This invention relates to an inhibitory RNA for modulating the molecular function of ZFAT gene and, more particularly, to an inhibitory RNA for inhibiting the expression of ZFAT gene capable of inhibiting cell proliferation or inducing apoptosis, an expression vector containing the same, a method for inhibiting the expression of ZFAT gene using the same, a cell proliferation inhibiting method, and an apoptosis induction method by inhibiting the expression of ZFAT gene, as well as a medical composition containing the inhibitory RNA for inhibiting the expression of ZFAT gene.
The understanding of molecular identification and molecular mechanisms regarding proliferation, apoptosis, differentiation, etc., leads to the understanding of malignant transformation and pathological conditions of cells. Modifications of molecular function (molecular target) focusing on particular genes or signal channels are one of methods for artificially controlling dynamics of cells and normalizing pathological conditions of cells.
Many anti-cancer agents as have been applied so far are derived from natural substances and chemical compounds. They, however, have drawbacks that they may react to cancer cells and to normal cells, too, causing severe side effects. Recent years, therefore, trials have been made of clinical applications of molecular target agents with the attempt to reduce such side effects, however, the current state of such molecular target agents is not yet satisfactory in terms of efficacy and reduction of side effects.
ZFAT gene (zinc-finger gene in autoimmune thyroid disease susceptibility region: ZNF406) identified as a gene associated with autoimmune thyroid diseases is a Zn finger protein coding for a protein composed of 1,243 amino acid residues and having one AT hook domain and 18 Zn finger domains. The distribution of the ZFAT proteins in mouse tissues is characterized in that it is strongly expressed in the thymus gland and the spleen yet the expressing cells are limited to B cells and T cells (Non-Patent Literature No. 1). The mechanism of ZFAT protein is not yet identified.
RNAi (RNA interference) is noted as an effective method for analyzing genetic functions. RNAi is a phenomenon in that a double-stranded RNA (dsRNA) consisting of a sense RNA and an anti-sense RNA, homologous to a gene, destroys a homologous portion of a transcription product (mRNA) of the gene, thus leading to inhibition of the expression of a gene having a sequence identical thereto, that is, to inhibition of the synthesis of a protein (for example, Non-patent Literature Nos. 2, 3, 4). In other words, RNAi is a unique method that can search for functions of genes by performing a knock-out of (destroying) mRNA of a target gene having a known sequence.
Further, RNAi can confirm results at a cell level so that it is advantageous to achieve result in more precise way, at lower costs and in shorter time, compared to conventional gene knock-out method that confirms results using animals. In addition thereto, the RNAi method has the merits that results can be expected to be achieved at a low concentration, and only mRNA can be destroyed without affecting any adverse influence on the genomic gene itself.
As a result of extensive review and study of ZFAT gene, the present inventors have conducted a further analysis utilizing RNAi techniques assuming that a ZFAT protein plays a significant function in immune reaction on the basis that it is expressed strongly in human leukemia cells and at the time of immunoresponse (at the time of stimulation of blast formation) found that siRNA (small interfering RNA), i.e., a double-stranded RNA composed of a sense RNA homologous to the ZFAT gene and an anti-sense RNA, inhibits the expression of ZFAT gene. It has further been found that inhibition of the expression of the ZFAT gene induces apoptosis of cancer cells and inhibits immunoresponse in naïve CD4 positive T cells. As a result, the present inventors have found that the inhibitory RNA for inhibiting the expression of ZFAT gene may be used as a molecular target agent or an immunosuppressive agent having a higher selectivity by controlling an amount and function of the ZFAT gene. This invention has been completed based on these findings.
Therefore, this invention has an object to provide an inhibitory RNA for inhibiting the expression of ZFAT gene comprising a double-stranded RNA composed of a sense RNA homologous to ZFAT gene and an anti-sense RNA thereto.
This invention, as its preferred embodiment, has another object to provide the inhibitory RNA for inhibiting the expression of ZFAT gene including a double-stranded RNA consisting of a sense RNA with a base sequence of contiguous 20 to 30 bases, preferably 23 to 27 bases, of ZFAT gene and the anti-sense RNA with a base sequence complementary to the base sequence of the sense RNA, and a shRNA (short hairpin RNA) having a hairpin structure formed by connecting the sense RNA to the anti-sense RNA through a spacer oligonucleotide.
This invention, as its another aspect, has a further object to provide an expression vector capable of expressing the inhibitory RNA for inhibiting the expression of ZFAT gene.
This invention, as its another aspect, has a further object to provide a method for inhibiting the expression of ZFAT gene in cells by introducing the inhibitory RNA for inhibiting the expression of ZFAT gene or the expression vector into cells.
This invention, as its another aspect, has a further object to provide a cell proliferation inhibiting method for inhibiting the cell proliferation of cells such as cancer cells and an apoptosis induction method for inducing apoptosis of cells such as cancer cells by introducing the inhibitory RNA for inhibiting the expression of ZFAT gene or the expression vector into cells.
This invention, as its another aspect, has a further object to provide an immunoresponse inhibiting method for inhibiting an immunoresponse in living bodies by inhibiting the immunoresponse in cells by inhibition of the expression of the ZFAT gene.
Furthermore, this invention has an object to provide a medical composition that contains the inhibitory RNA for inhibiting the expression of ZFAT gene or the expression vector and is effective for treatment or prevention of diseases including cancers by inhibiting the immunoresponse in cells by the inhibition of cell proliferation or apoptosis induction.
In order to achieve the objects as described above, this invention provides the inhibitory RNA for inhibiting the expression of ZFAT gene comprising a double-stranded RNA containing a sense RNA having a base sequence of contiguous 20 to 30 bases, preferably 23 to 27 bases, of ZFAT mRNA and an anti-sense RNA having the base sequence complementary to the base sequence of the sense RNA.
It is to be understood herein that the term “double-stranded RNA” as used herein is used to mean a siRNA composed of a sense RNA with a base sequence of contiguous 20 to 30 bases, preferably 23 to 27 bases, of ZFAT mRNA and an anti-sense RNA with a base sequence complementary to the base sequence of the sense RNA, and a shRNA with a hairpin structure formed by connecting the sense RNA to the anti-sense RNA in the form of a loop sequence through a spacer oligonucleotide as well as a dsRNA containing the siRNA or shRNA. Therefore, it is to be understood herein that the terms “inhibitory RNA for inhibiting the expression of ZFAT gene”, “double-stranded RNA”, “siRNA”, and “shRNA”, as well as their relating terms are used exchangeably herein so as to include all such meanings even if they are used singly or in combination with others, unless otherwise stated.
This invention, as its preferred embodiment, provides the inhibitory RNA for inhibiting the expression of ZFAT gene comprising a double-stranded RNA with one to six nucleotides added to each strand at its 3′-terminus.
This invention, as its further preferred embodiment, provides the inhibitory RNA for inhibiting the expression of ZFAT gene comprising a siRNA composed of the sense RNA with the base sequence of contiguous 20 to 30 bases, preferably 23 to 27 bases, of ZFAT mRNA or a shRNA consisting of the double-stranded RNA with the sense RNA and the anti-sense RNA which are connected together in the form of a loop sequence.
This invention, as its another preferred embodiment, provides the inhibitory RNA for inhibiting the expression of the ZFAT gene, comprising a human ZFAT (hZFAT) siRNA having the base sequences as indicated by SQ ID NO. 1 and 2, 3 and 4, 5 and 6, or 7 and 8, or a mouse ZFAT (mFAT) siRNA having the base sequences as indicated by SQ ID NO. 17 and 18, 19 and 20, or 21 and 22, respectively.
This invention, as its another aspect, provides an expression vector for expressing the inhibitory RNA for inhibiting the expression of ZFAT gene.
This invention, as its another aspect, provides a method for inhibiting the expression of ZFAT gene in cells by introducing the inhibitory RNA for inhibiting the expression of ZFAT gene or the expression vector into the cells.
This invention, as its further another aspect, provides a cell proliferation inhibiting method for inhibiting cell proliferation and an apoptosis induction method for inducing apoptosis of cells by introducing the inhibitory RNA for inhibiting the expression of ZFAT gene and the expression vector into the cells.
This invention, as its further another aspect, provides an immunoresponse inhibiting method for inhibiting the immunoresponse in living bodies by inhibiting the expression of ZFAT gene by introducing the inhibitory RNA for inhibiting the expression of ZFAT gene or the expression vector into cells.
This invention, as its further another aspect, provides a medical composition which contains the inhibitory RNA for inhibiting the expression of ZFAT gene or the expression vector as an active ingredient and which is effective for treatment or prevention of diseases such as cancers by inhibiting the cell proliferation of cancer cells or inducing apoptosis by inhibition of the immunoresponse in living bodies.
The inhibitory RNA for inhibiting the expression of ZFAT gene or the expression vector according to this invention have the effects on inhibition of the expression of ZFAT gene in living bodies, as well as on inhibition of the proliferation of cells such as cancer cells, or induction of apoptosis, or inhibition of an immunoresponse. Therefore, this invention can provide a medical composition having the effects on inhibition of cell proliferation, apoptosis induction, or inhibition of immunoresponse.
The inhibitory RNA for inhibiting the expression of ZFAT gene according to this invention comprises an oligonucleotide consisting of a double-stranded RNA composed of a sense RNA homologous to the ZFAT gene and an anti-sense RNA thereof. More specifically, the inhibitory RNA for inhibiting the expression of ZFAT gene according to this invention is an inhibitory RNA for inhibiting the expression of ZFAT gene comprising a double-stranded RNA composed of the sense RNA having a contiguous base sequence of ZFAT nRNA and the anti-sense RNA having the base sequence complementary to the base sequence of the sense RNA.
More specifically, the inhibitory RNA for inhibiting the expression of ZFAT gene according to this invention comprises a siRNA consisting of the double-stranded RNA containing the sense RNA with the contiguous base sequence of ZFAT mRNA and the anti-sense RNA with the base sequence complementary to the base sequence of the sense RNA, or a shRNA capable of producing the siRNA by cleavage with a biogenic enzyme in living bodies, which has a stem loop structure with the sense RNA and the anti-sense RNA connected to each other through a loop sequence and forms a structure in which the 3′-terminus of the sense RNA is connected to the 5′ terminus of the anti-sense RNA through a loop sequence (a hairpin loop sequence). Furthermore, the inhibitory RNA for inhibiting the expression of ZFAT gene according to this invention may contain a double-stranded RNA (dsRNA) including the siRNA or the shRNA.
More specifically, the inhibitory RNA for inhibiting the expression of ZFAT gene according to this invention is basically an oligonucleotide comprising a double-stranded RNA with the base sequences complementary to each other, and a sense RNA structuring one strand of the double-stranded RNA contains an optional region of the base sequence of mRNA corresponding to a target structuring gene portion of the ZFAT gene. This region may be preferably selected so as to contain a region in which the GC content accounts for, e.g., approximately 30 to 60%, preferably less than approximately 50%, and have a base length with, e.g., contiguous 20 to 30 bases, preferably 23 to 27 bases, of ZFAT mRNA. The anti-sense RNA structuring the other strand of the double-stranded RNA has a base sequence complementary to the base sequence of the sense RNA. The sense RNA or the anti-sense RNA may have an additional base sequence consisting of one to six bases at its 3′-terminus.
As shown in
The base sequences of the siRNAs as indicated by SQ ID NO. 1 to 8 are respectively indicated as follows:
As controls, there are prepared scramble base sequences having base sequences different from the above siRNAs corresponding thereto, respectively. Control base sequences corresponding to LFAT #1 as indicated by SQ ID NO. 1 and 2 are referred to herein as scramble #1 and indicated by SQ ID NO. 9 and 10, respectively; control base sequences corresponding to ZFAT #2 as indicated by SQ ID NO. 3 and 4 are referred to herein as scramble #2 and indicated by SQ ID NO. 11 and 12, respectively; control base sequences corresponding to ZFAT #4 as indicated by SQ ID NO. 5 and 6 are referred to herein as scramble #4 and indicated by SQ ID NO. 13 and 14, respectively; as well as control base sequences corresponding to ZFAT #5 as indicated by SQ ID NO. 7 and 8 are referred to herein as scramble #1 and indicated by SQ ID NO. 15 and 16, respectively.
It is also to be noted herein that this invention includes siRNAs from mouse ZFAT gene (NM—198644). As shown in
The base sequences of the siRNAs of mouse ZFAT gene as indicated by SQ ID NO. 17 to 22 are respectively indicated as follows:
As negative control base sequences corresponding to mZFAT siRNAs as indicated by SQ ID NO. 17 to 20, there have been prepared control sense RNA (SQ ID NO. 23 and 24) and anti-sense RNA (SQ ID NO. 24 and 26), respectively.
As described above, the inhibitory RNA for inhibiting the expression of ZFAT gene according to this invention includes a small molecular double-stranded hairpin RNA (shRNA) producing siRNA by cleavage with an intracellular enzyme (dicer) in living bodies. The shRNA has a stem loop structure in which the sense RNA containing the double-stranded portion is connected to the anti-sense RNA through a loop sequence, i.e., the 3′-terminus of the sense RNA is connected to the 5′-terminus of the anti-sense RNA through a loop sequence (a hairpin loop sequence). It is to be noted herein, however, that the hairpin loop sequence is not limited to the above particular one and may include a base sequence composed of four to seven bases including, for example, CGAA
For example, as shown in
As the siRNAs of the mouse ZFAT gene according to this invention, there may be mentioned, for example, siRNA (mZFAT siRNA1) consisting of sense RNA (556 Top: SQ ID NO. 27) and anti-sense RNA (556 Bottom: SQ ID NO. 28) having each a base sequence homologous to the corresponding base sequence from base 556 of the mouse ZFAT gene, as shown in
The base sequences of the above shRNA (SQ ID NO. 27 and 28) and the negative control double-stranded RNA (SQ ID NO. 29 and 30) may be respectively indicated as follows:
CTTGC-3′
CTTGC-3′
GTCTT-3′
GTCTT-3′
Among the base sequences as indicated above, the base sequence portion underlined on the left side indicates the sense RNA portion and the base sequence portion underlined on the right side indicates the anti-sense RNA portion. The base sequence between the sense RNA and the anti-sense RNA indicates a loop sequence, and the base sequence at the 5′-terminus of the sense RNA indicates a BsmB1 connecting sequence.
The inhibitory RNA for inhibiting the expression of ZFAT gene according to this invention as indicated by the above base sequences may be prepared, for example, by chemically synthesizing the sense RNA and the anti-sense RNA in a manner known to the art, annealing the sense RNA and the anti-sense RNA synthesized above, and purifying them in a manner known to the art.
The inhibitory RNA for inhibiting the expression of ZFAT gene according to this invention may be introduced into living bodies such as, for example, cells, tissues, individuals, etc., by an expression vector capable of expressing the inhibitory RNA for inhibiting the expression of ZFAT gene. The inhibitory RNA for inhibiting the expression of ZFAT gene introduced by the expression vector into living bodies is expressed in the living bodies resulting to inhibition of the expression of the ZFAT gene. For this invention, the inhibitory RNA for inhibiting the expression of ZFAT gene according to this invention may be introduced into living bodies as it is, however, it may be preferably introduced through an expression vector in terms of persistent effects.
The expression vectors according to this invention may be produced in accordance with conventional processes. Tandem-type expression vectors may be produced, for example, by amplifying a promoter portion with primers containing a sense sequence and an anti-sense sequence by PCR, cleaving the amplified fragment with a restriction enzyme, and introducing it into a downstream portion of a promoter, such as U6 promoter, of a vector. For the tandem-type expression vectors, the sense RNA and the anti-sense RNA are transcribed in cells from two U6 promoters. On the other hand, stem-loop type expression vectors may be produced, for example, by synthesizing DNAs corresponding to the sense RNA and the anti-sense RNA, respectively, synthesizing and annealing a template DNA of a double-stranded RNA consisting of oligonucleotides containing a sense-loop-antisense sequence having a structure with the sense RNA and the anti-sense RNA connected to each other through a loop sequence, and introducing the template DNA into a downstream portion of a promoter of an expression vector, e.g., a PolIII type promoter such as U6 promoter, H1 promoter, etc. The template DNA introduced into the expression vector may produce a siRNA by transcribing a short hairpin type RNA (shRNA) with an RNA polymerase and processing it with an enzyme. As the vectors, there may be used, for example, plasmid vectors, virus vectors, and so on. The plasmid vectors may include, for example, pBAsi vector, pSUPER vector, and so on. The virus vectors may include, for example, adenovirus vectors, lentivirus vectors, retrovirus vectors, and so on.
In this invention, the siRNAs may be produced by introducing the above sense and anti-sense DNAs into respectively different expression vectors, expressing them individually in cells, and annealing them in cells. When these expression vectors are used, it is preferred that the siRNAs can be produced continuously in living bodies or cells.
It is preferred that the extent to which the inhibitory RNA for inhibiting the expression of ZFAT gene according to this invention causes an inhibition (silencing) of the expression of a target gene by RNAi (RNA interference) reaches a rate at which an amount of the expression of mRNA or protein of the target gene is inhibited by 100%, compared to the case where no RNA is introduced. Even if the inhibition rate would not be 100%, however, it may be acceptable, depending upon objects or uses, etc., in some cases where the inhibition of the expression of the target gene could be judged to occur. Therefore, the inhibition rate is not limited to a particular one. Furthermore, the rate of expression of mRNAs and proteins may be measured in accordance with conventional processes. For example, mRNAs may be measured by northern hybridization, RT-PCR, etc., and proteins may be measured by western blotting, ELISA, etc.
The inhibitory RNA for inhibiting the expression of ZFAT gene according to this invention can inhibit the expression of ZFAT protein in cancer cells (
In this invention, a difference in effects of target sites of ZFAT siRNA on apoptosis and cell cycle can be recognized (
Further, the inhibitory RNA for inhibiting the expression of ZFAT gene according to this invention indicates a remarkable activation of caspase 9 inducing apoptosis, compared to a control, by inhibiting the expression of ZFAT gene in naïve CD4 positive T cells of mouse peripheral blood at the time of immunoresponse (at the time of blastogenesis stimulation) by anti-CD3 antibody and anti-CD28 antibody (
Therefore, this invention as another aspect provides a medical composition such as a molecular target agent or an immunosuppressive agent, which contains the inhibitory RNA for inhibiting the expression of ZFAT gene according to this invention as an active ingredient capable of inhibiting the expression of ZFAT gene. From the point of view of containing the inhibitory RNA for inhibiting the expression of ZFAT gene as an active ingredient, the expression vector for inhibiting the expression of ZFAT gene with the inhibitory RNA introduced thereinto is also encompassed within the scope of this invention.
In the event where the inhibitory RNA for inhibiting the expression of ZFAT gene or the expression vector for expressing the inhibitory RNA is applied to the medical composition such as molecular target agents or immunosuppressive agents, the inhibitory RNA or the expression vector may be administered in various routes including an oral route or a parenteral route. The preparation types for oral administration may include, for example, tablets, pills, capsules, powders, granules, syrups, and the like. The preparation types for parenteral administration may include, for example, injections, external preparations, suppositories, and the like. Whether it is administered orally or parenterally may be appropriately chosen depending upon its uses or the like. The medical composition may further contain, for example, a pharmacologically acceptable carrier including without limitation a diluent such as water, ethylalcohol, macrogol, propylene glycol, citric acid, acetic acid, phosphoric acid, lactic acid, sodium lactate, sulfuric acid, sodium hydroxide, etc., a pH adjusting agent or a buffer such as sodium citrate, sodium acetate, sodium phosphate, etc., and a stabilizer such as sodium pyrosulfite, ethylenediaminetetraacetic acid, thioglycolic acid, thiolactic acid, etc. It may also contain a sufficient amount of a compound for preparing an isotonic solution, such as salt, glucose, mannitol or glycerin. Monosacchardies such as glucose, etc., disaccharides such as saccharose, maltose, etc., sugar alcohols such as mannitol, sorbitol, etc., neutral salt such as sodium chloride, etc., amino acids such as glycine, non-ionic surfactants such as polyethylene glycol, polyoxyethylene-polyoxypropylene copolymer, polyoxyethylene sorbitan aliphatic acid ester, etc., human albumin, and so on may also be used as stabilizers. As a dose of administration may vary depending upon kinds of the active ingredients, administration routes, and ages, body weights, conditions, etc. of objects or patients, it cannot be limited to a particular one, and a daily dose may be in the range of approximately several mg to 2 grams, preferably several 10 mg, and it may be preferred to be administered once or several times per day.
The medical composition according to this invention may be prepared into formulations that can keep its stability and be delivered in a stable manner in living bodies by applying the technology of coupling with a delivery carrier such as liposome, etc., a site-specific antibody, or a cell-type specific functional peptide. Further, in order to facilitate uptake into cells, the RNA or its expression vector according to this invention may be used by preparing it in the form of a liposome.
This invention will be described more specifically by the following examples, but it is to be noted that they are solely illustrative for this invention and it is not limited in any respect to them.
The double-stranded RNAs of human ZFAT (NM—020863) and mouse ZFAT (NM—198644) were prepared as follows. First, a sense RNA and an anti-sense RNA structuring the double-stranded RNA were chemically synthesized each in the form of oligoribonucleotide using ribonucleoside 3′-phosphoramidite and DNA/RNA synthesizer (Applide Biosystems 394 Model). After completion of the synthesis, the resulting oligoribonucleotides connected to CPG (controlled pore glass) were treated at room temperature for 2 hours in a mixed solution of concentrated ammonium water:ethanol (3:1 v/v) and then cut off from the CPG resin, and warmed at 55° C. for another 15 hours. Thereafter, the solvent was removed off and the resulting residue was mixed with a 1M TBAF (tetrabutylammounium fluoride)/THF (tetrahydrofuran) solution and stirred at 37° C. for 16 hours. To the resulting mixture was added 0.1M triethylammonium acetate (pH 7.0), and the resulting mixture was subjected to column chromatography in a concentration gradient manner using a 5%-40% acetonitrile and 50 mM triethylammonium bicarbonate squcous solution. Fractions coloring with dimethoxytrityl were collected from the elute with an approximately 30% acetonitrile, and they were mixed with 0.01N hydrochloric acid and stirred to remove off the dimethoxytrityl group. Then, they were neutralized with 0.1N ammonium water, and the resulting aqueous layer was washed with ethyl acetate. After the solvent was removed off, they were dissolved in fertilized water. The oligoribonucleotide was separated and fractionated from the fractions by reverse HPLC and purified by subjecting it to ion-exchange HPLC, separating and fractionating it. The resulting oligoribonucleotide was used for experiments as will be described hereinunder. The sequence of the resulting oligoribonucleotide was determined by Block-it RNAi desiner (Invitrogen), and siRNA and shRNA each as indicated by the respective SQ ID NOs were annealed and purified in conventional manner.
The shRNA expression vector as indicated by
The proteins extracted fractions of B lymphocytes and T lymphocytes as well as other leukocytes separated from human peripheral blood were subjected to western blotting analysis.
The human peripheral blood was centrifuged with Ficoll to recover leukocytes and further treated with human CD 19 magnetic beads (Miltenyi Biotec) and human CD3 magnetic beads (Miltenyi Biotec) of MACS system (Miltenyi Biotec) to continuously separate B lymphocytes and T lymphocytes to give effluents containing neither B cell nor T cells. The resulting cells were in turn subjected to western blotting analysis, confirming that the ZFAT protein was shown to be expressed by B lymphocytes and T lymphocytes, respectively, derived from human peripheral blood (
Two kinds of siRNA oligonucleotides (ZFAT#1 and ZFAT#2) and two kinds of control siRNA oligonucleotides (Scramble #1 and Scramble #2), each prepared by the above example, were introduced into MOLT-4 cells, and the proteins extracted therefrom at hour 24 and hour 48 after introduction were determined by subjecting them to western blotting analysis. The MOLT-4 cells were incubated at 37° C. in the presence of 5% CO2 in a RPMI-1640 culture medium (GIBCO) with 10% bovine serum added thereto. The introduction of each of the siRNA oligonucleotides was performed by introducing 100 pmol of siRNA into 1×106 cells with MicroPorator MP-100 (Digital Bio). The proteins were extracted by using a protease inhibitor cocktel containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% deoxycholate, and 0.1% SDS. As an antibody, there was used anti-ZFAT rabbit polyclonal antibody produced in accordance with non-patent literature #1. As a result of western blotting analysis, it was confirmed that ZFAT expression was inhibited in MOLT-4 cells by the two siRNA oligonucleotides (ZFAT#1 and ZFAT#2) (
Experiments were carried out as to whether the inhibition of ZFAT expression causes a decrease in the proliferation rate of MOLT-4 cells.
The siRNA oligonucleotides (ZFAT#1, ZFAT#2, Scramble#1, and Scramble#2) prepared in Example 1 were introduced each into MOLT-4 cells which were in turn inoculated into a 24-well plate at the rate of 5×104 cells per well, and the number of cells was counted periodically (hour 24, 48 and 72 after inoculation) with cell counting kit (DOJIN). The results are shown in
Analysis of a cell cycle (an increase in the Sub-G1-entry) in cells inhibiting the expression of ZFAT was carried out in the manner as will be described below.
The siRNA oligonucleotides (ZFAT#1, ZFAT#2, Scramble#1, and Scramble#2) prepared in Example 1 were introduced each into MOLT-4 cells and fixed with 70% ethanol (48 hours, −20° C.) at hour 48. Then, the cells were treated at 37° C. for 1 hour with 1 □g/ml RNase and stained with propidium iodide (10 □g/ml). Thereafter, the cell cycle analysis was carried out by measuring the amount of DNA with flow cytometry. As a result, it was confirmed that the group of cells inhibiting the expression of ZFAT increased a rate of the Sub-G1 entry, compared to the control group, but it did not recognize any increase in other cell cycles (
Experiments were carried out to confirm apoptosis induction in cells inhibiting the expression of ZFAT.
The siRNA oligonucleotides (ZFAT#1, ZFAT#2, Scramble#1, and Scramble#2) prepared in Example 1 were introduced each into MOLT-4 cells and stained with anexin V at hour 24 and hour 48 after introduction. Then, the annexin V positive cells was subjected to flow cytometry using TACS Annexin V Kit (TREVIGEN) and counted the number of the apoptosis positive cells. As a result, it was found that the group of the cells inhibiting the expression of ZFAT caused an increase apoptosis to a significant extent, compared to the control group, as shown in
This example is directed to experiments for detecting active-type caspase 3 in cells inhibiting the expression of ZFAT.
The siRNA oligonucleotides (ZFAT#1 and ZFAT#2) and the corresponding scramble sequences (Scramble#1 and Scramble#2), each prepared in Example 1, were introduced each into MOLT-4 cells, and the cells were collected at hour 24 and 48 after introduction and then stained with active-type caspase 3 antibody, detecting caspase 3 positive cells by flow cytometry using Active Caspase-3 PE MAb Apoptosis Kit (BD Pharmingen). As a result, it was found that the group of the cells inhibiting the expression of ZFAT increased the number of the caspase 3 positive cells compared to the control group (
This example was carried out to determine activity of caspase 8 and caspase 9 in cells inhibiting the expression of ZFAT.
The siRNA oligonucleotides (ZFAT#1 and ZFAT#2) and the corresponding scramble sequences (Scramble#1 and Scramble#2), each prepared in Example 1, were introduced each into MOLT-4 cells, and the activity of caspase 8 (
This example was carried out to show that apoptosis to be induced by inhibition of the expression of ZFAT is inhibited by caspase inhibitor Z-VAD.
The siRNA oligonucleotides (ZFAT#2 and Scramble#2), each prepared in Example 1, were introduced each into MOLT-4 cells in the presence of the caspase inhibitor Z-VAD or DMSO as a control. The cells were then stained with annexin V to determine the apoptosis posive cells at hour 24 after introduction. As a result, it was observed that apoptosis was inhibited to a significant extent in the presence of the caspase inhibitor Z-VAD (
The siRNA oligonucleotides (ZFAT#1, ZFAT#2, Scramble#1 and Scramble#2), each prepared in Example 1, were introduced each into MOLT-4 cells in the presence of caspase inhibitor Z-VAD or DMSO, as a control, and the number of the cells was counted periodically by cell counting kit (DORN). As a result, it was observed that the inhibition of cell proliferation by inhibition of the expression of ZFAT was hindered thoroughly by the caspase inhibitor Z-VAD Z (
This example was caned out to conduct a cell cycle analysis at the time of inhibition of the expression of ZFAT with ZFAT siRNA targeting the C-terminal region of ZFAT gene.
Human ZFAT (hZFAT) siRNA #5 (and Scramble sequence as a control) prepared in Example 1 and targeting the C-terminal region of ZFAT were introduced each into MOLT-4 cells and immunostained with anti-ZFAT antibody (M16) at hour 48 after introduction. As a result, the inhibition of the expression of ZFAT with ZFAT siRNA was confirmed (
ZFAT (hZFAT) siRNA #5 (and Scramble sequence as a control) targeting the C-terminal region of ZFAT were introduced each into MOLT-4 cells, and subjected to a cell cycle analysis with BrdU Flow Kit (BD Pharmingen) at hour 48 after introduction. This analysis result indicates that a transferal to the early S-entry for control was inhibited at the time of inhibition of ZFAT (
This example was carried out to show transduction of shRNA expression vector constructed in Example 2 above. The double-stranded synthetic oligonucleotide DNA synthetized by annealing the oligonucleotide DNA prepared in Example 1 was cloned in the vector (pLenti6-U6+term) constructed above and transfected in 293FT cells with lyofectamin™ 2000 (Invitrogen), together with VitraPower™ packaging plasmid (Invitrogen). After 48 hours, a medium supernatant containing virus was collected, a titer was measured, and the CD4 positive cells were transducted in CD4 positive cells which were in turn separated from mouse spleen cells using mouse CD4 (L3T4) magnetic beads.
The separated CD4 positive cells were incubated in a RPMI-1640 medium (10% FCS, 50 □M 2-ME) containing IL-7 (5 ng/ml), and they were treated for 3 hours with 8 ug/ml of polybrene, followed by transduction with a virus supernatant of MOI=1, while IL-7 was administered continuously. After another 72 hours, the CD4 positive cells were inoculated on a plate coated at the rate of 1 ug/ml with anti-CD3 antibody (145-2C11; BD Pharmingen), and stimulation with RPMI-1640 containing anti-CD28 antibody (37.51; BD Pharmingen) was started.
This example was carried out to determine inhibition of the expression of ZFAT by the shRNA expessing vector formed in Example 2. The shRNA expressing vector was transducted into CD4 positive cells and stimulated with anti-CD3 antibody and anti-CD28 antibody at hour 48 after transduction. Then, the nuclear staining was carried out for an immunostained pattern and DAPI pattern with anti-ZFAT antibody (m16) at day 2 after stimulation. The expression of ZFAT with ZFAT shRNA was inhibited for a control (eGFP shRNA) (
This example was carried out to show an influence of the shRNA expression vector constructed in Example 2 on inhibition of the expression of ZFAT at the time of stimulation of CD4 positive cells with anti-CD3 antibody and anti-CD28 antibody. At hour 72 after transduction of the shRNA expression vector into CD4 positive cells, the transducted expression vector was stimulated with anti-CD3 antibody and anti-CD28 antibody, and the DAPI pattern and immunostained pattern were investigated at hour 0.24 after stimulation (
This example was carried out in the manner similar to that of Example 12 to determine an influence on the number of T cells by the shRNA expression vector formed in Example 2 at the time of stimulation of CD4 positive cells with anti-CD3 antibody and anti-CD28 antibody. After the shRNA expression vector was transducted in CD4 positive cells, it was stimulated with anti-CD3 antibody and anti-CD28 antibody, and an optical microscopic pattern was observed at hour 72 after stimulation. This result indicates that apoptosis can be induced by inhibiting the expression of ZFAT because ZFAT shRNA decreases the number of cells compared to the control (
The inhibitory RNA for inhibiting the expression of ZFAT gene and the expression vector containing the same according to this invention can inhibit the expression of ZFAT gene and induce apoptosis by introducing them into living bodies such as cells, tissues, individuals, etc., in which their expression is intended to be inhibited. Therefore, the inhibitory RNA for inhibiting the expression of ZFAT gene according to this invention can be used as a medical composition for treatment or prevention of diseases such as cancers and as a molecular target agent or an immunosuppressant having a high selectivity.
Filing Document | Filing Date | Country | Kind | 371c Date |
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PCT/JP2008/066155 | 9/8/2008 | WO | 00 | 5/24/2011 |