WO2016021673A1 - Method for predicting drug efficacy of pharmaceutical composition containing small rna - Google Patents

Abstract

　Provided is a method for evaluating the target-gene knockdown effect of a pharmaceutical composition containing a small RNA. Through the present invention, the target-gene knockdown effect of a pharmaceutical composition containing a small RNA can be evaluated by isolating an AGO protein-small RNA complex from a sample derived from a subject to which the pharmaceutical composition containing a small RNA is administered, and measuring the quantity of the small RNA included in the complex.

Description

A method for predicting the efficacy of a small RNA-containing pharmaceutical composition

Methods for predicting the efficacy of small RNA administered to a subject by measuring the amount of small RNA contained in an AGO protein-small RNA complex isolated from a sample derived from a subject administered with a pharmaceutical composition containing small RNA About.

A biomarker for evaluating drug efficacy in developing a nucleic acid drug has not yet been established, and if a drug evaluation marker for small RNA can be obtained, it can be a useful technique in the development of a nucleic acid drug.
Currently, there are various approaches to study the construction of biomarker technology for small RNA. Specifically, biomarker technology for analysis of biological tissue, plasma or serum isolated from blood, exosomes, etc. has been studied. (Patent Document 1). However, the method of collecting biological tissue (biopsy) is highly invasive and difficult to put into practical use. On the other hand, there are many attempts to detect miRNA and mRNA expression fluctuations via plasma, serum, and exosomes, but they still have practical problems due to poor reproducibility and complicated operation (Non-Patent Documents). 1).

As a method of evaluating the target expression suppression effect of siRNA and miRNA, it is common to check the presence or absence of target mRNA expression fluctuation and the expression fluctuation of the protein translated by the target mRNA. In order to perform monitoring, it is necessary to collect a part of a living tissue or organ from the subject, and the burden on the patient is large.
siRNA and miRNA bind to Argonaute protein (hereinafter referred to as AGO) and form an RNA-induced silencing complex (RISC) to repress target mRNA. In mammals, there are four types of AGO (AGO1, AGO2, AGO3, AGO4). Of these four types, only AGO2 is known to have the activity of cleaving target mRNA (slicer activity).

Recently, it has been reported that AGO2 protein leaks into the blood from the cell in a state where it forms a complex with miRNA (AGO2 / miRNA) (Non-patent Document 2). However, the presence of AGO2 / miRNA in blood in healthy individuals has only been confirmed, and the relationship between AGO2 / miRNA and disease and the availability of AGO2 / miRNA as a biomarker are not known. In addition, it is not known that a complex in which siRNA and AGO protein are bound leaks from the cell into the blood.

International Publication No. 2010/056337 Pamphlet

Blood, 121, 4977-4984-4 (2013) Proc. Natl. Acad. Sci., 108, 5003-5008 (2011)

The present invention has the effect of knocking down a target gene of a small RNA-containing pharmaceutical composition by quantifying or identifying small RNA contained in an AGO protein-small RNA complex derived from a subject administered with the small RNA-containing pharmaceutical composition. Evaluation method, method for predicting the efficacy of small 含有 RNA-containing pharmaceutical composition, method for selecting effective small RNA-containing pharmaceutical composition, evaluation of knockdown effect of target cells of small RNA-containing pharmaceutical composition, and selection of patients The present invention relates to a method, a method for selecting a cancer type in which a small RNA-containing pharmaceutical composition exhibits a medicinal effect, and the like.

The present invention relates to the following (1) to (5).
(1) The small RNA-containing pharmaceutical composition obtained by isolating an AGO protein-small RNA complex from a sample derived from a subject who has been administered the small RNA-containing pharmaceutical composition and measuring the amount of small RNA contained in the complex. Of evaluating the knockdown effect of a target gene.
(2) The small RNA-containing pharmaceutical composition obtained by isolating an AGO protein-small RNA complex from a sample derived from a subject administered with the small RNA-containing pharmaceutical composition and measuring the amount of small RNA contained in the complex. Of predicting the efficacy of drugs.
(3) Isolating an AGO protein-small RNA complex from a sample derived from a subject administered with a small RNA-containing pharmaceutical composition, measuring the amount of small RNA contained in the complex, and comparing the amount of small RNA According to the method for selecting an effective pharmaceutical composition containing small RNA.
(4) Isolating an AGO protein-small RNA complex from a sample derived from a subject administered with a small RNA-containing pharmaceutical composition, measuring the amount of small RNA contained in the complex, and comparing the amount of small RNA To evaluate the knockdown effect of target cells of the small RNA-containing pharmaceutical composition and to select patients.
(5) Isolating an AGO protein-small RNA complex from a sample derived from a subject administered with a small RNA-containing pharmaceutical composition, measuring the amount of small RNA contained in the complex, and comparing the amount of small RNA According to the method for selecting a cancer type in which a small RNA-containing pharmaceutical composition exhibits a medicinal effect.

The present invention provides a method for predicting the efficacy of a small RNA-containing drug and a method for evaluating the knockdown effect of a target gene of a small RNA-containing drug. Further, according to the present invention, it becomes possible to select an effective small-RNA-containing drug, and to select patients who effectively exhibit the medicinal effect of the small-RNA-containing drug.

FIG. 1 shows the amount of FVII in mouse blood administered with Formulations 1 and 2 and physiological saline, respectively, as a ratio to the amount of FVII in mouse blood administered with physiological saline. FIG. 2 shows 40-Ct values in qPCR for FVII siRNA isolated from AGO2 / siRNA in the blood of mice administered with Formulations 1, 2 and saline, respectively. Note that the (40-Ct value) was calculated as 0 for samples that were below the detection limit and whose Ct value exceeded 40. A larger 40-Ct value means that the amount of FVII siRNA contained in AGO2 / siRNA in mouse blood is larger. FIG. 3 shows the amount of FVII in mouse blood administered with preparations 3 and 4 and physiological saline, respectively, as a ratio to the amount of FVII in mouse blood administered with physiological saline. FIG. 4 shows 40-Ct values in qPCR for FVII siRNA isolated from AGO2 / siRNA in the blood of mice administered with preparations 3 and 4 and saline, respectively. Note that the (40-Ct value) was calculated as 0 for samples that were below the detection limit and whose Ct value exceeded 40. A larger 40-Ct value means that the amount of FVII siRNA contained in AGO2 / siRNA in mouse blood is larger. FIG. 5 shows changes in tumor volume in cancer-bearing mice to which Formulation 5 and physiological saline were respectively administered. The horizontal axis indicates the number of days since administration, the vertical axis indicates the tumor volume ratio (V / V0) when the tumor volume on day 0 after administration is 1, ● indicates formulation 5 and ○ indicates physiological saline The tumor volume ratios of the administered mice are shown respectively. FIG. 6 shows 40-Ct values in qPCR for CKAP5 siRNA isolated from AGO2 / siRNA in blood of tumor-bearing mice or non-tumor-bearing mice to which physiological saline and preparation 5 were administered, respectively.

1. Small RNA-containing pharmaceutical composition used in the present invention The small RNA-containing pharmaceutical composition used in the present invention is a pharmaceutical composition containing small RNA as an active ingredient.
(1) Small RNA used in the present invention
In the small RNA-containing pharmaceutical composition used in the present invention, the small RNA that can be used as an active ingredient includes a nucleic acid containing a base sequence complementary to the mRNA sequence of the target gene and / or the base sequence of the nucleic acid. As long as the nucleic acid has a complementary base sequence and can suppress the expression of the target gene, it may be composed of any nucleotide or derivative thereof.

Such small RNA includes a nucleic acid comprising a base sequence complementary to the mRNA sequence of the target gene, a nucleic acid comprising a base sequence complementary to the base sequence of the nucleic acid, and the target gene. SiRNA, shRNA or miRNA that suppresses expression is preferably used.
In addition, the nucleotide derivative in the small RNA used in the present invention may be any molecule as long as the molecule is a modified nucleotide. For example, in order to improve or stabilize nuclease resistance, In order to increase affinity, increase cell permeability, etc., a molecule having a modified nucleotide can be used.

Examples of the molecule in which the nucleotide is modified include a sugar moiety-modified nucleotide, a phosphodiester bond-modified nucleotide, a base-modified nucleotide, and a nucleotide in which at least one of the sugar moiety, phosphodiester bond and base is modified.
As the sugar-modified nucleotide, any or all of the chemical structure of the sugar of the nucleotide may be modified or substituted with any substituent, or substituted with any atom. '-Modified nucleotides are preferably used.

Examples of 2'-modified nucleotides include the 2'-OH group of ribose consisting of H, OR, R, R'OR, SH, SR, NH2, NHR, NR2, N3, CN, F, Cl, Br and I A nucleotide substituted with a substituent selected from the group (R is alkyl or aryl, preferably alkyl having 1 to 6 carbon atoms and R ′ is alkylene, preferably alkylene having 1 to 6 carbon atoms), preferably Is a nucleotide in which the 2′-OH group is substituted with H, F or a methoxy group, more preferably a nucleotide in which the 2′-OH group is substituted with F or a methoxy group. In addition, 2'-OH group is 2- (methoxy) ethoxy group, 3-aminopropoxy group, 2-[(N, N-dimethylamino) oxy] ethoxy group, 3- (N, N-dimethylamino) propoxy group, 2- A substituent selected from the group consisting of [2- (N, N-Dimethylamino) ethoxy] ethoxy group, 2- (methylamino) -2-oxoethoxy group, 2- (N-methylcarbamoyl) ethoxy group and 2-cyanoethoxy group Examples thereof include substituted nucleotides.

Examples of sugar-modified nucleotides include cross-linked artificial nucleic acids (BridgedridgeNucleic Acid, BNA) having two cyclic structures by introducing a cross-linked structure into the sugar portion, and specifically, an oxygen atom at the 2 ′ position And 4'-position carbon atoms cross-linked via methylene (Locked Nucleic Acid, LNA), ethylene-bridged artificial nucleic acid (Ethylene bridged nucleic acid, ENA) [Nucleic Acid Research, 32, In addition, peptide nucleic acids (PNA) [Acc. Chem. Res., 32, 624 (1999)], oxypeptide nucleic acids (OPNA) [J. Am. Chem. Soc., 123, 4653 (2001) ], Peptide ribonucleic acid (PRNA) [J. Am. Chem. Soc., 122, 6900 (2000)].

The phosphodiester bond-modified nucleotide is any nucleotide that has been modified or substituted with an arbitrary substituent for a part or all of the chemical structure of the phosphodiester bond of the nucleotide, or with any atom. For example, a nucleotide in which a phosphodiester bond is replaced with a phosphorothioate bond, a nucleotide in which a phosphodiester bond is replaced with a phosphorodithioate bond, a nucleotide in which a phosphodiester bond is replaced with an alkylphosphonate bond, a phosphate Examples thereof include nucleotides in which a diester bond is substituted with a phosphoramidate bond.

As the base-modified nucleotide, any or all of the nucleotide base chemical structure modified or substituted with an arbitrary substituent or substituted with an arbitrary atom may be used. In which oxygen atoms are substituted with sulfur atoms, hydrogen atoms are substituted with alkyl groups having 1 to 6 carbon atoms, halogens, etc., methyl groups are hydrogen, hydroxymethyl, alkyl groups with 2 to 6 carbon atoms, etc. And those in which the amino group is substituted with an alkyl group having 1 to 6 carbon atoms, an alkanoyl group having 1 to 6 carbon atoms, an oxo group, a hydroxy group, or the like.

Examples of the nucleotide derivative include a peptide, protein, sugar, lipid, phospholipid, phenazine, folate, phenanthridine, anthraquinone, acridine, a nucleotide derivative in which at least one of nucleotide or sugar moiety, phosphodiester bond or base is modified. Other chemicals such as fluorescein, rhodamine, coumarin, and dye may be added directly or via a linker. Specific examples include 5'-polyamine-added nucleotide derivatives, cholesterol-added nucleotide derivatives, and steroid-added nucleotide derivatives. Bile acid addition nucleotide derivatives, vitamin addition nucleotide derivatives, Cy5 addition nucleotide derivatives, Cy3 addition nucleotide derivatives, 6-FAM addition nucleotide derivatives, biotin addition nucleotide derivatives, etc. It is.

The nucleotide derivative may form a crosslinked structure such as an alkylene structure, a peptide structure, a nucleotide structure, an ether structure, an ester structure, and a structure obtained by combining at least one of these with other nucleotides or nucleotide derivatives in the nucleic acid.
The small RNA used in the present invention includes those in which some or all atoms in the nucleic acid molecule are substituted with atoms (isotopes) having different mass numbers.

The small RNA used in the present invention is a double strand consisting of a nucleic acid comprising a base sequence complementary to the mRNA sequence of the target gene and a nucleic acid comprising a base sequence complementary to the base sequence of the nucleic acid. Any length can be used as long as it can be used, but such length is usually 11 to 35 bases, preferably 15 to 30 bases, more preferably 17 to 25 bases, more preferably 17 to 23 bases, Preferably, 19 to 23 bases can be mentioned.

Furthermore, as the small RNA used in the present invention, a nucleic acid comprising a base sequence complementary to the mRNA sequence of the target gene and / or a nucleic acid comprising a base sequence complementary to the base sequence of the nucleic acid is used. Among these nucleic acids, 1 to 3 bases, preferably 1 to 2 bases, more preferably 1 base deleted, substituted or added may be used.
In the present invention, siRNA refers to a nucleic acid in which two strands pair and have a double-stranded region. A double-stranded region refers to a portion where nucleotides constituting a double-stranded nucleic acid or a derivative thereof constitute a base pair to form a double strand. The double-stranded region is usually 11 to 27 base pairs, preferably 15 to 25 base pairs, more preferably 15 to 23 base pairs, still more preferably 17 to 21 base pairs, and particularly preferably 17 to 19 base pairs.

The single-stranded nucleic acid constituting the siRNA usually comprises 11 to 30 bases, preferably 15 to 29 bases, more preferably 15 to 27 bases, and further preferably 15 to 25 bases. Preferably, it consists of 17 to 23 bases, most preferably 19 to 21 bases.
In addition, when the small RNA used in the present invention is miRNA, the length is usually 16 to 25 bases, preferably 20 to 25 bases. The forms of pri-miRNA, pre-miRNA and miRNA / miRNA ^* which are precursors of the miRNA may also be used.

When the siRNA used in the present invention has an additional nucleotide or nucleotide derivative that does not form a duplex on the 3 ′ end side or the 5 ′ end side following the duplex region, this is referred to as an overhang. Call. In the case of having an overhang, the nucleotide constituting the overhang may be ribonucleotide, deoxyribonucleotide, or a derivative thereof.

As the siRNA having a protruding portion, one having a protruding portion consisting of 1 to 6 bases, usually 1 to 3 bases at the 3 ′ end or 5 ′ end of at least one strand is used. Those having a protrusion are preferably used, and examples thereof include those having protrusions made of dTdT or UU. The overhang can be present only in the antisense strand, only in the sense strand, and in both the antisense strand and the sense strand, but siRNA having the overhang on both the antisense strand and the sense strand is preferably used.

Furthermore, as siRNA used in the present invention, for example, a nucleic acid molecule (WO2005 / 089287) that generates siRNA by the action of a ribonuclease such as Dicer, or a blunt end is formed without a 3 ′ or 5 ′ end overhang. siRNA, siRNA with a protruding sense strand (US2012-0040459), single-stranded siRNA reported by Walt et al. (Cell, 150, 883-894, 2012), and the like can also be used.
As the siRNA used in the present invention, a nucleic acid having the same sequence as the base sequence of the target gene or its complementary strand may be used, but the 5 ′ end or 3 ′ end of at least one strand of the nucleic acid is An siRNA comprising a nucleic acid from which 1 to 4 bases have been deleted and a nucleic acid having a base sequence complementary to the base sequence of the nucleic acid may be used.

As the small RNA used in the present invention, a single-stranded nucleic acid in which the antisense strand and the sense strand constituting the double-stranded nucleic acid are linked via a spacer sequence (spacer oligonucleotide) may be used. The spacer oligonucleotide is preferably a 6- to 12-base single-stranded nucleic acid molecule, and the sequence at the 5 'end is preferably 2 Us. An example of the spacer oligonucleotide is a nucleic acid having a UUCAAGAGA sequence. Either the antisense strand or the sense strand connected by the spacer oligonucleotide may be on the 5 'side. As such a single-stranded nucleic acid, for example, an shRNA having a duplex forming part by a stem loop structure can be mentioned. Single-stranded nucleic acids such as shRNA are usually 50 to 70 bases in length.

The small RNA used in the present invention is further designed to generate the above-mentioned single-stranded nucleic acid or double-stranded nucleic acid by the action of ribonuclease or the like, 70 base length or less, preferably 50 base length or less, more preferably A nucleic acid having a length of 30 bases or less may be used.
The method for producing small RNA used in the present invention is not particularly limited, and examples thereof include a method using known chemical synthesis, an enzymatic transcription method, and the like. Examples of methods using known chemical synthesis include phosphoramidite method, phosphorothioate method, phosphotriester method, CEM method [Nucleic Acid Research, 35, 3287 (2007)]. For example, ABI3900 high-throughput nucleic acid synthesis Can be synthesized by a machine (Applied Biosystems). The synthesized small RNA is used after desorption from the solid phase, deprotection of the protecting group, purification of the target product, and the like. It is desirable to obtain small RNA having a purity of 90% or more, preferably 95% or more by purification.

Examples of the enzymatic transcription method for producing small RNA used in the present invention include a transcription method using phage RNA polymerase, for example, T7, T3, or SP6 RNA polymerase, using a plasmid or DNA having the target nucleotide sequence as a template. It is done.
When the small RNA is a double-stranded nucleic acid, it can be used after annealing as necessary from the synthesized and purified sense strand and antisense strand.

In the present invention, small RNA can be used in combination with any delivery element. For example, it can be used in the form of a conjugated nucleic acid in which the 5 ′ end, 3 ′ end and / or the inside of the sequence is modified with one or more ligands.
At the time of extension reaction on the solid phase, the 5 ′ end, 3 ′ end and / or the inside of the sequence can be modified by reacting a modifying agent capable of reacting on the solid phase. Alternatively, a conjugated nucleic acid can be obtained by previously synthesizing and purifying a nucleic acid into which a functional group such as an amino group, a mercapto group, an azide group, or a triple bond has been introduced, and allowing a modifying agent to act on them. The ligand may be any molecule having affinity for a biomolecule, such as lipids such as cholesterol, fatty acid, tocopherol, and retinoid, N-acetylgalactosamine (GalNAc), galactose (Gal), mannose (Man), etc. Antibody fragments such as saccharides, antibodies, Fab, VHH, low density lipoprotein (LDL), proteins such as human serum albumin, peptide fragments such as RGD, NGR, R9, CPP, small molecules such as folic acid, synthetic polyamino acids, etc. A synthetic polymer, a nucleic acid aptamer, etc. are mentioned, These can also be used in combination.

Instead of small RNA, a vector that can be introduced into cells to express small RNA may be used. Specifically, the small RNA or the like can be expressed by inserting a sequence encoding small RNA downstream of the promoter in the expression vector, constructing the expression vector, and introducing it into a cell.
Expression vectors that can be used as such include pCDNA6.2-GW / miR (Invitrogen), pSilencer 4.1-CMV (Ambion), pSINsi-hH1 DNA (Takara Bio), pSINsi-hU6DNA ( (Takara Bio) and pENTR / U6 (Invitrogen).

A recombinant viral vector produced by inserting a sequence encoding small RNA downstream of a promoter in a viral vector and introducing the vector into a packaging cell can also be used. Examples of virus vectors include retrovirus vectors, lentivirus vectors, adenovirus vectors, adeno-associated virus vectors, and the like.
(2) Small RNA-containing pharmaceutical composition used in the present invention The small RNA-containing pharmaceutical composition used in the present invention is a pharmaceutical composition containing small RNA such as siRNA, shRNA or miRNA of (1) as an active ingredient. .

Such a pharmaceutical composition may contain a pharmaceutically acceptable carrier or diluent. A pharmaceutically acceptable carrier or diluent is a composition that is essentially chemically inert and harmless, and has no influence on the biological activity of the small RNA-containing pharmaceutical composition used in the present invention. It is. Examples of such carriers or diluents include but are not limited to salt solutions, sugar solutions, glycerol solutions, ethanol and the like.

The pharmaceutical composition used in the present invention contains an amount of small RNA effective for treating or preventing a disease, and is provided in a form that can be appropriately administered to a patient. The preparation form of the pharmaceutical composition of the present invention may be, for example, injections, eye drops, liquids for inhalation, etc., for example, external preparations such as ointments, lotions and the like.
In the case of a liquid, the concentration range of the pharmaceutical composition used in the present invention is usually 0.001 to 25% (w / v), preferably 0.1 to 10% (w / v), more preferably 0.5 to 5%. (w / v). The pharmaceutical composition used in the present invention may contain an appropriate amount of any pharmaceutically acceptable additive, for example, an emulsification aid, a stabilizer, a tonicity agent, a pH adjuster and the like.

The freeze-dried preparation can be prepared by freeze-drying a solution containing the pharmaceutical composition used in the present invention. The lyophilization treatment can be performed by a conventional method. For example, a solution containing the pharmaceutical composition used in the present invention is aseptically dispensed into vials and pre-dried at about −40 to −20 ° C. for about 2 hours. Primary drying can be performed at 10 ° C. under reduced pressure, followed by secondary drying under reduced pressure at about 15-25 ° C. and lyophilized. Then, for example, by replacing the inside of the vial with nitrogen gas and stoppering, a freeze-dried preparation of the pharmaceutical composition used in the present invention can be obtained.

The pharmaceutical composition used in the present invention can be redissolved and used by adding any appropriate solution. Examples of such a solution include electrolytes such as water for injection and physiological saline, glucose solution, and other general infusion solutions. The amount of this solution varies depending on the application and is not particularly limited, but it is preferably 0.5 to 2 times the amount before lyophilization, or 500 ml or less.

The pharmaceutical composition used in the present invention can be administered to animals, including humans, for example, intravenous administration, intraarterial administration, oral administration, tissue administration, transdermal administration, transmucosal administration, or rectal administration. It is preferable to administer by an appropriate method according to the symptoms. In particular, intravenous administration, transdermal administration, and transmucosal administration are preferably used. Moreover, local administration, such as local administration in cancer, can also be performed. Examples of dosage forms suitable for these administration methods include various injections, oral preparations, drops, absorbents, eye drops, ointments, lotions, suppositories and the like.

The dosage of the pharmaceutical composition used in the present invention is preferably determined in consideration of the drug, dosage form, patient condition such as age and weight, administration route, nature and degree of disease, etc. The mass is 0.1 mg to 10 g / day, preferably 1 mg to 500 mg / day per day for an adult. In some cases, this may be sufficient, or vice versa. It can also be administered once to several times a day, and can be administered at intervals of 1 to several days.

The pharmaceutical composition used in the present invention can further contain a carrier effective for transferring the nucleic acid into the cells, in addition to the components that can be contained in the above-mentioned ordinary pharmaceutical composition.
Examples of carriers effective for transferring nucleic acids into cells include cationic carriers. Examples of the cationic carrier include a complex using a cationic lipid.

Examples of complex forms using cationic lipids include a complex of a nucleic acid and a membrane (reverse micelle) composed of a single lipid (single molecule) layer, a complex of a nucleic acid and a liposome, and a complex of a nucleic acid and a micelle. Preferred examples include a complex of a nucleic acid and a membrane composed of a lipid monolayer or a complex of a nucleic acid and a liposome.
Examples of the composition containing a complex and a lipid membrane that encapsulates the complex include liposomes that encapsulate the complex and the complex with a lipid bilayer. One or more kinds of cationic lipids may be used in the composition.

Examples of the cationic lipid include N- [1- (2,3-dioleyloxy) propyl] -N, N disclosed in JP-A-61-161246 or US Pat. No. 5,049,386. , N-trimethylammonium chloride (DOTMA), N- (2,3-di- (9- (Z) -octadecenoyloxy))-prop-1-yl-N, N, N-trimethylammonium chloride ( DOTAP) and the like, disclosed in WO 91/16024 and WO 97/019675, N- [1- (2,3-dioleyloxypropyl)]-N, N-dimethyl- N-hydroxyethyl ammonium bromide (DORIE), 2,3-dioleyloxy-N- [2- (sperminecarboxamido) ethyl] -N, N-dimethyl-1-propanaminium trifluoroacetic acid (DOSPA), etc. DLinDMA, etc., disclosed in WO 2005/121348 pamphlet, DLin-K-DMA, WO 2011/136368, disclosed in WO 2009/086558 pamphlet (3R, 4R) -3,4-bis ((Z) -hexadec-9-enyloxy) -1-methylpyrrolidine, N-methyl-N, N-bis (2-(( Z) -octadec-6-enyloxy) ethyl) amine and the like, preferably DOTMA, DOTAP, DORIE, DOSPA, 1,2-dilinoleyloxy- N, N-dimethylaminopropane (DLinDMA), 2,2 A tertiary amine moiety with two unsubstituted alkyl groups, such as 2-dilinoleyl-4-dimethylaminomethyl- [1,3] -dioxolane (DLin-K-DMA) or a quaternary ammonium moiety with three unsubstituted alkyl groups And more preferably, a cationic lipid having the tertiary amine moiety. The unsubstituted alkyl group of the tertiary amine moiety and the quaternary ammonium moiety is more preferably a methyl group.

In addition, a cationic polymer or a carrier utilizing a virus envelope can be used as a cationic carrier as an effective carrier for transferring a nucleic acid into a cell. Examples of the cationic polymer include JetSI (Qbiogene), Jet- PEI (polyethyleneimine, Qbiogene) is preferably used. As a carrier using a virus envelope, for example, GenomeOne (HVJ-E liposome; Ishihara Sangyo Co., Ltd.) is preferably used.

A pharmaceutical composition containing a carrier effective for transferring nucleic acid into cells and containing small RNA as an active ingredient can be prepared by methods known to those skilled in the art. For example, it can be prepared by mixing a carrier dispersion having an appropriate concentration and a nucleic acid solution. When a cationic carrier is used, the nucleic acid is usually negatively charged in an aqueous solution, and therefore can be easily prepared by mixing in an aqueous solution by a conventional method.

Examples of the aqueous solvent used for preparing the pharmaceutical composition include electrolyte solutions such as water for injection, distilled water for injection and physiological saline, and sugar solutions such as glucose solution and maltose solution. In addition, conditions such as pH and temperature when preparing the pharmaceutical composition can be appropriately selected by those skilled in the art.
The pharmaceutical composition can be made into a uniform composition by carrying out a dispersion treatment using an ultrasonic dispersion device or a high-pressure emulsification device, if necessary. A person skilled in the art can select the most suitable method for the carrier to be used in preparing the composition containing the carrier and small RNA, without being limited to the above method.

In addition, the pharmaceutical composition includes, for example, a composite particle composed of small RNA and lead particles and a lipid membrane, and the composite particle and the lipid membrane are contained in a state dispersible in a polar organic solvent. It is preferably used (WO 2006/080118 pamphlet).
Examples of the lead particles include lipid aggregates, liposomes, emulsion particles, polymers, metal colloids, fine particle preparations, and the like, and cationic liposomes are preferably used. The lead particles may be composed of a composite of two or more lipid aggregates, liposomes, emulsion particles, polymers, metal colloids, fine particle formulations, etc., and lipid aggregates, liposomes, emulsion particles, polymers, A complex formed by combining a metal colloid, a fine particle preparation, and the like with another compound (eg, sugar, lipid, inorganic compound, etc.) may be used as a constituent component.

Examples of the lipid membrane include non-cationic lipids, lipids that prevent aggregation of particles, and cationic lipids as constituent components.
The composition can be prepared according to the method described in, for example, International Publication No. 2006/080118 Pamphlet.
Examples of the pharmaceutical composition used in the present invention include the compositions described in International Publication No. 2009/086558 and International Publication No. 2010/042877, and Invivofectamine (registered trademark) 2.0 (Invitrogen) A composition using a commercially available reagent typified by (manufactured) etc. can also be suitably used.

The mixing ratio of the small RNA and the carrier contained in the small RNA-containing pharmaceutical composition used in the present invention is suitably 1 to 200 parts by weight of the carrier with respect to 1 part by weight of the nucleic acid. The amount is preferably 2.5 to 100 parts by weight, more preferably 7 to 25 parts by weight, based on 1 part by weight of the nucleic acid.
The small RNA-containing pharmaceutical composition used in the present invention preferably has an average particle size of about 10 nm to 300 nm, more preferably about 30 nm to 200 nm, and even more preferably about 50 nm to 150 nm.
2. Method for Evaluating Target Gene Knockdown Effect of Small RNA-Containing Pharmaceutical Composition of the Present Invention Using the small RNA-containing pharmaceutical composition described in 1 above, the target gene knockdown effect of the small RNA-containing pharmaceutical composition is evaluated. be able to.

Specifically, an AGO / small RNA complex is isolated from a sample derived from a subject to which a small RNA-containing pharmaceutical composition has been administered, the amount of small RNA contained in the complex is measured, and the measured amount of small RNA is determined as small RNA. The knockdown effect of the target gene by the small RNA-containing drug can be evaluated by comparing with the non-administration group of the containing drug composition.
The AGO protein may be any of AGO1 to AGO4 as long as it can form a complex with small RNA in vivo. However, if the small RNA is siRNA, the AGO2 protein will be used. If the small RNA is miRNA, the AGO1 protein will be used. preferable.

The subject to which the small RNA-containing pharmaceutical composition is administered may be any animal, such as a human, mouse, monkey or the like, as long as it can isolate a sample described later, and preferably includes a human.
The sample derived from the subject administered with the small RNA-containing pharmaceutical composition may be any sample as long as it can isolate the AGO / small RNA complex. Specifically, bodily fluid samples that can be isolated from subjects such as blood, saliva, urine, lymph, sweat, cerebrospinal fluid, tears, breast milk, amniotic fluid, and the like can be used, and blood can be preferably used.
Isolation method of AGO / small RNA complex Isolation of AGO / small RNA complex from a sample derived from a subject who has been administered a pharmaceutical composition containing small RNA isolates and purifies the target protein from a normal biological sample. It can be done according to the method of the case.

For example, isolation of an AGO / small RNA complex from a blood sample derived from a subject administered with a small RNA-containing pharmaceutical composition can be performed as follows.
First, plasma is prepared from a blood sample. Plasma can be prepared by a method using ordinary centrifugation. An AGO / small RNA complex is further isolated from the obtained plasma from the subject.

The AGO / small RNA complex is isolated by immunosorbent capture using, for example, an anti-AGO antibody. More specifically, an anti-AGO antibody specific for the AGO molecule constituting the AGO / small RNA to be isolated, for example, an AGO2-specific antibody is used when isolating an AGO2 / small RNA complex.
AGO / small RNA complexes can also be isolated using size exclusion chromatography. The chromatographic fraction may in one embodiment be a void volume fraction.
In addition, AGO / small RNA complexes can be isolated by affinity chromatography.

A specific method for isolating and purifying the AGO / small RNA complex by immunoadsorption capture is, for example, an immunoprecipitation method using magnetic beads. Beads obtained by binding an antibody (for example, anti-AGO antibody) to magnetic beads can be obtained according to the protocol attached to Dynabeads (registered trademark) (manufactured by Veritas). The bead and AGO / small RNA complex can be bound by mixing an appropriate amount of the obtained beads and plasma, and the AGO / small RNA complex can be isolated by removing the supernatant.

As size exclusion chromatography, for example, each component can be fractionated according to size by subjecting plasma to Sephacryl S-500 resin column (GE Healthcare) (Proc. Natl. Acad. Sci., 108 , 5003-5008 (2012)).
Similarly, plasma components can be fractionated by using ultrafiltration using a VIVASPIN 20 column (Sartorius Stedim Japan) (Nucleic acids research, 39, 7223-7233 (2011)) .

Fractions fractionated by these techniques may be used as they are, or may be combined with immunoprecipitation.
The purified AGO / small RNA complex can be used for immunoblotting (Western blotting), ELISA, ultraviolet absorption, BCA, Bradford, surface plasmon resonance (SPR), or any protein quantification method or commercially available kit. Can be detected.

The small RNA contained in AGO / small RNA can be isolated from the complex by denaturing and dissolving the AGO protein, a method using a surfactant, a method using heating, cooling, and a degrading enzyme such as proteinase , Acid, organic solvent, phenol / chloroform extraction method, or any combination thereof.
Method for quantifying small RNA The quantification of small RNA contained in an AGO / small RNA complex isolated from a blood sample derived from a subject by the above method can be performed as follows.

Small RNA quantification methods include polymerase chain reaction including quantitative PCR (qPCR) such as real-time PCR, hybridization using specific probes, microarray analysis, enzyme reaction, chemical cleavage of mismatch, mass spectrometry, next generation Nucleic acid sequencing using a sequencer (deep sequencer), denaturant concentration gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE), etc. are not limited to the above, but suitable for detecting small RNAs It can be measured using any technique.

As a preferable means for quantifying the target small RNA, a technique using real-time PCR can be mentioned. Small RNA is converted into cDNA using a 96-well or 384-well plate, by reverse transcription with a primer specific to the target RNA or by reverse transcription with total RNA. Taqman® probe (Life Technologies), SYBR 、 Green And other fluorescent dyes, and a real-time PCR assay using any technique similar to them.

The detected small RNA level may be as it is, or may be calculated with correction using endogenous small RNA (for example, miRNA) or RNA added as an external standard.
More specifically, when using the above-described technique using real-time PCR, the Ct value obtained, the difference obtained by subtracting the Ct value from the Ct value of endogenous small RNA or RNA added as an external standard, or a constant value The difference obtained by subtracting the Ct value can be used as an indicator of the abundance of small RNA to be detected. Here, the constant numerical value is an integer equal to or less than the number of times of PCR (usually up to 40), and an appropriate numerical value can be set and used according to the sample and experimental conditions.
Method for evaluating knockdown effect of target gene The amount of small RNA contained in a sample derived from a subject administered with a pharmaceutical composition containing small RNA and the amount of small RNA contained in a control sample were quantified by the above method. By comparing the two, the knockdown effect of the target gene of the small RNA-containing pharmaceutical composition can be evaluated.

Examples of the control sample include a sample derived from a subject before administration of the small RNA-containing pharmaceutical composition, a sample derived from a subject not administered with the small RNA-containing pharmaceutical composition, and the like.
Based on the result of comparing the amount of small RNA contained in the sample derived from the subject administered with the small RNA-containing pharmaceutical composition and the amount of small RNA contained in the control sample, the target gene of the small RNA-containing pharmaceutical composition was determined. Determine if there is a knockdown effect.

Specifically, the amount of small RNA contained in a sample derived from a subject administered with a small RNA-containing pharmaceutical composition quantified using various indicators as described above is more than the amount of small RNA contained in a control sample. When it exceeds, the small RNA-containing pharmaceutical composition has an effect of knocking down the target gene.
In addition, when quantifying small RNA using the above-described method using real-time PCR, for example, when comparing using Ct value as an index, control is performed from the Ct value of a sample derived from a subject administered with a small RNA-containing pharmaceutical composition. If the difference (ΔCt value) obtained by subtracting the Ct value of the sample is 0 or less, more preferably 1 or less, the small RNA-containing pharmaceutical composition has a target gene knockdown effect. it can.

Alternatively, when quantifying small RNA using the above-described technique using real-time PCR, for example, when compared using 40-Ct value as an index, a sample derived from a subject administered with a pharmaceutical composition containing small RNA If the difference obtained by subtracting the 40-Ct value of the control sample from the Ct value (Δ40-Ct value) is 0 or more, preferably 1 or more, the small RNA-containing pharmaceutical composition has a knockdown effect on the target gene. It can be said that it has.
3. Method for Predicting Drug Effect of Small RNA-Containing Pharmaceutical Composition of the Present Invention The drug effect of the small RNA-containing pharmaceutical composition can be predicted using the small RNA-containing pharmaceutical composition described in 1 above.

Specifically, by using the sample derived from the subject to which the small RNA-containing pharmaceutical composition was administered, by evaluating the knockdown effect of the target gene of the small RNA-containing pharmaceutical composition by the above method 2, The efficacy of the pharmaceutical composition can be predicted.
In the present invention, the drug effect means an effect of ameliorating or curing a disease symptom by knocking down a target gene possessed by a small RNA-containing pharmaceutical composition. Specifically, for example, when the disease is solid cancer, administration of a small RNA-containing pharmaceutical composition to a subject means that the tumor becomes smaller than before administration.

When the knockdown effect of the target gene of the small RNA-containing pharmaceutical composition is evaluated according to the method described in 2 above for the sample derived from the subject administered with the small RNA-containing pharmaceutical composition, It can be said that the small RNA-containing pharmaceutical composition has a medicinal effect on the symptoms of the disease possessed by the subject.
4). Method for selecting effective small RNA-containing pharmaceutical composition of the present invention The effective small RNA-containing pharmaceutical composition can be selected using the small RNA-containing pharmaceutical composition described in 1 above.

Specifically, by using a sample derived from a subject administered with a small RNA-containing pharmaceutical composition, the target gene knockdown effect of the small RNA-containing pharmaceutical composition is evaluated by the above-mentioned method 2, thereby being effective in the subject. It is possible to determine whether the pharmaceutical composition is a small RNA-containing pharmaceutical composition that can exert its medicinal effects, and to select an effective small RNA-containing pharmaceutical composition.
In the present invention, the drug effect means an effect of ameliorating or curing a symptom of a disease possessed by a small RNA-containing pharmaceutical composition. Specifically, for example, when the disease is solid cancer, administration of a small RNA-containing pharmaceutical composition to a subject means that the tumor becomes smaller than before administration.

When the knockdown effect of the target gene of the small RNA-containing pharmaceutical composition is evaluated according to the method described in 2 above for the sample derived from the subject administered with the small RNA-containing pharmaceutical composition, It can be said that the small RNA-containing pharmaceutical composition is an effective small RNA-containing pharmaceutical composition in the subject.
Further, in the method for selecting an effective small RNA-containing pharmaceutical composition of the present invention, the small RNA-containing pharmaceutical composition having a different formulation composition, or the small RNA-containing pharmaceutical composition having a small RNA combined with a different delivery element as an active ingredient It is also possible to compare a plurality of different small RNA-containing pharmaceutical compositions, such as products, and select those having a higher target gene knockdown effect as effective small RNA-containing pharmaceutical compositions.

As a specific embodiment, for example, using a sample derived from a subject to which a plurality of small 対 象 RNA-containing pharmaceutical compositions each having the same small RNA as an active ingredient and having a different formulation composition are used, according to the above method 2, By evaluating the knockdown effect of the target gene of the small-RNA-containing pharmaceutical composition, the small-RNA-containing pharmaceutical composition that exhibits the most effective drug effect in the subject is selected, and is most effective as the small-RNA-containing pharmaceutical composition. Specific formulation can be determined.

In another specific embodiment, according to the method of 2 above, a sample derived from a subject to which a plurality of small RNA-containing pharmaceutical compositions each containing the same small RNA combined with different delivery elements as an active ingredient is administered. By evaluating the knockdown effect of the target gene of the small RNA-containing pharmaceutical composition, the small RNA-containing pharmaceutical composition that can exhibit the most effective drug effect in the subject is selected, and the smallest RNA-containing pharmaceutical composition is selected. An effective delivery element can be determined.
5. Method for Selecting Patients to be Administered with Small RNA-Containing Pharmaceutical Composition of the Present Invention Using the small RNA-containing pharmaceutical composition described in 1 above, patients to be administered with a small RNA-containing pharmaceutical composition can be selected.

Specifically, by using the sample derived from a patient administered with a small RNA-containing pharmaceutical composition, the target gene knockdown effect of the small RNA-containing pharmaceutical composition is evaluated by the above method 2, thereby It can be determined whether or not the pharmaceutical composition can effectively exert its efficacy in the patient. If it is determined in the determination that the small RNA-containing pharmaceutical composition can effectively exert its efficacy in the patient, the patient can be selected as a subject to be administered the small RNA-containing pharmaceutical composition.

In the present invention, the drug effect means an effect that a small RNA-containing pharmaceutical composition ameliorates or cures a disease symptom of a patient. Specifically, for example, when the disease is solid cancer, administration of a small RNA-containing pharmaceutical composition to a subject means that the tumor becomes smaller than before administration.
In the method for selecting a patient to be administered with the small RNA-containing pharmaceutical composition of the present invention, the patient is a human.

When the knockdown effect of the target gene of the small RNA-containing pharmaceutical composition was evaluated according to the method described in 2 above for a sample derived from a patient administered with the small RNA-containing pharmaceutical composition, It can be said that the small RNA-containing pharmaceutical composition is a small RNA-containing pharmaceutical composition effective in the patient.
6). Method for screening for cancer types in which the small RNA-containing pharmaceutical composition of the present invention exhibits drug efficacy

Using the small RNA-containing pharmaceutical composition described in 1 above, it is possible to select cancer types for which the small RNA-containing pharmaceutical composition exhibits a medicinal effect.
Specifically, by using a sample derived from a subject administered with a small RNA-containing pharmaceutical composition, the target gene knockdown effect of the small RNA-containing pharmaceutical composition is evaluated by the above-mentioned method 2, thereby being effective in the subject. It is possible to determine whether the subject is a small RNA-containing pharmaceutical composition that can exert a medicinal effect, and to select the cancer type of the cancer of the subject as a cancer type that the small RNA-containing pharmaceutical composition exhibits a medicinal effect .

The small RNA-containing pharmaceutical composition used in the method for selecting a cancer type of the present invention is a small RNA-containing pharmaceutical composition having a gene related to cancer as a target gene. Examples of such small RNA-containing pharmaceutical compositions include CKAP5 siRNA-containing pharmaceutical compositions.
In the method for selecting a cancer type of the present invention, a subject to which a small RNA-containing pharmaceutical composition is administered is a human cancer patient, a cancer-bearing mouse in which a cancer cell line or a cancer cell derived from a patient is transplanted into an immunodeficient mouse, Or it is restricted to the object which has developed cancer, such as the model animal of cancer illustrated by the mouse | mouth with cancer spontaneously developing.

The drug effect in the method for selecting a cancer type of the present invention refers to an effect of ameliorating or curing a cancer symptom possessed by a small RNA-containing pharmaceutical composition. Specifically, for example, when the cancer is a solid cancer, administration of a small RNA-containing pharmaceutical composition to a subject means that the tumor becomes smaller than before administration.
When the knockdown effect of the target gene of the small RNA-containing pharmaceutical composition is evaluated according to the method described in 2 above for the sample derived from the subject administered with the small RNA-containing pharmaceutical composition, The cancer type of the subject can be selected as a cancer that is effective as the cancer type of the subject to which the small RNA-containing pharmaceutical composition is administered.

Hereinafter, the present invention will be described with reference to examples. However, the present invention is not limited to these examples.

Detection of siRNA contained in blood AGO2 protein in liver-directed preparation-administered mice (1) Preparation of FVII siRNA-containing preparation siRNA (FVII siRNA) that suppresses expression of blood coagulation factor 7 (Coagulation Factor VII or FVII) gene Formulation 1 and Formulation 2 containing cationic lipid (Compound 1 or Compound 2) and lipid particles were prepared according to the following procedure. The structural formulas of Compound 1 and Compound 2 are shown in Formula (I) and Formula (II).

　　　　　　　　　　　　　　　　　　　　　　　(I)

(I)

　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　(II)

(II)

The concentration of compound 1 and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N- (methoxy (polyethylene glycol) -2000) sodium salt (PEG-DMPE Na, NOF Corporation) was 57.3 mmol each. / L, suspended in an aqueous solution containing hydrochloric acid and ethanol so as to be 5.52 mmol / L, and stirred and heated repeatedly with a vortex stirring mixer to obtain a uniform suspension. This suspension was passed through a 0.2 μm polycarbonate membrane filter at room temperature, and further passed through a 0.05 μm polycarbonate membrane filter to obtain a dispersion of Compound 1 / PEG-DMPE Na particles (liposomes).

The average particle size of the liposomes obtained with a dynamic light scattering (DLS) particle size measuring device (Zetasizer Nano ZS, manufactured by Malvern) was measured and confirmed to be in the range of 30 nm to 100 nm. The resulting liposome dispersion and the FVII siRNA solution prepared by dissolving the double-stranded nucleic acid consisting of the sense strand and antisense strand shown in Table 1 in distilled water to a concentration of 24 mg / mL are mixed at a ratio of 3: 1. Furthermore, a dispersion of Compound 1 / PEG-DMPE Na / FVII siRNA complex was prepared by adding and mixing 3 times the amount of distilled water.

On the other hand, the concentrations of Compound 1, PEG-DMPE Na, distearoyl phosphatidylcholine (DSPC, NOF Corporation) and cholesterol (NOF Corporation) are 8.947 mmol / L, 1.078 mmol / L, 5.707 mmol / L, 13.698 mmol, respectively. A solution of lipid membrane constituents was prepared by dissolving in 90 vol% ethanol so as to be / L.
After heating the resulting lipid membrane component solution, mix with the resulting compound 1 / PEG-DMPE Na / FVII siRNA complex dispersion at a ratio of 1: 1, and then several times the amount of distillation Water was mixed to obtain a crude preparation.

The obtained crude preparation was concentrated using Amicon Ultra (manufactured by Millipore), diluted with physiological saline, and filtered in a clean bench using a 0.2 μm filter (manufactured by Toyo Roshi Kaisha, Ltd.). Formulation 1 was obtained by measuring the FVII siRNA concentration of the obtained composition and diluting with physiological saline so that the siRNA concentration was 0.06 mg / mL. In addition, Preparation 2 containing Compound 2 instead of Compound 1 of Preparation 1 was prepared in the same manner as Preparation 1.

(2) Evaluation of FVII gene knockdown effect by preparation containing FVII siRNA The knockdown effect of preparations 1 and 2 containing FVII siRNA prepared in (1) was evaluated by the following method. Balb / cA mice were intravenously administered with each preparation in an amount corresponding to 0.3 mg / kg siRNA or physiological saline in the same amount. Mice were weighed before administration and 2 days after administration. After measurement 2 days after administration, blood was collected from the cheeks, and then Balb / cA mice were anesthetized with isoflurane, and the abdomen using a 1 ml syringe filled with 100 μl of anticoagulant (3.2% (w / w) citric acid, 5 mM glucose). Whole blood was collected from the vena cava.

The blood collected from the cheeks was converted into plasma using a BD Microtina (registered trademark) micro blood collection tube BD Microguard (registered trademark) with a cap (lithium heparin / plasma separator) (Nippon Becton Dickinson catalog number 365985). The obtained plasma was measured for the knockdown effect of each preparation by measuring the amount of FVII in the blood using BIOPHEN VII (CHROMOGENIC VII) (Anaira, catalog number A221304) (FIG. 1). The measurement procedure followed the attached instruction manual. As shown in FIG. 1, the amount of FVII in the blood was small in the mice administered with Formulation 1, and a knockdown effect was observed.
(3) Measurement of the amount of siRNA contained in AGO2 / siRNA in mouse blood AGO2 protein in the blood was purified from blood collected from Balb / cA mice in (2) by the following method, and siRNA contained in AGO2 / siRNA Was isolated.
Blood obtained from the abdominal vena cava of Balb / cA mice administered with Formulation 1, 2 or saline is transferred to a 1.5 mL centrifuge tube and centrifuged at 1900 xg for 10 min at 4 ° C to collect the supernatant. After that, it was further centrifuged at 16000 xg for 10 minutes at 4 ° C to obtain plasma.

The beads used for immunoprecipitation of AGO2 protein were prepared according to the following procedure. According to the attached instruction manual, 200 μL of Dynabeads (registered trademark) M-270 Epoxy (Invitrogen Cat # 143-01) resuspended in N, N-dimethylformamide was removed, and 400 μL of PBS was added. After vortexing for 30 seconds, sputum incubation was performed for 10 minutes, and the supernatant was removed. Further, 400 μL of PBS was added and the above operation was repeated. Thereafter, 200 μL of 2 mol / L ammonium sulfate / PBS, 200 μL of 1 mg / mL anti-mouse AGO2 monoclonal antibody (2D4, manufactured by WAKO, catalog number 018-22021) was added and incubated at 37 ° C. for 16 hours. Thereafter, the supernatant was removed, washed 4 times with 400 μL of 0.5% PBS BSA / PBS solution, and suspended in 0.6 mL of 0.5% BSA / PBS. At the time of use, the supernatant was removed, washed twice with 2 volumes of PBS relative to the initial solution, and then suspended in 1 volume of PBS to form a bead solution.

Balb / cA mouse plasma 80 μL and bead solution 5 μL were mixed and incubated at 4 ° C. for 2 hours. Then remove the supernatant and wash 3 times with 150 μL of wash buffer (1% Nonidet P-40, 25 mM Tris-HCl (pH 7.4), 1 M NaCl, 1 mM NaCl, 5% Glycerol) to obtain the AGO2 protein fraction. Isolated. Add 20 μL of Lysis® solution (Ambion, TaqMan® Gene® Expression® Cells-to-CT Kit, catalog number AM1728) to the washed beads, and stir at room temperature for 10 minutes, and then 2 μL of Stop solution (Ambion) Manufactured by TaqMan (registered trademark) Gene Expression Cells-to-CT Kit, catalog number AM1728), and the supernatant was transferred to another well to isolate FVII siRNA that binds to AGO2 protein.

Quantification of the isolated FVII siRNA was performed by the following method using the primer and probe described in Table 2. First, 5 μL of the sample solution was added to 5 μL of 45 nmol / L RT RT primer, and the denaturation reaction was performed by heating at 95 ° C. for 5 minutes and at 16 ° C. for 10 minutes. Thereafter, reverse transcription reaction was performed using TaqMan (registered trademark) MicroRNA RT Kit (Applied Biosystems, catalog number 4366597). The conditions for the reverse transcription reaction were in accordance with the attached instruction manual.

QPCR reactions for quantitation of FVII に は siRNA include 15μmol / L FP 2μL, 7μmol / L RP 2μL, 2μmol / L Taqman®probe 2μL, TaqMan® 4369016) 4 μL of a sample solution diluted 3-fold with distilled water was added to 10 μL. Applied Biosystems 7900HT Fast real-time PCR system (Applied Biosystems) was used for the PCR reaction, and the reaction conditions were in accordance with the attached instruction manual. The obtained Ct values are shown in Table 3.

Also, the value obtained by subtracting the average value of Ct values listed in Table 3 from 40 (40-Ct value) is shown as a bar graph (FIG. 2). The larger the 40-Ct value, the greater the amount of siRNA contained in blood AGO2 / siRNA. As shown in FIG. 2, the value is increased only in the formulation 1 administration group showing the knockdown effect, and the strength of the knockdown effect of the FVII siRNA-containing preparation correlates with the amount of FVII siRNA contained in the blood AGO2 / siRNA. I found out.

Detection of siRNA contained in AGO2 protein in blood of liver-directed preparation-administered mice (1) Evaluation of FVII gene knockdown effect by preparation containing FVII siRNA siRNA (FVII siRNA) that suppresses expression of FVII gene and cationic lipid (compound As a preparation containing 3 or compound 4) and lipid particles, preparation 3 containing compound 3 and preparation 4 containing compound 4 were prepared instead of compound 1 of preparation 1 of Example 1.
Formulation 3 was prepared as follows: Compound 3, PEG-DMPE Na, DSPC and cholesterol concentrations were 8.947 mmol / L, 0.147 mmol / L, 5.981 mmol / L, and 14.355 mmol / L, respectively. Except that it was dissolved in 100 vol% ethanol and used at room temperature without heating the lipid membrane components before mixing with the dispersion of Compound 3 / PEG-DPME Na / FVII siRNA complex. It was prepared in the same manner as formulation 1. Formulation 4 was prepared in the same manner as Formulation 1 except that compound 4 was used instead of compound 1. The structural formulas of Compound 3 and Compound 4 are shown in Formula (III) and Formula (IV).

　　　　　　　　　　　　　　　　　　　　　　　　(III)

(III)

　　　　　　　　　　　　　　　　　　　　　　　　　　　(IV)

(IV)

The knockdown effect of formulation 3 and formulation 4 was evaluated by the following method. Balb / cA mice were each administered with each preparation in an amount equivalent to 0.3 mg / kg siRNA or physiological saline in the same amount. Mice were weighed before administration and 2 days after administration. Two days after administration, cheek blood was collected after weighing, then anesthetized Balb / cA mice with isoflurane, and using a 1 ml syringe filled with 100 μl of anticoagulant (3.2% (w / w) citric acid, 5 mM glucose) Whole blood was collected from the abdominal vena cava.

The blood collected from the cheeks was converted into plasma using a BD Microtina (registered trademark) micro blood collection tube BD with micro guard cap (heparin lithium / plasma separator) (Nippon Becton, Dickinson catalog number 365985). The knockdown effect of each preparation was measured by measuring the amount of FVII in blood by BIOPHEN VII (CHROMOGENIC VII) (Anaira catalog number A221304) (FIG. 3). The measurement procedure followed the attached instruction manual. As shown in FIG. 3, in the mice administered with Formulation 4, the amount of FVII in the blood was small and a knockdown effect was observed.
(2) Measurement of the amount of siRNA contained in AGO2 / siRNA in mouse blood AGO2 protein in blood was purified by the following method, and FVII siRNA contained in AGO2 protein was isolated. Blood obtained from the abdominal vena cava of Balb / cA mice administered with Formulations 4 and 5 or physiological saline was transferred to a 1.5 mL centrifuge tube and centrifuged at 1900 xg for 10 minutes at 4 ° C to collect the supernatant. Thereafter, the mixture was further centrifuged at 16000 × g for 10 minutes at 4 ° C. to obtain plasma.

350 μL of plasma and 5 μL of the bead solution used in Example 1 (3) were mixed and incubated at 4 ° C. for 2 hours. Then remove the supernatant and resuspend in 150 μL wash buffer (1% Nonidet P-40, 25 mM Tris-HCl (pH 7.4), 1 M NaCl, 1 mM EDTA, 5% Glycerol). Transferred to a 96-well plate. Thereafter, the supernatant was removed and the plate was further washed twice with 150 μL of washing buffer to isolate the AGO2 fraction.

Add 20 μL of Lysis® solution (Ambion, TaqMan (registered trademark) Gene® Expressions, Cells-to-CT Kit, catalog number AM1728) to the washed beads, stir at room temperature for 10 minutes, and 2 μL of Stop solution (Ambion) TaqMan (registered trademark) Gene Expression Cells-to-CT Kit (catalog number AM1728) was added, and the supernatant was transferred to another well to isolate FVII siRNA that binds to AGO2 protein.

Quantification of the isolated FVII siRNA was performed in the same manner as in Example 1 using the primer and probe described in Table 2. The obtained Ct values are shown in Table 4.

FIG. 4 shows a bar graph representing a value obtained by subtracting the average value of Ct values described in Table 4 from 40 (40-Ct value). It shows that the amount of siRNA contained in AGO2 protein in blood is so large that a value is large. As shown in FIG. 4, the value is increased only in the formulation 4 administration group showing the knockdown effect, and the strength of the knockdown effect of the FVII siRNA-containing preparation correlates with the amount of FVII siRNA contained in the blood AGO2 protein. I found out.

Detection of siRNA contained in blood AGO2 protein of cancer-directed mice (1) Preparation of CKAP5 siRNA-containing preparations Preparation containing siRNA that suppresses CKAP5 gene expression (CKAP5 siRNA), cationic lipid, and lipid particles 5 was prepared according to the following procedure.
Compound 1 and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- (methoxy (polyethylene glycol) -2000) sodium salt (PEG-DSPE Na, manufactured by NOF Corporation) were each 57.3 mmol / The suspension was suspended in an aqueous solution containing hydrochloric acid and ethanol so that L and 5.52 mmol / L were obtained, and stirring and heating were repeated with a vortex stirring mixer to obtain a uniform suspension.

This suspension was passed through a 0.2 μm polycarbonate membrane filter and a 0.05 μm polycarbonate membrane filter at room temperature to obtain a particle (liposome) dispersion of Compound 1 / PEG-DSPE Na. The average particle size of the liposomes obtained with a DLS soot particle size measuring device was measured and confirmed to be within the range of 30 nm to 100 nm. Mix the obtained dispersion of liposomes and CKAP5 siRNA 溶解 solution prepared by dissolving double-stranded nucleic acid consisting of sense strand and antisense strand shown in Table 5 in distilled water to 24mg / mL at a ratio of 3: 1. Further, a dispersion of Compound 1 / PEG-DSPE Na / CKAP5 siRNA complex was prepared by adding and mixing 3 times the amount of distilled water.

On the other hand, 90 vol% so that compound 1, compound 5, PEG-DSPE Na, DSPC, and cholesterol would be 2.98 mmol / L, 5.97 mmol / L, 2.94 mmol / L, 5.71 mmol / L, 11.8 mmol / L, respectively. A solution of lipid membrane constituents was prepared by dissolving in ethanol.
The resulting lipid membrane component solution is heated and then mixed with the previously prepared dispersion of Compound 1 / PEG-DSPE Na / CKAP5 siRNA complex at a ratio of 1: 1, followed by several times the amount of distillation. Water was mixed to obtain a crude preparation.

The obtained crude preparation was concentrated using Amicon Ultra (manufactured by Millipore), further substituted with physiological saline, and filtered in a clean bench using a 0.2 μm filter (manufactured by Toyo Roshi Kaisha, Ltd.).
Formulation 5 was obtained by measuring the CKAP5 siRNA concentration of the obtained formulation and diluting with physiological saline so that the siRNA concentration was 1.0 mg / mL. The structural formula of Compound 5 is shown in Formula (V).

　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　(V)

(V)

(2) Evaluation of CKAP5 gene knockdown effect by preparation containing CKAP5 siRNA The efficacy of preparation 5 prepared in (1) was evaluated by the following method.
A human pancreatic cancer-derived cell line, MIA PaCa-2 (JCRB cell bank No. JCRB0070), 10% inactivated fetal bovine serum (GIBCO) and 1 vol% penicillin-streptomycin (GIBCO, catalog number 15140-122) ) And a high glucose-containing DMEM medium (GIBCO, catalog number 11995-065) at 37 ° C. and 5% CO 2.

MIA PaCa-2 was suspended in PBS to a concentration of 2 × 10 ⁸ cells / mL, and 50 μL of this cell suspension was immunized with CB-17 / Icr-scid / scidJc mice (CLEA Japan). ) (1 × 10 ⁷ cells / 0.05 mL PBS / head). Ten days after transplantation, the tumor volume was used as an index to group 9 mice per group to produce tumor-bearing mice.
Formulation 5 corresponding to siRNA 10 mg / kg was intravenously administered to cancer-bearing mice. In the physiological saline administration group, the same amount of physiological saline as that of the preparation 5 was administered. The tumor diameter and body weight of the tumor-bearing mice were measured before administration and 7 days after administration.

Compared with the physiological saline administration group, the preparation 5 administration group showed a remarkable cell growth inhibitory effect, that is, a knockdown effect by CKAP5 siRNA (FIG. 5).
(3) Measurement of amount of siRNA contained in mouse blood AGO2 / siRNA AGO2 in blood was purified by the following method, and siRNA contained in AGO2 / siRNA was isolated.
7 days after administration of Formulation 5 or physiological saline (2) above, mice were anesthetized with isoflurane and abdomen using a 1 ml syringe filled with 100 μl of anticoagulant (3.2% (w / w) citric acid, 5 mM glucose). Whole blood was collected from the vena cava.

Blood collected from each of the tumor-bearing mice or non-tumor-bearing mice administered with Formulation 5 or physiological saline is transferred to a 1.5 mL centrifuge tube, centrifuged at 1900 xg for 10 minutes at 4 ° C, and the supernatant is collected. Plasma was obtained by centrifugation at 16000 xg for 10 minutes at ° C.
The beads used for immunoprecipitation of AGO2 protein were prepared according to the following procedure.
Dynabeads (registered trademark) M-270 Epoxy (Catalog No. 143-01, manufactured by Invitrogen) resuspended in N, N-dimethylformamide was removed. 200 μL of the supernatant was removed, and 400 μL of PBS was added. After vortexing for 30 seconds, incubation was performed for 10 minutes, and the supernatant was removed. Further, 400 μL of PBS was added and the above operation was repeated.

Thereafter, 200 μL of 2 mol / L ammonium sulfate / PBS and 200 μL of 1 mg / mL anti-human AGO2 monoclonal antibody (4G8, manufactured by WAKO Co., Ltd., catalog number 015-22031) were added and incubated at 37 ° C. for 16 hours. Thereafter, the supernatant was removed, washed 4 times with 400 μL of 0.5% BSA / PBS solution, and suspended in 0.6 mL of 0.5% BSA / PBS. At the time of use, the supernatant was removed, washed twice with 2 volumes of PBS relative to the initial solution, and then suspended in 1 volume of PBS to form a bead solution.

The plasma obtained above (300 μL) and the bead solution (5 μL) were mixed and incubated at 4 ° C. for 2 hours. Then remove the supernatant and wash 3 times with 200 μL of wash buffer (1% Nonidet P-40, 25 mM Tris-HCl (pH 7.4), 500 mM NaCl, 1 mM mM EDTA, 5% Glycerol). Purified. Add 20 μL of Lysis® solution (Ambion, TaqMan (registered trademark) Gene® Expressions, Cells-to-CT Kit, catalog number AM1728) to the washed beads, stir at room temperature for 10 minutes, and 2 μL of Stop solution (Ambion) , TaqMan (registered trademark) Gene Expression Cells-to-CT Kit (catalog number AM1728) was added, and the supernatant was transferred to another well to isolate siRNA that binds to AGO2 protein.

Quantification of the isolated siRNA was performed by the following method using the primers and probes described in Table 6. First, 5 μL of the sample solution was added to 5 μL of 45 nM RT RT primer, and the denaturation reaction was performed by heating at 95 ° C. for 5 minutes and at 16 ° C. for 10 minutes. Thereafter, reverse transcription reaction was performed using TaqMan (registered trademark) MicroRNA RT Kit (Applied Biosystems, catalog number 4366597). The conditions for the reverse transcription reaction were in accordance with the attached instruction manual. qPCR reactions for siRNA quantification include 15 μM 2 μL, 7 μM RP 2 μL, 2 μM Taqman® probe 2 μL, TaqMan® Gene® Expression® Master® Mix (Life Technologies, catalog number 4369016) with 10 μL in distilled water. 4 μL of sample solution diluted twice was added. Applied Biosystems 7900HT Fast real-time PCR system (Applied Biosystems) was used for the PCR reaction, and the reaction conditions were in accordance with the attached instruction manual. The obtained Ct values are shown in Table 7.

 

 

FIG. 6 shows a value (40-Ct value) obtained by subtracting the average value of Ct values described in Table 7 from 40. The larger the 40-Ct value, the greater the amount of siRNA contained in blood AGO2. The value was increased only in the group in which the preparation 5 was administered to cancer-bearing mice, and it was found that the amount of siRNA contained in AGO2 in the blood was large in the mice in which the tumor diameter decreased and the medicinal effect was recognized in FIG.

According to the present invention, by quantifying or identifying small RNA contained in an AGO protein-small RNA complex derived from a subject administered with a small RNA-containing pharmaceutical composition, the target gene knockdown effect of the small RNA-containing pharmaceutical composition can be reduced. Evaluation method, method for predicting the efficacy of small RNA-containing pharmaceutical composition, method for selecting effective small RNA-containing pharmaceutical composition, evaluation of knockdown effect of target cells of small RNA-containing pharmaceutical composition, and selection of patients And a method for selecting cancer types in which a small RNA-containing pharmaceutical composition exhibits a medicinal effect.
This application is based on a patent application No. 2014-160487 filed in Japan (filing date: August 6, 2014), the contents of which are hereby incorporated by reference.

The present invention provides a method for predicting the efficacy of a small RNA-containing drug and a method for evaluating the knockdown effect of a target gene of a small RNA-containing drug. Further, according to the present invention, it becomes possible to select an effective small-RNA-containing drug, and to select patients who effectively exhibit the medicinal effect of the small-RNA-containing drug.

SEQ ID NO: 1 shows the base sequence of the sense strand of FVII siRNA.
SEQ ID NO: 2 shows the base sequence of the antisense strand of FVII siRNA.
SEQ ID NO: 3 shows the base sequence of RT primer for FVII siRNA quantification.
SEQ ID NO: 4 shows the base sequence of FP primer for quantification of FVII siRNA.
SEQ ID NO: 5 shows the base sequence of RP primer for FVII siRNA quantification.
SEQ ID NO: 6 shows the base sequence of Taqman probe for FVII siRNA quantification.
SEQ ID NO: 7 shows the base sequence of the sense strand of CKAP5 siRNA.
SEQ ID NO: 8 shows the base sequence of the antisense strand of CKAP5 siRNA.
SEQ ID NO: 9 shows the nucleotide sequence of RT primer for quantifying CKAP5 siRNA.
SEQ ID NO: 10 shows the base sequence of the FP primer for quantifying CKAP5 siRNA.
SEQ ID NO: 11 shows the base sequence of the RP primer for quantifying CKAP5 siRNA.
SEQ ID NO: 12 shows the base sequence of Taqman probe for quantifying CKAP5 siRNA.

Claims (5)

A target of the small RNA-containing pharmaceutical composition by isolating an AGO protein-small RNA complex from a sample derived from a subject administered with the small RNA-containing pharmaceutical composition and measuring the amount of the small RNA contained in the complex A method for evaluating the knockdown effect of a gene. The efficacy of the small 医 薬 RNA-containing pharmaceutical composition by isolating an AGO protein-small RNA complex from a sample derived from a subject administered with the small RNA-containing pharmaceutical composition and measuring the amount of small RNA contained in the complex How to predict. Effective by isolating an AGO protein-small RNA complex from a sample derived from a subject administered with a small RNA-containing pharmaceutical composition, measuring the amount of small RNA included in the complex, and comparing the amount of the small RNA For selecting a small small RNA-containing pharmaceutical composition. An AGO protein-small RNA complex is isolated from a sample derived from a subject administered with a small RNA-containing pharmaceutical composition, the amount of small RNA included in the complex is measured, and the small RNA is compared by comparing the small RNA amounts. A method for screening a patient by evaluating the knockdown effect of a target cell of an RNA-containing pharmaceutical composition. By isolating an AGO protein-small RNA complex from a sample derived from a subject administered with a small RNA-containing pharmaceutical composition, measuring the amount of small RNA contained in the complex, and comparing the amount of small RNA, A method for selecting cancer types in which an RNA-containing pharmaceutical composition exhibits a medicinal effect.

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