JP2024026903A - siRNA AND PHARMACEUTICAL COMPOSITION, AND PROPHYLACTIC AND/OR THERAPEUTIC AGENT - Google Patents

Abstract

To provide a pharmaceutical composition containing siRNA, as an active ingredient, that suppresses the BAZF expression, specifically, a prophylactic and/or therapeutic agent that, through suppressing the BAZF expression, prevents and/or treats diseases associated with the VEGF action that promotes neoangiogenesis, e.g., various intraocular neoangiogenesis diseases including diabetic retinopathy such as proliferative diabetic retinopathy, retinopathy of prematurity, retinal branched vein occlusion, central retinal vein occlusion, neovascular glaucoma, neovascular maculopathy, diabetic macular edema, and age-related macular degeneration.SOLUTION: Provided are: siRNA characterized in that a nucleotide sequence represented by SEQ ID NO: 1 is the target gene sequence; and a pharmaceutical composition that contains the siRNA as an active ingredient.SELECTED DRAWING: None

Description

The present invention provides a novel siRNA (small interfering RNA) that suppresses the expression of BAZF (BCL6 associated zinc finger protein), a prophylactic and/or therapeutic agent for intraocular angiogenesis diseases, etc., containing the siRNA as an active ingredient, and The present invention relates to a pharmaceutical composition as an active ingredient.

Intraocular angiogenesis diseases such as proliferative diabetic retinopathy, retinopathy of prematurity, retinal vein occlusion, and age-related macular degeneration are diseases caused by pathological angiogenesis in the retina and are irreversible. It is known that vascular endothelial growth factor (VEGF) is involved in this angiogenesis (Non-patent Document 1). ).
Patent Document 1 describes that BAZF is the key to controlling the angiogenesis promoting effect of VEGF.

Further, for example, Patent Document 2 describes specific sequences of double-stranded siRNA that can suppress the expression of BAZF (SEQ ID NO: 1 and 2 of Patent Document 2).

However, the siRNA described in Patent Document 2 targets the vicinity of positions 1827 to 1845, which is further downstream of the nucleotide sequence of SEQ ID NO: 1 (positions 1468 to 1486) in the present invention, and is different from the siRNA of the present invention. differ.

WO2010/18764 publication Patent No. 5665213

Japanese Pharmacological Journal 135, 149-152 (2010)

The present inventors conducted intensive studies to find a drug useful for the prevention and/or treatment of intraocular angiogenic diseases, and as a result, discovered an siRNA sequence that effectively inhibits the expression of BAZF, and further developed a method for using it as an active ingredient. The present invention has been completed by discovering that a drug that suppresses the angiogenesis-promoting action of VEGF, particularly intraocular blood vessel angiogenesis, can be suppressed by suppressing the expression of BAZF. The purpose of the present invention is to provide novel siRNAs, pharmaceutical compositions, and preventive and/or therapeutic agents for intraocular neovascular diseases.

The above object is achieved by the following first to eleventh inventions.

<First invention>
siRNA characterized by having the nucleotide sequence shown in SEQ ID NO: 1 as a target gene sequence.

Sequence number 1: ACACCAAAGUGCACUACCA

<Second invention>
The siRNA according to the first invention, which has an overhang sequence of one or several bases at the 3' end.

<Third invention>
The siRNA according to the first invention or the second invention, wherein the 2'-position hydroxyl group of at least some of the sugars (ribose) constituting the siRNA is chemically modified.

<Fourth invention>
siRNA according to any one of the first to third inventions, wherein the chemical modification is substitution of a hydroxyl group with a methoxy group.

<Fifth invention>
siRNA according to any one of the first to fourth inventions, which is a double-stranded RNA composed of any combination of sequence numbers listed below.
i) SEQ ID NO: 2 (sense strand) and 3 (antisense strand)
ii) SEQ ID NO: 4 (sense strand) and 5 (antisense strand)
iii) SEQ ID NO: 6 (sense strand) and 7 (antisense strand)

<Sixth invention>
siRNA according to the fifth invention, which is a double-stranded RNA composed of a combination of SEQ ID NOs: 4 and 5.

<Seventh invention>
siRNA according to the fifth invention, which is a double-stranded RNA composed of a combination of SEQ ID NOs: 6 and 7.

<Eighth invention>
A pharmaceutical composition comprising the siRNA according to any one of the first to seventh inventions.

<Ninth invention>
A prophylactic and/or therapeutic agent for diseases caused by angiogenesis, characterized by containing the siRNA according to any one of the first to seventh inventions.

<Tenth invention>
A prophylactic and/or therapeutic agent for intraocular angiogenic diseases, characterized by containing the siRNA according to any one of the first to seventh inventions.

<Eleventh invention>
The intraocular neovascular disease is selected from diabetic retinopathy, retinopathy of prematurity, branch retinal vein occlusion, central retinal vein occlusion, neovascular glaucoma, neovascular maculopathy, diabetic macular edema, and age-related macular degeneration. The preventive and/or therapeutic agent according to the tenth invention, which is a disease.

The siRNA of the present invention can be used to treat diseases associated with the angiogenesis-promoting effect of VEGF, such as diabetic retinopathy such as proliferative diabetic retinopathy, retinopathy of prematurity, branch retinal vein occlusion, central retinal vein occlusion, neovascular glaucoma, Neovascular maculopathy, diabetic macular edema, age-related macular degeneration, and the like can be prevented and/or treated by suppressing the expression of BAZF.

FIG. 1 is a diagram showing the suppressive effect of BAZF expression by siRNA of the present invention (siBAZF#5, siBAZF#5-4, and siBAZF#5-6).

The present invention will be explained in detail below.

[siRNA of the present invention]
The siRNA of the present invention has a target gene sequence that is a nucleotide sequence common to humans and monkeys shown in SEQ ID NO: 1, and is characterized by significantly suppressing the angiogenesis-promoting effect of VEGF. .

The inventors of the present application designed siRNA targeting various sites in the nucleic acid sequence of BAZF (BCL6B; Ref Seq (mRNA); NM 181844), and as a result of various studies, the siRNA shown in SEQ ID NO: 1 was obtained. We have found that siRNA directed against site #5 below (hereinafter sometimes referred to as siBAZF#5 etc.) is extremely effective.

#5; positions 1468 to 1486 (SEQ ID NO: 1)

《siRNA sequence》
Therefore, siRNA (double-stranded) whose target gene sequence is the nucleotide sequence shown in SEQ ID NO: 1 of the present invention,
A) A sequence (sense strand) consisting of a 19 base sequence corresponding to positions 1468 to 1486 (SEQ ID NO: 1) in the nucleic acid sequence of the BAZF (BCL6B) gene,
B) A sequence that can form a double strand with A) (antisense strand)
Examples include those consisting of.

In addition, the siRNA of the present invention includes those in which the following various modifications and modifications are made to A) and/or B) above, as long as A) and B) can form a double strand. .

Note that being able to form a double strand means being able to hybridize under stringent conditions, and corresponding constituent bases do not necessarily need to form a completely complementary pair.

(Modification of nucleic acid sequence)
More specifically, the above-mentioned modifications of A) and/or B) include substitutions, deletions, additions, and additions of one to several bases (nucleotides) to the nucleic acid sequences of A) and/or B). It means that / or insertion etc. (hereinafter sometimes referred to as modification) has been made.

The base to be substituted, added, and/or inserted may not only be one that binds to ribose, but also one that binds to deoxyribose, such as dTdT, which will be described later.

However, substitutions, deletions, additions, and/or insertions are preferably those that do not impair the siRNA effect; for example, a polynucleotide that can hybridize with the target RNA sequence of SEQ ID NO: 1 under stringent conditions. Examples include nucleotides.

Stringent conditions refer to conditions under which specific hybrids are formed and nonspecific hybrids are not formed; for example, J. Sambrook et al., Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press ( 1989) (in particular Section 11.45 “Conditions for Hybridization of oligonucleotide Probes, etc.”) and Elbashir SM, Lendeckel W, Tuschl T., Genes Dev. 2001 Jan 15;15(2):188-200. doi: 10.1101/gad.862301 Examples include the conditions described.

An example of the above addition is the overhang arrangement described below.

(Adding array for overhang)
The siRNA of the present invention preferably has an overhang sequence of one or several bases at the 3' end of the siRNA described above.

An overhanging sequence is a sequence that is used to make the 3' end of the sense strand and/or antisense strand protrude when the double-stranded siRNA consists of a sense strand and an antisense strand through annealing. means an array.

Specifically, one or several bases generally includes two bases, but is not limited to this.

As the overhanging arrangement, any known arrangement can be used, but dTdT, for example, is preferable because its overhanging effect has been established.

(Modification of sugar moieties constituting nucleic acids)
The siRNA of the present invention has the advantage of improving the ability to form a stable hybrid with a nucleic acid having a complementary base sequence and/or improving nuclease resistance. It is preferable that the 2'-position hydroxyl group is chemically modified.

In the present invention, chemical modification means substituting the 2'-position hydroxyl group of sugar (ribose) with the following substituent.

・Methoxy group ・2-methoxyethoxy group ・3-aminopropyloxy group ・2-(N,N-dimethylaminooxy)ethoxy group ・Fluoro group

Among these, in order to avoid an immune response mediated by, for example, TLR (Toll-like receptor), it is preferable to substitute with a methoxy group (conversion of OH to OMe).

In addition, in order to fully demonstrate RNAi activity (siRNA effect), only a part of the 2'-position hydroxyl group of the sugar (ribose) constituting the target siRNA molecule must be chemically modified. is preferred.

As a method for chemically modifying only a part, for example, when siRNA is synthesized using an automatic nucleic acid synthesizer using the phosphoramidite method, part or all of the amidite containing a specific base is used as the raw material amidite. By converting only the 2'-position hydroxyl group of the siRNA to OMe, it is possible to prevent the 2'-position hydroxyl group of all the sugars (ribose) constituting the siRNA molecule from being chemically modified.

Preferred examples of the above-mentioned specific bases include U and G, since they are known to effectively suppress TLR activation.

The chemical modification may be carried out in at least one of the above A) and B).

As specific examples of the siRNA of the present invention, for example, double-stranded RNAs composed of any of the following combinations are listed as preferred, but are not necessarily limited to these, and these double-stranded RNAs In addition, as long as the sense strand and antisense strand can form a double strand, the above-mentioned modification of the base itself (substitution, deletion, addition and/or insertion of one to several bases (nucleotides)) This also includes those that have been given.

In the sequences in Table 1 above, the small letters represent RNA, and the bold letters dT represent DNA (sequence for overhang).

In addition, Um and Gm in Table 1 mean that the hydroxyl group at the 2' position of the sugar (ribose) to which the respective bases are bonded is substituted with a methoxy group; (or 2'-O-Methyl), etc.

《SiRNA production method》
The above-described siRNA (siBAZF) of the present invention can be synthesized artificially using, for example, an automatic nucleic acid synthesizer or transformed cells;
a method for extracting naturally occurring polynucleotides;
A method of deleting, substituting, adding and/or inserting a part of the base of a polynucleotide extracted from nature,
A method of synthesizing the desired sequence using reverse transcriptase, RNA polymerase, etc. using a sequence complementary to the desired sequence;
Alternatively, it can be manufactured by a method that combines these methods.

Specific examples of methods using the automatic nucleic acid synthesizer include, but are not limited to, the phosphoramidite method using amidites.

In addition, the siRNA of the present invention, in which the hydroxyl group at the 2' position of at least some of the sugars (ribose) constituting the siRNA has been chemically modified, comprises the sugar (ribose) in the amidite material or the nucleotide after synthesis. Part or all of the 2'-position hydroxyl group of the sugar (ribose) is substituted (protected) with the above-mentioned substituent according to a known method, and if necessary, some of the substituents are deprotected as appropriate. This can be done by

《Applications of siRNA》
The siRNA of the present invention can be used not only as a pharmaceutical composition of the present invention and as a preventive and/or therapeutic agent, which will be described later, but also as an experimental/research reagent (BAZF expression inhibitor or angiogenesis inhibitor) in the laboratory. It is of high value.

[Pharmaceutical composition of the present invention]
The pharmaceutical composition of the present invention is characterized by containing the above-described siRNA of the present invention.
Specifically, examples of pharmaceutical compositions include those detailed in the section of the prophylactic and/or therapeutic agent of the present invention below.

[Prophylactic and/or therapeutic agent for diseases of the present invention]
The preventive and/or therapeutic agent for diseases of the present invention is characterized by containing the above-mentioned siRNA of the present invention, and is suitable for diseases involving the angiogenesis-promoting effect of VEGF, such as diabetic retina such as proliferative diabetic retinopathy. By suppressing the expression of BAZF, we can treat diseases such as retinopathy of prematurity, branch retinal vein occlusion, central retinal vein occlusion, neovascular glaucoma, neovascular maculopathy, diabetic macular edema, and age-related macular degeneration. It can be prevented and/or treated.

(Additive)
The pharmaceutical composition of the present invention or the prophylactic and/or therapeutic agent of the present invention (hereinafter sometimes referred to as a pharmaceutical composition, etc.) contains the siRNA of the present invention as an active ingredient alone, or siRNA in a pharmaceutically acceptable manner. They can be used together with additives to formulate their respective formulations.

Examples of pharmaceutically acceptable additives include, but are not limited to, those listed below.

Organic excipients such as sucrose and starch Binder such as cellulose and methyl cellulose Disintegrants such as starch and carboxymethyl cellulose Lubricants such as magnesium stearate and Aerosil
Aromatic agents such as menthol Preservatives such as sodium benzoate and sodium bisulfite Stabilizers such as citric acid and sodium citrate Suspension agents such as methylcellulose and polyvinylpyrrolidone Dispersants such as surfactants Dilution agents such as water and physiological saline agent

(Reagent for nucleic acid introduction)
In order to promote the introduction of the siRNA of the present invention into target cells, a pharmaceutical composition or an experimental reagent containing the siRNA of the present invention may further contain a known reagent for nucleic acid introduction.

Specific examples of nucleic acid introduction reagents include, but are not limited to, those listed below.

Atelocollagen, liposome, nanoparticle, lipofectin, lipofectamine (registered trademark), DOGS (transfectam: dioctadecylamidoglycylspermine), DOPE (L-α-dioleoylphosphatidylethanolamine), DOTAP (1, 2-dioleoyl-3-trimethylammoniumpropane), DDAB (dimethyldioctadecylammonium bromide), DHDEAB (N,N-di-n-hexaadecyl-N,N-dihydroxyethylammonium bromide), HDEAB (N-n-hexaadecyl- Cationic lipids such as N,N-dihydroxyethylammonium bromide), polybrene, or polyethyleneimine (PEI) can be used.

Furthermore, the pharmaceutical composition of the present invention can also be used in combination with the following molecular target drugs related to angiogenesis control, or in a combination that takes advantage of their respective properties.

Human monoclonal antibody medicines against VEGF, RNA aptamer medicines against VEGF, various nucleic acid medicines against Notch1, Notch1 gene, CBF-1, CBF-1 gene, HEY1, HEY1 gene, HEY2, HEY2 gene, HES1, HES1 gene, etc.

(Dosage form)
Examples of the dosage form of the pharmaceutical composition of the present invention include injections such as intravitreal injections, eye drops, and inhalation preparations.

(Content of active ingredients)
The content of the active ingredient siRNA in the pharmaceutical composition etc. of the present invention varies depending on the dosage form and cannot be absolutely limited, and may be determined as appropriate in relation to the dosage described below, within the range that allows various dosage forms. All you have to do is choose.

(Production method)
The pharmaceutical composition of the present invention can be formulated using the above-mentioned components by a well-known method.

Note that methods for encapsulating siRNA into liposomes include, for example, the following known methods, but are not limited to these.

Lipid film method (vortex method), reversed phase evaporation method, surfactant removal method, freeze-thaw method, remote loading method, etc.

(Route of administration)
Administration routes for the pharmaceutical composition of the present invention include systemic administration and local administration, and specifically, intravenous administration such as intravenous injection, intramuscular administration such as intramuscular injection, nasal administration, intradermal administration, and subcutaneous administration. Examples include administration, mucosal administration, inhalation, intraocular administration, etc., and can be appropriately selected depending on the disease, symptoms, etc. to be treated. For example, in the case of a disease such as age-related macular degeneration where the focal area is specified, intravitreal administration or local administration such as eye drops is preferred.

(Administration method)
The administration method is not particularly limited, and conventionally known delivery systems can be used as appropriate.

For example, local administration and systemic administration using atelocollagen are preferred.

The atelocollagen has already been clinically applied, for example, as a local hemostatic agent, and the local administration of siRNA using the atelocollagen has been shown to be effective in animal experiments using VEGF as a molecular target (Takei et al. Y et al., Cancer Res 64(10):3365-3370, 2004).

In addition, as other delivery systems, chemical modification to prevent in vivo degradation and increase intracellular permeability (Rossi JJ., Nature 432 (7014): 155-156, 2004; Soutschek J et al., Nature 432 ( 7014): 173-178, 2004) or a delivery system using cationic liposomes (Yano J et al., Clin Cancer Res 10(22): 7721-7726, 2004).

Furthermore, if necessary, an siRNA expression vector that expresses the double-stranded siRNA of the present invention can be constructed and a delivery system using gene therapy technology can be used.

(Dose)
The dosage of the pharmaceutical composition of the present invention varies depending on the administration route, symptoms, age, sex, body weight, form of the pharmaceutical composition, etc., and cannot be unconditionally prescribed. It is preferable to administer the component (siRNA) in an amount of 0.0001 to 100 mg, preferably 0.001 to 10 mg, about once every few days to several months.

The contents of the present invention will be explained in further detail in the following test examples and examples, but the present invention is not limited to the contents.

Test example 1
Confirmation test for suppressing BAZF expression in human retinal capillary endothelial cells

《Materials and methods》
Human retinal microvascular endothelial cells (manufactured by HRMEC, Cell Systems) were seeded at 3.0 x ¹⁰⁴ cells/ml and cultured at 37°C and 5% _CO2 for 24 hours. .

For cell culture, CS-C Complete Medium (manufactured by Cell Systems) containing Culture boost (registered trademark), a type of mitogen, was used. After culturing for 24 hours, control siRNA (siRNA that does not target BAZF, MISSION (registered trademark) siRNA Universal Negative Control (SIC001) manufactured by Sigma-Aldrich, hereinafter referred to as siCont) was prepared using Lipofectamine (registered trademark) RNAiMAX (Thermo Fisher Scientific). ), siBAZF #5, siBAZF #5-4, and siBAZF #5-6 listed in Table 1 were added at a final concentration of 25 nM and cultured for 24 hours.
Furthermore, the medium was exchanged to a 10% FBS-containing medium (not containing Culture boost (registered trademark)), and cultured for 6 hours.
Recombinant human VEGF (10 ng/ml, manufactured by R&D Systems) dissolved in PBS was added, and the cells were further cultured for 1 hour.

In addition, PBS was added to a group to which VEGF was not added for comparison.

RNA was extracted using NucleoSpin (registered trademark) RNA kit (manufactured by Takara), and cDNA was produced using PrimeScript (registered trademark) RT reagent kit (manufactured by Takara).

RT-PCR was performed using TB Green Premix Ex Taq (registered trademark) II (manufactured by Takara) by one cycle of 95°C for 30 seconds and 40 cycles of 95°C for 5 seconds and 60°C for 30 seconds. Ta.

The sequences of the primers used are as follows.
BAZF forward: CGGGAAGTGAATTTTTCAGC (SEQ ID NO: 8)
BAZF reverse: TGGTAAGGCTTTTCCCCTGT (SEQ ID NO: 9)

GAPDH forward: CCTGCACCACCAACTGCTTA (SEQ ID NO: 10)
GAPDH reverse: GGCCATCCACAGTCTTCTGAG (SEQ ID NO: 11
(Made by Eurofins Genomics (Tokyo))

The siRNAs used were siBAZF#5, siBAZF#5-4, and siBAZF#5-6 listed in Table 1 above.

"result"
The results are shown in Figure 1.
As shown in FIG. 1, siBAZF#5, siBAZF#5-4, and siBAZF#5-6 significantly suppressed the expression of BAZF.

Therefore, siBAZF (siBAZF#5, siBAZF#5-4, and siBAZF#5-6) targeting SEQ ID NO: 1 (#5), which is a part of the nucleotide sequence of BAZF, especially siBAZF#5-4 , diseases involving the angiogenesis-promoting effect of VEGF, such as diabetic retinopathy such as proliferative diabetic retinopathy, retinopathy of prematurity, branch retinal vein occlusion, central retinal vein occlusion, neovascular glaucoma, neovascular maculopathy, Diabetic macular edema, age-related macular degeneration, and the like can be prevented and/or treated by suppressing the expression of BAZF.

Example 1
siBAZF#5
Double-stranded siRNA (siBAZF#5) was created as follows.

Oligonucleotides for the sense and antisense strands of siRNA are synthesized using an automatic nucleic acid synthesizer by the phosphoramidite method using the corresponding amidite reagents for RNA synthesis, and then the base portion is deprotected and 2 The hydroxyl group at the ' position was deprotected, the single strand was purified by column, desalted, the sense strand and the antisense strand were annealed, and lyophilized to produce the siRNA shown in Table 1.

Example 2
siBAZF#5-4
Double-stranded siRNA (siBAZF#5-4) was created as follows.

Oligonucleotides for the sense and antisense strands of siRNA are synthesized using an automatic nucleic acid synthesizer by the phosphoramidite method using the corresponding amidite reagent for RNA synthesis and a 2'-O-Methyl modified amidite reagent, and then , deprotect the base part and deprotect the substituent at the 2'-position hydroxyl group of the ribose in accordance with conventional methods, column purify in a single-stranded state, desalt, then anneal the sense strand and antisense strand, and freeze-dry. Then, the siRNAs listed in Table 1 were produced.

Example 3
siBAZF#5-6
Double-stranded siRNA (siBAZF#5-6) was used in the same manner as in Example 2 to produce the siRNA listed in Table 1.

The medicine of the present invention containing siRNA as an active ingredient that suppresses the expression of BAZF can be used to treat diseases related to the angiogenesis-promoting effect of VEGF, such as diabetic retinopathy such as proliferative diabetic retinopathy, retinopathy of prematurity, and branch retinal vein occlusion. By suppressing the expression of BAZF, diseases such as central retinal vein occlusion, neovascular glaucoma, neovascular maculopathy, diabetic macular edema, and age-related macular degeneration can be prevented and/or treated.

Claims (11)

siRNA characterized by having the nucleotide sequence shown in SEQ ID NO: 1 as a target gene sequence. siRNA according to claim 1, characterized in that it has a sequence for overhanging one or several bases at the 3' end. The siRNA according to claim 1 or 2, wherein the hydroxyl group at the 2' position of at least part of the ribose constituting the siRNA has been chemically modified. The siRNA according to any one of claims 1 to 3, wherein the chemical modification is substitution of a hydroxyl group with a methoxy group. The siRNA according to any one of claims 1 to 4, which is a double-stranded RNA composed of any combination of sequence numbers listed below.
i) SEQ ID NO: 2 (sense strand) and 3 (antisense strand)
ii) SEQ ID NO: 4 (sense strand) and 5 (antisense strand)
iii) SEQ ID NO: 6 (sense strand) and 7 (antisense strand) 6. The siRNA according to claim 5, which is a double-stranded RNA composed of a combination of SEQ ID NOs: 4 and 5. 6. The siRNA according to claim 5, which is a double-stranded RNA composed of a combination of SEQ ID NOs: 6 and 7. A pharmaceutical composition comprising the siRNA according to any one of claims 1 to 7. A prophylactic and/or therapeutic agent for diseases caused by angiogenesis, comprising the siRNA according to any one of claims 1 to 7. A prophylactic and/or therapeutic agent for intraocular neovascular disease, comprising the siRNA according to any one of claims 1 to 7. The intraocular neovascular disease is selected from diabetic retinopathy, retinopathy of prematurity, branch retinal vein occlusion, central retinal vein occlusion, neovascular glaucoma, neovascular maculopathy, diabetic macular edema, and age-related macular degeneration. The prophylactic and/or therapeutic agent according to claim 10, which is a disease.

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