCl buffer, pH
8.4; 50 mM KCl; 1.5 mM MgCl ₂ ;
01% gelatin; 10 mM dATP; 10 mM dAT
P; 10mM dCTP; 10mM dGTP; 0.5 unit), add 100 units of reverse transcriptase to ribonuclease inhibitor) and react at 37 ° C for 60 minutes, and then at 94 ° C
For 15 minutes to inactivate the reverse transcriptase and then c
DNA was obtained.

Next, a sense primer (C) is designed further upstream from the above-mentioned sense primer (A), and PCR is carried out using this and the antisense primer (B) to detect the sense cDNA among HCV cDNAs. A base sequence portion including a portion sandwiched between the primer (A) and four types of genotype-specific antisense primers was amplified. The gene amplification reaction by PCR is carried out by PCR reaction solution (10 mM) containing 10 μl of the above-mentioned reverse transcription reaction solution, 100 pmol of sense primer (C) and 100 pmol of antisense primer (B).
Tris-HCl buffer, pH 8.4; 50 mM KCl;
1.5 mM MgCl ₂ ; 0.01% gelatin; 2 mM d
ATP; 2 mM dATP; 2 mM dCTP; 2 mM dG
TP) 50 μl of Taq DNA polymerase 1.25
Unit added, 94 ℃, 1 minute → 55 ℃, 1 minute → 72
The reaction temperature change was repeated 35 times at 1 ° C. for 1 minute, and after the reaction at 72 ° C. for 7 minutes, the temperature of 4 ° C. was maintained.

Next, PCR was performed to determine the genotype. A PCR reaction solution containing 5 μl of a reaction solution containing the PCR product obtained in the above reaction, a sense primer (A), and 100 pmol of each of four types of genotype-specific antisense primers (the composition is the same as the PCR reaction solution described above. ) 5
Taq DNA Polymerase (1.25 units) was added to 0 μl and PCR was performed under the same reaction conditions as described above.
An R product was obtained. This was electrophoresed on a 4% agarose gel, detected by ethidium bromide staining, and the PCR product DN
The genotype was determined from the size of A. To prove the activity of ribozymes this time, genotype II type HCV
-RNA was obtained. The above-described sense primer (A), antisense primer (B), sense primer (C), and four types of genotype-specific antisense primers are all described in the above-mentioned literature (J. Gen. Viro.
l. Vol 73, 673-679, 1992). In the following, reverse transcription of RNA and PCR
Performing is referred to as RT-PCR.

(3) Patient serum HCV-by ribozyme
Cleavage reaction of RNA According to the above method, the genotype II HCV-RNA is
According to the method already described, from 100 μl of the determined patient serum,
The RNA thus obtained was dissolved in 15 μl of sterilized water.
Examination of cleavage activity by ribozyme using 5 μl each
did. 35 μg of ribozyme and patient blood obtained as described above
Reaction solution containing 5 μl of sterilized water containing clear RNA (50 mM
Tris-HCl buffer, pH 7.5; 10 mM MgC
l ₂) After reacting 15 μl at 37 ° C. for 3 hours, reverse transcription
A reaction was performed to prepare cDNA. As a control
Reactions without ribozyme were performed similarly.

(4) Generation of cDNA by reverse transcription reaction of HCV-RNA As a primer for reverse transcription, HC by ribozyme was used.
8 after the sequence of GUC which is a specific cleavage site of V-RNA
An oligonucleotide having the base sequence shown in FIG. 6 was synthesized at the position shown in FIG. 5 with a DNA synthesizer interposed between the 5th and 86th bases as a primer for reverse transcription. Among the two primers for reverse transcription, c upstream of the cleavage site
The DNA primer was designated as D, and the downstream cDNA primer was designated as E. The number in parentheses in the figure indicates the base number from the protein translation initiation site. When all HCV-RNA is cleaved When the downstream cDNA primer E is used, reverse transcription is stopped at the cleavage site, so that DNA as a gene amplification product will not be generated even if PCR is performed upstream from this cleavage site. Not generated. On the other hand, when the upstream cDNA primer D was used, cDNA was synthesized by reverse transcription, and when PCR was carried out with the primer sets (a and b) shown in FIGS. 5 and 6, DNA of 215 base pairs was obtained as a gene amplification product. Is generated. The reverse transcription reaction was carried out in 40 μl containing 7.5 μl obtained by dividing 15 μl of the above ribozyme reaction solution into two equal parts.
Reverse transcription reaction solution (50 mM Tris-HCl buffer, pH
8.3; 75 mM KCl; 3 mM MgCl ₂ ; 10 mM D
TT; 500 μM dATP; 500 μM dATP;
500 μM dCTP; 500 μM dGTP; 30 units ribonuclease inhibitor) 40 μl of reverse transcriptase 300 units and cDNA primer A or cDNA
A primer B (250 pmol each) was added to each of them, and each was reacted at 37 ° C. for 60 minutes, and then kept at 94 ° C. for 15 minutes to inactivate the reverse transcriptase, and then HC
V cDNA (HCV-cDNA) was obtained. In addition, the reaction was similarly performed for the control.

(5) Detection of HCV-cDNA by PCR PCR was performed using the two oligonucleotides of FIG. 6 prepared as described above. That is, 50 pmol of each of these two oligonucleotides and a reaction solution (10 μl) containing 10 μl of the above reverse transcription reaction solution in which HCV cDNA was produced.
mM Tris-HCl buffer, pH 8.4; 50 mM KC
1; 1.5 mM MgCl ₂ ; 400 μM dATP; 40
0 μM dATP; 400 μM dCTP; 400 μM
2.5 units of Taq DNA polymerase was added to 50 μl of dGTP), and 94 ° C., 1 minute → 55 ° C., 1 minute →
The reaction temperature change was repeated 35 times at 72 ° C. for 1 minute as one cycle, and after the reaction at 72 ° C. for 7 minutes, the temperature of 4 ° C. was maintained. In addition, the reaction was similarly performed for the control.

(6) Confirmation of HCV-RNA Cleavage by Electrophoresis The gene amplification product by PCR was electrophoresed on a 5% polyacrylamide / TBE gel and then detected by silver staining. As shown in FIG. 7, in the case where the lanes 1 and 2-ribozyme were not added, the reverse transcription reaction was performed by the downstream cDNA primer E even when the reverse transcription reaction was performed by the upstream cDNA primer D (lane 1). Even when it was carried out (lane 2), a gene amplification product of 215 base pairs by PCR was clearly observed. On the other hand, when ribozyme was added as shown in lanes 3 and 4, cDNA primer D upstream of 3 was added.
A gene amplification product was clearly observed when PCR was carried out after reverse transcription using, but a faint gene amplification product was only observed when the reverse transcription reaction was carried out using the cDNA primer E downstream of 4. Absent. This indicates that the ribozyme cleaved full-length HCV-RNA in serum at the target site.

E) Preparation of sequence-specific RNA cleaving drug that is specifically taken up by human hepatocytes: (1) Preparation of asialoorosomucoid (ASOR) A method known in the literature (J. Biol. Chem. Vol. 2)
57, 12563-12572, 1982),
Human orosomucoid (also known as human α ₁ -acid
Glycoprotein) in 0.05M sulfuric acid at 80 ℃
The reaction was carried out for 1 hour to convert into asialo, followed by dialysis and freeze-drying to obtain ASOR.

(2) Asialoorosomucoid-poly-L
-Preparation of lysine conjugates Methods known in the literature (Biochem. J. Vol 17
3, 723-737, 1978), coupling was carried out by introducing a disulfide bridge after modification with SPDP. ASOR with a 2-pyridyl sulfide group introduced: 0.2
6 mM ASOR, containing 0.1 M sodium chloride.
100 ml of 40 mM ethanol solution of SPDP was mixed with 1.5 ml of 1M phosphate buffer solution under shaking, and ASOR:
The mixture was reacted at a ratio of SPDP = 0.4 μmol: 4 μmol for 1 hour at room temperature with occasional stirring. The reaction mixture was subjected to gel filtration using Biogel A1.5 with 0.1 M phosphate buffer (pH 7.5) containing 0.1 M sodium chloride to remove excess reagents and decomposed products.

Thiolated poly-L-lysine: First, poly-L-lysine having a 2-pyridyl sulfide group introduced therein was prepared in the same manner as above. 13.62 mg (approximately 0.4 mM) of poly-L-lysine having an average molecular weight of 68100 was added to
It was dissolved in 0.5 ml of 0.1 M phosphate buffer containing 1 M sodium chloride, and 50 μl of 40 mM ethanol solution of SPDP was mixed with shaking to give poly-L-lysine: SPD.
The mixture was reacted at room temperature for 1 hour at a ratio of P = about 0.2 μmol: 2 μmol with occasional stirring. The reaction mixture was subjected to gel filtration with 0.1 M sodium acetate buffer (pH 4.5) containing 0.1 M sodium chloride using Biogel A1.5 to remove excess reagents and decomposition products, 2-pyridyl sulfide. A poly-L-lysine fraction having a group introduced was obtained.
This fraction was added to a buffer solution (0.1 M sodium acetate buffer solution (pH 4.5); 0.1 M sodium chloride; 50 mM dithiothreiol) containing dithiothreiol at a final concentration of 50 mM at room temperature for 20 minutes. Let react for minutes.
Then, in order to remove the reducing agent and its reaction product, pyridine-2-thione, 0.1 M using Biogel A1.5.
0.1M phosphate buffer containing sodium chloride (pH 7.
Gel filtration was performed in 5) to obtain a fraction of thiolated poly-L-lysine.

Asialo-orosomucoid-poly-L-lysine conjugate: The 2-pyridyl sulfide group-introduced ASOR obtained as described above was mixed with thiolated poly-L-lysine to obtain 0.1M. 12 in 0.1M phosphate buffer (pH 7.5) containing sodium chloride
After reaction for 0.1 hours, 0.1M containing 0.1M sodium chloride
Gel filtration was performed with a phosphate buffer (pH 7.5) to obtain a fraction of asialo-orosomucoid-poly-L-lysine conjugate. This was dialyzed and then freeze-dried to obtain asialoorosomucoid-poly-L-lysine conjugate.

(3) Asialoorosomucoid-poly-L
-Preparation of lysine / ribozyme complex Nucleic acid such as DNA or RNA is negatively charged, and poly-L-lysine constituting the asialoorosomucoid-poly-L-lysine conjugate is positively charged. ,
These electrostatic interactions cause an electrostatic bond between the two, resulting in asialoorosomucoid-poly-L-lysine /
A nucleic acid complex is formed (J. Biol. Chem. V
ol 262,4429-4432,1987; Pro.
c. Natl. Acad. Asi. USA Vol 8
7, 3655-3659, 1990). Asialoorosomucoid-poly-L-lysine conjugate 1000
μg was dissolved in 150 μl of water, added to 150 μl of 0.3 M NaCl solution containing 200 μg ribozyme with stirring, and dissolved, and allowed to stand at room temperature for 30 minutes to obtain asialoorosomucoid-poly-L-lysine / ribozyme. 300 μl of 0.15 M NaCl solution containing the complex was obtained. As a control, a 300 μl 0.15 M NaCl solution containing only asialo-orosomucoid-poly-L-lysine prepared as described above except for ribozyme was used.

F) Demonstration of the presence of the asialoglycoprotein receptor (AGPR) in human hepatocytes: The human hepatocyte cell line (HepG2) has been biochemically demonstrated to have AGPR (J. Biol. Chem. Vol 2
56,8878-8881,1981), while the presence of AGPR has not been reported in the human pancreatic cancer cell line (PANC-1). However, in this study, since it is necessary to prove by immunohistochemistry that AGPR is surely present on the surface of the hepatocyte membrane, the presence of AGPR using an anti-human AGPR antibody (manufactured by Daiichi Pure Chemicals) It was confirmed. First, a human hepatocyte cell line (HepG2) and a human pancreatic cancer cell line (PANC-1) were respectively subjected to LAB-TEC chamber (Nunc Inc., Naperville, I).
L) was cultured on a slide glass, fixed in 4% paraformaldehyde / 0.01 M phosphate buffered saline (pH 7.2) at 4 ° C for 10 minutes, dried in a dryer for about 1 hour, and then placed in an oven. It was dried at 45 ° C. for about 4 hours and used for immunohistochemical staining.

0.01M phosphate buffered saline (pH 7.
2) After hydrophilic treatment with (PBS) and blocking with normal goat IgG, PBS containing 20 μg / ml of mouse anti-human AGPR antibody IgG as the primary antibody
For 1 hour, then washed 3 times with PBS, further reacted with horseradish peroxidase-labeled goat IgG (anti-mouse IgG) which is a secondary antibody for 1 hour, and then washed 3 times with PBS. Diaminobenzidine (DA
Color was developed using B) to confirm the presence of AGPR.

As a result, ASPR was detected in the cell membrane and cytoplasm only in the human hepatocyte cell line (HepG2), and no AGPR was detected in the human pancreatic cancer cell line (PANC-1). No staining was observed when normal mouse IgG was used instead of the primary antibody.

G) Demonstration of hepatocyte-specific ribozyme uptake via the asialoglycoprotein receptor: (1) Cell culture Human hepatocyte cell line (HepG2) and human pancreatic cancer cell line (PA)
NC-1) LAB-TEC chamber (N
unc Inc. , Naperville, IL) on glass slides and then serum-free medium (RPM
I 1640) was used in the experiment by adding 300 μl per chamber.

(2) Asialoorosomucoid-poly-L
-Addition of lysine / ribozyme complex to culture solution Based on the above-mentioned asialo-orosomucoid-poly-L-lysine / ribozyme complex preparation method, 200 μg of asialo-orosomucoid-poly-L-lysine conjugate was added to water 3
0.3 μl dissolved in 0 μl and containing 160 μg ribozyme
A 0.15M NaCl solution containing an asialo-orosomucoid-poly-L-lysine / ribozyme complex (a ribozyme concentration of 2.67 μg) was added to 30 μl of an M NaCl solution with stirring to dissolve it, and the mixture was allowed to stand at room temperature for 30 minutes.
/ Μl) 60 μl was obtained. As a control, asialoorosomucoid-prepared as described above except for ribozyme
A 60 μl 0.15 M NaCl solution containing only poly-L-lysine was used. 20 μm of asialo orosomucoid-poly-L-lysine / ribozyme complex solution (0.15 M NaCl) containing ribozyme prepared as described above
1 and asialoorosomucoid-poly-as a control
20 μl of L-lysine solution (0.15M NaCl),
The aforementioned serum-free medium (RPMI 1640) 30
The human hepatocyte cell line (HepG2) containing 0 μl was added to each of the cultured chambers by pipetting, and the mixture was cultured at 37 ° C. for 1 hour. After 1 hour, PBS
Washed 3 times with 4% paraformaldehyde / 0.01
It was fixed in M phosphate buffered saline (pH 7.2) at 4 ° C for 10 minutes, then dried in a dryer for about 1 hour, and then dried in an oven at 45 ° C for about 4 hours at high temperature, and the liver was dried. Used for intracellular ribozyme RNA detection. As for the human pancreatic cancer cell line (PANC-1), 20 μ of an asialoorosomucoid-poly-L-lysine / ribozyme complex solution (0.15 M NaCl) containing ribozyme was also used.
1 and asialoorosomucoid-poly-as a control
The same experiment was performed using 20 μl of L-lysine solution (0.15 M NaCl), and cells were similarly fixed.

(3) Detection of intracellular ribozyme RNA The detection of ribozyme RNA in the cytoplasm of hepatocyte strain HepG2 was carried out by the immunohistological in situ hybridization method (ISH). The probe for detecting ribozyme RNA in ISH (A-RZ-35) is a DN which becomes a complementary sequence to the base sequence of ribozyme as shown in FIG.
An oligonucleotide having a sequence in which a base sequence of TTATTA on the 5 ′ side of the A sequence and a −ATT base sequence on the 3 ′ side is bound is synthesized by a DNA synthesizer according to a standard method. UV irradiation (254
nm, 10000 J / m ² )
A thymine dimer is formed in the base sequence portion of T, and the localization of the oligonucleotide can be known by using an antibody against this thymine dimer (Histo).
chem. J. Vol 20,551-557,198
8; Acta Histochem. Cytoche
m. Vol 22, 295-307, 1989; Act.
a Pathol. Jpn. Vol 40, 793-8
07, 1990). The slide glass fixed as above and dried at high temperature was added with 0.01M phosphate buffered saline (pH
7.2) After hydrophilic treatment with (PBS), 0.2N hydrochloric acid treatment 1 is performed to remove the protein around the nucleic acid and expose the target nucleic acid.
1 μg / ml Proteinase K treatment was carried out for 0 minutes at 37 ° C. for 10 minutes, and transferred to hybridization. Hybridization solution containing 3 μg / ml thymine dimerized synthetic DNA probe (10 mM Tris-HCl buffer, pH 7.3; 1 mM EDTA; 0.6 MN)
aCl; 1 × Denhardt's solution; 40% deionized formamide; 250 μg / ml yeast transfer RNA; 1
25 μg / ml ultrasonically disrupted salmon sperm DNA; 10% dextran sulfate) was placed on a slide glass and reacted with the cells for about 15 hours. After the reaction, the hybridization solution was washed, and normal goat I was used according to the usual enzyme-indirect method.
Non-specific binding was suppressed with gG, and rabbit IgG as an anti-thymine dimer antibody was used as the primary antibody, horseradish peroxidase (HRP) -labeled goat IgG (anti-rabbit IgG) was used as the secondary antibody, and the secondary antibody was used. It was detected by HRP activity labeled with. The localization of HRP was detected in the presence of diaminobenzidine (DAB) and cobalt and nickel ions (J. Histochem. Cytoch.
em. Vol 29, 775, 1981). This HRP
Will indicate the localization of ribozyme RNA. As a result, in the human hepatocyte cell line (HepG2), positive findings showing the presence of ribozymes in granular form in the cytoplasm were observed. On the other hand, such a positive finding was not observed at all in the human pancreatic cancer cell line (PANC-1).

H) HCV in human hepatocytes of ribozyme incorporated via asialoglycoprotein receptor
-Proof of RNA cleaving activity (1) Preparation of human hepatocyte suspension A non-cancerous liver tissue obtained from a surgical material of an HCV antibody-positive liver cancer patient was washed with PBS, and serum-free medium (RPMI) was used.
1640) and finely cut with ophthalmic scissors and scalpel,
Furthermore, the liver parenchymal cells are separated by pipetting, and P
The cells were washed 3 times with BS and centrifuged to obtain a cell mass, which was suspended in 3 ml of serum-free medium (RPMI 1640) to prepare a human hepatocyte suspension.

(2) Asialoorosomucoid-poly-L
-Lysine / ribozyme complex uptake into hepatocytes and extraction of hepatocyte RNA Asialoorosomucoid-poly-L-lysine / ribozyme complex solution containing 0.1 μg of ribozyme (0.15
M NaCl) 150 μl and a solution of asialoorosomucoid-poly-L-lysine alone as a control (0.1
150 μl of 5M NaCl) was added to 3 ml of the human hepatocyte suspension prepared as above by pipetting, and the mixture was incubated at 37 ° C. for 1 hour with occasional stirring. After 1 hour, the cells were washed 3 times with PBS, and RNA was immediately extracted from the cell mass obtained by centrifugal precipitation based on the acidic guanidine / phenol / chloroform method described above.

(3) Examination of Ribozyme Activity The examination of the cleavage activity of HCV-RNA in human hepatocytes by ribozyme was carried out by RT-based on the method of d)-(4), (5) and (6) described above. After PCR, electrophoresis was performed and silver staining was examined. As shown in FIG. 9, when there is no ribozyme, the reverse transcription reaction is performed even when the reverse transcription reaction is carried out with the upstream cDNA primer D as in lanes 1 and 2 (lane 1).
Even when the reverse transcription reaction was performed with the primer E of A (lane 2), a gene amplification product of 215 base pairs by PCR was clearly confirmed. On the other hand, lanes 3 and 4 show asialoorosomucoid-poly-L-lysine / containing ribozyme.
When a ribozyme complex pair was added to the culture medium, a gene amplification product was clearly observed when PCR was performed after reverse transcription using the cDNA primer A upstream from the cleavage site by the ribozyme (lane 3). In the case where PCR was carried out by reverse transcription using cDNA primer B downstream from the cleavage site by (lane 4), no gene amplification product was confirmed. This is because the ribozyme which constitutes the asialo-orosomucoid-poly-L-lysine / ribozyme complex is the asialoglycoprotein receptor (A
It is shown that it was introduced into actual human hepatocytes infected with HCV via GPR) and cleaved HCV-RNA in the cells.

[Brief description of drawings]

FIG. 1 is a diagram showing two PCR primers for producing a DNA template for transcription of ribozyme RNA, a double-stranded DNA as a gene amplification product by PCR performed using the same, and a structure thereof.

FIG. 2 DNA for transcription of HCV-RNA fragment as a substrate
FIG. 3 is a diagram showing two PCR primers for producing a template, a double-stranded DNA as a gene amplification product by PCR performed using the same, and a structure thereof.

FIG. 3: Ribozyme prepared this time and H as substrate RNA
It is a figure which shows the complementarity between a CV-RNA fragment.

FIG. 4 shows that ribozyme RZ was used as a substrate RNA (T
It is a figure which shows having activity with respect to HCV).

FIG. 5 is a diagram showing the structure of HCV-RNA, the cleavage site of HCV-RNA targeted by ribozyme, and the relative positions of the reverse transcription primer (D, E) and the PCR primer (a, b). .

FIG. 6 shows the nucleotide sequences of reverse transcription primers (D, E) and PCR primers (a, b).

FIG. 7 shows the in vitro activity of ribozyme RZ on HCV-RNA in the serum of HCV-infected patients.

FIG. 8 shows the nucleotide sequence of a probe used for ISH to detect RNA of ribozyme RZ in cells.

FIG. 9 shows in vivo activity of ribozyme RZ against HCV-RNA in HCV-infected human hepatocytes.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Internal reference number FI Technical display location C12N 15/09 ZNA // C07H 21/02 (72) Inventor Shuhei Yamada 1 Fukubashi, Matsumoto City, Nagano Prefecture No. 2-8 Maison No. 306 Hanaoka (72) Inventor Kendo Kiyosawa 270-10 Sousha, Matsumoto City, Nagano Prefecture

Claims (5)

[Claims] 1. General formula (1) 3'-X ₁ -Y-X ₂ -5 '(1) (wherein X ₁ and X ₂ are the base sequences of the nucleic acid complementary to the base sequence of the target RNA. , Y represents a nucleotide sequence having an RNA cleaving activity) and a ribozyme represented by the general formula (2) ALB (2) (where A is a (glyco) protein having a receptor on the cell membrane surface of an animal) Or a polypeptide, B is a polycation, and L is a linker that forms a covalent bond with the amino group terminals of A and B) (glyco) protein or polypeptide-polycation conjugate complex An RNA cleaving agent comprising: 2. The RNA cleaving agent according to claim _1, wherein X ₁ and X ₂ are nucleotide sequences complementary to the core region of hepatitis C virus. 3. The RNA cleaving agent according to claim 2, wherein the ribozyme is represented by the following formula (3): 3'-CGCCACCA-AAGCAGGAGUGCCUGAGUAGUC-UCUAAGCAA-5 '(3). 4. A has a receptor on the surface of hepatocyte membrane (sugar)
The RNA cleaving agent according to claim 1, which is a protein. 5. A is asialo-orosomucoid, B is poly-L-lysine, and L is a disulfide group.
5. The RNA cleaving agent according to any one of 4 to 4.