JP2005312397A - NEW CONJUGATED TYPE siRNA - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new siRNA having an improved enzyme resistance, without being decomposed by enzymes in cells, also localized in cytoplasm, exhibiting a high activity and capable of being suitably utilized as a genetic medicine. <P>SOLUTION: This new conjugated type siRNA has a feature that a chemically modified base is introduced at a 5'-terminal of at least one of a sense chain and an antisense chain constituting a double strand, at a dangling end of the antisense chain or at both of them, the chemically modified base is contained at a non-terminal position of at least one of the sense chain and antisense chain, an extracellular transporting signal peptide or a membrane fusion peptide is introduced at the 5'-terminal of at least one of the sense chain and antisense chain constituting the double strand, at the dangling end of the antisense chain or at both of them through a bifunctional linker, or the extracellular transporting signal peptide or the membrane fusion peptide is introduced to at least one of the sense chain and antisense chain at its non-terminal position through the bifunctional linker. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a novel siRNA with enhanced enzyme resistance, which is difficult to be degraded by an enzyme in a cell, and a highly active novel siRNA that can be introduced into a cell and can be localized in the cytoplasm.

Since RNA interference has a much stronger ability to control gene expression than antisense RNA and ribozymes, there is an increasing expectation as a tool for elucidating gene drugs and gene functions.
However, siRNA cannot be put into practical use because it is difficult to introduce into cells and has a low resistance to degrading enzymes in cells and a stable effect cannot be obtained.

  On the other hand, gene drugs are known to exhibit higher potency in the cytoplasm than intracellular nuclei. Therefore, if it is possible to achieve localization of the gene drug in the cytoplasm, it is excellent. However, no such gene drug has been known so far.

  Under such circumstances, the present invention provides a novel enzyme that increases enzyme resistance, is not degraded by the enzyme in the cell, is localized in the cytoplasm, exhibits high activity, and can be suitably used as a gene drug. It was made for the purpose of providing siRNA.

  The present inventors have conducted various studies on siRNA to enable its introduction into cells and to increase enzyme resistance in the cells or to enable localization to the cytoplasm. By introducing a chemically modified base capable of imparting cationic or lipophilic properties, it is possible to introduce into the cell and to increase enzyme resistance in the cell, and through this chemically modified base, It was found that if a transition signal peptide (hereinafter abbreviated as NES peptide) was bound, it could be localized in the cytoplasm, and the present invention was made based on this finding.

That is, the present invention is characterized in that a chemically modified base is introduced into the 5 ′ end of at least one of the sense strand and the antisense strand constituting the double strand or the dangling end of the antisense strand or both. Conjugated siRNA, and the 5 'end of at least one of the sense strand and the antisense strand constituting the double strand and / or the dangling end of the antisense strand, or both, via a bifunctional linker The present invention provides a conjugated siRNA characterized by introducing a transition signal peptide or a membrane fusion peptide.
Here, the dangling end means a portion that is non-complementary to the anti-strand of the antisense strand constituting the double strand, that is, a single-stranded portion.

  In the conjugated siRNA of the present invention, a chemically modified base is introduced at the 5 ′ end of at least one of the sense strand and the antisense strand constituting it or the dangling end of the antisense strand or both.

As such, for example, the formula 5′-sequence listing SEQ ID NO: 1-3 ′
A sense strand represented by the formula 5′-sequence listing SEQ ID NO: 2-3 ′
In the conjugated siRNA composed of the antisense strand represented by the formula, a chemically modified base is introduced only at the 5 ′ end of the sense strand, and a chemically modified base is introduced only at the 5 ′ end of the antisense strand. In which a chemically modified base is introduced into both the 5 ′ end of the sense strand and the 5 ′ end of the antisense strand, the formula 5′-sequence listing SEQ ID NO: 1-3 ′
A sense strand represented by the formula 5′-sequence listing SEQ ID NO: 2-3 ′
In which a chemically modified base is introduced into both the 5 ′ end of the sense strand and the dangling end of the antisense strand, and the antisense strand dangling Examples thereof include those in which a chemically modified base is introduced only at the end.

（式中のＡ及びｎは前記と同じ）
で表わされる環状有機塩基をもつカルボン酸アミドのアルキレンジアミン付加物残基などの水溶性を付加し得る基を挙げることができる。
これらの中で特に好ましいのは、Ａがエチレン基でｎが１又は２の化合物である。 As the chemically modified base introduced at this time, for example, the general formula — (OA) _n —NH ₂ (I)
(In the formula, A is an alkylene group, and n is an integer of 1 or more)
An ether residue of alkylene glycol or polyalkylene glycol and hydroxyalkylamine represented by the general formula

(A and n in the formula are the same as above)
The group which can add water solubility, such as the alkylenediamine adduct residue of the carboxylic acid amide which has the cyclic organic base represented by these can be mentioned.
Particularly preferred among these are compounds in which A is an ethylene group and n is 1 or 2.

Next, as a conjugated siRNA in which at least one of the sense strand and the antisense strand contains a chemically modified base at a non-terminal position, for example, the formula 5′-sequence listing SEQ ID NO: 1-3 ′
A sense strand represented by the formula 5′-sequence listing SEQ ID NO: 2-3 ′
In which a second t from the 3 ′ end of the sense strand is replaced with a chemically modified base, a dangling end of the antisense strand, ie 3 ′ Only the second t from the end is replaced with a chemically modified base, the second t from the 3 ′ end of the sense strand and the dangling end of the antisense strand, ie the second t from the 3 ′ end, are respectively chemically modified. Replaced with a base, 6th and 15th u from the 5 'end of the sense strand replaced with a chemically modified base, 5th, 7th, 10th from the 3' end of the antisense strand And the 15th u is replaced with a chemically modified base, the 6th and 15th u from the 5 'end of the sense strand and the 5th, 7th, 10th and 15th from the 3' end of the antisense strand U In which is replaced with a chemically modified base.

  There are no particular limitations on the number of bases of these sense strands and antisense strands and their binding order, and any of the known siRNA structures can be selected. Also, the structure of the chemically modified base is not particularly limited, and the size, type of functional group, etc. can be arbitrarily selected.

  Thus, siRNA into which a chemically modified base is introduced has enhanced enzyme resistance in cells and no cytotoxicity is observed.

On the other hand, in the present invention, by conjugating an NES peptide or a membrane fusion peptide to an oligonucleotide, it can be introduced into a cell, is not degraded into an enzyme in the cell, and can be localized in the cytoplasm. Highly active siRNA can be obtained.
This NES peptide or membrane fusion peptide is introduced through a bifunctional linker, and as this bifunctional linker, two functional groups that can react with an active hydrogen-containing group to form a stable bond are used. The compound which has is used.

（ロ）Ｎ‐スクシンイミジル＝３‐（２‐ピリジルジチオ）プロピナート

（ハ）ヨードアセトキシスクシンイミド

（ニ）ジチオイソシアナトアルカン
ＳＮＣ−（ＣＨ₂）_n−ＮＣＳ
（ホ）ジイソシアナトアルカン
ＯＮＣ−（ＣＨ₂）_n−ＮＣＯ
（ただしｎは１〜１０の整数）
（ヘ）２‐（２‐ピリジルジチオ）エチルイソシアネート

（ト）Ｎ‐（４‐マレイミドブチリルオキシ）スクシンイミド

（チ）Ｎ‐４‐マレイミド酪酸

Examples of such compounds include the compounds shown in the following (a) to (h).
(I) S- (2-pyridyldithio) cysteamine

(B) N-succinimidyl = 3- (2-pyridyldithio) propinate

(C) Iodoacetoxysuccinimide

(D) Dithioisocyanatoalkane SNC- (CH ₂ ) _n -NCS
(E) diisocyanato alkane ONC- (CH _{2) n} -NCO
(Where n is an integer from 1 to 10)
(F) 2- (2-Pyridyldithio) ethyl isocyanate

(G) N- (4-maleimidobutyryloxy) succinimide

(H) N-4-maleimidobutyric acid

  Examples of NES peptides introduced into siRNA via this bifunctional linker include HIV-1 Rev (SEQ ID NO: 3), PKIα (SEQ ID NO: 4), MAPKK (SEQ ID NO: 5), Dsk -1 (SEQ ID NO: 6), TFIIIA (SEQ ID NO: 7) and the like, but other NES peptides can also be used.

  Examples of peptides other than the NES peptide include HIV-1 tat C-terminus membrane fusion peptide (SEQ ID NO: 8), gp-41 membrane fusion peptide (SEQ ID NO: 9), SV40 T antigen ( antigen) nuclear localization signal (SEQ ID NO: 10), HIV-1 tat nuclear localization signal (SEQ ID NO: 11), amphipathic artificial design peptide α-helix (SEQ ID NO: 12) ), Amphipathic artificial design peptide β-sheet (SEQ ID NO: 13), amphipathic artificial design peptide β-sheet (SEQ ID NO: 14), amphiphilic artificial design peptide β-sheet (SEQ ID NO: 13) 15).

  Furthermore, polyamines such as spermine, spermidine, glucosamine, and galactosamine can be introduced. When such a polyamine is introduced, enzyme resistance in cells is improved.

  In order to introduce the above-described peptide or polyamine into siRNA via a bifunctional linker, first, the bifunctional linker is reacted with the sense strand or antisense strand of siRNA. This is based on the solid phase fragment condensation method, in which the sense strand or the antisense strand is first condensed on a solid phase carrier such as porous glass, controlled porous glass (CPG), polyethylene glycol / polystyrene, and the active hydrogen-containing group. For example, an amino group, a carboxyl group, a thiol group, or a hydroxyl group is reacted with a bifunctional linker.

  As the solvent in this case, polar solvents such as acetonitrile, dimethylformamide, dimethylacetamide, and dimethylsulfoxide are preferable. The reaction temperature at this time is 10 to 40 ° C., and the reaction time varies depending on the kind of the bifunctional linker to be reacted, the kind of peptide or polyamine, the reaction temperature, etc., but is generally 2 to 10 hours. The concentration of the bifunctional linker in the solvent is suitably from 0.1 to 1 mol / liter. After completion of the reaction, it is thoroughly washed with the same solvent to remove impurities.

Next, while the condensation product of the sense strand or antisense strand of the siRNA thus obtained and the bifunctional linker is supported on the solid phase carrier, the peptide or polyamine is added to the organic solvent such as acetonitrile or dimethyl. It is dissolved in formamide and added, and reacted by stirring at 10 to 40 ° C. for 2 to 10 hours. After completion of the reaction, washing with the same solvent and removal of impurities yields siRNA bound to the solid support.
Next, if it is treated with an alkali and desorbed from the solid phase carrier and purified by chromatography or the like, the desired conjugate-type siRNA can be obtained in a yield of 2 to 50%.
As the alkali used at this time, concentrated aqueous ammonia or 0.5M sodium carbonate aqueous solution is preferable.

で表わされるアミンがある。 Further, as the bifunctional linker, it is particularly preferable to use a compound capable of introducing a chemically modified base represented by the above general formula (I) or (II). Such compounds include, for example, the formula HO—CH ₂ —CH ₂ —O—CH ₂ —CH ₂ —NH ₂
An amine represented by

There is an amine represented by

（ただし、Ｒ¹はＮＥＳペプチド、膜融合ペプチド、核局在化シグナルペプチドなどのペプチド又はスペルミン、スペルミジン、グルコサミン、ガラクトサミン、トリス（２‐アミノエチル）アミン、トリエチレンテトラミンなどのポリアミンである）
又は一般式

（ただし、Ｒ²はスペルミン、スペルミジン、グルコサミン、ガラクトサミンなどのポリアミンである）
で表わされる化学修飾塩基が導入される。 After reacting such a bifunctional linker with a sense strand or an antisense strand and then reacting with various peptides or polyamines, the general formula

(However, R ¹ is a peptide such as NES peptide, membrane fusion peptide, nuclear localization signal peptide or polyamine such as spermine, spermidine, glucosamine, galactosamine, tris (2-aminoethyl) amine, triethylenetetramine)
Or general formula

(However, R ² is a polyamine such as spermine, spermidine, glucosamine, galactosamine)
A chemically modified base represented by is introduced.

  The conjugated siRNA of the present invention can be obtained by forming a double strand according to a conventional method using the sense strand and / or antisense strand into which the chemically modified base produced as described above is introduced. it can.

This double strand is formed by introducing an unmodified sense strand and an antisense strand into which a chemically modified base is introduced, an antisense strand into which a chemically modified base is introduced and an unmodified antisense strand, or a chemically modified base. The sense strand and the antisense strand into which the chemically modified base is introduced can be dissolved in a polar organic solvent such as acetonitrile, dimethylformamide, dimethylacetamide, dimethylsulfoxide, and stirred for 1 to 10 hours. At this time, if desired, it can be heated to an elevated temperature, for example, 30 to 60 ° C. in order to promote the reaction.
The conjugated siRNA thus obtained is purified using reverse phase high performance liquid chromatography or the like according to a conventional method.

  According to the present invention, a novel highly active siRNA that can be easily introduced into a cell, is not degraded by an enzyme in the cell, and is localized in the cytoplasm is provided.

  Next, the best mode for carrying out the present invention will be described by way of examples, but the present invention is not limited to these.

Production Example 1
Using a DNA / RNA automatic synthesizer (product name “PS250” manufactured by KruAchem, Inc.) on a controlled porous glass (hereinafter referred to as “CPG”), the sense strand of RNA (5′-sequence list) is obtained by a solid phase method according to a conventional method. SEQ ID NO: 1-3 ') (hereinafter referred to as RNA ₁ ) or antisense strand (5'-Sequence Listing SEQ ID NO: 2-3') (hereinafter referred to as RNA ₂ ) amination reagent (Glen Research) And chemically modified with the product name “5′-amino modifier 5”).

Next, 28% strength aqueous ammonia was added to the obtained reaction product, and the mixture was stirred for 6 hours at 50 ° C., so that the chemically modified product was removed from the solid phase carrier and the protecting group was removed. Purified by reverse phase high performance liquid chromatography. The obtained chemically modified RNA was identified by mass spectrometry (MALDI TOF-MS). In this way, two kinds of RNA having the following chemically modified ends were produced.
RNA ₃ 5′-sequence listing SEQ ID NO: 16-3 ′ (sense)
RNA ₄ 5′-sequence listing SEQ ID NO: 17-3 ′ (antisense)
However, n = —O—CH ₂ CH ₂ —O—CH ₂ CH ₂ NH _{2 in} the base sequence
Table 5 shows the MALDI TOF-MS of the RNA thus obtained.

このようにして得たＲＮＡのＭＡＬＤＩ ＴＯＦ−ＭＳを表５に示す。 Production Example 2
Using an amino reagent for intermediate position substitution (manufactured by Glen Research, product name “Amino modifier C ₂ dT”), t or u at the non-terminal position in the base sequence is set to X in the same manner as in Production Example 1. Four kinds of RNA shown below were produced.
RNA ₅ 5'-sequence listing SEQ ID NO: 18-3 '(sense)
RNA ₆ 5′-sequence listing SEQ ID NO: 19-3 ′ (antisense)
RNA ₇ 5′-sequence listing SEQ ID NO: 20-3 ′ (sense)
RNA ₈ 5'-sequence listing SEQ ID NO: 21-3 '(antisense)
Where X =

Table 5 shows MALDI TOF-MS of the RNA thus obtained.

Production Example 3
After chemically modifying the 5 ′ end of RNA _{1 in} the same manner as in Production Example 1, the monoaminotrityl group (MMT), which is a protecting group for the terminal amino group, was treated with a 3% trichloroacetic acid acetonitrile solution for 1 minute. Desorbed.
Next, after adding a 0.5 molar solution prepared by dissolving hexamethoxydiisocyanate in acetonitrile and reacting at 20 ° C. for 5 hours, the product was separated, and this was separated into various polyamines, sugars or various peptides in dimethylformamide. Those having a free amino group were continuously reacted.

Next, the reaction product is treated in concentrated ammonia water at 50 ° C. for 6 hours according to a conventional method to cleave from the solid phase carrier and remove the protective group, and then the 5′-end conjugated RNA obtained. Was purified by reverse phase high performance liquid chromatography and identified by MALDI TOF-MS.
Thus, RNA having polyamine, saccharide or peptide introduced at the 5′-end shown in Table 1 was obtained.
Table 5 shows the MALDI TOF-MS of the RNA thus obtained.
Sense strand 5'-sequence listing SEQ ID NO: 22-3 '
However, n = —O—CH ₂ CH ₂ —O—CH ₂ CH ₂ —NH—R ^{1 in} the base sequence

Production Example 4
In the same manner as in Production Example 2, a sense strand containing a chemically modified base X at the non-terminal position is produced, and the trifluoroacetyl group, which is a protecting group of the amination reagent, is treated with a 20% acetonitrile solution of ethylene glycol for 1 minute. Was released. Next, in the same manner as in Production Example 3, first, hexamethylene diisocyanate is reacted as an acetonitrile solution, and various polyamines are reacted as a dimethylformamide solution. RNA having a chemically modified base Y at such a non-terminal position was obtained.
Table 5 shows the MALDI TOF-MS of the RNA thus obtained.
Sense strand = 5′-sequence listing SEQ ID NO: 23-3 ′
Y =

  Using these sense strands and the antisense strand having the chemically modified base Y at the non-terminal position obtained in Production Example 3, conjugate siRNA was produced. The conjugated siRNA thus obtained was identified by MALDI TOF-MS.

Production Example 5
By combining Production Example 3 and Production Example 4 with RNA, the RNA ₁ shown in Table 3 is produced by chemically modifying the 5 ′ end of RNA ₁ with n and substituting t at the non-terminal position of the base sequence with X. did. Table 5 shows MALDI TOF-MS of the conjugate RNA thus obtained.
Sense strand = 5′-sequence listing SEQ ID NO: 24-3 ′
However, n = —O—CH ₂ CH ₂ —O—CH ₂ CH ₂ —NH—R ^{1 in} the base sequence
X =

Production Example 6
In the same manner as in Production Example 5, RNA 5 shown in Table 4 was produced by chemically modifying the 5′-end of RNA ₁ with n, and substituting multiple u or t at the non-terminal position of the base sequence with X. did. Table 5 shows MALDI TOF-MS of the conjugate RNA thus obtained.

Examples 1-14
Conjugated siRNA was formed using RNA ₉ and RNA _{15 to} RNA ₂₇ as the sense strand and RNA ₂ as the antisense strand.
1 μM of the conjugated siRNA thus obtained was added to RPMI medium containing 10% by mass of fetal bovine serum (FBS), incubated at 37 ° C., and then 2 hours, 4 hours, 6 hours, 12 hours. The degradation rate of the conjugated siRNA after 24 hours was measured. This measurement was performed by separating siRNA by 20% by mass polyacrylamide gel electrophoresis and detecting it using a silver staining method. The results are shown in Table 6.
In addition, the degradation rate about siRNA formed using sense strand RNA ₁ which does not introduce | transduce a chemically modified base and antisense strand RNA ₂ which does not introduce | transduce a chemically modified base is also written together as a control | contrast.

As can be seen from this table, for siRNA using a sense strand having a chemically modified base introduced at the 5 ′ end and an antisense strand having a chemically modified base introduced at the 5 ′ end, spermine conjugate (RNA ₉ ) When the sense strand was used, the resistance to the enzyme was low, but in other cases, the resistance was clearly significantly improved. In particular, those using a sense strand conjugated with 7 LeuArg or LeuLys (RNA ₂₆ or RNA ₂₇ ) showed high resistance.

Examples 15-19
In the same manner as in Examples 1 to 14, siRNA was formed from a sense strand having a chemically modified base at the non-terminal and an antisense strand not having a chemically modified base, and the degradation rate by the enzyme was measured. The results are shown in Table 7.

  Most of siRNAs formed from a sense strand having a chemically modified base at the non-terminal and an antisense strand not having a chemically modified base have a degradation rate of 50% or less even after 24 hours.

Examples 20-32
In the same manner as in Examples 1 to 14, siRNA was formed from a sense strand in which a chemically modified base was introduced at the 5 ′ end and a non-terminal at the same time and an antisense strand having no chemically modified base, and the degradation rate by the enzyme was measured. . The results are shown in Table 8.

As is apparent from this table, for siRNAs using sense strands in which chemically modified bases are introduced simultaneously at the 5 ′ end and non-end, PKIα nuclear export signal peptide (RNA ₃₇ ), Dsk-1 nuclear export signal peptide (RNA ₃₉ ), SV40 T antigen nuclear localization signal peptide (RNA ₄₂ ), artificial peptide (RNA ₄₃ ), and artificial peptide (RNA ₄₄ ) are conjugated and show particularly high resistance to enzymatic degradation.

In addition, for siRNA in which 5 ′ end and a plurality of non-terminals are conjugated at the same time, HIV-1 Rev nuclear export signal peptide (RNA ₄₅ ), PKIα nuclear export signal peptide (RNA ₄₆ ) and MAPKK nuclear export signal All of the conjugates of peptides (RNA ₄₇ ) exhibit high resistance with a degradation rate of 5% or less.

Reference example 1
Leukemia cells (Jurkat: 0.5 × 10 ⁶ cells / ml) were added to 100 μmM of siRNA obtained in Examples 1 to 32, and in an RPMI medium containing 10% by mass of FBS in an atmosphere containing 5% by volume of CO ₂ . After incubating at 37 ° C. for 48 hours, cytotoxicity was measured using a cell viability kit (Promega).
As a result, the siRNA obtained in Example 21 and Example 26 showed a slight toxicity with a cell survival rate of 90% after 48 hours, but almost no toxicity was observed for other siRNAs. It was.

Reference example 2
Fluorescently labeled conjugate siRNA (1 μM) was added to leukemia cells (Jurkat: 1 × 10 ⁶ cells / ml) and incubated in RPMI medium containing 10% FBS for 48 hours at 37 ° C. in the presence of 5% CO ₂ . . Thereafter, the cells were washed three times with PBS (−), and cell introduction property and subcellular localization were observed with a confocal laser fluorescence microscope.
As a result, it was obtained in Example 2, Example 3, Example 4, Example 5, Example 9, Example 10, Example 11, and Example 13 in which various peptides were conjugated to the 5′-end of the sense strand. All of the siRNAs significantly enhanced cellular uptake (by observation with confocal laser-excited fluorescence microscope).
In addition, the siRNA obtained in Example 11 conjugated with the artificial RNA and the siRNA obtained in Example 2, Example 3, Example 4, and Example 5 conjugated with the nuclear export signal peptide were added to the cytoplasm, It was found that the siRNA obtained in Example 9 and Example 10 conjugated with the transition signal peptide and Example 13 conjugated with the artificial peptide were localized in the nucleus.

Conjugate siRNA (100 nM) was added to leukemia cells (K-562: 1 × 10 ⁶ cells / ml) and incubated in RPMI medium containing 10% FBS at 37 ° C. for 48 hours in the presence of 5% CO ₂ . Subsequently, the tyrosine kinase inhibitory activity was measured by the protein tyrosine kinase assay and expressed as a percentage.
As a result, the natural siRNA (RNA ₁ / RNA ₂ ) showed only a 6% inhibitory effect, whereas the conjugated siRNA showed a high inhibitory effect of 90% or more. In particular, the siRNA (RNA ₃₆ / RNA ₂ ) obtained in Example 21 in which the sense strand 5′-end and the vicinity of the 3′-end are simultaneously conjugated with the HIV-1 Rev nuclear export signal peptide have an almost 100% inhibitory effect. Achieved.

  According to the present invention, since the efficacy of gene medicine can be improved, the utility in the pharmaceutical field is high.

Claims (6)

  A conjugated siRNA, wherein a chemically modified base is introduced at the 5 ′ end of at least one of a sense strand and an antisense strand constituting the double strand or a dangling end of the antisense strand, or both.   A conjugated siRNA, wherein at least one of the sense strand and the antisense strand contains a chemically modified base at a non-terminal position.   The conjugated siRNA according to claim 1 or 2, wherein the chemically modified base binds a polyamine molecule.   A nuclear export signal peptide or a membrane fusion peptide via a bifunctional linker at the 5 'end of at least one of the sense strand and the antisense strand constituting the double strand or the dangling end of the antisense strand or both Conjugated siRNA, characterized in that is introduced.   A conjugated siRNA, wherein at least one of a sense strand and an antisense strand has a nuclear export signal peptide or a membrane fusion peptide introduced through a bifunctional linker at a non-terminal position. The conjugated siRNA according to claim 4 or 5, wherein the bifunctional linker is a divalent chemically modified base residue.