JPH11285391A - Anti-hiv medicine - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new antisense oligonucleotide which specifically hybridizes to a chromosomal DNA and/or RNA coding for CXCR4 protein to inhibit the expression of the protein, and useful, for example, as a medicine for treatment and prophylaxis of the HIV infection. SOLUTION: This is a new antisense oligonucleotide which specifically hybridizes to a chromosomal DNA and/or RNA coding for CXCR4 protein to inhibit the expression of CXCR4 protein, contains at least one of sequences shown by formulas I-III, and is useful as an anti-HIV medicine which is highly effective, for example, as medicines for prophylaxis and treatment of human immunodeficiency viruses(HIV). This antisense oligonucleotide is obtained by synthesizing a base sequence complementary to a base sequence from +429 to +758 (gene transcription initiation point of mRNA coding for CXCR4 protein being +1) on a DNA synthesizer by the phosphoramidite method, followed by purifying by reversed phase HPLC and so on.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to an antisense oligonucleotide against chromosomal DNA and / or RNA encoding CXCR4 protein and an anti-HIV containing the same.
It relates to the agent. More specifically, the present invention relates to C-cell receptors that are involved in AIDS infection.
The present invention relates to an antisense oligonucleotide capable of inhibiting HIV infection by specifically hybridizing to chromosomal DNA and / or RNA of XCR4 protein and suppressing its expression, and an anti-HIV agent containing the same.

[0002]

2. Description of the Related Art AIDS is known as the human immunodeficiency virus (HI).
V) A disease that is caused by infection and results in significant impairment of cellular immunity, resulting in various opportunistic infections, lymphomas, neuropathies, etc., and finally death. Current treatments include azidothymidine (AZ
T) A single or combination therapy of a reverse transcriptase inhibitor such as dideoxyinosine (ddI) and dideoxycytidine (ddC) and a protease inhibitor such as sacinavir, ritonavir, indinavir has been reported (Hamme).
r, SM et al. , New Engl. J. Me
d. 335, 1081-1090, 1996). Nucleoside reverse transcriptase inhibitors prevent the virus from integrating into the chromosome by acting on the stage in which the virus's own reverse transcriptase converts viral genetic information from RNA to DNA after HIV enters cells. Is what you do. However, there are cases where these reverse transcriptase inhibitors and protease inhibitors readily develop resistant viruses by long-term administration and render the drugs ineffective (Shirasaka, Te.).
tal., Proc. Natl. Acad. Sci. USA,
92, 2398-2402, 1995; Condra, J. et al.
H. et al., Nature, 374, 569-571,
1995). Furthermore, since it also causes abnormalities in DNA metabolism on the cell side, many side effects such as anemia, leukopenia, nausea, headache, malaise, stupor and myositis due to long-term administration have been reported, and the development of new treatments is strongly expected. It is rare.

[0003] The main target cells of HIV are CD4-positive T cells and macrophages. HIV is roughly divided into C
A strain that infects D4-positive T cells but does not infect macrophages (T cell-tropic HIV), a strain that infects macrophages but does not infect CD4-positive T cells (macrophage-tropic HIV), and infects any cells It is classified into three types of strains that can be obtained (bidirectional HIV). CD4 has been known as a receptor on the cell side of HIV for some time. A second receptor (second receptor) involved in the T cell tropism of HIV was identified in 1996 and named CXCR4 (Feng, Y., et a
l., Science, 272, 872-877 (199).
6)). At the same time, CCR5 was identified as a second receptor for macrophage-tropic HIV (Al
khatib, G. et al., Science, 272,
1955-1958, 1996). These second receptors are essential cell factors for HIV infection together with CD4, but are originally receptors for chemokines secreted in vivo, and CXCR4 ligand is SD
The ligands of F1 and CCR5 are MIP-1α, MIP-1
It has also been shown to be β and RANTES.

[0004] With respect to the treatment of HIV infection,
Although there have been many reports, since mutations in the genomic gene of HIV occur at a much higher probability than in the genomic gene of mammalian cells, we have devised a drug that specifically acts on HIV. However, it has a disadvantage that it cannot act on the mutated HIV. Such HIV
With respect to the direction of research directed to factors on the side, attempts have been made to treat HIV infection by controlling factors on the cell side involved in HIV infection. According to this method, since the genomic gene of the cell is hardly mutated, there is little possibility that the drug becomes ineffective due to the emergence of a resistant virus such as a reverse transcriptase inhibitor. In contrast to conventional anti-HIV drugs, which act on the virus itself after infection of cells, this method inhibits the HIV infection stage, so that HIV does not enter the cells themselves, and the survival of cells does not occur. The likelihood of compromising the rate and its function is low.

[0005] In recent years, many basic studies on the treatment of HIV infection focusing on these second receptors have been reported. For example, SDF1, MIP-1α, M
IP-1β and RANTES inhibit infection of T cell-directed HIV and macrophage-directed HIV, respectively, in a manner of competitive inhibition by competing for the receptor (Bleul, CC, et al., Natur.
e, 829-833, 1996; Cocchi, F., et.
al., Science, 270, 1811-1815, 1
995), a method in which the antagonist inhibits HIV infection or overexpresses a chemokine in a target cell so that the receptor is not expressed on the cell surface is considered (Chen, JD, et al.). al., Natu
re Medicine, 3,1110-1116,199
7). However, large doses of chemokines can release large amounts of HIV as a result of stimulating HIV-infected cells (Schmidtmayerova, H., et al.,
Nature, 382, 767, 1996). However, there is a doubt about its effect when actually trying to apply it to therapy.

[0006]

DISCLOSURE OF THE INVENTION The present invention has been made in view of the above circumstances, and is intended to suppress the expression of CXCR4 protein in a target cell when infected with HIV.
An object is to provide an antisense oligonucleotide capable of inhibiting IV infection and preventing and treating AIDS infection, and an anti-HIV agent containing the same.

[0007]

Means for Solving the Problems The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, have found that antisense which specifically suppresses the expression of CXCR4 protein and inhibits HIV infection of cells. Succeeded in finding the gene sequence,
The present invention has been completed. The antisense nucleic acid sequence is
A gene encoding CXCR4 protein as a target gene,
When the target gene partially forms a higher-order structure, the target gene is in a single-stranded state in consideration of the fact that the antisense nucleic acid is unlikely to hybridize to the portion forming the higher-order structure. A sequence part having a high probability is used for the “antisense adjustment method” developed by Uchida et al.
No. 85, which is referred to as “antisense nucleic acid target site search software (PAS ^™ )”, and the present invention has been completed.

[0008] The present invention provides a sequence of any one or more of the following (A) to (C) which specifically hybridizes to chromosomal DNA and / or RNA encoding CXCR4 protein and inhibits expression of CXCR4 protein. The present invention relates to an antisense oligonucleotide comprising: (A) The sequence described in SEQ ID NO: 1 in the sequence listing (B) The sequence described in SEQ ID NO: 2 in the sequence listing (C) The sequence described in SEQ ID NO: 3 in the sequence listing The present invention further provides the antisense oligonucleotide And anti-HIV agents.

[0009]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail.
The antisense oligonucleotide of the present invention comprises CXCR
Chromosomal DNA and / or RNA encoding 4 proteins
Is an antisense oligonucleotide complementary to a part of the base sequence. The antisense oligonucleotide of the present invention may be DNA or RNA. The antisense oligonucleotide of the present invention has complementarity to the base sequence from +429 to +758, and specifically and stably to the base sequence from +429 to +758, when the transcription start point of the mRNA encoding the CXCR4 protein is +1. As a result of hybridization and blocking translation into protein, CXCR4
It has the effect of inhibiting protein biosynthesis.

[0010] The antisense oligonucleotide of the present invention comprises any one of the sequences described in SEQ ID NOs: 1 to 3 in the sequence listing.
It is preferable to include the above. The sequences described in SEQ ID NOs: 1 to 3 show the antisense DNA strand of the CXCR4 protein gene, and SEQ ID NO: 1 corresponds to SEQ ID NO: 5 (Nomur
a, H., et al., Int. Immunol., 5,123.
9-1249, 1993), the cXCR4 protein c
SEQ ID NO: 2 corresponds to +715 to +736 of SEQ ID NO: 5, and SEQ ID NO: 3 corresponds to +735 to 758 of SEQ ID NO: 5, respectively.

[0011] The upper limit of the length of the antisense oligonucleotide of the present invention is preferably 100 bases or less, because the efficiency of gene synthesis in a DNA / RNA synthesizer decreases with an increase in the number of bases and the cost of synthesis increases. Yes, more preferably 30 bases or less. The lower limit of the length of the gene is preferably at least 8 bases, more preferably at least 12 bases, and particularly preferably at least 15 bases, in order to maintain the specificity of the antisense oligonucleotide.

The method for synthesizing the antisense oligonucleotide of the present invention is not particularly limited. For example, a phosphorothioate-type or phosphotriester-type oligonucleotide can be obtained by a phosphoramidite method using a general oligonucleotide synthesizer. it can. Examples of antisense oligonucleotides obtained by such a synthesis method include phosphodiester-type oligonucleotides, phosphorothioates in which a phosphate group is covalently modified with a sulfur atom to prevent degradation by nucleases when administered to a living body. -Type oligonucleotides, methylphosphonate-type oligonucleotides in which the phosphate skeleton is methylated, morpholine-skeleton oligonucleotides in which morpholine is introduced in place of the phosphate skeleton, and oligos modified with the lipophilic substance geraniol to enhance membrane permeability Nucleotides and the like.

[0013] The antisense oligonucleotide of the present invention can be used as an anti-HIV agent. in this case,
Although the antisense oligonucleotide of the present invention can be used alone, it can be used as a composition in combination with a pharmaceutically acceptable carrier to reliably introduce it into target cells and prevent degradation in vivo. It is. The carrier is not particularly limited as long as it is a pharmaceutically acceptable substance. For example, a polymer having a positive charge, a liposome, a microsphere, or the like can be suitably used. As the polymer having a positive charge, TfX-50 (−10, −2
0) (manufactured by Promega), Transfectam (manufactured by Wako Pure Chemical Industries), ExGen 500 (manufactured by Wako Pure Chemical Industries), a synthetic polyamino acid or a derivative thereof, specifically, described in WO95 / 09909 Poly-lysine: serine (PLS), P-lysine described in JP-A-9-176038
Examples include modified LS PEG blocks.
As liposomes, lipofectin (manufactured by GIBCO), lipofectamine (manufactured by GIBCO), cellfectin (manufactured by GIBCO), DMRIE-C (GIBCO)
Co., Ltd.). Examples of the microsphere include Superfect (manufactured by QIAGEN) and the like. These pharmaceutically acceptable carriers and the antisense oligonucleotide of the present invention can form a complex by a known method. In addition, the method of using the antisense oligonucleotide and the anti-HIV agent of the present invention is not particularly limited.
Any of o, ex vivo and the like can be used.

[0014] The antisense oligonucleotide of the present invention can also be used as an antisense RNA expression construct for gene therapy by inserting it into a gene expression vector containing a gene sequence that promotes gene expression. As the gene expression vector, a plasmid, a recombinant virus, or the like can be used. When a plasmid is selected as the gene expression vector, the plasmid can be used alone, but in order to prevent degradation by nuclease and improve gene expression efficiency, a complex with a pharmaceutically acceptable carrier was formed. Preferably, it is used as a composition. Also, the recombinant virus
It can be used without particular limitation as long as it can infect mammal-derived cells, preferably human-derived cells,
Mouse leukemia virus, adenovirus, adeno-associated virus, human immunodeficiency virus, Sindbis virus,
Herpes virus, Sendai virus, Epstein-Barr virus can be selected. Among them, mouse leukemia virus, adenovirus, adeno-associated virus, and human immunodeficiency virus are preferable, and human immunodeficiency virus is particularly preferable. The method for using the antisense RNA-expressing gene construct is not particularly limited.
Both tro and ex vivo can be used.

[0015]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Examples, but these Examples are for the purpose of assisting the understanding of the present invention and do not limit the scope of the present invention.
In an example, SEQ ID NO: 5 is the cD of the CXCR4 protein.
NA base sequence (Nomura, H., et al., In
t. Immunol., 5, 1239-1249, 199
3) is shown. In addition, SEQ ID NO: 4 is a negative control group C
2 shows the sequence of the antisense DNA strand of the CR5 gene (Comparative Example).

(Example 1) (Synthesis and purification of antisense oligonucleotide)
The antisense oligonucleotides of SEQ ID Nos. 1 to 3 in the sequence listing were synthesized using a DNA synthesizer (manufactured by Expedite PerSeptive) under a known condition by a phosphoramidite method under a known condition.
(Waters) and purified under known conditions. SEQ ID NOs: 1 to 3 are antisense DNA strands of the CXCR4 protein gene, and SEQ ID NO: 1 is SEQ ID NO: 5
SEQ ID NO: 2 from +429 to +452, SEQ ID NO: 2 from +715 to +736 of SEQ ID NO: 5, SEQ ID NO: 3
735 to 758, respectively.

Example 2 Preparation of Complex (Anti-HIV Agent) of Antisense Oligonucleotide and Gene Transfer Reagent GI as a gene transfer preparation (pharmaceutically acceptable carrier)
DMRIE-C reagent manufactured by BCO was used. 500 μl
The antisense oligonucleotides of SEQ ID NOs: 1 to 3 were each prepared at 2 μM in an Opti-MEM medium (manufactured by GIBCO) to obtain solution A. Also, 500
A DMRIE-C reagent was prepared in 20 μl of Opti-MEM medium at a concentration of 20 μg / ml, and used as solution B. Solution B was added to Solution A, and the mixture was gently shaken and allowed to stand at room temperature for 30 minutes to give the antisense oligonucleotide /
A DMRIE-C complex was obtained. As a comparative example, a similar complex was obtained using the antisense oligonucleotide of SEQ ID NO: 4.

(Test Example 1) CXCR4 protein expression inhibitory effect test (A) Use of antisense oligonucleotide on cultured cells
Introduction C of the antisense oligonucleotides of SEQ ID NOs: 1-4
Investigation of XCR4 protein expression inhibitory effect in cultured cell lines
did. Cells constitutively express endogenous CXCR4 protein
The CD4 protein so that the CD4 protein is constantly expressed.
HeLa cells incorporating the encoding gene were used
(Hereinafter referred to as CD4HeLa cells). CD4HeLa
Cells are grown at 37 ° C., 5% CO ₂10% fetal calf serum under conditions
(FCS: manufactured by GIBCO) and antibiotics were added.
Dulbecco's Modified Eagle Medium (DMEM: GIBCO)
). This CD4HeLa cell is
5 × 10 in 6-well plate⁵Pieces / well
After seeding and culturing overnight, cells were plated in Opti-MEM for 2 hours.
Washed twice and prepared the antisense oligonucleotide prepared in Example 2.
1 ml of Otide / DMRIE-C complex was added. 37
℃, 5% CO₂After culturing for 4 hours under the conditions of
5 ml of DMEM containing CS was added, and 37 ° C., 5% CO 2 was added.
₂The cells were further cultured under the following conditions.

(B) Detection of CXCR4 Protein by Fluorescent Antibody Method Cells into which the antisense oligonucleotide was introduced in the above (A) were collected every 6, 12, 24 and 48 hours, and a monoclonal antibody against CXCR4 (PHAR) was collected.
MINGEN) and FITC-labeled 2
Secondary antibody (Jackson ImmunoResearch)
h company). The stained cells were subjected to FACS Ca
libur flow cytometer (Becton Di)
The ratio of CXCR4-positive cells was analyzed using Cinson Corporation. The results are shown in FIG. In the antisense oligonucleotide non-administration group and SEQ ID NO: 4 (Comparative Example), the ratio of CXCR4-positive cells did not change during the 48-hour culture period. After time, the ratio of CXCR4 positive cells decreased significantly. From these results, the antisense oligonucleotide of the present invention,
It was found that the effect of suppressing the expression of CXCR4 protein was very excellent.

[0020]

As described above, the antisense oligonucleotide of the present invention can suppress the expression of CXCR4 protein and inhibit HIV infection. Therefore, the antisense oligonucleotide of the present invention and an anti-HIV agent containing the same are very effective as preventive and therapeutic agents for HIV infection.

[Brief description of the drawings]

FIG. 1 shows the C of the antisense oligonucleotide of the present invention.
It is a graph which shows the XCR4 protein expression suppression effect.

[Sequence list] SEQ ID NO: 1 Sequence length: 24 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Antisense: YES Sequence characteristics: SEQ ID NO: Corresponds to +429 to +452 in 5 Sequence TGAGGACACTGCTGTAGAGGTTGA SEQ ID NO: 2 Sequence length: 22 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: YES Sequence features: Corresponds to +715 to +736 in SEQ ID NO: 5 Sequence GCAATAGCAGGACAGGATGACA SEQ ID NO: 3 Sequence length: 24 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other Nucleic acid synthetic DNA Antisense: YES Sequence characteristics: +735 to +758 of SEQ ID NO: 5 Sequence GTGACAGCTTGGAGATGATAATGC SEQ ID NO: 4 Sequence length: 26 Sequence type: Nucleic acid Number of strands: Single strand Topology: Straight Chain Sequence type: Other nucleic acid Synthetic DNA Antisense: YES sequence TAAAGT CCTAGAATGTATTTAGTTGC SEQ ID NO: 5 Sequence length: 1664 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: cDNA antisense: NO Sequence characteristics: CXCR4 cDNA sequence CGGCAGCAGG TAGCAAAGTG ACGCCGAGGG CCTGAGTGCT CCAGTAGCCA CCGCATCTGG 60 AGAACCAGCG GTTACCATGG AGGGGATCAG TATATACACT TCAGATAACT ACACCGAGGA 120 AATGGGCTCA GGGGACTATG ACTCCATGAA GGAACCCTGT TTCCGTGAAG AAAATGCTAA 180 TTTCAATAAA ATCTTCCTGC CCACCATCTA CTCCATCATC TTCTTAACTG GCATTGTGGG 240 CAATGGATTG GTCATCCTGG TCATGGGTTA CCAGAAGAAA CTGAGAAGCA TGACGGACAA 300 GTACAGGCTG CACCTGTCAG TGGCCGACCT CCTCTTTGTC ATCACGCTTC CCTTCTGGGC 360 AGTTGATGCC GTGGCAAACT GGTACTTTGG GAACTTCCTA TGCAAGGCAG TCCATGTCAT 420 CTACACAGTC AACCTCTACA GCAGTGTCCT CATCCTGGCC TTCATCAGTC TGGACCGCTA 480 CCTGGCCATC GTCCACGCCA CCAACAGTCA GAGGCCAAGG AAGCTGTTGG CTGAAAAGGT 540 GGTCTATGTT GGCGTCTGGA TCCCTGCCCT CCTGCTGACT ATTCCCGACT TCATCTTTGC 600 CAACGTCAGT GAGGCAGATG ACAGATATAT CTGTGACCGC TTCTACCCATGTGTG CCAGTTTC AGCACATCAT GGTTGGCCTT ATCCTGCCTG GTATTGTCAT 720 CCTGTCCTGC TATTGCATTA TCATCTCCAA GCTGTCACAC TCCAAGGGCC ACCAGAAGCG 780 CAAGGCCCTC AAGACCACAG TCATCCTCAT CCTGGCTTTC TTCGCCTGTT GGCTGCCTTA 840 CTACATTGGG ATCAGCATCG ACTCCTTCAT CCTCCTGGAA ATCATCAAGC AAGGGTGTGA 900 GTTTGAGAAC ACTGTGCACA AGTGGATTTC CATCACCGAG GCCCTAGCTT TCTTCCACTG 960 TTGTCTGAAC CCCATCCTCT ATGCTTTCCT TGGAGCCAAA TTTAAAACCT CTGCCCAGCA 1020 CGCACTCACC TCTGTGAGCA GAGGGTCCAG CCTCAAGATC CTCTCCAAAG GAAAGCGAGG 1080 TGGACATTCA TCTGTTTCCA CTGAGTCTGA GTCTTCAAGT TTTCACTCCA GCTAACACAG 1140 ATGTAAAAGA CTTTTTTTTA TACGATAAAT AACTTTTTTT TAAGTTACAC ATTTTTCAGA 1200 TATAAAAGAC TGACCAATAT TGTACAGTTT TTATTGCTTG TTGGATTTTT GTCTTGTGTT 1260 TCTTTAGTTT TTGTGAAGTT TAATTGACTT ATTTATATAA ATTTTTTTTG TTTCATATTG 1320 ATGTGTGTCT AGGCAGGACC TGTGGCCAAG TTCTTAGTTG CTGTATGTCT CGTGGTAGGA 1380 CTGTAGAAAA GGGAACTGAA CATTCCAGAG CGTGTAGTTA ATCACGTAAA GCTAGAAATG 1440 ATCCCCAGCT GTTTATGCAT AGATAATCTC TCCATTCCCG TGGAACGTTT TTCCTGTTCT 1500 TAAGACGTGA TTTTGCTGTA GA AGATGGCA CTTATAACCA AAGCCCAAAG TGGTATAGAA 1560 ATGCTGGTTT TTCAGTTTTC AGGAGTGGGT TGATTTCAGC ACCTACAGTG TACAGTCTTG 1620 TATTAAGTTG TTAATAAAAG TACATGTTAA ACTTAAAAAA AAAA 1664

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Takashi Shimada 1-20-6-801 Mukooka, Bunkyo-ku, Tokyo (72) Inventor Kiyoshi Uchida 2 Okubo Tsukuba, Ibaraki Pref.

Claims (2)

[Claims] 1. A chromosome DN encoding a CXCR4 protein
Specifically hybridizes with A and / or RNA,
The following (A) to (C) which inhibit the expression of CXCR4 protein
An antisense oligonucleotide comprising any one or more of the following sequences: (A) Sequence described in SEQ ID NO: 1 in Sequence Listing (B) Sequence described in SEQ ID NO: 2 in Sequence Listing (C) Sequence described in SEQ ID NO: 3 in Sequence Listing 2. An anti-HIV agent comprising the antisense oligonucleotide according to claim 1.