WO2013191223A1 - 核酸複合体および核酸多糖複合体 - Google Patents
核酸複合体および核酸多糖複合体 Download PDFInfo
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- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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- C12N2320/51—Methods for regulating/modulating their activity modulating the chemical stability, e.g. nuclease-resistance
Definitions
- the present invention relates to a technique for improving the stability of a nucleic acid complex composed of DNA and RNA in serum.
- siRNA Small interfering RNA
- RNAi RNA interference
- mRNA messenger RNA
- phosphate-type RNA which is a natural nucleic acid
- phosphate-type RNA which is a natural nucleic acid
- nucleolytic enzymes and proteins for this reason, natural nucleic acid pharmaceuticals have not had a significant effect in human clinical research.
- many chemically modified nucleic acids obtained by chemically modifying natural nucleic acids have been proposed.
- the hydroxyl group at the 2′-position of ribose is a methoxy group (2′-O-methyl) (see Non-Patent Document 2), fluorine (F) (see Non-Patent Document 3), locked nucleic acid (LNA) (non-Patent Document 3).
- Those chemically modified in Patent Document 4) are particularly known. It has also been reported that such chemical modification improves the binding affinity between siRNA and target mRNA.
- nucleic acid analogs Such chemically modified nucleic acids are called nucleic acid analogs.
- Nucleic acid analogs have succeeded in greatly extending the inactivation time compared to natural nucleic acids. This is because the nucleolytic enzyme cannot recognize the nucleic acid analog.
- non-naturally occurring toxicity such as nonspecific adsorption to proteins in vivo and causing unexpected physiological activity and severe liver damage, has become a problem.
- a technique has also been proposed in which a natural nucleic acid is encapsulated in a biocompatible compound, the membrane permeability is improved while protecting the nucleic acid from degradation, and the nucleic acid is introduced into the cell.
- Retroviruses see non-patent document 5
- adenoviruses see non-patent document 6
- retroviruses gave extremely promising results in vitro as gene carriers, but the inflammatory and immunogenicity of these naturally-derived viruses
- the nature and risk of mutagenesis and integration into the cellular genome has been pointed out and their use in vivo is limited.
- Non-Patent Document 7 polyethylene glycol-modified polycations (see Non-Patent Document 8), polyethyleneimines (see Non-Patent Document 9), and cationic polymer block copolymers (Non-Patent Document 10) have been used as non-viral artificial nucleic acid carriers. And dendrimers (see Non-Patent Document 11) have been developed. However, the safety of such cationic polymers has not been confirmed. In order to have a cationic property, the presence of an amino group is indispensable, but the amino group has a high physiological activity and has a risk of toxicity in the body.
- ⁇ -1,3-glucan as a gene carrier, and ⁇ -1,3-glucan forms a new type of complex with a nucleic acid drug (antisense DNA, CpG DNA).
- a nucleic acid drug antisense DNA, CpG DNA.
- ⁇ -1,3-glucan that exists in triplicate in nature is dissolved in an aprotic polar organic solvent such as dimethyl sulfoxide (DMSO) or an alkaline solution of 0.1 N or more to be dissociated into a single strand.
- DMSO dimethyl sulfoxide
- alkaline solution of 0.1 N or more alkaline solution of 0.1 N or more
- the nucleic acid to be complexed with ⁇ -1,3-glucan is a single-stranded nucleic acid, and in particular, polydeoxyadenine (poly dA) and polycytosine (poly C) are ⁇ , including schizophyllan (SPG). It has been reported to have a strong affinity with 1,3-glucan.
- ⁇ -1,3-glucan as a siRNA carrier to RNAi is also being studied.
- siRNA is a double-stranded nucleic acid, it cannot form a complex with ⁇ -1,3-glucan as it is. Therefore, a DNA-RNA heteronucleic acid in which poly (dA) is added to the siRNA sense strand to be complexed with ⁇ -1,3-glucan such as SPG is prepared, and the siRNA antisense strand is annealed with it.
- Poly (dA) -siRNA is produced. Thereafter, the poly (dA) portion is used to make a composite with SPG.
- siRNAs applications in functional genomics and potential as therapeutics.
- Potent gene-specific inhibitor properties of mixed-backbone antisense oligonucleotides comprised of 2'-deoxy-2'-fluoro-D-arabinose and 2'-deoxyribose nucleotides.
- RNA interference-mediated gene silencing of pleiotrophin through polyethylenimine-complexed small interfering RNAs in vivo exerts antitumoral effects in glioblastoma xenografts.M. Grzelinski, B. Urban-Klein, T.Martens, K. F. Czubayko and A. Aigner, Hum. Gene. Ther., 17, 751-766 (2006) Monomolecular Assembly of siRNA and Poly (ethylene glycol) -Peptide Copolymers. J. DeRouchey, C. Schmidt, G.F. Walker, C. Koch, C. Plank, E. Wagner and J. O.
- DNA-siRNA nucleic acid complex is unstable with respect to ribonucleases widely present in the living body, there is a problem that it is susceptible to rapid degradation in the living body like RNA.
- changes in molecular weight were confirmed using acrylamide gel electrophoresis. A band cleaved at the binding site of was confirmed.
- the present invention has been made in view of the above problems, and an object thereof is to provide a nucleic acid complex that is not decomposed at a binding portion between DNA and RNA even in a living body.
- the 3 ′ position of the deoxyribonucleotide residue at the 3 ′ end of the single-stranded DNA and the ribonucleotide residue at the 5 ′ end of one ribonucleotide strand of the double-stranded RNA A nucleic acid complex bound at the 5′-position, wherein the 2′-position hydroxyl group in the 5′-end nucleotide of the ribonucleotide chain bound to the single-stranded DNA is substituted with an alkoxy group or a halogen atom.
- the 3 ′ position of the deoxyribonucleotide residue at the 3 ′ end of the single-stranded DNA and the ribonucleotide residue at the 5 ′ end of one ribonucleotide strand of the double-stranded RNA A nucleic acid complex having a 5′-position bound thereto, wherein a phosphorothioate is a phosphorothioate group between the 3′-position of the first ribonucleotide bound to the single-stranded DNA and the 5′-position of the adjacent ribonucleotide.
- the present invention solves the above problems by providing a nucleic acid complex substituted with either -O-) or a trithiophosphoric acid diester group (-O-PS (S) -S-).
- the number of nucleotides of the single-stranded DNA may be 10 or more.
- the single-stranded DNA may be polydeoxyadenine.
- At least a part of the phosphoric acid diester group of the single-stranded DNA may be substituted with any of a phosphorothioate group, a dithiophosphoric acid diester group, and a trithiophosphoric acid diester group.
- at least 50% or more of the phosphoric acid diester group of the single-stranded DNA may be substituted with any of a phosphorothioate group, a dithiophosphoric acid diester group, and a trithiophosphoric acid diester group.
- the double-stranded RNA may be siRNA.
- the siRNA may be a 21-mer type or a 27-mer type.
- the siRNA target gene may be a gene expressed in a Dectin-1 expressing cell.
- the above-mentioned problems are solved by providing a nucleic acid polysaccharide complex that forms a triple helical structure.
- the polysaccharide having a ⁇ -1,3-glucan skeleton is preferably schizophyllan.
- the fourth aspect of the present invention solves the above problem by providing a pharmaceutical composition comprising the nucleic acid polysaccharide complex according to the third aspect of the present invention.
- the 3 ′ position of the deoxyribonucleotide residue at the 3 ′ end of the single-stranded DNA and the ribonucleotide residue at the 5 ′ end of one ribonucleotide strand of the double-stranded RNA A method for increasing the stability of a nucleic acid complex bound at the 5 ′ position to an RNase, comprising a hydroxyl group at the 2 ′ position in the 5 ′ terminal nucleotide of the ribonucleotide chain bound to the single-stranded DNA.
- the sixth aspect of the present invention includes the 3 ′ position of the deoxyribonucleotide residue at the 3 ′ end of the single-stranded DNA and the ribonucleotide residue at the 5 ′ end of one ribonucleotide strand of the double-stranded RNA.
- a method for increasing the stability of a nucleic acid complex bound to a 5 ′ position to an RNase comprising the 3 ′ position of the first ribonucleotide bound to the single-stranded DNA and the 5 ′ position of a ribonucleotide adjacent thereto.
- the present invention solves the above-mentioned problems by providing a method for stabilizing a nucleic acid complex in which the phosphoric acid diester group between the two positions is substituted with any of a phosphorothioate group, a dithiophosphoric acid diester group, and a trithiophosphoric acid diester group.
- DNA-RNA in which the 3 ′ position of the deoxyribonucleotide residue at the 3 ′ end of single-stranded DNA and the 5 ′ position of the ribonucleotide residue at the 5 ′ end of one ribonucleotide strand of double-stranded RNA are bound In the nucleic acid complex, the hydroxyl group at the 2 ′ position in the 5 ′ terminal nucleotide of the ribonucleotide strand bound to the single-stranded DNA is replaced with an alkoxy group or a halogen atom, or the first ribonucleotide bound to the single-stranded DNA
- replacing the phosphodiester group between the 3 ′ position of the ribonucleotide and the 5 ′ position of the adjacent ribonucleotide By replacing the phosphodiester group between the 3 ′ position of the ribonucleotide and the 5 ′ position of the adjacent ribonucleotide with one of
- nucleic acid complex of the present invention can maintain its original function without being rapidly degraded in vivo and losing its function.
- a nucleic acid complex in which a polydeoxynucleotide that forms a stable complex with ⁇ -1,3-glucan (such as schizophyllan) such as poly (dA) is bound to the 5 ′ end of siRNA and ⁇ -1
- a nucleic acid complex of the present invention is applied to a nucleic acid polysaccharide complex formed from 1,3-glucan, siRNA can be reliably delivered to the cell nucleus without being degraded in the cytoplasm, and RNA interference can be prevented. It is expected to increase the usefulness and therapeutic effect of nucleic acid drugs that suppress the expression of genes that cause diseases.
- Example 2 is a gel fluorescence image showing the results of a dA40-siRNA degradation experiment in serum (Example 1). It is a gel fluorescence image which shows the result of the identification experiment (Example 2) of the degradation product in the serum of the fluorescence modification dA10-siRNA prepared using the antisense strand which added FITC to the 5 'terminal. It is a gel fluorescence image which shows the result of the identification experiment (Example 2) of the degradation product in the serum of fluorescence modification dA10-siRNA prepared using the antisense strand which added FITC to the 3 'terminal. It is an image figure which shows decomposition
- 2 is a gel fluorescence image showing the effect of 2'-O-methyl modification on RNA degradation (Example 3).
- 2 is a gel fluorescence image showing the effect of 2'-O-methyl modification on the degradation of single-stranded RNA (Example 4).
- FIG. 6 is a gel fluorescence image showing the effect of 2′-O-methyl modification positions on the degradation of nucleic acid complexes in serum (Example 5).
- FIG. It is a gel fluorescence image which shows the influence (Examples 6 and 7) of the kind of base exerted on DNA degradation.
- It is a gel fluorescence image which shows the effect (Example 8) of phosphorothioate modification on degradation of RNA.
- It is a gel fluorescence image which shows the influence (Example 9) when DNA has joined to the 3 'terminal of RNA.
- the nucleic acid complex according to the first embodiment of the present invention includes a 3′-position of a deoxyribonucleotide residue at the 3′-end of a single-stranded DNA and a 5′-side of one ribonucleotide strand of a double-stranded RNA.
- the nucleic acid complex according to the present embodiment corresponds to the case where R 2 is an alkoxy group or a halogen atom in the following general formula (I).
- R 1 is any one of adenine (A), guanine (G), uracil (U) and cytosine (C), and R 3 and R 5 are phosphate ester groups (—PO 2 -O-).
- R 3 and R 5 are phosphate ester groups (—PO 2 -O-).
- the alkoxy group include linear or branched alkoxy groups having 1 to 5 carbon atoms, arylalkyl groups having 5 to 15 carbon atoms, and alkenylalkyl groups such as O-allyl groups.
- halogen atom include a fluorine atom (F), a chlorine atom (Cl), a bromine atom (Br), and an iodine atom (I), with a fluorine atom being particularly preferred.
- a polyribonucleotide portion bonded to a polydeoxynucleotide portion may form a double strand with RNA having a complementary base sequence to constitute siRNA.
- siRNA is a short double-stranded RNA that participates in a phenomenon called RNA interference (RNAi), destroys mRNA containing a complementary base sequence, and has a function of suppressing gene expression in a sequence-specific manner. .
- RNAi RNA interference
- the siRNA may be a 21-mer type that is widely used, but may be a 27-mer type in which the specificity for dicer is further improved.
- SiRNA base sequence design can be carried out by any known method. If a sequence complementary to a sequence highly conserved among multiple genes is selected, it may be difficult to suppress gene-specific expression. For example, a sequence complementary to a sequence specific to the target gene is selected.
- siRNA can be obtained using any known method based on the known sequence data obtained from databases such as GenBank, EMBL, PDB, and DDBJ. Can be designed.
- the single-stranded DNA (polydeoxynucleotide) part in the nucleic acid complex may itself have a unique function, and is intended to improve the stability of the double-stranded RNA (polyribonucleotide) part.
- a specific base for improving the complex-forming ability when a complex (nucleic acid polysaccharide complex) of a nucleic acid complex and ⁇ -1,3-glucan is formed. It may have a sequence (including repetitive sequences).
- the number of nucleotides of single-stranded DNA is not particularly limited, but is preferably 10 or more.
- a phosphodiester group (also referred to as phosphodiester bond, phosphodiester bond, etc.) in single-stranded DNA and a part of R 5 in the above general formula (I) More preferably, 50% or more is a phosphorothioate group (thiophosphoric acid ester group: —O—PO (S) —O—: a structure in which P ⁇ O 2 of the phosphoric acid group is substituted with P ⁇ S), dithiophosphoric acid diester It may be substituted with either a group (—O—PS (S) —O—) or a trithiophosphoric acid diester group (—O—PS (S) —S—).
- thiophosphoric acid ester group —O—PO (S) —O—: a structure in which P ⁇ O 2 of the phosphoric acid group is substituted with P ⁇ S
- dithiophosphoric acid diester It may be substituted with either a group (—O—PS (S) —O—) or a
- the polynucleotide having the base sequence as described above can be synthesized using any known method such as a chemical synthesis method or a genetic engineering method.
- the nucleic acid complex according to the second embodiment of the present invention includes the 3 ′ position of the deoxyribonucleotide residue at the 3 ′ end of the single-stranded DNA and the 5 ′ side of one ribonucleotide strand of the double-stranded RNA.
- the phosphoric acid diester group is substituted with any of a phosphorothioate group, a dithiophosphoric acid diester group and a trithiophosphoric acid diester group. That is, the nucleic acid complex according to the present embodiment corresponds to the case where R 3 is any one of a phosphorothioate group, a dithiophosphate diester group, and a trithiophosphate diester group in the following general formula (I).
- R 1 is any one of adenine (A), guanine (G), uracil (U) and cytosine (C)
- R 2 is a hydroxyl group
- R 5 is a phosphate ester.
- R 2 may be an alkoxy group or a halogen atom
- R 5 may be any one of a phosphorothioate group, a dithiophosphoric acid diester group, and a trithiophosphoric acid diester group.
- the nucleic acid complex obtained as described above is allowed to interact with ⁇ -1,3-glucan, a single-stranded DNA site of one molecule of the nucleic acid complex, and two polysaccharide molecules having a ⁇ -1,3-glucan skeleton. Forms a nucleic acid polysaccharide complex forming a triple helical structure.
- the polynucleotide can be protected from hydrolysis, and the half-life in blood and body fluid can be greatly increased (for example, about 10 times). Therefore, for example, a nucleic acid complex containing siRNA can be reliably delivered to a target cell.
- a polysaccharide whose main chain is composed of ⁇ -1,3-glucan and ⁇ -1,3-xylan has a helix parameter that approximates a nucleic acid such as poly (C) (for example, Takahashi, Kominato, Suzuki, Prog Polym.Phys.Jpn.27, p.767, and “Conformation of Carbohydrates”, Sharwood academic publisher, 1998), which has a hydroxyl group capable of hydrogen bonding with a nucleobase, and thus interacts with a nucleic acid. It is known to form a stable complex having a triple helical structure.
- C poly
- ⁇ -1,3-glucan examples include schizophyllan, curdlan, lentinan, parkan, glyphoran, scleroglucan and the like. These are natural polysaccharides whose main chains are linked by ⁇ -linkages ( ⁇ -D-linkages) and have different side chain frequencies. These ⁇ -1,3-glucans may be used as they are without undergoing treatment such as chemical modification, but their solubility can be reduced by thinning out their side chains appropriately using a normal periodate oxidation method. It can also be controlled.
- the molecular weight of ⁇ -1,3-glucan is appropriately adjusted according to the base length of the polynucleotide used for the preparation of the therapeutic agent for inflammatory bowel disease, the base length of the repetitive sequence, and the like. However, when the molecular weight is small, the so-called cluster effect (polymeric cooperative phenomenon) is difficult to be exhibited, which is not preferable.
- the weight average molecular weight of ⁇ -1,3-glucan capable of forming a complex with a nucleic acid varies depending on the type of nucleobase and the higher order structure, but is preferably 2000 or more, more preferably 4000 or more, and more preferably Is 6000 or more.
- the number of hydroxyl groups that form hydrogen bonds with nucleobases on the polynucleotide is usually 5 or more, preferably 8 or more, and more preferably 10 or more.
- the weight average molecular weight of ⁇ -1,3-glucan can be determined by using any known method such as a light scattering method, a sedimentation velocity method (ultracentrifugation method) or the like.
- ⁇ -1,3-glucan is generally produced by fungi and eubacteria, after culturing these microorganisms, the cells are homogenized, and impurities such as cell elution and insoluble residues are used to obtain ultracentrifugation and other methods. It can be obtained by isolation.
- ⁇ -1,3-glucan obtained in this way has a high molecular weight (weight average molecular weight of several hundreds of thousands) and a triple helical structure. However, it may be used as it is, and it may be reduced in molecular weight as necessary. It may be used.
- the molecular weight reduction is performed by appropriately selecting appropriate methods and conditions from any methods and conditions such as enzymatic degradation and acid hydrolysis according to the type of ⁇ -1,3-glucan and the desired molecular weight.
- any methods and conditions such as enzymatic degradation and acid hydrolysis according to the type of ⁇ -1,3-glucan and the desired molecular weight.
- schizophyllan single-chain schizophyllan having various molecular weights can be obtained by hydrolysis with 80% DMSO-sulfuric acid.
- ⁇ -1,3-glucan such as schizophyllan usually has a triple helical structure in water. Therefore, in order to form a complex with the polynucleotide, it is dissolved in a solvent such as DMSO (dimethyl sulfoxide) to dissolve the association state due to intermolecular hydrogen bonding and hydrophobic interaction to form a single strand.
- a solvent such as DMSO (dimethyl sulfoxide)
- a solvent such as DMSO (dimethyl sulfoxide)
- the association of the polynucleotide and the polysaccharide is formed within and between the molecules while associating and taking up the molecular chain of the polynucleotide.
- a complex having a triple helix structure composed of one molecule of polynucleotide and two molecules of ⁇ -1,3-glucan molecule is formed.
- the formation of the complex can be confirmed by examining the conformational change, for example, by measuring a CD (circular dichroism) spectrum.
- the resulting complex is generally water soluble and dissociates and recombines with temperature and pH changes. Furthermore, the complex is resistant to nucleolytic enzymes and the polynucleotide is not destroyed.
- the single-stranded DNA portion of the nucleic acid complex is either a poly (dA) sequence or a poly (dT) sequence. It preferably has such a repeating sequence.
- the types of bases and nucleotides constituting the repetitive sequence and the number of bases are appropriately determined according to the length of the ribonucleotide portion, the type and molecular weight of ⁇ -1,3-glucan used.
- the polydeoxynucleotide portion preferably has a poly (dA) sequence as a repeating sequence.
- the length of the repeating sequence is, for example, The length is preferably 10 bases or more, and more preferably 10 to 80 bases in length.
- any known method can be used without particular limitation.
- FBS fetal bovine serum
- 10 Prepare RPMI1640 medium to contain% FBS, add DNA-siRNA nucleic acid complex to it, incubate at 37 ° C for 1 hour, stain the nucleic acid with SYBR Gold after 12% acrylamide gel electrophoresis, and observe with gel imaging device The method of doing is mentioned.
- the nucleic acid polysaccharide complex can be used as an active ingredient in the production of a pharmaceutical composition for gene therapy including RNAi.
- a pharmaceutical composition for gene therapy including RNAi.
- any known ingredients any carrier, excipient and additive acceptable for pharmaceutical use
- formulation methods can be used.
- therapeutic agents for inflammatory bowel disease can take the form of tablets, suppositories, capsules, syrups, microcapsules such as nanogels, sterile solutions, suspensions, and the like.
- the pharmaceutical composition can be administered to humans or warm-blooded animals (mouse, rat, rabbit, sheep, pig, cow, horse, chicken, cat, dog, monkey, etc.) by either oral or parenteral routes.
- parenteral administration routes include subcutaneous and intramuscular injection, intraperitoneal administration, rectal administration, enteral administration using an endoscope and the like.
- the dose of the complex of the active ingredient nucleic acid complex and ⁇ -1,3-glucan molecule is determined depending on the activity, the disease to be treated, the type of animal to be administered, the body weight, sex, age, and severity of the disease. It varies depending on the degree and administration method. Taking an adult with a body weight of 60 kg as an example, in the case of oral administration, the daily dose is usually about 0.1 to about 100 mg, preferably about 1.0 to about 50 mg, more preferably about 1.0 to about 20 mg. In the case of parenteral administration, the daily dose is usually about 0.01 to about 30 mg, preferably about 0.1 to about 20 mg, more preferably about 0.1 to about 10 mg. When administered to other animals, the dose obtained by converting the above dose to a dose per unit body weight and multiplying by the body weight of the animal to be administered is used.
- the base sequence of the nucleic acid complex is appropriately selected depending on the disease to be treated and the type of target gene, but is expressed in Dectin-1 expressing cells. It is preferable that siRNA targeting the gene to be targeted is included.
- Dectin-1 is a membrane protein belonging to the C-type lectin expressed in dendritic cells and macrophages, and has the property of binding to ⁇ -glucan, and therefore is suitable for specifically introducing a nucleic acid polysaccharide complex.
- the nucleic acid complex (including those according to the first and second embodiments described above) that can be used for the production of the nucleic acid polysaccharide complex has a partial base sequence represented by the following formula (A). ing.
- the nucleic acid complex may consist of only the base sequence represented by the formula (A), or may have the base sequence as a partial base sequence.
- dRN represents deoxyribonucleotide
- AN represents a ribonucleotide derivative or peptide obtained by chemically modifying one or both of the 2′-position hydroxy group and the 5′-position phosphate group of ribonucleotide.
- RN represents ribonucleotide
- x and z are each independently an integer of 1 or more
- y is an integer of 1-10.
- the polydeoxynucleotide part (dRN) x located on the 5 ′ end side in the base sequence represented by the formula (A) is represented by the following formula (III), wherein the base B 1 is adenine (A ), Guanine (G), thymine (T) or cytosine (C).
- the 3 ′ terminal polyribonucleotide moiety (RN) z in the base sequence represented by the formula (I) is represented by the following formula (IV), in which the base B 2 is adenine (A ), Guanine (G), uracil (U) or cytosine (C).
- each base sequence of the polydeoxynucleotide part (dRN) x and the polyribonucleotide part (RN) z may be a gene encoding some biological function or a part thereof, or a primer sequence. Those having no biological function, such as one in which a certain number of bases are arranged and one in which a plurality of bases are regularly arranged, may be used.
- repeating unit constituting (AN) y located between the polydeoxynucleotide part and the polyribonucleotide part in the base sequence represented by the formula (I) are represented by the following general formula (II):
- PNA peptide nucleic acid
- V glycol nucleic acid
- VI locked nucleic acid
- VIII threose nucleic acid
- IX morpholino nucleic acid represented by the following formula (IX), and the like.
- R 1 in the formula (II) and B 3 to B 7 in the formulas (V) to (IX) are adenine (A), guanine (G), cytosine (C), uracil (U) and an unnatural base ( Thymine, 8-oxoguanine, 2-amino-6-dimethylaminopurine, 2-amino-6-thienylpurine, pyridin-2-one, 4-acetylcytidine, 5- (carboxyhydroxymethyl) uridine, 2′-O -Methylcytidine, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluridine, dihydrouridine, 2'-O-methyl pseudouridine, ⁇ -D-galactosyl cueosine, 2'-O- Methyl guanosine, inosine, N6-isopentenyl adenosine, 1-methyl adenosine, 1-methyl pseudo
- R 2 is a hydrogen atom (H), a halogen atom (F, Cl, Br, I), a hydroxyl group, a linear or branched alkoxy group having 1 to 5 carbon atoms, a carbon number of 5 to Any one of 15 arylalkyl groups, O-allyl groups and the like
- R 5 is any one of a phosphoric diester group, a phosphorothioate group (thiophosphoric diester group), a dithiophosphoric diester group, and a trithiophosphoric diester group (Except when R 2 is a hydroxyl group and R 5 is a phosphate group).
- repeating units of the y moiety particularly preferred are those in the formula (II) where R 2 is an O-methyl group and R 5 is a phosphate group, and R 2 is a hydroxyl group and R 5 is a phosphorothioate. This is the case.
- the base sequence of the y portion may constitute a part of the base sequence encoding a gene having a biological function, continuously with the base sequence of the polydeoxyribonucleotide portion and / or the polyribonucleotide portion, It may have a base sequence having some function independently, and may be a regular or random base sequence having no particular function.
- y is an integer of 1 or more and 10 or less, preferably 1 or more and 4 or less, particularly preferably 1.
- Example 1 Confirmation of degradation of (dA) 40 -siRNA in serum siRNA sense strand (5′-CAAAGACAACCAACUAGUGGU-3 ′: SEQ ID NO: 1.
- RNA DNA-RNA nucleic acid complex in which deoxyadenine (hereinafter sometimes abbreviated as “dA”) 40-mer (dA40) is added to the 5 ′ end of the strand and the antisense strand of siRNA (5 '-ACCACUAGUUGGUUGUCUUUG-3': SEQ ID NO: 2) is annealed to add a DNA-siRNA nucleic acid complex (dA40-siRNA.
- x-mer of deoxyadenine x is a natural number
- the DNA-siRNA nucleic acid complex obtained by annealing the DNA-RNA nucleic acid complex and the antisense strand was abbreviated as “dAx-siRNA”). All the nucleic acids used were purchased from Hokkaido System Science Co., Ltd.
- DNA-siRNA nucleic acid complex was added to RPMI1640 medium containing 10% FBS, incubated at 37 ° C. for 1 hour, and subjected to 12% acrylamide gel electrophoresis (100 V, 1 hour). The gel was stained with SYBR Gold (Life Technologies, California, USA), and a fluorescent image of the gel was taken with a fluorescent imager.
- the obtained gel fluorescence image is shown in FIG.
- the band of the dA40-siRNA nucleic acid complex before the incubation is detected on the higher molecular side than the band of the control dA40 and siRNA (21 bp).
- two bands are observed on the low molecular side. From the position of the band, it is considered that the DNA-siRNA nucleic acid complex was cleaved into DNA and siRNA by the action of the enzyme contained in FBS. Similar results were obtained when similar experiments were performed using RPMI 1640 medium containing mouse serum (MS) instead of FBS.
- Example 2 Examination of cleavage site of DNA-siRNA nucleic acid complex It was examined how a DNA-siRNA nucleic acid complex was cleaved by an enzyme in serum using a fluorescence-modified nucleic acid.
- Each of the antisense strands prepared by FITC modification on the 5 ′ end side or 3 ′ end side is prepared, and deoxyadenine decamer (on the 5 ′ end side of the sense strand having the base sequence represented by SEQ ID NO: 1)
- the DNA-RNA nucleic acid complex to which dA10) was added and the sense strand to which dA10 was not added were each annealed. Both were incubated in serum, followed by fluorescence observation with FITC after gel electrophoresis, followed by SYBR Gold staining and fluorescence observation in the same manner.
- FIGS. 2A and 2B The obtained fluorescence images are shown in FIGS. 2A and 2B.
- siRNA obtained by annealing a sense strand to which dA10 is not added and an antisense strand whose FITC is modified on the 5 ′ end side
- both fluorescence bands are completely observed in the fluorescence image after FITC and SYBR Gold staining. You can see that they match.
- RNA double strand hardly decomposes in serum (FIG. 2A, lanes 1 and 2).
- siRNA in which the 3 ′ end of the antisense strand is FITC-modified the position of the fluorescent band derived from FITC and SYBR Gold staining is the same as in the case where the 5 ′ end of the antisense strand is FITC-modified. It does not change before and after incubation of FITC and FBS (FIG. 2B, lanes 1 and 2). This indicates that siRNA is not degraded by incubation in serum regardless of the FITC introduction site (5′-end side and 3′-end side), whereas dA10-siRNA does not significantly degrade after incubation with FBS. In FIG. 2, a fluorescent band is observed on the low molecule side (FIG.
- RNA duplex was shorter than 21 bp, and not only the sense strand but also the antisense strand was cleaved by the enzyme protein in FBS.
- dA10-siRNA is recognized in the vicinity of RNA at the junction with DNA in serum and undergoes degradation. After the degradation, it is considered that the double-stranded RNA shorter than 21 bp, the single strand obtained by adding the quantifier RNA to dA, and the complementary strand of the RNA are separated (FIG. 2C shows a schematic diagram). ).
- Example 3 Avoidance of DNA-siRNA nucleic acid complex degradation by 2'-O-methylation of RNA residues in siRNA From the results of Example 2, it is considered that the DNA and siRNA are degraded in the vicinity of the junction. Therefore, the first one, two, three, and four RNA residues counted from the 5 ′ end of the siRNA (sense strand) joined to the 3 ′ end of the DNA (dA40) are each 2 A '-O-methylated dA40-siRNA nucleic acid complex was prepared, and the stability in serum (FBS) was evaluated using gel electrophoresis of the sample before and after incubation as in Example 2. .
- FBS stability in serum
- the obtained gel fluorescence image is shown in FIG.
- the previously unmethylated DNA-siRNA was cleaved after incubation with serum for 1 hour, but it was confirmed that the position of the band did not change even after 20 hours when methylated. It was also found that degradation could be sufficiently prevented by only 2'-O-methylating only the first RNA (5'-end side) RNA site to join DNA.
- Example 4 Comparison of DNA-siRNA Nucleic Acid Complex and DNA-RNA Nucleic Acid Complex From the previous experiment, the degradation of the DNA-siRNA nucleic acid complex by incubation in serum showed that In order to investigate whether this degradation is a phenomenon specific to RNA duplex, it is considered that this occurs at the point of attachment, but siRNA (sense strand) joined to the 3 ′ end of DNA (dA40) Using dA40-RNA (single stranded) nucleic acid complex in which the first 1, 2, 3, 4 RNA residues counted from the 5 ′ end are 2′-O-methylated, respectively. The same experiment as in Example 3 was performed.
- RNA-siRNA nucleic acid complexes In the case of double-stranded RNA, degradation is sufficient if the first RNA residue counted from the 5 ′ end of the RNA sense strand joined to the 3 ′ end of DNA is 2′-O-methylated. Although it could be avoided, in the case of single-stranded RNA, 2'-O-methylation of the RNA residue in the RNA joined to the 3 'end of DNA may cause degradation of other RNA moieties. all right. In other words, avoiding degradation by 2′-O-methylation of RNA residues adjacent to the 3 ′ end of DNA is effective for DNA-siRNA nucleic acid complexes but not for DNA-RNA nucleic acid complexes. It was confirmed not to have.
- Example 5 Examination of 2'-O-methylation site of RNA residue effective for avoiding degradation of DNA-siRNA nucleic acid complex From the result of Example 3, in order to prevent degradation of DNA-siRNA nucleic acid complex It was found that one RNA residue is sufficient for 2′-O-methylation, but the sense strand where the 2′-O-methylation site is joined to the 3 ′ end side of the DNA In order to confirm whether it is necessary to be the first RNA counted from the 5 ′ end side, a similar experiment was performed by changing the location of the RNA residue to be 2′-O-methylated. 2′-O-methylation was performed on the RNA residue at the 5 ′ end or the second RNA from the 5 ′ end. In the above-described embodiment, the length of dA is made into a 40-mer considering that it is combined with SPG. However, whether the same phenomenon is observed even if the length of dA is shortened. The same was confirmed.
- Example 6 Examination of stability of DNA-dsDNA nucleic acid complex in serum
- FBS degradation in serum
- siRNA sequences both sense strand and antisense strand
- Example 7 Relationship between DNA base sequence in DNA-siRNA nucleic acid complex and stability of DNA-siRNA nucleic acid complex
- the DNA portion was dA10 or dA40.
- deoxycytosine (dC) deoxyguanine (dG)
- deoxythymidine A nucleic acid complex with siRNA was prepared using a 10-mer of (dT), and the same experiment was performed.
- the obtained gel fluorescence image is shown in FIG.
- dT10-siRNA and dC10-siRNA as with dA10-siRNA, a decrease in molecular weight (fluorescence band corresponding to the same molecular weight as that of siRNA) was observed after incubation with FBS. It is thought that it has been cut. Since dG10-siRNA has the property of aggregating at the dG10 portion, a fluorescence band is seen on the higher molecular weight side of the sample before incubation than other DNA-siRNA nucleic acid complexes, but after incubation with FBS. From the fact that a fluorescent band is observed at the same position as siRNA, it was found that dG10-siRNA was also subjected to the same degradation.
- Example 8 Avoiding degradation of the DNA-siRNA nucleic acid complex by chemical modification other than 2'-O-methylation
- the first ribonucleotide that joins the DNA is 2' It was found that -O-methyl modification (replacement of the hydroxyl group at the 2 'position of ribose with a methoxy group) can avoid degradation in serum, but there are other initial ribozymes that join the 3' end of DNA.
- a similar experiment was carried out by preparing a phosphorothioate group in which the phosphodiester group between the 3 ′ position of the nucleotide and the 5 ′ position of the second ribonucleotide was substituted.
- the obtained gel fluorescence image is shown in FIG.
- the phosphorothioate compound is obtained by replacing one of the oxygen atoms bonded to the phosphate group in the phosphodiester skeleton with a sulfur atom, and is obtained as a racemic mixture. Therefore, from the fact that half is decomposed and half remains without being decomposed, it is considered that only one of the S-form or R-form is a substrate for a degrading enzyme in serum.
- Example 9 When DNA is joined to the 3 ′ end of RNA DNA has been joined to the 5 ′ end of the sense strand of siRNA so far, but DNA is joined to the 3 ′ end of the sense strand. In the same way, in order to examine whether the first RNA that is joined to DNA undergoes degradation, the previous sequence was reversed (on the 3 ′ end side of the oligoribonucleotide represented by SEQ ID NO: 1). An experiment was conducted by preparing a nucleic acid complex to which poly (dA) was bound.
- the obtained gel fluorescence image is shown in FIG. No change in molecular weight was observed in serum even when DNA was joined to the 3 'end of the siRNA sense strand (Fig. 8, lanes 3 and 4). From this, it is considered that the degrading enzyme accurately recognizes DNA extending from the 5 'end of the DNA to the 3' end or from the 5 'end of the double-stranded RNA to the 3' end.
- the hydroxyl group at the 2 ′ position of the first RNA that joins DNA (the second and subsequent RNA residues that join DNA do not have an effect) is replaced with an alkoxy such as a methoxy group.
- a phosphorothioate group or a phosphodiester group between the 3 ′ position of the first ribonucleotide and the 5 ′ position of the second ribonucleotide joined to the 3 ′ end of DNA It is necessary to substitute with one of a group, a dithiophosphoric acid diester group and a trithiophosphoric acid diester group.
- Example 10 Preparation of complex of polynucleotide and schizophyllan (1) Preparation of triple helix schizophyllan (A.C.S.38 (1), 253 (1997), Carbohydrate Research, 89, 121-135 (1981) )) A triple helix Schizophyllan was prepared according to the standard method described. That is, using a minimal medium, Schizophyllum commune., Obtained from ATCC (American Type Culture Collection). After culturing Fries (ATCC 44200) for 7 days, the supernatant obtained by centrifuging cell components and insoluble residues was sonicated, and this was subjected to ultrafiltration to replace the solution with water and frozen. It was dried to obtain a triple helix schizophyllan having a molecular weight of 450,000.
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Abstract
Description
本発明の第一の実施の形態に係る核酸複合体は、一本鎖DNAの3’側末端のデオキシリボヌクレオチド残基の3’位と、二本鎖RNAの一方のリボヌクレオチド鎖の5’側末端のリボヌクレオチド残基の5’位が結合した核酸複合体であって、一本鎖DNAと結合したリボヌクレオチド鎖の5’側末端ヌクレオチドにおける2’位のヒドロキシル基がアルコキシ基またはハロゲン原子で置換されている。すなわち、本実施の形態に係る核酸複合体は、下記の一般式(I)において、R2がアルコキシ基またはハロゲン原子である場合に相当する。なお、式(I)において、R1はアデニン(A)、グアニン(G)、ウラシル(U)およびシトシン(C)のいずれかであり、R3およびR5はリン酸エステル基(-PO2-O-)である。なお、アルコキシ基の具体例としては、炭素数1~5の直鎖または分岐鎖アルコキシ基、炭素数5~15のアリールアルキル基、O-アリル(allyl)基等のアルケニルアルキル基等が挙げられ、好ましくは炭素数1~3のアルコキシ基、特に好ましくはメトキシ基である。また、ハロゲン原子の具体例としては、フッ素原子(F)、塩素原子(Cl)、臭素原子(Br)およびヨウ素原子(I)が挙げられ、特に好ましくはフッ素原子である。
なお、β-1,3-グルカンの重量平均分子量は、光散乱法、沈降速度法(超遠心法)等の任意の公知の方法を用いて決定することができる。
実施例1:(dA)40 -siRNAの血清中での分解の確認
siRNAのセンス鎖(5’-CAAAGACAACCAACUAGUGGU-3’:配列番号1。以下、「siRNA」、「siRNAのセンス鎖」または「センス鎖」という。)の5’側末端にデオキシアデニン(以下「dA」と略称する場合がある。)40量体(dA40)を付加させたDNA-RNA核酸複合体とsiRNAのアンチセンス鎖(5’-ACCACUAGUUGGUUGUCUUUG-3’:配列番号2)をアニーリングさせ、DNA-siRNA核酸複合体(dA40-siRNA。以下、デオキシアデニンのx(xは自然数)量体をセンス鎖の5’末端側に付加させたDNA-RNA核酸複合体とアンチセンス鎖をアニーリングさせて得られるDNA-siRNA核酸複合体を「dAx-siRNA」と略称する。)を得た。
使用した核酸は全て北海道システムサイエンス株式会社より購入した。
DNA-siRNA核酸複合体が血清中の酵素によりどのように切断されているか蛍光修飾核酸を用いて検討した。アンチセンス鎖の5’側末端側あるいは3’側末端側にFITC修飾したものをそれぞれ用意し、配列番号1で表される塩基配列を有するセンス鎖の5’末端側にデオキシアデニン10量体(dA10)を付加させたDNA-RNA核酸複合体およびdA10を付加させていないセンス鎖と、それぞれアニーリングさせた。両者について、血清中でインキュベーションさせた後、ゲル電気泳動後にFITCで蛍光観察を行い、次いでSYBR Gold染色し、同様に蛍光観察を行った。
実施例2の結果より、DNAとsiRNAの連結部付近で分解されていると考えられるため、DNA(dA40)の3’末端側と接合するsiRNA(センス鎖)の、5’末端側から数えて最初の1つ、2つ、3つ、4つのRNA残基を、それぞれ、2’-O-メチル化させたdA40-siRNA核酸複合体を用意し、実施例2と同様に、血清(FBS)中での安定性を、インキュベーション前後の試料のゲル電気泳動法を用いて評価した。
先の実験より、血清中でのインキュベーションによるDNA-siRNA核酸複合体の分解が、DNAとRNA二本鎖部分との結合点で起きていると考えられるが、この分解がRNA二本鎖に特異的な現象であるかを検討するために、DNA(dA40)の3’末端側と接合するsiRNA(センス鎖)の、5’末端側から数えて最初の1つ、2つ、3つ、4つのRNA残基を、それぞれ、2’-O-メチル化させたdA40-RNA(1本鎖)核酸複合体を用いて、実施例3と同様の実験を行った。
実施例3の結果より、DNA-siRNA核酸複合体の分解を阻止するためには、2’-O-メチル化するRNA残基数は1つで十分であることがわかったが、2’-O-メチル化する箇所が、DNAの3’末端側と接合するセンス鎖の、5’末端側から数えて最初のRNAである必要があるかを確認するために、2’-O-メチル化するRNA残基の場所を変えて同様の実験を行った。2’-O-メチル化は、5’末端のRNA残基または5’末端から2番目のRNAについて行った。また、上述の実施例では、SPGと複合化させることを考慮し、dAの長さを40量体にしていたが、dAの長さを短くしても同様の現象が観察されるかについても同様に確認した。
上述の実施例において、血清(FBS)中での分解が確認されたのは、DNA-二本鎖RNAの核酸複合体であるが、DNA-二本鎖DNA(dsDNA)核酸複合体の場合でも同様の分解が観測されるか検討するために、siRNAの配列(センス鎖およびアンチセンス鎖の両者)を全てDNAに置き換えたものを用意し、同様の実験を行った。
これまでの実験では、SPGとの複合化を考慮し、DNA部分はdA10またはdA40に統一していたが、DNA部分の塩基配列がDNA-siRNA核酸複合体の安定性に及ぼす影響を検討するために、dA10の代わりに、デオキシシトシン(dC)、デオキシグアニン(dG)、デオキシチミジン(dT)の10量体を用いてsiRNAとの核酸複合体を調製し、同様の実験を行った。
DNAと接合する最初のリボヌクレオチド(DNAの3’末端側に隣接するリボヌクレオチド)を2’-O-メチル修飾(リボースの2’位のヒドロキシル基をメトキシ基に置換)することで血清中での分解を回避できることがわかったが、その他にもDNAの3’末端と接合する最初のリボヌクレオチドの3’位と2番目のリボヌクレオチドの5’位との間のリン酸ジエステル基をホスホロチオエート基に置換したものを用意して同様の実験を行った。
これまでDNAはsiRNAのセンス鎖の5’側末端に接合させていたが、センス鎖の3’側末端にDNAが接合した場合も同様にDNAと接合する最初のRNAのところで分解を受けるかを検討するために、これまでの配列を逆にしたもの(配列番号1で表されるオリゴリボヌクレオチドの3’側末端側にポリ(dA)が結合した核酸複合体)を用意して実験を行った。
(1)三重らせんシゾフィランの調製
文献(A.C.S.38(1),253(1997)、Carbohydrate Research,89,121-135(1981))記載の定法にしたがい、三重らせんシゾフィランを調製した。すなわち、最少培地を用いて、ATCC(American Type Culture Collection)から入手したSchizophyllum commune.Fries(ATCC 44200)を7日間振盪培養した後、細胞成分および不溶残渣を遠心分離して得られた上清を超音波処理して、これを限外ろ過にて溶液を水に置換し、凍結乾燥させ、分子量45万の三重らせんシゾフィランを得た。
上記のようにして得られたシゾフィランを、濃度が15g/dLとなるように0.25NのNaOH水溶液に溶解した。この溶液10μLに、リン酸緩衝液(pH=4.5)10μLと、3g/dLのポリヌクレオチド溶液30μLを混合し、複合体の水溶液を調製した。得られた溶液は透明で均一であった。
本出願は、2012年6月20日に出願された日本国特許出願2012-139250号に基づくものであり、その明細書、特許請求の範囲、図面および要約書を含むものである。上記日本国特許出願における開示は、その全体が本明細書中に参照として含まれる。
Claims (14)
- 一本鎖DNAの3’側末端のデオキシリボヌクレオチド残基の3’位と、二本鎖RNAの一方のリボヌクレオチド鎖の5’側末端のリボヌクレオチド残基の5’位が結合した核酸複合体であって、前記一本鎖DNAと結合した前記リボヌクレオチド鎖の5’側末端ヌクレオチドにおける2’位のヒドロキシル基がアルコキシ基またはハロゲン原子で置換されている核酸複合体。
- 一本鎖DNAの3’側末端のデオキシリボヌクレオチド残基の3’位と、二本鎖RNAの一方のリボヌクレオチド鎖の5’側末端のリボヌクレオチド残基の5’位が結合した核酸複合体であって、前記一本鎖DNAと結合した最初のリボヌクレオチドの3’位と、それに隣接するリボヌクレオチドの5’位との間のリン酸ジエステル基がホスホロチオエート基、ジチオリン酸ジエステル基ジチオリン酸ジエステル基およびトリチオリン酸ジエステル基のいずれかで置換されている核酸複合体。
- 前記一本鎖DNAのヌクレオチド数が10以上である請求項1または2に記載の核酸複合体。
- 前記一本鎖DNAがポリデオキシアデニンである請求項1から3のいずれか1項に記載の核酸複合体。
- 前記一本鎖DNAのリン酸ジエステル基のうち少なくとも一部がホスホロチオエート基、ジチオリン酸ジエステル基およびトリチオリン酸ジエステル基のいずれかで置換されている、請求項1から4のいずれか1項に記載の核酸複合体。
- 前記一本鎖DNAのリン酸ジエステル基のうち少なくとも50%以上がホスホロチオエート基、ジチオリン酸ジエステル基およびトリチオリン酸ジエステル基のいずれかで置換されている請求項5に記載の核酸複合体。
- 前記二本鎖RNAがsiRNAである請求項1から6のいずれか1項に記載の核酸複合体。
- 前記siRNAが21mer型または27mer型である、請求項7に記載の核酸複合体。
- 前記siRNAの標的遺伝子が、Dectin-1発現細胞において発現している遺伝子である請求項7または8に記載の核酸複合体。
- 請求項1から9のいずれか1項に記載の核酸複合体1分子の一本鎖DNA部位と、β-1,3-グルカン骨格を有する多糖2分子とが、3重らせん構造を形成している核酸多糖複合体。
- 前記β-1,3-グルカン骨格を有する多糖が、シゾフィランである請求項10に記載の核酸多糖複合体。
- 請求項10または11に記載の核酸多糖複合体を含む医薬組成物。
- 一本鎖DNAの3’側末端のデオキシリボヌクレオチド残基の3’位と、二本鎖RNAの一方のリボヌクレオチド鎖の5’側末端のリボヌクレオチド残基の5’位が結合した核酸複合体のRNA分解酵素に対する安定性を増大させる方法であって、前記一本鎖DNAと結合した前記リボヌクレオチド鎖の5’側末端ヌクレオチドにおける2’位のヒドロキシル基をアルコキシ基またはハロゲン原子で置換する核酸複合体の安定化方法。
- 一本鎖DNAの3’側末端のデオキシリボヌクレオチド残基の3’位と、二本鎖RNAの一方のリボヌクレオチド鎖の5’側末端のリボヌクレオチド残基の5’位が結合した核酸複合体のRNA分解酵素に対する安定性を増大させる方法であって、前記一本鎖DNAと結合した最初のリボヌクレオチドの3’位と、それに隣接するリボヌクレオチドの5’位との間のリン酸ジエステル基をホスホロチオエート基、ジチオリン酸ジエステル基およびトリチオリン酸ジエステル基のいずれかで置換する核酸複合体の安定化方法。
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CN201380032032.3A CN104471063A (zh) | 2012-06-20 | 2013-06-19 | 核酸复合物及核酸多糖复合物 |
US14/408,577 US20150148529A1 (en) | 2012-06-20 | 2013-06-19 | Nucleic acid complex and nucleic acid-polysaccharide complex |
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WO2019230898A1 (ja) * | 2018-05-30 | 2019-12-05 | ナパジェン ファーマ,インコーポレテッド | 化学修飾siRNAとシゾフィランとの複合体を含む医薬組成物 |
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CN110121558A (zh) * | 2016-11-28 | 2019-08-13 | 日商那帕洁制药股份有限公司 | 化学修饰siRNA |
JP7100367B2 (ja) | 2016-11-28 | 2022-07-13 | ナパジェン ファーマ,インコーポレテッド | 化学修飾siRNA |
CN110121558B (zh) * | 2016-11-28 | 2023-06-13 | 日商那帕洁制药股份有限公司 | 化学修饰siRNA |
WO2019230898A1 (ja) * | 2018-05-30 | 2019-12-05 | ナパジェン ファーマ,インコーポレテッド | 化学修飾siRNAとシゾフィランとの複合体を含む医薬組成物 |
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