Patent Application
20080071068
Next, the best mode for carrying out the present invention will be described with reference to Examples.
Cytoplasmic localization, enzymatic degradation resistance, degradation resistance in serum, telomerase inhibition activity, and tyrosine kinase activity inhibition in each Example are evaluated as follows.
A fluorescently labeled, modified DNA is suspended at a concentration of 1 μM in a physiological saline to prepare a sample. Separately, leukemia cells (Jurkat) are added at a concentration of 106 cells/ml to a standard nutrient medium, to which the suspension of the modified DNA is in turn added, and cultured for 24 hours under conditions of 5% CO2 and 37° C. After culture, the cells are centrifuged, then washed three times with a PBS (−) buffer solution, and evaluated by use of flow cytometry (manufactured by BECKMAN COULTER, product code: “Epics XL”) and a fluorescence and laser scanning confocal microscope (manufactured by BIO-RAD, product code: “Radiance 2000”).
A nutrient medium containing a modified DNA at a concentration of 1 μM is supplemented with 100 units of an enzyme (DNase 1) and cultured at 37° C. for 10 minutes, followed by analysis by RP-HPLC to determine the degradation rate of the modified DNA.
A modified DNA with a concentration of 1 μM and fetal bovine serum (FBS) with a concentration of 10% are added into a nutrient medium and cultured at 37° C. for 2 hours, followed by analysis by RP-HPLC to determine the degradation rate of the modified DNA.
A modified DNA is added at a concentration of 1 μM to an RPMI medium containing leukemia cells (Jurkat) at a concentration of 1×106 cells/ml and cultured at 37° C. for 48 hours under conditions of 5% CO2 and 37° C. to determine telomerase activity inhibition in terms of IC50 (nM) by TRAP assay.
A sample is added at a concentration of 5 μM to leukemia cells K-562 at a cell concentration of 1×106 cells/ml and cultured for 48 hours under conditions of 5% CO2 and 37° C. to determine its inhibition rate by protein tyrosine kinase assay.
An automatic DNA synthesizer (manufactured by Cruachem, product name: “PS250”) was used to chemically modify the 5′-terminus of an oligonucleotide HIV-1 Rev (5′-SEQ ID NO: 16 in Sequence Listing-3′) with an O-aminoethoxyethyl-O′-cyanoethylphosphoric ester residue on a CPG support according to a routine method.
Subsequently, the oligonucleotide was supplemented and reacted at 20° C. for 5 hours with 0.5 M solution prepared by dissolving hexamethylene diisocyanate in acetonitrile and then reacted with a peptide fragment HIV-1 Rev (SEQ ID NO: 1 in Sequence Listing) having a free N-terminal amino group with the protected amino acid side chain, thereby binding the NES peptide to the 5′-terminus of the oligonucleotide via the hexamethylene diisocyanate.
Next, this reaction product was supplemented with ammonia water with a concentration of 28% and stirred at 55° C. for 5 hours, thereby cleaving the produced conjugate from the solid support and removing the protecting group from the peptide.
A DNA localized in the cytoplasm represented by the formula 3′-SEQ ID NO: 16 in Sequence Listing-5′-O—CO—NH—CH2CH2NH—CO—NH—(CH2)6—NH-SEQ ID NO: 1 in Sequence Listing, wherein the first Ala in SEQ ID NO: 1 in Sequence Listing is β-alanine, was thus obtained at a yield of 10.7%.
The enzymatic degradation resistance of the DNA localized in the cytoplasm thus obtained was 29.1%, the degradation resistance in serum thereof was 42.3%, the telomerase inhibition activity thereof was 120 nM, and the tyrosine kinase activity inhibition thereof was approximately 50%. The enzymatic degradation resistance of the raw material DNA used as a control was 49.2%, the degradation resistance in serum thereof was 56.9%, the telomerase inhibition activity thereof was 40 nM, and the tyrosine kinase activity inhibition thereof was approximately 25%. The heat of fusion between the DNA localized in the cytoplasm and its complementary DNA or RNA was almost the same as the melting point of the raw material DNA used as a control.
Next, the DNA localized in the cytoplasm thus obtained was dissolved in acetonitrile and supplemented and reacted with an equimolar amount of fluorescein isothiocyanate to fluorescently label the DNA localized in the cytoplasm.
This fluorescently labeled DNA localized in the cytoplasm was examined for its cytoplasmic localization. For comparison, the fluorescently labeled raw material DNA used as a control was also examined for its cytoplasmic localization. When they were compared, the former was shown to have higher cytoplasmic localization.
A DNA localized in the cytoplasm represented by the formula 5′-SEQ ID NO: 17 in Sequence Listing-3′-PO(OH)—O—CH2CH2OCH2CH2—NH—CO-SEQ ID NO: 1 in Sequence listing, wherein the first Ala in SEQ ID NO: 1 in Sequence Listing is β-alanine, was obtained at a yield of 2.7% in the same way as in Example 1 except that 5′-SEQ ID NO: 17 in Sequence Listing-3′ was used as a DNA to introduce the NES peptide (SEQ ID NO: 1 in Sequence Listing) via a residue as a linker represented by the formula
The enzymatic degradation resistance thereof was 29.1%, the degradation property in serum was 42.3%, and the telomerase inhibition activity in a system using a cell lysis solution was 120 nM. In a cell system, approximately 12% telomerase activity inhibition was confirmed.
The observed telomerase inhibition activity of the raw material DNA used as a control was 400 nM or higher in a non-cell system and 0% in a cell system.
Next, this DNA localized in the cytoplasm was fluorescently labeled in the same way as in Example 1 and examined for its cytoplasmic localization. For comparison, the fluorescently labeled raw material DNA used as a control was also examined for its cytoplasmic localization. When they were compared, the former was shown to have higher cytoplasmic localization.
A DNA localized in the cytoplasm represented by the formula 5′-SEQ ID NO: 18 in Sequence Listing-3′-PO(OH)—O—CH2CH2OCH2CH2—NH—CO-SEQ ID NO: 3 in Sequence listing, wherein the first Ala in SEQ ID NO: 1 in Sequence Listing is β-alanine, was obtained in the same way as in Example 2 except that 5′-SEQ ID NO: 18 in Sequence Listing-3′ and MAPKK (SEQ ID NO: 3 in Sequence Listing) were used as a DNA and an NES peptide, respectively.
The enzymatic degradation resistance thereof was 34.2%, the degradation resistance in serum was 41.4%, and tyrosine kinase activity inhibition was approximately 46.2%. The enzymatic degradation resistance of the raw material DNA used as a control was 49.2%, the degradation resistance in serum thereof was 56.9%, and the tyrosine kinase activity inhibition thereof was approximately 21.8%.
Next, this DNA localized in the cytoplasm was fluorescently labeled in the same way as in Example 1 and examined for its cytoplasmic localization. For comparison, the raw material DNA used as a control was also examined for its cytoplasmic localization.
When they were compared, the DNA localized in the cytoplasm of the present invention exhibited cytoplasmic localization. Moreover, the DNA localized in the cytoplasm was shown to have more excellent enzymatic degradation resistance, degradation resistance in serum, and tyrosine kinase inhibition activity than those exhibited by the raw material DNA.
A DNA modified with an NLS peptide SV40 T antigen (SEQ ID NO: 12 in Sequence Listing) used instead of the NES peptide HIV-1 Rev in Example 1 in the same way as above was fluorescently labeled and examined for its cytoplasmic localization. As a result, its photomicrograph was exactly the same as that of the raw material DNA, wherein no cytoplasmic localization was observed.
An automatic DNA/RNA synthesizer (manufactured by Cruachem, product name: “PS250”) was used to chemically modify the 5′-terminus of the sense strand (5′-SEQ ID NO: 1 in Sequence Listing-3′) (hereinafter, referred to as RNA1) or the antisense strand (5′-SEQ ID NO: 2 in Sequence Listing-3′) (hereinafter, referred to as RNA2) of an RNA with an aminating reagent (manufactured by Glen Research, product name: “5′-Amino Modifier 5”) on controlled pore glass (hereinafter, referred to as CPG) by a solid phase method according to a routine method.
Subsequently, the obtained reaction product was supplemented with ammonia water with a concentration of 28% and stirred at 50° C. for 6 hours, thereby cleaving the chemically modified product from the solid support and removing the protecting group, followed by purification by reverse-phase high-performance liquid chromatography. The obtained chemically modified RNA was identified by mass spectrometry (MALDI TOF-MS). The following RNAs of two types having the chemically modified terminus were thus produced:
RNA3 5′-SEQ ID NO: 19 in Sequence Listing-3′ (sense); and
RNA4 5′-SEQ ID NO: 20 in Sequence Listing-3′ (antisense),
wherein n=—O—CH2CH2—O—CH2CH2NH2 in the nucleotide sequences.
Results of MALDI TOF-MS of the RNAs thus obtained are shown in Table 5.
An aminating reagent for substitution at intermediate positions (manufactured by Glen Research, product name: “Amino Modifier C2dT”) was used to produce, in the same way as in Production Example 1, the following RNAs of four types in which t or u located at a non-terminal position in the nucleotide sequence was substituted by X:
RNA5 5′-SEQ ID NO: 21 in Sequence Listing-3′ (sense);
RNA6 5′-SEQ ID NO: 22 in Sequence Listing-3′ (antisense);
RNA7 5′-SEQ ID NO: 23 in Sequence Listing-3′ (sense); and
RNA8 5′-SEQ ID NO: 24 in Sequence Listing-3′ (antisense),
wherein
Results of MALDI TOF-MS of the RNAs thus obtained are shown in Table 5.
The 5′-terminus of the RNA, was chemically modified in the same way as in Production Example 1. The monomethoxytrityl (MMT) group, a protecting group for a terminal amino group, was treated for 1 minute with a solution of 3% trichloroacetic acid acetonitrile and thereby removed.
Subsequently, the RNA, was supplemented and reacted at 20° C. for 5 hours with 0.5 M solution prepared by dissolving hexamethoxy diisocyanate in acetonitrile, to remove a product, which was in turn sequentially reacted in dimethylformamide with polyamines or saccharides of various types or peptides of various types having a free amino group.
Next, this reaction product was treated at 50° C. for 6 hours in concentrated ammonia water according to a routine method, thereby achieving cleavage from the solid support and the removal of the protecting group. The obtained 5′-terminal conjugate RNA was purified by reverse-phase high-performance liquid chromatography and identified by MALDI TOF-MS.
The RNAs shown in Table 1 in which the polyamine, saccharide, or peptide was introduced into the 5′-terminus were thus obtained.
Results of MALDI TOF-MS of the RNAs thus obtained are shown in Table 5.
Sense strand 5′-SEQ ID NO: 25 in Sequence Listing-3′,
wherein n=—O—CH2CH2—O—CH2CH2—NH—R1 in the nucleotide sequence.
A sense strand containing a chemical modification group X at a non-terminal position was produced in the same way as in Production Example 2. The trifluoroacetyl group, a protecting group of the aminating reagent, was treated for 1 minute with 20% acetonitrile solution of ethylene glycol and thereby removed. Subsequently, the RNA was reacted with an acetonitrile solution of hexamethylene diisocyanate in the same way as in Production Example 3 and then with dimethylformamide solution of polyamines of various types and treated in the same way as in Production Example 3, thereby obtaining RNAs shown in Table 2 in which the chemical modification group Y was introduced at the non-terminal position.
Results of MALDI TOF-MS of the RNAs thus obtained are shown in Table 5.
Sense strand=5′-SEQ ID NO: 26 in Sequence Listing-3′.
These sense strands and the antisense strands having the chemical modification group Y at the non-terminal position obtained in Production Example 3 were used to produce siRNAs localized in the cytoplasm. The siRNAs localized in the cytoplasm thus obtained were identified by MALDI TOF-MS.
Production Examples 3 and 4 were combined to produce RNAs shown in Table 3 in which the 5′-terminus of the RNA1 was chemically modified with n and in which t located at a non-terminal position of the nucleotide sequence was substituted by X. Results of MALDI TOF-MS of the conjugate RNAs thus obtained are shown in Table 5.
Sense strand=5′-SEQ ID NO: 27 in Sequence Listing-3′,
wherein n=—O—CH2CH2—O—CH2CH2—NH—R1 in the nucleotide sequence.
RNAs shown in Table 4 in which the 5′-terminus of the RNA1 was chemically modified with n and in which a plurality of u or t located at non-terminal positions of the nucleotide sequence was substituted by X were produced in the same way as in Production Example 5. Results of MALDI TOF-MS of the conjugate RNAs thus obtained are shown in Table 5.
siRNAs localized in the cytoplasm were formed by using the RNA9 and RNA15 to RNA27 as sense strands and the RNA2 as an antisense strand.
A 1-μM portion each of the siRNAs localized in the cytoplasm thus obtained was added to an RPMI medium containing 10% by mass of fetal bovine serum (FBS) and incubated at 37° C. to measure the degradation rates of the conjugate siRNAs after a lapse of 2, 4, 6, 12, and 24 hours. This measurement was performed by separating the siRNA by electrophoresis of 20% by mass of polyacrylamide gel and detecting it by use of a silver impregnation method. This result is shown in Table 6.
The degradation rate of an siRNA formed with the sense strand RNA1 having no chemical modification group introduced and the antisense strand RNA2 having no chemical modification group introduced was also written as a control.
As can be seen from this table, the siRNAs using the sense strand having the chemical modification group introduced at the 5′-terminus and the antisense strand having the chemical modification group introduced at the 5′-terminus obviously exhibited considerably improved resistance to enzymes except that the siRNA using the sense strand of the spermine conjugate (RNA9) had low resistance. Particularly, those using the sense strand having seven LeuArg or LeuLys sequences conjugated (RNA26 or RNA27) exhibited high resistance.
siRNAs were formed in the same way as in Examples 4 to 17 from the sense strands having the chemical modification group at the non-terminal position and the antisense strand having no chemical modification group to measure their degradation rates by enzymes. This result is shown in Table 7.
Most of the siRNAs formed from the sense strands having the chemical modification group at the non-terminal position and the antisense strand having no chemical modification group had 50% or lower degradation rates even after a lapse of 24 hours.
siRNAs were formed in the same way as in Examples 4 to 17 from the sense strands having the chemical modification groups simultaneously introduced at the 5′-terminus and the non-terminal position(s) and the antisense strand having no chemical modification group to measure their degradation rates by enzymes. This result is shown in Table 8.
As can be seen from this table, the siRNAs using the sense strands having the chemical modification groups simultaneously introduced at the 5′-terminus and the non-terminal position exhibited high enzymatic degradation resistance particularly when conjugated with the PKIα nuclear export signal peptide (RNA37), Dsk-1 nuclear export signal peptide (RNA39), SV40 T antigen nuclear localization signal peptide (RNA42), artificial peptide (RNA43), and artificial peptide (RNA44).
Moreover, all the siRNA simultaneously conjugated with the chemical modification groups at the 5′-terminus and a plurality of non-terminal positions exhibited high resistance such as 5% or lower degradation rate when conjugated with the HIV-1 Rev nuclear export signal peptide (RNA45), PKIα nuclear export signal peptide (RNA46), and MAPKK nuclear export signal peptide (RNA47).
Leukemia cells (Jurkat: 0.5×106 cells/ml) was added to a 100-μmM portion each of the siRNAs obtained in Examples 4 to 35 and incubated at 37° C. for 48 hours in an atmosphere containing 5% by volume of CO2 in an RPMI medium containing 10% by mass of FBS to measure cytotoxicity by use of a cell viability kit (manufactured by Promega).
As a result, slight cytotoxicity as much as 90% cell viability after 48 hours was observed in the siRNAs obtained Examples 24 and 29, whereas almost no cytotoxicity was observed in the remaining siRNAs.
The fluorescently labeled conjugate siRNAs (1 μM each) were added to leukemia cells (Jurkat: 1×106 cells/ml) and incubated at 37° C. for 48 hours in the presence of 5% CO2 in an RPMI medium containing 10% FBS. Then, the cells were washed three times with PBS (−) and observed for their introduction into the cells and intracellular localization by use of a fluorescence and laser scanning confocal microscope.
As a result, cellular uptake was significantly promoted in all the siRNAs obtained in Examples 5, 6, 7, 8, 12, 13, 14, and 17, which were conjugated with the peptides of various types at the 5′-terminus of the sense strand (under observation with the confocal laser induced fluorescence microscope).
The siRNAs obtained in Examples 5, 6, 7, and 8, which were conjugated with the nuclear export signal peptide, and the siRNA obtained in Example 14, which was conjugated with the artificial peptide, were shown to be respectively localized in the cytoplasm, whereas the siRNAs obtained in Examples 12 and 13, which were conjugated with the nuclear export signal peptide, and the siRNA obtained in Example 16, which was conjugated with the artificial peptide, were shown to be respectively localized in the nucleus.
The conjugate siRNAs (100 nM each) were added to leukemia cells (K-562: 1×106 cells/ml) and incubated at 37° C. for 48 hours in the presence of 5% CO2 in an RPMI medium containing 10% FBS. Subsequently, their tyrosine kinase inhibition activities were measured by protein tyrosine kinase assay to indicate them in terms of percentage.
As a result, only 6% inhibition effect was observed in the natural siRNA (RNA1/RNA2), whereas inhibition effects as high as 90% or more were observed in the conjugate siRNAs. Particularly the siRNA (RNA36/RNA2) obtained in Example 24, which was simultaneously conjugated with the HIV-1 Rev export signal peptides in the 5′-terminus and the proximity of the 3′-terminus of the sense strand, achieved almost 100% inhibition effect.
According to the present invention, efficacy of genetic medicine can be improved. Therefore, the present invention is highly usable in medical fields.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2004-045488 | Feb 2004 | JP | national |
| 2004-136228 | Apr 2004 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP05/02743 | 2/21/2005 | WO | 00 | 11/1/2006 |
