WO2021085592A1

WO2021085592A1 - Nucleic acid for suppressing gene expression of disease-specific col3a1 mutant allele and pharmaceutical composition for treating vascular ehlers-danlos syndrome

Info

Publication number: WO2021085592A1
Application number: PCT/JP2020/040786
Authority: WO
Inventors: 哲郎吉田; 原　光信
Original assignee: ナノキャリア株式会社
Priority date: 2019-10-31
Filing date: 2020-10-30
Publication date: 2021-05-06
Also published as: JP2022177340A

Abstract

The present invention provides a pharmaceutical composition for use in treatment of a disease caused by a mutation in collagen 3A1. This pharmaceutical composition is for treating a disease caused by a collagen 3A1 mutant in a subject having a gene encoding the mutant and a gene encoding the wild type, said pharmaceutical composition containing a nucleic acid selected from siRNAs and shRNAs. The mutant is a gene encoding collagen 3A1 in which the base G at position 755 in the base sequence represented by SEQ ID No:27 is substituted with T or a gene encoding collagen 3A1 in which the base G at position 547 in the base sequence represented by SEQ ID No:27 is substituted with A. The nucleic acid suppresses the expression of the mutant more strongly than the expression of the wild type.

Description

Nucleic acid that suppresses gene expression of disease-specific COL3A1 mutant allele and pharmaceutical composition for treating vascular Ehlers-Danlos syndrome

The present invention relates to a nucleic acid that suppresses gene expression of a disease-specific COL3A1 mutant allele and a pharmaceutical composition for treating vascular Ehlers-Danlos syndrome.

Ehlers-Danlos syndrome (EDS) is a hereditary disease based on the fragility of systemic connective tissues such as skin, joints, and blood vessels. Based on the causes and symptoms, it is classified into 6 main disease types (classical type, joint type, vascular type, kyphoscoliosis type, multiple joint relaxation type, skin fragile type), and the estimated frequency of all disease types combined is It is said to be about 1 / 5,000. The cause of EDS is based on genetic mutations in collagen molecules or enzymes involved in the collagen maturation process.

Vascular Type EDS (VEDS) (MIM 130050), which is a category of EDS, is the most severe type EDS with serious complications that also cause sudden death such as rupture of arteries, gastrointestinal tract, and uterus during pregnancy. And the mode of inheritance is autosomal dominant inheritance. Vascular EDS develops due to an abnormality in the type III collagen molecule due to a mutation in the type III collagen gene (COL3A1). At present, there is no radical cure for vEDS, and symptomatic treatment is the main treatment.

WO2011 / 019793A

The present invention provides a nucleic acid that suppresses gene expression of a disease-specific COL3A1 mutant allele and a pharmaceutical composition for treating vascular Ehlers-Danlos syndrome.

According to the present invention, the following inventions can be provided.
[1] A pharmaceutical composition for treating a disease caused by a mutant type of collagen 3A1 in a subject having a gene encoding a mutant type and a gene encoding a wild type, respectively.
Contains nucleic acids selected from siRNA or shRNA
The variant is a gene encoding collagen 3A1 having a G-to-T substitution of the 755th corresponding base in the nucleotide sequence of SEQ ID NO: 27, or 547 in the nucleotide sequence of SEQ ID NO: 27. A gene encoding collagen 3A1 having a G-to-A substitution of the second corresponding base.
The nucleic acid can strongly suppress the expression of the mutant form rather than the expression of the wild type.
Pharmaceutical composition.
[2] The pharmaceutical composition according to the above [1], wherein the subject is a subject suffering from vascular Ehlers-Danlos syndrome (vEDS).
[3] The above-mentioned [1] or [2], wherein the variant is a gene encoding collagen 3A1 having a G-to-T substitution of the base corresponding to the 755th base in the base sequence shown in SEQ ID NO: 27. The pharmaceutical composition described.
[4] The above-mentioned [1] or [2], wherein the variant is a gene encoding collagen 3A1 having a G-to-A substitution of the base corresponding to the 547th in the base sequence shown in SEQ ID NO: 27. The pharmaceutical composition described.
[5] The base sequence of the sense strand and antisense strand of siRNA or shRNA has a base sequence selected from the group consisting of SEQ ID NOs: 1 and 2, SEQ ID NOs: 3 and 4, and SEQ ID NOs: 5 and 6, respectively. The pharmaceutical composition according to [3].
[6] The pharmaceutical composition according to the above [5], wherein the base sequences of the sense strand and the antisense strand of siRNA have the base sequences shown in SEQ ID NOs: 3 and 4, respectively.
[7] The base sequences of the sense strand and antisense strand of siRNA or shRNA are the combination of SEQ ID NOs: 7 and 8, the combination of SEQ ID NOs: 9 and 10, the combination of SEQ ID NOs: 11 and 12, and the combination of SEQ ID NOs: 13 and 14, respectively. , A base sequence selected from the group consisting of combinations of SEQ ID NOs: 15 and 16, combinations of SEQ ID NOs: 17 and 18, combinations of SEQ ID NOs: 51 and 52, combinations of SEQ ID NOs: 53 and 54, and combinations of SEQ ID NOs: 55 and 56. The pharmaceutical composition according to the above [4], which has the combination of.
[8] The base sequences of the sense strand and the antisense strand of siRNA are a combination of the base sequences shown in SEQ ID NOs: 9 and 10, respectively, or a combination of the base sequences shown in SEQ ID NOs: 13 and 14, respectively. The pharmaceutical composition according to the above [7].
[9] SiRNA or shRNA whose sense strand and antisense strand are combinations of SEQ ID NOs: 7 and 8, combinations of SEQ ID NOs: 9 and 10, combinations of SEQ ID NOs: 11 and 12, and SEQ ID NOs: 13 and 14, respectively. A base selected from the group consisting of combinations, combinations of SEQ ID NOs: 15 and 16, combinations of SEQ ID NOs: 17 and 18, combinations of SEQ ID NOs: 51 and 52, combinations of SEQ ID NOs: 53 and 54, and combinations of SEQ ID NOs: 55 and 56. SiRNA or shRNA having a combination of sequences.
[10] SiRNA or shRNA whose sense and antisense strands are a combination of SEQ ID NOs: 7 and 8, a combination of SEQ ID NOs: 9 and 10, a combination of SEQ ID NOs: 11 and 12, and SEQ ID NOs: 13 and 14, respectively. A base selected from the group consisting of combinations, combinations of SEQ ID NOs: 15 and 16, combinations of SEQ ID NOs: 17 and 18, combinations of SEQ ID NOs: 51 and 52, combinations of SEQ ID NOs: 53 and 54, and combinations of SEQ ID NOs: 55 and 56. SiRNA or shRNA having a base sequence with an additional mismatch of 1 to several bases for a combination of sequences.
[11]
The siRNA or shRNA of the above [10], the sense strand and the antisense strand thereof are the combination of SEQ ID NOs: 33 and 34, the combination of SEQ ID NOs: 35 and 36, the combination of SEQ ID NOs: 37 and 38, SEQ ID NO: 39 and, respectively. Selected from the group consisting of 40 combinations, SEQ ID NOs: 41 and 42 combinations, SEQ ID NOs: 43 and 44 combinations, SEQ ID NOs: 45 and 46 combinations, SEQ ID NOs: 47 and 48 combinations, and SEQ ID NOs: 49 and 50 combinations. SiRNA or shRNA having a combination of base sequences.
[12] A composition for use in suppressing the expression of a mutant type in cells having a gene encoding a mutant type of collagen 3A1 and a gene encoding a wild type, respectively.
Contains nucleic acid selected from the siRNA or shRNA according to any one of the above [9] to [11].
The variant is a gene encoding collagen 3A1 having a G-to-A substitution of the 547th corresponding base in the nucleotide sequence set forth in SEQ ID NO: 27.
Composition.

FIG. 1 shows the knockdown efficiency of various siRNAs against wild-type (548G and 755G) and mutant (755T) or mutant (548A) collagen 3A1 in HeLa cells. FIG. 2 shows the concentration dependence (1-100 nM) of knockdown efficiency on the wild type (548G and 755G) and mutant (755T) or mutant (548A) of collagen 3A1 by various siRNAs in HeLa cells. In FIG. 2, the number at the end of the siRNA name and the concentration are linked by a hyphen and displayed. FIG. 3 shows the concentration dependence (1-100 nM) of knockdown efficiency of collagen 3A1 by various siRNAs on wild type (548G and 755G) and mutant type (755T) or mutant type (548A) in HEK293 cells. Is shown. In FIG. 3, the number at the end of the siRNA name and the concentration are linked by a hyphen and displayed. FIG. 4 shows the concentration dependence (0.01 to 10 nM) of knockdown efficiency for the wild type (548G) and the mutant type (548A) of collagen 3A1 by various siRNAs in HeLa cells. In FIG. 4, the number at the end of the siRNA name and the concentration are linked by a hyphen and displayed. FIG. 5 shows the concentration dependence (0.01-10 nM) of knockdown efficiency on the wild type (548G) and mutant type (548A) of collagen 3A1 by various siRNAs in HEK293 cells. In FIG. 5, the number at the end of the siRNA name and the concentration are linked by a hyphen and displayed. FIG. 6 shows the knockdown efficiency of collagen 3A1 by various siRNAs in HeLa cells against the wild type (548G) and the mutant type (548A). FIG. 7 shows the knockdown efficiency of collagen 3A1 by various siRNAs in HeLa cells against the wild type (548G) and the mutant type (548A). FIG. 8 shows the knockdown efficiency of collagen 3A1 by various siRNAs in HeLa cells against the wild type (547G) and the mutant type (547A). FIG. 9 shows the knockdown efficiency of collagen 3A1 by various siRNAs in HEK293 cells against the wild type (547G) and the mutant type (547A).

Specific description of the invention

In the present specification, the "subject" is a mammal, and may be a human in particular.

As used herein, "treatment" means therapeutic and prophylactic treatment. As used herein, "treatment" means the treatment, cure, prevention, improvement of remission, or reduction of the rate of progression of a disease or disorder. As used herein, "prevention" means reducing the likelihood of developing a disease or condition, or delaying the onset of a disease or condition.

As used herein, the term "disease" means a symptomatology for which treatment is beneficial.

As used herein, the term "therapeutically effective amount" means the amount of a drug effective for treating (preventing or treating) a disease or condition. A therapeutically effective amount of a drug slows the rate of exacerbation of a symptom of a disease or condition, stops the exacerbation of the symptom, ameliorates the symptom, cures the symptom, or develops or develops the symptom. It is possible to suppress.

In the present specification, "collagen 3A1" is also called type III collagen α-1 or COL3A1 and is a fibrogenic collagen containing three α-1 (III) chains. Collagen 3A1 is expressed throughout early embryonic and embryonic development. In adults, collagen 3A1 is a major component of the extracellular matrix of internal organs and skin. Examples of the gene encoding human collagen 3A1 include a base sequence registered with NCBI reference number: NM_0000900.3, and may have, for example, a base sequence corresponding to the base sequence set forth in SEQ ID NO: 27. The gene encoding human collagen 3A1 may have, for example, the nucleotide sequence set forth in SEQ ID NO: 27.

As used herein, "vascular Ehlers-Danlos syndrome (vEDS)" is a hereditary disease based on the fragility of systemic connective tissues such as skin, joints, and blood vessels. Symptoms include skin fragility (easy to tear, atrophic scarring), joint fragility (flexible, easy to dislocate), vascular fragility (easy to bleed internally), heart valve deviation / regurgitation, Presents with ascending aortic dilatation. Vascular EDS presents with serious complications such as arterial dissection / aneurysm / rupture, intestinal rupture, and uterine rupture, and has physical characteristics such as relaxation of small joints, characteristic facial features, and see-through of subcutaneous veins. The majority of the causes of vEDS are attributed to collagen 3A1 gene mutations. In particular, as the cause of vEDS, substitution of the base sequence of collagen 3A1 (substitution of the base corresponding to the 547th G in the base sequence shown in SEQ ID NO: 27 to A, and substitution of the base corresponding to the 755th G to T) Replacement of). vEDS develops only with mutations in one-sided alleles. This is because the mutant form forms a trimer with the wild type (normal type) and produces abnormal collagen. Selective knockdown of mutants is a treatment for vEDS because increasing the production of trimers consisting only of wild-type (normal) by reducing the expression of mutants reduces the symptoms of vEDS. It can be a strategy.

In the present specification, "siRNA" is a nucleic acid molecule used for knockdown of a gene, and is a nucleic acid molecule containing a double-stranded RNA having a length of about 19 to 25 mer. A siRNA designed complementary to the base sequence of a target mRNA can inhibit the translation of a protein from the mRNA by binding to the mRNA and degrading the mRNA. In addition to natural RNA, RNA used for siRNA includes stabilized nucleic acid analogs (for example, cross-linked artificial nucleic acids such as AmNA, 2', 4'cross-linked nucleic acids (locked nucleic acids), and the 2'position of ribose of RNA. 2'-OMe form modified with methoxy group, 2'-MOE form modified with methoxyethyl and 2'-F form modified with fluoro group, phosphorothioate bond instead of phosphate diester bond Nucleic acids having) or nucleic acids containing DNA (eg, gapmers and mixmers) can be used. Those skilled in the art can appropriately design siRNA using these stabilized nucleic acid analogs and DNA.

In the present specification, "SHRNA" is a nucleic acid molecule containing double-stranded RNA having a hairpin structure. The hairpin structure of the shRNA is cleaved intracellularly and converted to siRNA, which allows silencing of the target gene. The hairpin structure can be appropriately designed by those skilled in the art.

In vascular Ehlers-Danlos syndrome (vEDS), the mutation of collagen 3A1 is a dominant mutation. That is, collagen 3A1 functions by forming a trimer, and one of them is mutated to inhibit the function of collagen 3A1. In contrast, treatments that reduce the expression of mutant collagen 3A1 compared to wild-type can increase the proportion of functional collagen 3A1 trimers, which can treat vEDS. Be expected.

The present inventors have found a method that can be used for a treatment that reduces the expression of a mutant form of collagen 3A1 as compared with the wild type.

In certain aspects of the invention
A pharmaceutical composition for treating a disease caused by a mutant type of collagen 3A1 in a subject having a gene encoding a mutant type and a gene encoding a wild type, respectively.
Contains nucleic acids selected from siRNA or shRNA
Is the variant a gene encoding collagen 3A1 having a G-to-T substitution of the base corresponding to position 755 in the base sequence shown in SEQ ID NO: 27 (for example, the base sequence shown in SEQ ID NO: 29)? , Or a gene encoding collagen 3A1 having a G-to-A substitution of the 547th corresponding base in the base sequence of SEQ ID NO: 27 (for example, the base sequence of SEQ ID NO: 31).
The nucleic acid can strongly suppress the expression of the mutant form rather than the expression of the wild type.
Pharmaceutical compositions are provided. In this embodiment, the subject can be a subject suffering from vascular Ehlers-Danlos syndrome (vEDS). In this aspect, the variant may preferably be a gene encoding collagen 3A1 in which the variant has a G-to-T substitution of the 755th corresponding base in the nucleotide sequence set forth in SEQ ID NO: 27. Also in this aspect, the variant can preferably be a gene encoding collagen 3A1 having a G-to-A substitution of the base corresponding to position 547 in the base sequence set forth in SEQ ID NO: 27. Also, in some embodiments, the variant may be a gene encoding collagen 3A1 having a G-to-A substitution of the base corresponding to position 548 in the nucleotide sequence set forth in SEQ ID NO: 27.

In some embodiments of the invention, the nucleotide sequences of the sense and antisense strands of siRNA or shRNA are selected from the group consisting of SEQ ID NOs: 1 and 2, SEQ ID NOs: 3 and 4, and SEQ ID NOs: 5 and 6, respectively. Can have. In some embodiments of the invention, the base sequences of the sense and antisense strands of siRNA or shRNA can be SEQ ID NOs: 1 and 2, respectively. In some embodiments of the invention, the base sequences of the sense and antisense strands of siRNA or shRNA can be SEQ ID NOs: 3 and 4, respectively. In some embodiments of the invention, the base sequences of the sense and antisense strands of siRNA or shRNA can be SEQ ID NOs: 5 and 6, respectively.

In some embodiments of the invention, the base sequences of the sense and antisense strands of siRNA or shRNA are SEQ ID NOs: 7 and 8, SEQ ID NOs: 9 and 10, SEQ ID NOs: 11 and 12, SEQ ID NOs: 13 and 14, and SEQ ID NO: 15, respectively. And 16, may have a base sequence selected from the group consisting of SEQ ID NOs: 17 and 18, SEQ ID NOs: 51 and 52, SEQ ID NOs: 53 and 54, and SEQ ID NOs: 55 and 56. In some embodiments of the invention, the base sequences of the sense and antisense strands of siRNA or shRNA can be SEQ ID NOs: 7 and 8, respectively. In some embodiments of the invention, the base sequences of the sense and antisense strands of siRNA or shRNA can be SEQ ID NOs: 9 and 10, respectively. In some embodiments of the invention, the base sequences of the sense and antisense strands of siRNA or shRNA can be SEQ ID NOs: 11 and 12, respectively. In some embodiments of the invention, the base sequences of the sense and antisense strands of siRNA or shRNA can be SEQ ID NOs: 13 and 14, respectively. In some aspects of the invention, the base sequences of the sense and antisense strands of siRNA or shRNA can be SEQ ID NOs: 15 and 16, respectively. In some embodiments of the invention, the base sequences of the sense and antisense strands of siRNA or shRNA can be SEQ ID NOs: 17 and 18, respectively. In some embodiments of the invention, the base sequences of the sense and antisense strands of siRNA or shRNA can be SEQ ID NOs: 51 and 52, respectively. In some embodiments of the invention, the base sequences of the sense and antisense strands of siRNA or shRNA can be SEQ ID NOs: 53 and 54, respectively. In some embodiments of the invention, the base sequences of the sense and antisense strands of siRNA or shRNA can be SEQ ID NOs: 55 and 56, respectively.

In some embodiments of the invention, the nucleotide sequences of the sense and antisense strands of siRNA or shRNA are SEQ ID NOs: 7 and 8, SEQ ID NOs: 9 and 10, SEQ ID NOs: 11 and 12, SEQ ID NOs: 13 and 14, and SEQ ID NO: 15, respectively. 1 to several bases (eg, 1 or 2) for a base sequence selected from the group consisting of 16 and 16, SEQ ID NOs: 17 and 18, SEQ ID NOs: 51 and 52, SEQ ID NOs: 53 and 54, and SEQ ID NOs: 55 and 56. It may have a base sequence with one and three) additional mismatches. In this aspect, the siRNA or shRNA encodes collagen 3A1 having a G-to-A substitution of the 547th corresponding base in the nucleotide sequence set forth in SEQ ID NO: 27, rather than the gene encoding the wild-type of collagen 3A1. Strongly silence genes.

In some embodiments of the invention, the base sequences of the sense and antisense strands of siRNA or shRNA are SEQ ID NOs: 33 and 34, SEQ ID NOs: 35 and 36, SEQ ID NOs: 37 and 38, SEQ ID NOs: 39 and 40, SEQ ID NO: 41, respectively. And 42, SEQ ID NOs: 43 and 44, SEQ ID NOs: 45 and 46, SEQ ID NOs: 47 and 48, and a base sequence selected from the group consisting of SEQ ID NOs: 49 and 50. In some embodiments of the invention, the base sequences of the sense and antisense strands of siRNA or shRNA can be SEQ ID NOs: 33 and 34, respectively. In some embodiments of the invention, the base sequences of the sense and antisense strands of siRNA or shRNA can be SEQ ID NOs: 35 and 36, respectively. In some embodiments of the invention, the base sequences of the sense and antisense strands of siRNA or shRNA can be SEQ ID NOs: 37 and 38, respectively. In some embodiments of the invention, the base sequences of the sense and antisense strands of siRNA or shRNA can be SEQ ID NOs: 39 and 40, respectively. In some embodiments of the invention, the base sequences of the sense and antisense strands of siRNA or shRNA can be SEQ ID NOs: 41 and 42, respectively. In some embodiments of the invention, the base sequences of the sense and antisense strands of siRNA or shRNA can be SEQ ID NOs: 43 and 44, respectively. In some embodiments of the invention, the base sequences of the sense and antisense strands of siRNA or shRNA can be SEQ ID NOs: 45 and 46, respectively. In some embodiments of the invention, the base sequences of the sense and antisense strands of siRNA or shRNA can be SEQ ID NOs: 47 and 48, respectively. In some embodiments of the invention, the base sequences of the sense and antisense strands of siRNA or shRNA can be SEQ ID NOs: 49 and 50, respectively.

ShRNA can be obtained by linking the base sequences of the sense strand and the antisense strand with a linker loop. In shRNA, the sense strand and the antisense strand are hybridized within the molecule. Further, in siRNA, the base sequences of the sense strand and the antisense strand are present on separate nucleic acids, but the sense strand and the antisense strand are hybridized between the molecules.

According to the present invention, siRNA or shRNA whose sense and antisense strands are SEQ ID NOs: 7 and 8, SEQ ID NOs: 9 and 10, SEQ ID NOs: 11 and 12, SEQ ID NOs: 13 and 14, and SEQ ID NO: 15, respectively. And 16, siRNA or shRNA having a base sequence selected from the group consisting of SEQ ID NOs: 17 and 18, SEQ ID NOs: 51 and 52, SEQ ID NOs: 53 and 54, and SEQ ID NOs: 55 and 56 are provided. These siRNA or shRNA may be included in the pharmaceutical composition.

According to the present invention, siRNA or shRNA, the sense strand and the antisense strand thereof, are SEQ ID NOs: 7 and 8, SEQ ID NOs: 9 and 10, SEQ ID NOs: 11 and 12, SEQ ID NOs: 13 and 14, respectively. One to several bases (eg, one) for a base sequence selected from the group consisting of 15 and 16, SEQ ID NOs: 17 and 18, SEQ ID NOs: 51 and 52, SEQ ID NOs: 53 and 54, and SEQ ID NOs: 55 and 56. SiRNA or shRNA having a base sequence with two and three) additional mismatches may be included in the pharmaceutical composition.

According to the present invention, siRNA or shRNA, the sense strand and the antisense strand thereof, are SEQ ID NOs: 33 and 34, SEQ ID NOs: 35 and 36, SEQ ID NOs: 37 and 38, SEQ ID NOs: 39 and 40, and SEQ ID NOs, respectively. SiRNAs or shRNAs having a base sequence selected from the group consisting of 41 and 42, SEQ ID NOs: 43 and 44, SEQ ID NOs: 45 and 46, SEQ ID NOs: 47 and 48, and SEQ ID NOs: 49 and 50 are included in the pharmaceutical composition. May be.

In some aspects of the invention, mutant expression can be significantly reduced compared to wild-type expression. In a preferred embodiment, the expression of the variant can be reduced to 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, or 20% or less as compared to before treatment. Here, the rate of decrease means the rate of decrease after the treatment as compared with the rate before the treatment. On the other hand, wild-type expression can be reduced to 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, or 20% or less as compared to before treatment, but mutant expression. The rate of decrease can be 60% or less, 50% or less, 40% or less, 30% or less, or 20% or less.

The rate of decrease in gene expression can be determined by a method of analyzing gene expression levels well known to those skilled in the art, such as Northern blot and quantitative RT-PCR.

The pharmaceutical composition of the present invention may further contain a pharmaceutically acceptable excipient. Excipients are not particularly limited, but include, for example, diluents, buffers, salts, solvents (such as water), tonicity agents, preservatives, and production aids (eg, lubricants, talc, magnesium, calcium stearate). , Zinc stearate, or stearic acid). The pharmaceutical composition may have solution conditions suitable for storage of nucleic acids. The pharmaceutical composition has a formulation composition suitable for parenteral administration.
The pharmaceutical composition of the present invention may be encapsulated in vesicles such as micelles and liposomes. The micelles and liposomes can be polycationic polymers or cationic lipids as constituents, or lipid vesicles (lipid nanovesicles). Micelle or liposome is a targeting molecule (eg, a molecule that binds to a molecule expressed extracellularly in a target cell, tissue, or organ) that imparts targeting orientation to the target cell, tissue, or organ. It may be modified. Those skilled in the art can use known drug delivery systems to improve the targeting orientation of micelles and liposomes to target cells, tissues, or organs.
The pharmaceutical composition of the present invention can be administered by an appropriate route of administration such as intraperitoneal administration, intravenous administration, subcutaneous administration, or intramuscular administration. The pharmaceutical composition of the present invention may be administered topically in some embodiments.

According to the present invention
It is a method for treating a disease caused by the mutant type in a subject having a gene encoding a mutant type of collagen 3A1 and a gene encoding a wild type, respectively.
Including administering to the subject a therapeutically effective amount of nucleic acid selected from siRNA or shRNA.
The variant is a gene encoding collagen 3A1 having a G-to-T substitution of the 755th corresponding base in the nucleotide sequence of SEQ ID NO: 27, or 547 in the nucleotide sequence of SEQ ID NO: 27. A gene encoding collagen 3A1 having a G-to-A substitution of the second corresponding base.
A method is provided in which the nucleic acid can strongly suppress the expression of the mutant form rather than the expression of the wild type. The siRNA or shRNA may be encapsulated in micelles or liposomes.

According to the present invention
In the use of a nucleic acid selected from siRNA or shRNA in the manufacture of a drug for treating a disease caused by the variant in a subject having a gene encoding a variant of collagen 3A1 and a gene encoding the wild type, respectively. There,
The variant is a gene encoding collagen 3A1 having a G-to-T substitution of the 755th corresponding base in the nucleotide sequence of SEQ ID NO: 27, or 547 in the nucleotide sequence of SEQ ID NO: 27. A gene encoding collagen 3A1 having a G-to-A substitution of the second corresponding base.
The use is provided in which the nucleic acid can strongly suppress the expression of the mutant form rather than the expression of the wild type. The siRNA or shRNA may be encapsulated in micelles or liposomes.

According to the present invention, it is a nucleic acid selected from siRNA or shRNA for treating a disease caused by the variant in a subject having a gene encoding a variant of collagen 3A1 and a gene encoding a wild form, respectively. The variant is a gene encoding collagen 3A1 having a G-to-T substitution of the 755th corresponding base in the base sequence set forth in SEQ ID NO: 27, or the base sequence set forth in SEQ ID NO: 27. A gene encoding collagen 3A1 having a G-to-A substitution of the base corresponding to the 547th position in the above, wherein the nucleic acid can strongly suppress the expression of the mutant type rather than the expression of the wild type. Provided. As the nucleic acid, the nucleic acid specified in the present specification can be used. The siRNA or shRNA may be encapsulated in micelles or liposomes.

Example 1: Design of siRNA targeting mutant Col3A1 gene Design the following siRNA targeting two human mutant Col3A1 genes (NCBI RefSeq number: NM_0000900) responsible for vascular Ehlers-Danlos syndrome (vEDS) (Table 1). As a mutant, 755T and 548A of Col3A were prepared as model cases, respectively, and the silencing ability of the wild type and the mutant by siRNA was examined below.

In the targeted mutant Col3A1 allele, the 755th G from the start codon was mutated to A, and as a result, the 252nd Gly of the COL3A1 protein was mutated to Val (c.755G> T; pG252V), and from the start codon. The 548th G is mutated to A, and as a result, the 183rd Gly of the COL3A1 protein is mutated to Asp (c.548G> A; pG183D).

The siRNA was designed to include the mutation site in the sequence, the antisense side was made a completely complementary sequence with the mutant allele, and the sense side was made to be completely complementary to the sequence. The terminal overhang was 21mer (19mer double strand + 2 overhang residues) siRNA as dTdT. The synthesis was done by Gene Design.

Example 2: Preparation of luciferase reporter plasmid for evaluating siRNA targeting the mutant Col3A1 gene A plasmid for evaluating the siRNA designed and synthesized in Example 1 with the luciferase reporter system was prepared as follows.
Cleavage was performed at the restriction enzyme sites XhoI and NotI located downstream of N-Luc of pNFL1-N [CMV / Hygro] (Promega), a plasmid encoding the NanoLuc (Nluc) reporter gene downstream of the CMV promoter. A synthetic DNA consisting of 60 base pairs was inserted. The synthetic DNA was derived from the human Col3A1 sequence and was designed so that the mutant residue was located in the center. Mutation c. For 755G> T, wild-type pNLF1-N-755G was prepared using SEQ ID NOs: 19 and 20, and mutant pNLF1-N-755T was prepared using SEQ ID NOs: 21 and 22, respectively. Similarly, mutation c. For 548G> A, wild-type pNLF1-N-548G was prepared using SEQ ID NOs: 23 and 24, and mutant pNLF1-N-548A was prepared using SEQ ID NOs: 25 and 26 (Table 2).

Example 3: Evaluation of siRNA Targeting Mutant Col3A1 Gene by Reporter Assay The Col3A1 mutation-specific siRNA candidates designed and synthesized in Example 1 were evaluated using the luciferase reporter system prepared in Example 2. HeLa cells and HEK293 cells were used as cells (both obtained from RIKEN BRC).
HeLa cells and HEK293 cells were cultured in MEM (Sigma, M2279) + 10% FBS (Sigma) + 2 mM L-Glutamine (FUJIFILM Wako Pure Chemical Corporation) + 1% NEAA (MP Biomedicals). 8 × 10 ³ / well (HeLa) and 2 × 10 ⁴ / well (HEK293) cells were seeded on a 96-well plate. The next day, a reporter plasmid (one of those prepared in Example 2) 20 pg / well, an internal control vector pGL4.54 1.2 ng / well (HeLa experiments 1 and 2) encoding Fluc, 0.2 ng / well (HeLa experiments 1 and 2), 0.2 ng / well ( HEK293 Experiment 3) or pGL4.53 0.2 ng / well (Experiment 4 and 5), and final concentrations 100 nM (Experiment 1), 1-100 nM (Experiment 2 and 3), 0.01-10 nM (Experiment 4 and 5) SiRNA was introduced using a transfection reagent (Libofectoamine2000 (Thermo Experiment Scientific) 0.1 μL / Well).

After 24 hours, the luciferase activity was measured with a plate reader (TECAN Microplate Reader Infinity M1000 PRO) using a Nano-GloR Dual-LuciferaseR Reporter Assay System (Promega).

The Nluc value was corrected using the internal standard Fluc. The inhibitory activity of each siRNA was shown in comparison with the control (vector only) sample. The results of Experiments 1 to 5 are shown in FIGS. 1 to 5, respectively. All experiments were performed in triplets and the data were shown as mean ± SEM.

From the above results, siRNA 755T-6, 755T-8, 755T-10 is Col3A1 c. 755G> T mutation-selective siRNA, and siRNA 547A-5, 547A-6, 547A-7, 547A-8, 547A-11, 547A-15 are Col3A1 c. It was shown to be 547G> A mutation-selective siRNA.

Normal COL3A1 protein functions by forming a homotrimer. It is believed that the complex loses its function when even one mutated COL3A1 protein that has lost its function is mixed. Therefore, it is considered that when a missense mutation occurs in one of the alleles and COL3A1 that has lost its function is expressed, the ratio of the normal COL3A1 trimer is attenuated to 1/2 × 1/2 × 1/2 = 1/8. Be done.

On the other hand, in the haploinsufficidal (null mutant) COL3A1 in which one allele is deleted, the mutant COL3A1 protein is not expressed, so that 1/2 of the normal COL3A1 trimer is expressed. These account for about 5% of the total, but the results show that the prognosis of patients with haploinsufficiency is good, mild, and the expected life expectancy is the same as that of healthy subjects (Non-Patent Document 2).

Missense mutations account for 70% of the COL3A1 mutations, which is the majority, but since these are dominant negative mutations as described above, it is considered theoretically beneficial to specifically silence the missense mutations in the present invention. That is, when the expression level of mutant collagen 3A1 is halved with respect to normal collagen 3A1, the ratio of normal COL3A1 trimer theoretically recovers to about 30%, and the ratio of normal COL3A1 trimer is increased. It improves about 2.4 times as much as when no treatment is performed. Therefore, if the COL3A1 gene can be suppressed specifically for the mutant allele, it is considered that the effective normal COL3A1 trimer can be increased and the missense type can be converted to the haploinsufficiency type. A pharmaceutical composition that suppresses the COL3A1 gene is expected to improve various symptoms of EDS.

Example 4: Design of siRNA targeting the mutant Col3A1 gene (Part 2)
In Example 3, c. To further increase mutant selective suppression of the two siRNAs that showed particularly strong activity against 548G> A, 547A-6 (SEQ ID NOs: 9 and 10) and 547A-8 (SEQ ID NOs: 13 and 14). , SiRNAs with one additional mutation were designed respectively (Table 3). The synthesis was performed by Thermo Fisher Scientific.

Example 5: Evaluation of siRNA targeting the mutant Col3A1 gene by a reporter assay (Part 2)
The Col3A1 mutation-specific siRNA candidates designed and synthesized in Example 4 were evaluated using the luciferase reporter system prepared in Example 2. HeLa cells and HEK293 cells were used as cells (both obtained from RIKEN BRC).
HeLa cells were cultured in MEM medium (Sigma, M2279) containing 10% FBS (Sigma), 2 mM L-Glutamine (FUJIFILM Wako Pure Chemical Corporation), and 1% NEAA (MP Biomedicals). 8 × 10 ³ / well cells were seeded on a 96-well plate. The next day, a reporter plasmid (one of those prepared in Example 2) 20 pg / well, an internal control vector pGL4.53 0.2 ng / well encoding Fluc, and a siRNA having a final concentration of 1 nM were used as a transfection reagent (Lipofectoamine2000). (Thermo Fisher Scientific) 0.1 μL / Well was used for introduction.
After 24 hours, luciferase activity was measured with a plate reader (TECAN Microplate Reader Infinite M1000 PRO) using the Nano-GloR Dual-LuciferaseR Assay System (Promega).
The Nluc value was corrected using the internal standard Fluc. The inhibitory activity of each siRNA was shown in comparison with the control (vector only) sample. The results are shown in FIGS. 6 and 7. All experiments were performed in triplets and the data were shown as mean ± SEM.

As shown in FIGS. 6 and 7, when any of the siRNAs shown in Table 3 was used, silencing for wild-type Col3A1 gene expression was weakened, and silencing for mutant Col3A1 gene expression was stronger. did.

Example 6: Design of siRNA targeting the mutant Col3A1 gene (Part 2)
Similar to Example 1, the following siRNAs targeting the (c.547G>A; pG183S) mutations responsible for vascular Ehlers-Danlos syndrome (vEDS) were additionally designed (Table 4). The synthesis was done by Gene Design. In the following examples, "547A" means the actual replacement of the 547th G with A.

Example 7: Preparation of a luciferase reporter plasmid for evaluating siRNA targeting the mutant Col3A1 gene (Part 2)
A plasmid for evaluating the siRNA designed and synthesized in Example 6 with a luciferase reporter system was prepared in the same manner as in Example 2 as follows.
Similar to Example 2, pNFL1-N [CMV / Hygro] was cleaved with restriction enzyme sites XhoI and NotI, and synthetic DNA consisting of 60 base pairs was inserted therein. The synthetic DNA was derived from the human Col3A1 sequence and was designed so that the mutant residue was located in the center. Mutation c. Mutant pNLF1-N-547A-2 was prepared using SEQ ID NOs: 57 and 58 for 547G> A (Table 5).

Example 8: Evaluation of siRNA targeting the mutant Col3A1 gene by a reporter assay (Part 3)
The Col3A1 mutation-specific siRNA candidates designed and synthesized in Example 6 were evaluated using the luciferase reporter system prepared in Examples 2 and 7. HeLa cells and HEK293 cells were used as cells.
The culture conditions for HeLa cells and HEK293 cells are the same as in Example 3. 8 × 10 ³ / well (HeLa) and 2 × 10 ⁴ / well (HEK293) cells were seeded on a 96-well plate. The next day, a reporter plasmid (pNLF1-N-547G or pNLF1-N-547A-2) 20 pg / well, an internal control vector pGL4.53 0.2 ng / well encoding Fluc, and a final concentration of 1-10 nM (HeLa experiment 8). ), 0.1-10 nM (HEK293 Experiment 9) siRNA was introduced using a transfection reagent (Pofectamine2000 (Thermo Fisher Scientific) 0.1 μL / Well. PNLF1-N-547G was pNLF1-N-548G. It is a plasmid containing a gene encoding wild-type COL3A1 having the same base sequence as.
After 24 hours, luciferase activity was measured with a plate reader using the ^{Nano-Glo ™ Dual-Luciferase R Reporter Assay System (Promega).}
The Nluc value was corrected using the internal standard Fluc. The inhibitory activity of each siRNA was shown in comparison with the control (vector only) sample. The results are shown in FIGS. 8 and 9. All experiments were performed in triplets and the data were shown as mean ± SEM.

As shown in FIGS. 8 and 9, when any of the siRNAs shown in Table 4 is used, silencing for wild-type Col3A1 gene expression is weakened, and silencing for mutant Col3A1 gene expression is stronger. did.

Supplementary notes on the sequence listing SEQ ID NO: 27: Human collagen 3A1 gene and amino acid SEQ ID NO: 28: Amino acid sequence of human collagen 3A1 SEQ ID NO: 29: c.I. 547G> A mutant gene and amino acid sequence SEQ ID NO: 30: human collagen 3A1 c. Amino acid sequence of 547G> A variant SEQ ID NO: 31: c. Of human collagen 3A1. Gene and amino acid sequence of 755G> T mutant SEQ ID NO: 32: c. of human collagen 3A1. Amino acid sequence of 755G> T mutant

Claims

A pharmaceutical composition for treating a disease caused by a mutant type of collagen 3A1 in a subject having a gene encoding a mutant type and a gene encoding a wild type, respectively.
Contains nucleic acids selected from siRNA or shRNA
The variant is a gene encoding collagen 3A1 having a G-to-T substitution of the 755th corresponding base in the nucleotide sequence of SEQ ID NO: 27, or 547 in the nucleotide sequence of SEQ ID NO: 27. A gene encoding collagen 3A1 having a G-to-A substitution of the second corresponding base.
The nucleic acid can strongly suppress the expression of the mutant form rather than the expression of the wild type.
Pharmaceutical composition.
The pharmaceutical composition according to claim 1, wherein the subject is a subject suffering from vascular Ehlers-Danlos syndrome (vEDS).
The pharmaceutical composition according to claim 1 or 2, wherein the variant is a gene encoding collagen 3A1 having a G-to-T substitution of the base corresponding to the 755th in the base sequence shown in SEQ ID NO: 27.
The pharmaceutical composition according to claim 1 or 2, wherein the variant is a gene encoding collagen 3A1 having a G-to-A substitution of the base corresponding to the 547th in the base sequence shown in SEQ ID NO: 27.
3. The base sequence of the sense strand and antisense strand of siRNA or shRNA has a base sequence selected from the group consisting of SEQ ID NOs: 1 and 2, SEQ ID NOs: 3 and 4, and SEQ ID NOs: 5 and 6, respectively. The pharmaceutical composition according to the description.
The pharmaceutical composition according to claim 5, wherein the base sequences of the sense strand and the antisense strand of siRNA have the base sequences shown in SEQ ID NOs: 3 and 4, respectively.
The base sequences of the sense strand and antisense strand of siRNA or shRNA are the combination of SEQ ID NOs: 7 and 8, the combination of SEQ ID NOs: 9 and 10, the combination of SEQ ID NOs: 11 and 12, the combination of SEQ ID NOs: 13 and 14, and the SEQ ID NOs: Combinations of nucleotide sequences selected from the group consisting of combinations of 15 and 16, combinations of SEQ ID NOs: 17 and 18, combinations of SEQ ID NOs: 51 and 52, combinations of SEQ ID NOs: 53 and 54, and combinations of SEQ ID NOs: 55 and 56. The pharmaceutical composition according to claim 4.
7. Claim 7 where the base sequences of the sense strand and the antisense strand of siRNA are a combination of the base sequences shown in SEQ ID NOs: 9 and 10, respectively, or a combination of the base sequences shown in SEQ ID NOs: 13 and 14, respectively. The pharmaceutical composition according to.
A siRNA or shRNA whose sense and antisense strands are a combination of SEQ ID NOs: 7 and 8, a combination of SEQ ID NOs: 9 and 10, a combination of SEQ ID NOs: 11 and 12, a combination of SEQ ID NOs: 13 and 14, and a sequence. A combination of base sequences selected from the group consisting of combinations of numbers 15 and 16, combinations of SEQ ID NOs: 17 and 18, combinations of SEQ ID NOs: 51 and 52, combinations of SEQ ID NOs: 53 and 54, and combinations of SEQ ID NOs: 55 and 56. SiRNA or shRNA having.
A siRNA or shRNA whose sense and antisense strands are a combination of SEQ ID NOs: 7 and 8, a combination of SEQ ID NOs: 9 and 10, a combination of SEQ ID NOs: 11 and 12, a combination of SEQ ID NOs: 13 and 14, and a sequence. A combination of base sequences selected from the group consisting of combinations of numbers 15 and 16, combinations of SEQ ID NOs: 17 and 18, combinations of SEQ ID NOs: 51 and 52, combinations of SEQ ID NOs: 53 and 54, and combinations of SEQ ID NOs: 55 and 56. SiRNA or shRNA having a combination of base sequences having an additional mismatch of 1 to several bases with respect to.
The siRNA or shRNA of claim 10, wherein the sense strand and the antisense strand thereof are a combination of SEQ ID NOs: 33 and 34, a combination of SEQ ID NOs: 35 and 36, a combination of SEQ ID NOs: 37 and 38, and SEQ ID NOs: 39 and 40, respectively. Selected from the group consisting of combinations of SEQ ID NOs: 41 and 42, combinations of SEQ ID NOs: 43 and 44, combinations of SEQ ID NOs: 45 and 46, combinations of SEQ ID NOs: 47 and 48, and combinations of SEQ ID NOs: 49 and 50. SiRNA or shRNA having a combination of base sequences.
A composition for use in suppressing the expression of a mutant type in cells having a gene encoding a mutant type of collagen 3A1 and a gene encoding a wild type, respectively.
A nucleic acid selected from the siRNA or shRNA according to any one of claims 9 to 11 is included.
The variant is a gene encoding collagen 3A1 having a G-to-A substitution of the 547th corresponding base in the nucleotide sequence set forth in SEQ ID NO: 27.
Composition.