WO2021177419A1 - Method for screening antisense oligonucleotide drug - Google Patents

Abstract

The present invention provides: a method for screening a drug for treatment, prevention, and/or amelioration of calmodulinopathy that includes an antisense oligonucleotide; and a drug for treatment, prevention, and/or amelioration of calmodulinopathy that includes an antisense oligonucleotide for inhibiting expression of a calmodulin gene.

Description

Screening method for antisense oligonucleotide drugs

The present application relates to a screening method for antisense oligonucleotide drugs.

Calmodulin is a calcium detection protein expressed in ubiquitous, and three different genes (CALM1 to 3) encode calmodulin protein having the same amino acid sequence. Calmodulin regulates various proteins including multiple ion channels and is involved in various processes such as inflammation, metabolism, apoptosis, muscle contraction, intracellular migration, short-term memory, long-term memory, nerve growth, and immune response. .. Calmodulin, a calcium-detecting protein, regulates the inactivation of L-type calcium channels (LTCCs) expressed in the heart (Non-Patent Document 1).

Long QT Syndrome (LQTS) is an arrhythmia disorder that prolongs the QT interval on the electrocardiogram, causing fainting and sudden death due to ventricular arrhythmia. Mutations in the genes of ion channels or their regulatory proteins expressed in the heart have been reported as the cause of congenital LQTS.
In catecholaminergic polymorphic ventricular tachycardia (CPVT), delayed depolarization due to excess calcium in myocardial cells caused by exercise, emotional stress, etc. is involved in the development of arrhythmia, and suddenly in infants It is considered to be a cause of death syndrome and idiopathic ventricular fibrillation. (Non-Patent Document 2)
Conventionally, the RYR2 gene related to the ryanodine receptor has been known as a cause of CPVT. However, in recent years, it has become clear that the calmodulin (CALM) gene is a causative gene such as certain CPVT (CPVT4).

Heteromissense mutations in the CALM gene have been reported to be associated with the development of severe arrhythmias such as severe long QT syndrome and catecholamine-induced polymorphic ventricular tachycardia. A new disease concept collectively called (Non-Patent Document 3) was proposed. In carmodulinopathy, a missense mutation in one of the three different CALM genes is thought to exert a dominant negative effect and cause serious symptoms (Non-Patent Document 4).

In particular, many patients with carmodulinopathy who have arrhythmia as a symptom are severely ill and suddenly die in childhood, and there are many cases in which they cannot survive until adulthood. Implantable cardioverter-defibrillator treatment is considered as a treatment method, for example, to prevent sudden death, but it is a symptomatic treatment, many patients are children and the burden on the body and mind is heavy, and after invasiveness or implantation. There is a problem in that the complications of the above occur (Non-Patent Document 5).

The possibility of gene therapy using genome editing technology has also been reported. Non-Patent Document 6 describes that the action potential duration prolongation of iPS cell-differentiated cardiomyocytes prepared from LQT patients is shortened by non-specific knockdown of mutant alleles using the CRISPRi system. It has been shown that it can be a non-allelic gene therapy method. Similarly, Non-Patent Document 4 describes electrophysiological abnormalities of iPS cell-differentiated cardiomyocyte clones prepared from patients with carmodulinopathy by mutant allele-specific knockout using the CRISPR / Cas9 system (delayed inactivation of LTCC, Normalization of action potential duration) indicates that this method can be an effective gene therapy method for patients with carmodulinopathy due to the dominant negative effect of the CALM gene.
There are no clinical application examples of methods using either the CRISPRi system or the CRISPR / Cas9 system, and it is said that there are many problems in clinical application.
Although β-blockers have been administered to carmodulinopathy, it has been reported that the therapeutic effect is low (Non-Patent Document 5).

The use of antisense oligonucleotides (ASOs) is envisioned as a drug treatment for congenital diseases caused by gene mutations. However, no ASO has ever been attempted to treat carmodulinopathy, and the concept, design and evaluation system were completely unknown. In addition, the mechanism of carmodulinopathy has many unclear points compared to other pathological conditions, and it is unknown whether ASO is effective for this pathological condition, and a certain probability of success could not be expected.
For example, in LQT1, which is a typical pathological condition of long QT syndrome (LQTS), a mutation in the KCNQ1 gene is the cause. The KCNQ1 gene is an expression gene for IKs channel protein, which is a potassium channel expressed in the myocardium. Mutations in the KCNQ1 gene lead to mutations in myocardial potassium channel proteins and directly affect arrhythmias.
On the other hand, in calmodulinopathy, the calmodulin protein is first mutated, and as a result, for example, the affinity with calcium ions changes or the shape of the protein changes subtly, and as a result, it affects calcium signal transduction downstream. give. Therefore, the therapeutic effect of ASO is indirect, and it is quite unknown whether ASO actually changes the amount of calcium current involved in arrhythmia.
In addition, calmodulin has four calcium-binding sites, and it was unclear which binding site is involved in the pathological condition. In addition, at least 28 amino acids are known for the possibility of amino acid mutation related to carmodulinopathy (Non-Patent Document 5), and it was unclear which amino acid is involved in the pathological condition to what extent. Therefore, the concept and design of ASO were also quite unknown.

Drug therapy has the potential to overcome the challenges of conventional surgical or gene therapy, so there is a need for drugs to treat carmodulinopathy. In particular, the need for a therapeutic agent capable of preventing a fatal arrhythmia event and improving the prognosis is extremely high, and it is considered to have great clinical significance (Non-Patent Document 5).
Therefore, the task of the present application is to provide a novel screening method for an antisense oligonucleotide drug, which makes it possible to provide a new drug for treating carmodulinopathy, as well as a novel antisense oligonucleotide drug. Is to provide.

As a result of diligent studies in view of the above problems, the present inventors have determined.
(1) For the first time, we constructed an ASO evaluation system for carmodulinopathy by clarifying appropriate cells and conditions.
(2) For the first time, we constructed an ASO evaluation system, which is a pathological model derived from pluripotent stem cells prepared from patients with carmodulinopathy.
(3) For the first time, it was clarified that ASO can actually suppress gene expression related to carmodulinopathy.
(4) It was clarified for the first time that ASO can actually change the action potential duration of cardiomyocytes in a carmodulinopathy pathological model.
In addition, by adding ASO to cardiomyocytes derived from pluripotent stem cells prepared from somatic cells of carmodulinopathy patients, the present inventors reduce CALM gene expression and prolong the duration of action potentials. It was also found that the ASO can be corrected and that the ASO can be applied to a living body.
That is, the present application provides the gist and the following.

1. 1.
A method of screening for therapeutic, prophylactic and / or ameliorating agents of carmodulinopathy containing antisense oligonucleotides, wherein:
(1) A step of contacting the antisense oligonucleotide with cardiomyocytes derived from pluripotent stem cells having a mutation in the calmodulin gene.
The step of measuring the action potential duration of the contacted cardiomyocytes, and the case where the action potential duration measured in the step (3) step (2) is not contacted with the antisense oligonucleotide. A method comprising the step of comparing the action potential duration of cardiomyocytes.
2.
A method of screening for therapeutic, prophylactic and / or ameliorating agents of carmodulinopathy containing antisense oligonucleotides, wherein:
(1) A step of contacting the antisense oligonucleotide with cardiomyocytes derived from pluripotent stem cells having a mutation in the calmodulin gene.
(2) The step of measuring the expression level of the calmodulin gene in the contacted myocardial cells, and (3) the case where the expression level of the calmodulin gene measured in the step (2) was not contacted with the antisense oligonucleotide. A method comprising the step of comparing the expression level of the calmodulin gene in the myocardial cells.
3. 3.
A method of screening for therapeutic, prophylactic and / or ameliorating agents of carmodulinopathy containing antisense oligonucleotides, wherein:
(1) A step of contacting the antisense oligonucleotide with cardiomyocytes derived from pluripotent stem cells having a mutation in the calmodulin gene.
(2) The step of measuring the amount of calcium current in the contacted myocardial cells, and (3) the step (3) the amount of calcium current measured in the step (2) is not brought into contact with the antisense oligonucleotide. A method comprising a step of comparing with the amount of calcium current in.
4.
1. The pluripotent stem cells are iPS cells derived from patients with carmodulinopathy. ~ 3. The method described in any one of.
5.
A method of screening for therapeutic, prophylactic and / or ameliorating agents of carmodulinopathy containing antisense oligonucleotides, wherein:
(1) A step of contacting the antisense oligonucleotide with mammalian cells,
(2) The step of measuring the expression level of the carmodulin gene in the contacted cells, and (3) the case where the expression level of the carmodulin gene measured in the step (2) was not contacted with the antisense oligonucleotide. A method comprising the step of comparing the expression level of the carmodulin gene in the cell.
6.
5. The mammalian cells are human liver cancer-derived cells or mouse fibroblasts. The method described in.
7.
1. The calmodulin gene is at least one selected from the group consisting of CALM1, CALM2, and CALM3. ~ 6. The method described in any one of.
8.
1. The antisense oligonucleotide is a CALM2 antisense oligonucleotide. ~ 7. The method described in any one of.
9.
The antisense oligonucleotide inhibits the expression of the calmodulin gene. ~ 8. The method described in any one of.
10.
A therapeutic, prophylactic and / or ameliorating agent for calmodulinopathy, which comprises an antisense oligonucleotide that inhibits the expression of the calmodulin gene.
11.
10. The calmodulin gene is CALM2. Therapeutic, prophylactic and / or ameliorating agents described in.
12.
10. The antisense oligonucleotide inhibits the expression of the calmodulin gene in cardiomyocytes. Or 11. Therapeutic, prophylactic and / or ameliorating agents described in.
13.
10. The antisense oligonucleotide shortens the action potential duration in cardiomyocytes. ~ 12. The therapeutic, prophylactic and / or ameliorating agents according to any one of the above.
14.
10. The antisense oligonucleotide suppresses the amount of calcium current in cardiomyocytes. ~ 13. The therapeutic, prophylactic and / or ameliorating agents according to any one of the above.
15.
A method for treating, preventing and / or ameliorating calmodulinopathy, which comprises the step of administering to a patient with calmodulinopathy an effective amount of an antisense oligonucleotide that inhibits the expression of the calmodulin gene.
16.
The calmodulin gene is CALM2, 15. The method described in.
17.
15. The antisense oligonucleotide inhibits the expression of the calmodulin gene in cardiomyocytes. Or 16. The method described in.
18.
The antisense oligonucleotide shortens the action potential duration in cardiomyocytes, 15. ~ 17. The method described in any one of.
19.
The antisense oligonucleotide suppresses the amount of calcium current in cardiomyocytes, 15. ~ 18. The method described in any one of.
20.
A compound that is an antisense oligonucleotide that inhibits the expression of the calmodulin gene for use in the treatment, prevention and / or amelioration of calmodulinopathy.
21.
The calmodulin gene is CALM2, 20. The compound described in.
22.
The antisense oligonucleotide inhibits the expression of the calmodulin gene in cardiomyocytes, 20. Or 21. The compound described in.
23.
The antisense oligonucleotide shortens the action potential duration in cardiomyocytes, 20. ~ 22. The compound according to any one of.
24.
The antisense oligonucleotide suppresses the amount of calcium current in cardiomyocytes, 20. ~ 23. The compound according to any one of.
25.
Use of antisense oligonucleotides that inhibit the expression of the calmodulin gene in the manufacture of drugs to treat, prevent and / or improve calmodulinopathy.
26.
The calmodulin gene is CALM2, 25. Use as described in.
27.
The antisense oligonucleotide inhibits the expression of the calmodulin gene in cardiomyocytes, 25. Or 26. Use as described in.
28.
The antisense oligonucleotide shortens the action potential duration in cardiomyocytes, 25. ~ 27. Use described in any one of.
29.
The antisense oligonucleotide suppresses the amount of calcium current in cardiomyocytes, 25. ~ 28. Use described in any one of.

A method of screening for antisense oligonucleotides that can prevent, treat, and / or improve carmodulinopathy is provided. In addition, by using the screening method, it is possible to provide a novel drug for the prevention, treatment and / or improvement of carmodulinopathy.

It is a graph which shows the influence of ASO of calmodulin gene on the expression level of CALM1, CALM2 and CALM3 in human liver cancer-derived cells. It is a measurement result of the action potential in the human iPS cell differentiated cardiomyocyte derived from the calmodulinopathy patient in the presence and absence of ASO of the calmodulin gene. It is a graph which shows the influence of the calmodulin gene of ASO on the action potential duration in the human iPS cell differentiation cardiomyocyte derived from the calmodulinopathy patient. It is a graph which shows the influence of the calmodulin gene on the expression level of CALM2 in the differentiated cardiomyocytes of human iPS cells derived from the calmodulinopathy patient of ASO. It is a graph which shows the influence on the expression level of CALM2 in the heart of the C57BL / 6J mouse which administered the ASO of the calmodulin gene.

It is understood that both the above overview and the detailed description below are merely exemplary and descriptive and do not limit the claimed invention in any way. In the present specification, the matters indicated in the singular form include examples in the plural form unless the matter is clearly different from the context or otherwise specifically stated.
Also, in the present specification, the antisense oligonucleotide may be referred to as "ASO".

Nucleic acids in nature are most basically composed of adenosine (A), thymidine (T) (or uridine (U)), cytidine (C), and guanosine (G). Those basic nucleic acids are often referred to as AT (U) GC and the like. Therefore, in the present specification, for example, with respect to the sequence of the CALM2 gene and the like, when the sequences are indicated by "nucleic acid base sequence" or "SEQ ID NO:", they are basically sequences composed of A, G, C, T and U. Is.
On the other hand, the nucleic acids constituting the ASO of the present application include not only basic nucleic acids (AT (U) CG) but also those having undergone structural modification. Details of the modification will be described later, but include modification to a sugar moiety, an internucleoside bond, and / or a nucleobase.
Therefore, in the present specification, for example, ASO and the like of the present application describe the nucleic acid base sequence by A, G, C, T and U in the description of "compound" or "compound numbered with compound number (P number)". If so, A, G, C, T and U also include those that have undergone structural modification.

Hereinafter, it will be described in more detail.
Unless otherwise indicated, the following terms have the following meanings:

The "antisense effect" means that the function of the target RNA is controlled by hybridizing the target RNA selected corresponding to the target gene and, for example, an oligonucleotide having a sequence complementary to the partial sequence thereof. Means that. For example, when the target RNA is mRNA, the target RNA is degraded by inhibiting the translation of the target RNA by hybridization, the splicing function conversion effect such as exon skipping, and the recognition of the hybridized portion. Etc.

"Antisense oligonucleotide" (ASO) is an oligonucleotide that produces the antisense effect. For example, DNA, gapmer, mixmer and the like can be mentioned, but the present invention is not limited to these, and RNA or an oligonucleotide designed to normally produce an antisense effect may be used.

The ASO of the present application can be prepared by a person skilled in the art by appropriately selecting a known method. For example, a person skilled in the art designs a nucleoside sequence of ASO based on the information of the nucleoside sequence of the target RNA, and uses a commercially available nucleic acid automatic synthesizer (Applied Biosystems, Beckman, Genedesign, etc.). Can be synthesized. It can also be synthesized by a reaction using an enzyme. Examples of the enzyme include, but are not limited to, polymerases, ligases, restriction enzymes and the like.

"Calmodulin" is a calcium detection protein expressed in ubiquitous, and controls various proteins including multiple ion channels. Calmodulin is associated with various processes such as inflammation, metabolism, apoptosis, muscle contraction, intracellular migration, short-term memory, long-term memory, nerve growth, and immune response. In particular, it controls the inactivation of L-type calcium channels (LTCCs) expressed in the heart.

The "calmodulin gene" (CALM gene) is a gene encoding a calmodulin protein, and CALM1, CALM2 and CALM3 have been reported. The base sequence of the human calmodulin gene and the amino acid sequence of the human calmodulin protein are known. For example, the base sequence of the calmodulin genes CALM1, CALM2, CALM3 and the amino acid sequence of the human calmodulin protein are registered in GenBank and published as follows. There is.
Calmodulin protein: AAD45181
CALM1: NC_0000014
CALM2: NC_000002
CALM3: NC_0000019

"Carmodulinopathy" is an arrhythmia disease caused by a mutation in the CALM gene. For example, symptoms of carmodulinopathy include, for example, Long QT Syndrome (LQTS), catecholamine-induced polymorphic ventricular tachycardia (CPVT), and idiopathic ventricular fibrillation (IVF).

Mutations in the calmodulin gene that cause calmodulinopathy include mutations in the coding region (CDS) of the calmodulin gene and mutations in the intron region that causes splicing abnormalities. As a mutation of the CALM gene, a gene mutation is known in which one amino acid of the calmodulin protein, which is a gene product of CALM, is replaced with another amino acid. Currently, the following 28 types of such gene mutations have been reported:
F46K, N54I, F90L, D94N, D96H, D96V, D96G, N98I, N98S, A103V, E105K, E105A, G114W, D130A, D130G, D130V, D132E, D132F, D132H, D132G, D134H, D134N, Q136P, N138 E141V, E141K, F142L.
However, as long as it is a calmodulin gene mutation that causes calmodulinopathy, the gene mutation in the present application is not limited to the above 28 species.
It is also apparent to those skilled in the art that mutations reported in only one or two of CALM1, CALM2, and CALM3 can occur in the remaining two or one gene. .. For example, to date, D130G has been reported to occur in any of the CALM1, CALM2, and CALM3 genes.
The above-exemplified mutation means, for example, N98S is a mutation in which the 98th amino acid from the N-terminal of the calmodulin protein is changed from asparagine (N) to serine (S). That is, the number before the number indicates the type of amino acid before the mutation, the number indicates the position of the amino acid at which the mutation occurs from the N-terminal, and the number after the number indicates the type of the amino acid after the mutation.

for example,
N98S-CALM2 (mutation in which the 98th amino acid from the N-terminus of the calmodulin protein, which is the gene product of CALM2, is changed from asparagine (N) to serine (S)),
D130G-CALM2 (a mutation in which the 130th amino acid from the N-terminus of the calmodulin protein, which is the gene product of CALM2, is changed from aspartic acid (D) to glycine (G)),
N98S-CALM1 (mutation in which the 98th amino acid from the N-terminus of the calmodulin protein, which is the gene product of CALM1, is changed from asparagine (N) to serine (S)),
F90L-CALM1 (mutation in which the 90th amino acid from the N-terminus of the calmodulin protein, which is the gene product of CALM1, is changed from phenylalanine (F) to leucine (L)),
D130G-CALM1 (mutation in which the 130th amino acid from the N-terminus of the CALM2 protein, which is the gene product of CALM1, is changed from aspartic acid (D) to glycine (G))
D134H-CALM1 (mutation in which the 134th amino acid from the N-terminus of the CALM2 protein, which is the gene product of CALM1, is changed from aspartic acid (D) to histidine (H))
Is.

A "carmodulinopathy patient" is a mammal that currently or may have symptoms due to a mutation in the CALM gene, preferably presently or in the future due to a mutation in the CALM gene. It is a human being who can have a gene.

"Long-QT Syndrome" (LQTS) is an arrhythmic disorder in which the QT interval of the electrocardiogram is longer than that of a healthy person. LQTS causes fainting and sudden death due to ventricular arrhythmia. The QT interval is the time from the start of the QRS complex to the end of the T wave on the electrocardiogram, and indicates the contraction / relaxation time of the ventricle. Since the normal value of the QT interval changes with the heart rate, the corrected QT interval (QTc = QT / √RR), which is usually corrected by the heart rate in the Bazett equation, is used. The normal value of the correction QT interval is 360 ms to 440 ms. QT prolongation is the extension of the corrected QT interval from the normal value.
"LQT15" is a congenital long QT syndrome caused by a mutation in CALM2.
"LQT14" is a congenital long QT syndrome caused by a mutation in CALM1.
"LQT16" is a congenital long QT syndrome caused by a mutation in CALM3.

"Action potential" is a transient change in membrane potential that occurs in the cell membrane in response to an excitatory stimulus. Because the distribution of ions is different inside and outside the cell, the intracellular potential of an unactivated quiescent cell is usually negative (ie, said to be polarized) compared to the outside of the cell. Action potentials are mainly generated by the passive diffusion of sodium ions, potassium ions, calcium ions, etc. through ion channels according to the difference in concentration inside and outside the cell.

As the "action potential duration"(APD; Action Potential Duration), APD ₉₀ , APD _50, or the like is usually used. The average value of the potential level in the stationary state is set to the resting potential value Vm _0, and the potential value of the maximum amplitude is set to Vm _max .
Vm ₉₀ = (Vm _max- Vm ₀ ) x 0.1
Based on the Vm ₉₀ obtained in the above, the time from Vm ₉₀ at the time of depolarization to Vm ₉₀ at the time of repolarization is APD ₉₀ . (Biomedical Engineering 47 (6), pp. 514-521, 2009)
Similarly
Vm ₅₀ = (Vm _max- Vm ₀ ) x 0.5
Based on the Vm ₅₀ obtained in the above, the time from Vm ₅₀ at the time of depolarization to Vm ₅₀ at the time of repolarization is APD ₅₀ .

"Pluripotent stem cells" is a well-known term in the art and is endoderm (eg, internal stomach wall, gastrointestinal tract, lung), mesodermal (eg, muscle, bone, blood, urogenital) or ectogerm (eg, muscle, bone, blood, urogenital) or ectodermal (eg, muscle, bone, blood, urogenital) For example, it has both pluripotency that can differentiate into various types of cells that make up the living body, such as cells of the epidermal tissue or nervous system), and self-renewal ability that can maintain pluripotency even after division and proliferation. It is a cell. Examples include artificial multipotent stem (iPS) cells, ES cells, embryonic reproductive (EG) cells derived from progenitor germ cells, and multipotent germanium isolated during the establishment and culture process of GS cells from testis tissue. Examples include embryonic stem (mGS) cells, multipotent progenitor cells (MAPC) isolated from bone marrow, and the like.
ES cells are a well-known term in the art, and are pluripotent stem cells derived from embryos in the blastocyst stage, which is the early stage of animal development, and are ES cells generated by nuclear reprogramming from somatic cells. There may be.
iPS cells are a well-known term in the art, also called induced pluripotent stem cells or induced pluripotent stem cells, and are ES by introducing several types of nuclear reprogramming substances into somatic cells such as fibroblasts. It is a cell that has acquired pluripotency and self-renewal ability equivalent to that of a cell.

The pluripotent stem cells used in the present application are not particularly limited as long as they have an abnormality in the calmodulin gene that causes calmodulinopathy and have "pluripotency for differentiation" and "self-renewal ability". The pluripotent stem cells used in the present application may have an abnormality in the calmodulin gene in a homozygous form, but are preferably a heterozygous form. Some of such pluripotent stem cells have already been established (eg, human iPS cells with abnormalities in the calmodulin gene). The pluripotent stem cell may be any mammalian pluripotent stem cell, and examples thereof include pluripotent stem cells such as human, mouse, monkey, pig, rat, and dog, but human pluripotent is preferable. It is a sex stem cell. Pluripotent stem cells are preferably iPS cells or ES cells, and particularly preferably iPS cells. In another embodiment, mGS cells and MAPCs are also preferred in that they can be obtained from postnatal individuals.

Method for producing pluripotent stem cells Examples of production of iPS cells suitable as pluripotent stem cells used in the present application are shown below, but the method for producing pluripotent stem cells is not limited thereto.

Method for producing iPS cells Induced pluripotent stem (iPS) cells can be produced by introducing a specific nuclear reprogramming substance into somatic cells in the form of DNA or protein (K. Takahashi and S. Yamanaka (K. Takahashi and S. Yamanaka). 2006) Cell, 126: 663-676; K. Takahashi et al. (2007) Cell, 131: 861-872; J. Yu et al. (2007) Science, 318: 1917-1920; M. Nakagawa et al. (2008) Nat. Biotechnol., 26: 101-106; International Publication No. 2007/069666). The nuclear reprogramming substance may be a gene specifically expressed in ES cells, a gene that plays an important role in maintaining undifferentiated ES cells, or a gene product thereof, and is not particularly limited, but is, for example, Oct3 / 4, Klf4, Klf1, Klf2, Klf5, Sox2, Sox1, Sox3, Sox15, Sox17, Sox18, c-Myc, L-Myc, N-Myc, TERT, SV40 Large Gene, HPV16 E6, HPV16 , Nanog, Esrr b and Esrrg. These nuclear reprogramming substances may be used in combination when establishing iPS cells. For example, the combination of the nuclear reprogramming substances is a combination containing at least one, two, three or four, preferably a combination containing five, and particularly preferably Oct3 / 4, Sox2, Klf4. , L-Myc and Lin28. For the nucleotide sequences of the mouse and human cDNAs of each of the above nuclear reprogramming substances and the amino acid sequence information of the protein encoded by the cDNA, refer to the NCBI deposit number described in International Publication No. 2007/069666. A person skilled in the art can obtain the nucleotide sequence and the amino acid sequence information of each of the above nuclear reprogramming substances by referring to the NCBI deposit number. One of ordinary skill in the art can prepare a desired nuclear reprogramming substance by a conventional method based on the cDNA sequence or amino acid sequence information.

These nuclear reprogrammers may be introduced into somatic cells in the form of proteins, for example by methods such as lipofection, binding to cell membrane permeable peptides, microinjection, or in the form of DNA, eg, It can be introduced into somatic cells by techniques such as viruses, plasmids, vectors such as artificial chromosomes, lipofection, liposomes, and microinjection. Examples of viral vectors include retroviral vectors and lentiviral vectors (above, Cell, 126, pp.663-676, 2006; Cell, 131, pp.861-872, 2007; Science, 318, pp.1917-1920. , 2007), adenovirus vector (Science, 322, 945-949, 2008), adeno-associated virus vector, Sendai virus vector (Proc Jpn Acad Ser B Phys Biol Sci. 85, 348-62, 2009) and the like. Examples of the artificial chromosome vector include a human artificial chromosome (HAC), a yeast artificial chromosome (YAC), and a bacterial artificial chromosome (BAC, PAC). As the plasmid, a plasmid for mammalian cells can be used (Science, 322: 949-953, 2008).

The vector can contain regulatory sequences such as promoters, enhancers, ribosome binding sequences, terminators, polyadenylation sites, etc. so that nuclear reprogrammers can be expressed, and if desired, drug resistance genes (eg, eg, drug resistance genes). Canamycin resistance gene, ampicillin resistance gene, puromycin resistance gene, etc.), thymidine kinase gene, diphtheriatoxin gene and other selective marker sequences, green fluorescent protein (GFP), β-glucuronidase (GUS), FLAG® and other reporters It can include gene sequences and the like. In addition, in the above vector, after introduction into somatic cells, in order to excise both the gene encoding the nuclear reprogramming substance or the promoter and the gene encoding the nuclear reprogramming substance that binds to the promoter, the LoxP sequences before and after them are used. May have. Furthermore, in the above vector, the replication origin of the genomic DNA of EBNA-1 (Epstein-Barr virus nuclear antigen 1) and oriP (Epstein-Barr virus) is replicated even if it is not incorporated into the chromosome and is present episomatically. ) Or Large T (Simian virus 40 large T antigen) and SV40 ori (origin of replication of the genomic DNA of Simian virus 40 in mammalian cells) can also be included.

In addition to the above factors, for example, histone deacetylase (HDAC) inhibitors [eg, valproic acid (VPA) (Nat. Biotechnol., 2 6) 7): 795-797 (2008)), Tricostatin A, sodium butyrate, MC 1293 [3- (4-toluoil-1-methyl-1H-2-pyrrolyl) -N-hydroxy-2-propenamide], M344 [ Low molecular weight HDAC inhibitors such as 4- (dimethylamino) -N- [7- (hydroxyamino) -7-oxoheptyl] benzamide], siRNA and shRNA against HDAC (eg, HDAC1 siRNA Smartpool® (Millipole)) , HuSH 29mer shRNA Constructs against HDAC1 (OriGene), etc.), DNA methyltransferase inhibitors (eg, 5'-azacytide) (Nat. Biotechnol., 26 (7): 795-797 (2008): 795-797 )), G9a histone methyltransferase inhibitor [eg, BIX-01294 [N- (1-benzylpiperidin-4-yl) -6,7-dimethoxy-2- (4-methyl-1,4-diazepan-1-) Il) quinazoline-4-amine] (Cell Stem Cell, 2: 525-528 (2008)) and other small molecule inhibitors, siRNA and shRNA against G9a (eg, G9a siRNA (human) (Santa Cruz Biotechnology), etc.), etc. Inhibitors of nucleic acid expression, etc.], L-type calcium channel agent (for example, Bayk8644 [1,4-dihydro-2,6-dimethyl-5-nitro-4- [2- (trifluoromethyl) phenyl] -3- (trifluoromethyl) phenyl] -3- Ppyridinecarboxylic acid methyl ester]) (Cell Stem Cell, 3, 568-574 (20 08)), p53 inhibitors (eg siRNA and shRNA against p53) (Cell Stem Cell, 3, 475-479 (2008)), Wnt Signaling activator (eg, soluble Wnt3a) (Cell Stem Cell, 3, 132-135 (2008)), LIF (leukemia inhibitor) and bFGF (basic fiber) Kinases such as blastogenic factor), ALK5 inhibitors (eg SB431542 [4- [4- (1,3-benzodioxol-5-yl) -5- (2-pyridinyl) -1H-imidazole-2) -Il] -Benzamide]) (Nat Methods, 6: 805-8 (2009)), mitogen-activated protein kinase sinalling inhibitor, glycogen kinase kinase-3 inhibitor (PloS Biology, 6 (10), 2 2008)), miRNAs such as miR-291-3p, miR-294, and miR-295 (RL Judson et al., Nat. Biotech., 27: 459-461 (2009)) and the like can be used.

Examples of the culture medium for inducing iPS cells include the following (1) and (2).
(1) DMEM [Dulbecco-modified Eagle's medium], DMEM / F12 [DMEM nutritional mixture] and DME medium [Dulbecco-modified Eagle's medium] containing 10 to 15% FBS [fetal bovine serum] (where, these media include Further, LIF, penicillin / streptomycin, puromycin, L-glutamine, non-essential amino acids, β-mercaptoethanol and the like can be appropriately contained.)
(2) ES cell culture medium containing bFGF [basic fibroblast growth factor] or SCF [stem cell factor], mouse ES cell culture medium (for example, TX-WES medium, Thrombo X) and primate ES cells. Culture medium (eg, primate (human and monkey) ES cell medium, manufactured by Reprocell)
The culture medium for inducing iPS cells is particularly preferably a medium for primate ES cell culture (medium for primate (human and monkey) ES cells, manufactured by Reprocell).

Examples of culturing methods for iPS cells include contacting somatic cells with a nuclear reprogramming substance (DNA or protein) in DMEM or DMEM / F12 medium containing 10% FBS in the presence of 5% carbon dioxide at 37 ° C. After culturing for about 4 to 7 days, the cells are sprinkled on feeder cells (for example, mitomycin C-treated STO cells, SNL cells, etc.), and bFGF-containing primates about 10 days after contact between the somatic cells and the nuclear reprogramming substance. It can be cultured in ES cell culture medium to give rise to iPS-like colonies about 30-45 days or more after contact. Further, in order to increase the production efficiency of iPS cells, the cells may be cultured under the condition of an oxygen concentration as low as 5 to 10%. Alternatively, as an alternative culture method, DMEM medium containing 10% FBS in feeder cells (for example, mitomycin C-treated STO cells, SNL cells, etc.) (in addition, LIF, penicillin / threptomycin, puromycin, L-glutamine, etc.) , Non-essential amino acids, β-mercaptoethanol and the like can be appropriately contained), and ES cell-like colonies can be generated after about 25 to 30 days or more. During the above culture, the fresh medium and the medium are exchanged once a day from the second day after the start of the culture. The number of somatic cells used for nuclear reprogramming is not limited, but is in the range of about 5 × 10 ³ to 5 × 10 ^{6 cells per 100 cm 2 of the cultured dish.}

When a gene containing a drug resistance gene is used as a marker gene, a marker gene-expressing cell can be selected by culturing in a medium (selective medium) containing the corresponding drug. If the marker gene is a fluorescent protein gene, observe it with a fluorescence microscope, if it is a luciferase gene, add a luminescent substrate, or if it is a luciferase gene, add a chromogenic substrate to obtain marker gene-expressing cells. Can be detected.

As used herein, "somatic cells" may be any cells other than germ cells derived from mammals (eg, humans, mice, monkeys, pigs, rats, dogs, etc.) and, for example, keratinize. Epithelial cells (eg, keratinized epidermal cells), mucosal epithelial cells (eg, epithelial cells on the surface of the tongue), exocrine gland epithelial cells (eg, mammary cells), hormone-secreting cells (eg, adrenal medulla cells), for metabolism / storage Cells (eg, hepatocytes), luminal epithelial cells that make up the interface (eg, type I alveolar cells), luminal epithelial cells of the inner chain canal (eg, vascular endothelial cells), Certain cells (eg, airway epithelial cells), extracellular matrix secretory cells (eg, fibroblasts), contractile cells (eg, smooth muscle cells), blood and immune system cells (eg, T lymphocytes), sensation Cells (eg, rod cells), autonomic nervous system neurons (eg, cholinergic neurons), sensory organ and peripheral neuronal support cells (eg, companion cells), central nervous system nerve cells and glial cells (eg, stars) Glya cells), pigment cells (eg, retinal pigment epithelial cells), and their precursor cells (tissue precursor cells) and the like. There are no particular restrictions on the degree of cell differentiation or the age of the animal from which the cells are collected, and both undifferentiated progenitor cells (including somatic stem cells) and final differentiated mature cells are similarly used as somatic cells in the present application. can do. Examples of undifferentiated progenitor cells include tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells. In the present application, the mammal from which the somatic cells are collected is not particularly limited, but is preferably human, more preferably a calmodulinopathy patient or an unaffected human having the same calmodulin gene mutation as the patient. be.

Other methods of producing pluripotent stem cells In in vitro fertilization in fertility treatment, preimplantation genetic diagnosis (PGD) is performed on a family in which a pathogenic mutation has been identified, and if the fertilized egg has a pathogenic mutation, a surplus embryo is performed. May be treated as. Overseas, human embryonic stem (ES) cells established from surplus embryos having such pathogenic mutations are used in research as disease-specific ES cells. Therefore, disease-specific ES cells can be obtained from early embryos that have become surplus embryos due to the discovery of calmodulin gene abnormalities by PGD. As a method for producing ES cells, for example, a method for culturing an internal cell mass at the blastocyst stage of a mammal (see, for example, Manipulating the Mouse Embryo A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1994)). , Method of culturing early embryos produced by somatic cell nuclear transfer (Wilmut et al., Nature, 385, 810 (1997); Cibel li et al., Science, 280, 1256 (1998); Akira Iriya et al., Protein nucleic acid Enzymes, 44, 892 (1999); Baguisi et al., Nature Biotechnology, 17, 456 (1999); Wakayama et al., Nature, 3 94, 369 (1998); Wakayama et al., Nature Genetics, 22, 127 (1999); Wakayama et al., Proc. Natl. Acad. Sci. USA, 96, 14984 (1999); RideoutIII et al., Nature Genetics, 24, 109 (2000)). Not limited.
In the case of somatic cell nuclear transfer, the type of somatic cell and the animal from which the somatic cell is collected are the same as in the case of the above iPS cell.
EG cells can be induced by isolating primordial germ cells according to conventional methods and culturing them in the presence of LIF, bFGF and SCF.
mGS cells can be made from testicular cells according to the method described in WO 2005/100458.
Pluripotent adult progenitor cells (MAPCs) can be isolated from bone marrow according to the method described in J. Clin. Invest. 109: 337-346 (2002).

Method for Inducing Differentiation from Pluripotent Stem Cell to Cardiomyocyte The pluripotent stem cell obtained as described above can be differentiated into cardiomyocyte by any known differentiation induction method. As a method for inducing differentiation into cardiomyocytes, for example, a method using embryoid body (EB) formation, a method using a directional differentiation approach, and a two-dimensional differentiation induction method (GiWi method, etc.) have been reported ( For example, Nature 2008; 453: 524-8, J Clin Invest 2001; 108: 407-14, Circ Res 2002; 91: 501-8, Circ Res 2003; 93: 32-9, Nat Biotechnol 2007; 25: 1015- 24, Circulation 200 8; 118: 517, Circulation 2008; 118: 506, Nat Biotechnol 2005; 23: 611, Nat Protoc. 2013 Jan; 8 (1): 162-75 etc.). The medium can be used by adding an additive to the basal medium, and examples of the basal medium include Neurobasal (registered trademark) medium, NeuroProgenitor Basic medium, NeuroCult NS-A medium, and BME medium (Basal Medium for Eagle). BGJb medium, CMRL 1066 medium, Glasgo MEM medium (Grasgo's minimum essential medium), Improved MEM Zinc Option medium, IMDM medium (Iscover's Modified Dulvecco's Medium), Medium Eagle's medium, Medium Eagle's Medium, Medium Eagle's Medium Animal cells such as αMEM medium (Eagle's minimum essential medium α modified type), DMEM medium, DMEM / F12 medium, ham medium, RPMI 1640 medium (Roswell Park Memorial Laboratory medium), Fisher's medium, and a mixed medium thereof. The medium is not particularly limited as long as it can be used for culture. More preferably, it is RPMI 1640 medium. As additives, serum, retinoic acid, ascorbic acid, BMP (bone morphogenetic factors; BMP2, BMP4), Nodal, TGFβ1, Activin A, Dkk1, IGFBP-4, bFGF, EGF, HGF, LIF, amino acids, vitamins, inter Leukins, insulin, transferase, heparin, heparan sulfate, collagen, fibronectin, progesterone, selenite, B27®-supplements, N2-supplements, ITS-supplements, antibiotics. In addition, in the early stage of induction of differentiation, a GSK-3 inhibitor (for example, CHIR99021 [6- {2- [4- (2,4-dichloro-phenyl) -5- (5-methyl-1H-imidazol-2-yl) ) -Pyrimidine-2-ylamino] -ethylamino} -nicotinonitrile]) and Wnt inhibitors (eg, IWP-2 [N- (6-methyl-2-benzothiazolyl) -2-[(3,4,6) , 7-Tetrahydro-4-oxo-3-phenylthieno [3,2-d] pyrimidin-2-yl) thio] -acetamide]) is also preferred. Additives are preferably B27®-supplements and insulin, and more preferably these are used in combination.

The incubator can be coated with a coating agent such as collagen, gelatin, matrigel, poly-L-lysine, poly-D-lysine, fibronectin, laminin and the like. The concentration of pluripotent stem cells at the start of culturing can be appropriately set so as to efficiently form cardiomyocytes, and is not particularly limited, but is, for example, about 1 × 10 ³ to 1 × 10 ⁶ cells / mL, preferably about. 1 × a ¹⁰ 4 ~ ⁵ × ¹⁰ 5 cells / mL. Other culture conditions such as culture temperature and carbon dioxide concentration can be set as appropriate. The culture temperature is not particularly limited, but is, for example, about 30 to 40 ° C, preferably about 36 to 38 ° C. The carbon dioxide concentration is, for example, about 1 to 10%, preferably about 4 to 6%. The cardiomyocytes obtained as described above are characterized by carmodulinopathy represented by expression of the causative mutant gene, prolongation of action potential duration and / or dysfunction of various ion channels, decrease in contractile force, and the like. The prophylactic and / or therapeutic agents for carmodulinopathy can be screened using the improvement of these abnormalities as an index.

It is also possible to construct myocardial tissue from the cardiomyocytes obtained as described above. For example, cardiomyocytes can be purified from embryoid bodies treated with ascorbic acid with Percoll gradient and myocardial tissue can be constructed in the presence of type I collagen and Matrigel (Circulation 2006; 1 13: 2237). In the myocardial tissue thus obtained, pathological conditions such as abnormal conduction of electrical signals and a decrease in muscle contraction force are reproduced. Therefore, using these as indicators, prophylactic and / or therapeutic agents for carmodulinopathy can be screened.

Screening Method for Antisense Oligonucleotides Effective for Prevention and / or Treatment of Carmodulinopathy In the present application, the test antisense oligonucleotide is brought into contact with the pluripotent stem cell-derived myocardial cells or tissues obtained as described above. , Each index is used to provide a method for screening candidate antisense oligonucleotides for the prevention and treatment of carmodulinopathy. The preferred pluripotent stem cells used in the present application are pluripotent stem cells derived from patients who exhibit LQT and / or CPVT as phenotypes in carmodulinopathy, or pluripotency into which a mutant CALM is exogenously introduced. It is a stem cell. As the mutant CALM, for example, CALM1-F90L, CALM1-N98S, CALM1-D130G, CALM2-N98S, CALM2-D130G, CALM3-D130G and the like are known.

As another aspect, the pluripotent stem cell used in the present application is an iPS cell in which an action potential duration and an increase in calcium current amount are confirmed in cardiomyocytes prepared from the iPS cell. desirable. In the present application, as such iPS cells, iPS cells derived from patients whose LQT is shown as a phenotype in carmodulinopathy are exemplified.

As one embodiment of the present application, when the action potential duration of cardiomyocytes is used as an index, a method including the following steps may be used to screen for a therapeutic, prophylactic and / or ameliorating agent for carmodulinopathy, including ASO. can.
(1) A step of contacting ASO with cardiomyocytes derived from pluripotent stem cells having a mutation in the calmodulin gene (for example, pluripotent stem cells prepared from somatic cells of calmodulinopathy patients).
(2) The step of measuring the action potential duration of the cardiomyocytes that have been contacted, and (3) the action potential duration measured in step (2) of the cardiomyocytes when they are not contacted with the ASO. The step of comparing the action potential duration (control).
Then, ASO having an effect of shortening the duration of action potential is usually selected as a candidate for a therapeutic, prophylactic and / or ameliorating agent for carmodulinopathy.
The degree of shortening of the action potential duration is not particularly limited because it depends on the concentration of ASO, but for example, a shortening of 100 ms or more, preferably 200 ms or more, and more preferably 300 ms or more is observed. Those are preferred as candidates for the treatment, prevention and / or amelioration of carmodulinopathy. The concentration of ASO is not particularly limited, but can be appropriately set from the range of 0.1 nM to 10 μM (for example, 0.1 nM, 1 nM, 10 nM, 100 nM, 1 μM, 10 μM), and the action potential lasts at a lower concentration. Time-saving ASOs are preferably selected as candidates for treatment, prevention and / or amelioration of carmodulinopathy.
In addition, the selected candidates may be subjected to further screening to confirm their therapeutic, preventive and / or ameliorating effects, if necessary.

Contact between ASO and pluripotent stem cell-derived cardiomyocytes may or may not use a transfection reagent, but it is preferable not to use a transfection reagent. When a transfection reagent is used, examples of the transfection reagent include lipofectamine 3000.

The contact between the ASO and the pluripotent stem cell-derived cardiomyocytes is, for example, 30 to 40 ° C, preferably 35 to 40 ° C, and more preferably 36 to 38 ° C. The culture period after contact until the action potential duration is measured is, for example, 1 day to 1 week, preferably 4 days to 6 days. The culture temperature is, for example, 30-40 ° C, preferably 35-40 ° C, more preferably 36-38 ° C.

Examples of the method for measuring the action potential of cardiomyocytes include measurement by a patch clamp system, measurement by a multi-electrode array system, and measurement by imaging using a fluorescent dye probe. Preferably, measurement by imaging with a fluorescent dye probe is used. Examples of the fluorescent dye probe include a fluorescent calsim dye (for example, Fluo-8 (registered trademark)) and a voltage-sensitive dye (for example, Fluvolt (registered trademark)), and Fluvolt is preferably used. As the imaging system, a high-speed optical measurement system manufactured by Brainvision (MiCAM02, MiCAM03, etc.) is preferably used, and MiCAM03 is particularly preferably used.
The action potential of cardiomyocytes is preferably measured at, for example, 30-40 ° C, preferably 35-40 ° C, more preferably 36-38 ° C.
The action potential of cardiomyocytes is preferably measured under constant rhythmic electrical stimulation, eg, 0.1-2.0 Hz, preferably 0.2-1.0 Hz, more preferably 0.4. It is preferably measured at pacing of ~ 0.6 Hz, particularly preferably 0.5 Hz. As the electrical stimulation of a constant rhythm, for example, an electrical stimulation by an Electrical Stimulator (SEN-3301) manufactured by Nihon Kohden Co., Ltd. can be used.

The action potential duration can be determined from the action potential measured as described above. In this screening method, APD ₉₀ is preferably used as the action potential duration.

In another embodiment of the present application, when the expression level of the calmodulin gene in cardiomyocytes is used as an index, a therapeutic, preventive and / or ameliorating agent for calmodulinopathy including ASO is screened by a method including the following steps. Can be done.
(1) A step of contacting ASO with cardiomyocytes derived from pluripotent stem cells having a mutation in the calmodulin gene (for example, pluripotent stem cells prepared from somatic cells of calmodulinopathy patients).
The step of measuring the expression level of the calmodulin gene in the contacted cardiomyocytes, and the step of measuring the expression level of the calmodulin gene in the step (3) step (2), when the calmodulin gene expression level was not contacted with the ASO, the myocardium A step of comparing the expression level of the calmodulin gene in cells.
Then, ASO having an effect of reducing the expression level of the calmodulin gene is usually selected as a candidate for a therapeutic, prophylactic and / or ameliorating drug for calmodulinopathy.
The degree of decrease in the expression level of the calmodulin gene is not particularly limited because it depends on the concentration of ASO, but for example, a decrease of 20% or more, preferably 40% or more, and more preferably about 50% is observed. , Preferred as a candidate for treatment, prevention and / or amelioration of calmodulinopathy. The concentration of ASO is not particularly limited, but can be appropriately set from the range of 0.1 nM to 10 μM (for example, 0.1 nM, 1 nM, 10 nM, 100 nM, 1 μM, 10 μM), and the concentration of the calmodulin gene can be set at a lower concentration. ASOs that reduce expression are preferably selected as candidates for treatment, prevention and / or amelioration of calmodulinopathy.
In addition, the selected candidates may be subjected to further screening to confirm their therapeutic, preventive and / or ameliorating effects, if necessary.

Contact between ASO and pluripotent stem cell-derived cardiomyocytes may or may not use a transfection reagent. It is preferable not to use transfection reagents. When a transfection reagent is used, examples of the transfection reagent include lipofectamine 3000.

The contact between the ASO and the pluripotent stem cell-derived cardiomyocytes is, for example, 30 to 40 ° C, preferably 35 to 40 ° C, and more preferably 36 to 38 ° C. The culture period after contact until the expression level of the calmodulin gene is measured is, for example, 1 day to 1 week, preferably 4 days to 6 days. The culture temperature is, for example, 30-40 ° C, preferably 35-40 ° C, more preferably 36-38 ° C.

As a method for measuring the expression level of a gene, a real-time PCR method can be mentioned.
First, RNA was extracted from cells using NucleoSpin RNA Plus XS (Takarabio), and then cDNA was prepared by reverse transcription using Transscriptor First Strand cDNA Synthesis Kit (Roche), and TaqMan was used using the prepared cDNA. The expression level of the carmodulin gene can be measured by quantitative real-time PCR by the method.

In yet another embodiment of the present application, when calcium current levels in cardiomyocytes are used as indicators, screening for therapeutic, prophylactic and / or ameliorating agents for carmodulinopathy, including ASO, can be performed by methods involving the following steps. can.
(1) A step of contacting ASO with pluripotent stem cell-derived cardiomyocytes having a mutation in the calmodulin gene,
(2) The step of measuring the calcium current amount in the cardiomyocytes that have been contacted, and (3) the calcium current amount in the cardiomyocytes when the calcium current amount measured in step (2) is not contacted with the ASO. The process of comparing with quantity.
Then, ASO having an effect of reducing the amount of calcium current is usually selected as a candidate for a therapeutic, prophylactic and / or ameliorating agent for carmodulinopathy.
The degree of decrease in the amount of calcium current is not particularly limited because it depends on the concentration of ASO, but for example, a decrease of about 20%, preferably 40%, and more preferably about 50% is observed. Preferable as a candidate for treatment, prevention and / or amelioration of pachi. The concentration of ASO is not particularly limited, but can be appropriately set from the range of 0.1 nM to 10 μM (for example, 0.1 nM, 1 nM, 10 nM, 100 nM, 1 μM, 10 μM), and the calcium current amount can be set at a low concentration. ASOs with a large degree of reduction are preferably selected as candidates for treatment, prevention and / or amelioration of calcium carmodulinopathy.
In addition, the selected candidates may be subjected to further screening to confirm their therapeutic, preventive and / or ameliorating effects, if necessary.

A manual patch clamp system or an auto patch clamp system is used as a method for measuring the amount of calcium current in cardiomyocytes. As a manual patch clamp device, Multiclamp 700B amplifier, Digidata 1440 digitizer hardware, pclamp 10.4 software (manufactured by Molecular Devices), and as an auto patch clamp system device, QPatch system (manufactured by Sophion Bioscience) and System ) Is used. Those skilled in the art can measure the amount of calcium current using the device. For example, as a method of manual patch clamp, a pipette is manufactured by using a borosilicate glass manufactured by World Precision Instruments Co., Ltd. and using a Single Stage Glass Microeleductorode Puller manufactured by Narishige Scientific Instruments Research Institute Co., Ltd. Pipette solution, CsCl [cesium _{chloride] (120 mmol / L),} MgCl 2 · 6H 2 0 [ magnesium chloride hexahydrate] (3 mmol / L), TEA · Chloride [ tetraethylammonium chloride] (20 mol / L), EGTA [glycol ether diamine tetraacetic acid] (10 mmol / L), MgATP [magnesium adenosine triphosphate] (5 mmol / L), HEPES [hydroxyethylpiperazine ethanesulfonic acid] (5 mmol / L) Can be used. Extracellular fluid, TEA · HCl [triethylamine hydrochloride] (140 mol / L), CsCl (5.4 mol / L), CaCl 2 [ Calcium chloride] (1.8 mol / L), MgCl 2 · 6H 2 An aqueous solution of 0 (1.2 mmol / L), HEPES (5 mmol / L), and glucose (10 mmol / L) can be used. The amount of calcium current can be measured by the whole cell patch clamp method under fixed potential. For example, while the membrane potential is held at -80 mV, a -40 mV pulse stimulus is transiently applied, then a depolarizing pulse stimulus of 0 mV is applied for 0.3 seconds, and then a membrane potential of -80 mV is applied. The amount of calsim current can be measured by returning to.

The expression level of the calmodulin gene can be similarly measured using mammalian cells such as human cells or mouse cells instead of pluripotent stem cells having a mutation in the calmodulin gene. The mammalian cells are preferably human liver cancer-derived cells (eg, HepG2 cells) or mouse fibroblasts (eg, 3T3-L1 cells). When human liver cancer-derived cells are used, it is preferable to use, for example, lipofectamine 3000 as a transfection reagent. When mouse fibroblasts are used, it is preferable not to use a transfection reagent.

In the above screening method, the calmodulin gene is preferably at least one selected from the group consisting of CALM1, CALM2, and CALM3, and particularly preferably CALM2.
In the above screening method, the ASO may be selected from CALM1, CALM2, and CALM3, but preferably the ASO is the ASO of CALM2.

In a preferred embodiment, the ASO selected using the above screening method inhibits the expression of the calmodulin gene.

The present application also provides therapeutic, prophylactic and / or ameliorating agents for calmodulinopathy, including ASO, which inhibits the expression of the calmodulin gene.
Preferably, the calmodulin gene is CALM2.
Preferably, the ASO is an ASO that inhibits the expression of the calmodulin gene in cardiomyocytes.
Preferably, the ASO is an ASO that shortens the action potential duration in cardiomyocytes.
Preferably, the ASO is an ASO that suppresses the amount of calcium current in cardiomyocytes.
The effective amount of ASO is an amount of a compound sufficient to bring about the desired pharmacological effect in a carmodulinopathy patient, and the age, body weight of the patient whose carmodolinopathy should be treated, prevented and / or improved. , Symptoms, health conditions, specific types of compounds used or pharmacologically acceptable salts thereof, amounts thereof to be blended in pharmaceutical compositions, etc., and are appropriately selected.

Hereinafter, the present invention will be described in more detail based on Examples, but the embodiments of the present invention are not limited to the following Examples.

In Table (1) in the examples, "Compd No" means the compound number and "Chemical structure" means the chemical structure of the corresponding compound.
In the sequence notation in the examples, unless otherwise specified, "(L)" is LNA, lower alphabet is deoxyribonucleoside, "^" is phosphorothioate bond, and "5 (x)" is its deoxyribonucleoside. The nucleobase of the nucleoside is 5-methylcytosine, and the "5" in "5 (L)" means that the nucleobase of the nucleoside is 5-methylcytosine.

[Manufacturing Example 1]
The antisense oligonucleotides shown in Table 1 were prepared using an automatic nucleic acid synthesizer nS-8II (manufactured by Gene Design).

Each antisense oligonucleotide set forth in Table 1 is either a human CALM2 mRNA designated herein by SEQ ID NO: 1 and / or a human CALM2 pre-mRNA designated herein by SEQ ID NO: 2. Targeted at.
Both "CALM2 mRNA" and "CALM2 pre-mRNA" have nucleosides linked to each other by phosphodiester bonds, and thymine is usually replaced by uracil. "CALM2 mRNA" and "CALM2 pre-mRNA" do not have any other modifications for sugar, nucleobase, or nucleoside linkage.

Table 2 shows the target position of each antisense oligonucleotide shown in Table 1 in the sequence of human CALM2 mRNA or pre-mRNA, the SEQ ID NO: and the nucleobase sequence corresponding to each antisense oligonucleotide.
In the sequence notation of Table 2, "SEQ1 START" means "SEQ ID NO: 1 starting site" and the position number of the most 5'side nucleoside targeted by the antisense oligonucleotide in the human CALM2 mRNA sequence. show. "SEQ1 END" means "SEQ ID NO: 1 termination site" and indicates the position number of the most 3'-side nucleoside targeted by the antisense oligonucleotide in the human CALM2 mRNA sequence.
"SEQ2 START" means "SEQ ID NO: 2 start site" and indicates the position number of the most 5'side nucleoside targeted by the antisense oligonucleotide in the sequence of pre-mRNA of human CALM2. "SEQ2 END" means "SEQ ID NO: 2 termination site" and indicates the position number of the most 3'-side nucleoside targeted by the antisense oligonucleotide in the pre-mRNA sequence of human CALM2.
"SEQ No" indicates the SEQ ID NO: and "BASE SEQENCE" indicates the nucleic acid base sequence of the antisense oligonucleotide.

[Reference example 1]
Establishment of iPS cells Similar to Non-Patent Document 4, a method of collecting blood from a calmodulinopathy patient in which a calmodulin gene mutation (c.293A> G, p.N98S) has been identified and using an episomal vector (Stem). According to Cells 31: 458-466 (2013)), iPS cells derived from calmodulinopathy patients were established using Oct3 / 4, Sox2, Klf4, L-Myc, and Lin28 as nuclear reprogramming substances. At the time of establishment, a medium for primate ES cell culture (medium for primate (human and monkey) ES cells, manufactured by Reprocell) was used.

[Reference example 2]
Differentiation into cardiomyocytes The iPS cells established in Reference Example 1 were differentiated into myocardium using a two-dimensional differentiation induction method called the GiWi method (Lian et al, Nat Protoc. 2013 Jan; 8 (1): 162- 75). IPS cells cultured on plates coated with Matrigel Growth Factor Reduce® diluted in IMDM (Insulin Modified Dulvecco's Media) medium were added to RPMI 1640 with a B-27® supplement. CHIR99021 was added to the medium (insulin (−)) and cultured for 1 day. From the 3rd day after the start of differentiation, IWP-2 was added and cultured for 2 days, and after 7 days after the start of differentiation, the cells were cultured in RPMI 1640 supplemented with B-27 (registered trademark) supplement and insulin, and after 12 days. Cardiomyocytes that beat in insulin appeared.

[Evaluation Example 1] Antisense suppression of human CALM2 in HepG2 cells HepG2 cells seeded on a 96-well plate at a density of 10,000 cells / well were subjected to Lipofectamine (registered trademark) 3000 (Thermo Fisher Scientific) after about 24 hours. Was added (transfection) to an antisense oligonucleotide (produced in Production Example 1) so that the final concentration was 50 nM. Twenty-four hours later, RNA was isolated from cells using RNeasy Mini Kit (QIAGEN) and then reverse transcribed with PrimeScript® RT Master Mix (Perfect Real Time) (Takara Bio) to prepare cDNA. Using the prepared cDNA, the CALM2 gene expression level was measured by quantitative real-time PCR using TaqMan® Gene Expression Assays (Thermo Fisher Scientific). In real-time PCR, the mRNA amount of the housekeeping gene Peptidylprolyl isomerase A [PPIA] was also quantified at the same time, and the amount of CALM2 mRNA relative to the amount of PPIA mRNA was evaluated as the expression level of CALM2. The results are shown in Table 3 as the percentage expression of CALM2 relative to untreated control cells.

[Evaluation Example 2] Dose-dependent antisense suppression of human CALM1 / CALM2 / CALM3 in HepG2 cells HepG2 cells seeded on a 96-well plate at a density of 10,000 cells / well were subjected to Lipofectamine (registered trademark) after about 24 hours. ) 3000 (Thermo Fisher Scientific) was used to add P19710002 to a final concentration of 0.1 nM, 1 nM, 10 nM, 100 nM (transfection). Twenty-four hours later, RNA was isolated from cells using RNeasy Mini Kit (QIAGEN) and then reverse transcribed with PrimeScript® RT Master Mix (Perfect Real Time) (Takara Bio) to prepare cDNA. Using the prepared cDNA, the expression levels of CALM1, CALM2, and CALM3 genes were measured by quantitative real-time PCR using TaqMan® Gene Expression Assays (Thermo Fisher Scientific). In real-time PCR, the amount of mRNA of PPIA of the housekeeping gene was also quantified at the same time, and the amount of mRNA of CALM1, CALM2, and CALM3 with respect to the amount of mRNA of PPIA was evaluated as the expression level of CALM1, CALM2, and CALM3. The results are shown in FIG. 1 as the percentage expression of CALM2 relative to untreated control cells (cntrоl).

As is clear from FIG. 1, P19710002 suppressed the expression of CALM2 in a dose-dependent manner. On the other hand, it was shown that the expression of the family genes CALM1 and CALM3 was not suppressed.

[Evaluation Example 3] Antisense suppression of mouse CALM2 in 3T3-L1 cells The final concentration of 3T3-L1 cells seeded on a 96-well plate at a density of 10,000 cells / well was 0.1 nM after about 24 hours. P19710002 was added so as to be 1, 1 nM, 10 nM, and 100 nM (Free-Uptake). Five days later, RNA was isolated from cells using RNeasy Mini Kit (QIAGEN) and then reverse transcribed with PrimeScript® RT Master Mix (Perfect Real Time) (Takara Bio) to prepare cDNA. Using the prepared cDNA, mouse CALM2 gene expression levels were measured by quantitative real-time PCR using TaqMan® Gene Expression Assays (Thermo Fisher Scientific). In real-time PCR, the amount of PPIA mRNA of the housekeeping gene was also quantified at the same time, and the amount of CALM2 mRNA relative to the amount of PPIA mRNA was evaluated as the expression level of CALM2. The results are shown in Table 4 as the percentage expression of CALM2 relative to untreated control cells. In Table 4, "concentration" indicates the concentration of P19710002.

[Evaluation example 4]
Evaluation of the effect of antisense oligonucleotides on action potential duration in iPS cell-differentiated cardiomyocytes Suspend 50,000 cardiomyocytes differentiated in Reference Example 2 in 4 μL of culture medium and 4 μL of IMDM (Iscover's Modified). Sheet-shaped iPS cell-derived cardiomyocytes were prepared by seeding on 96-well plates coated with Matrigel Growth Factor Reduce (registered trademark) diluted in Dulvecco's Media) medium. After about 24 hours, P19710002 was added to a final concentration of 1 μM (Free-uptake). After about 120 hours, Fluovolt (Thermo Fisher Scientific), which is one of the voltage-sensitive dyes, was loaded at 37 ° C. for 30 minutes, and at 37 ° C. using an imaging system (microscope: Nikon, high-speed optical measurement system: Brainvision). The action potential was measured under 0.5 Hz pacing. The results are shown in FIGS. 2 and 3. In addition, even when P19710002 was not added, it was measured at the same time and shown in FIGS. 2 and 3 as ASO (−). In FIGS. 2 and 3, "ms" indicates milliseconds.

As is clear from FIGS. 2 and 3, the shortening of the action potential duration by the antisense oligonucleotide of CALM2 could be measured. P1971002 was also shown to reduce action potential duration (APD _{90) by 90%.}

[Evaluation example 5]
Evaluation of CALM2 Gene Expression Level by Antisense Oligonucleotide in iPS Cell Differentiated Cardiomyocytes The cardiomyocytes differentiated in Reference Example 2 were seeded on a 96-well plate at a density of 200,000 cells per well. After about 24 hours, P19710002 was added to a final concentration of 1 μM (Free-uptake). After about 120 hours, RNA was extracted from the cells using NucleoSpin RNA Plus XS (Takara Bio), and then cDNA was prepared by reverse transcription by Transscriptor First Strand cDNA Synthesis Kit (Roche). Using the prepared cDNA, the mouse CALM2 gene expression level was measured by quantitative real-time PCR by the TaqMan method. In real-time PCR, the amount of mRNA of the housekeeping gene GAPDH (Glyceraldehyde-3-phosphate Dehydogenase) was also quantified at the same time, and the amount of CALM2 mRNA relative to the amount of GAPDH mRNA was evaluated as the expression level of CALM2. The results are shown in FIG. In addition, ASO (-) shows the case where P19710002 was not added.

[Evaluation example 6]
Evaluation of intracardiac CALM2 gene expression level by antisense oligonucleotide in a mouse body P1971002 dissolved in physiological saline (Otsuka saline, Otsuka Pharmaceutical Factory) was added to C57BL / 6J mice (male 6 weeks old, Charles River Japan). , The dose per mouse was intravenously administered so as to be 1.9 μmol / kg or 9.5 μmol / kg in terms of antisense oligonucleotide amount. As a control, only physiological saline (Otsuka Raw Food Injection, Otsuka Pharmaceutical Factory) was administered. Five days after administration, the heart was collected after euthanasia by total blood sampling under isoflurane anesthesia. RNA was isolated from each organ using RNeasy Mini Kit (manufactured by Qiagen), and then cDNA was obtained using PrimeScript (registered trademark) RT Master Mix (Perfect Real Time) (Takara Bio). Using the prepared cDNA, mouse CALM2 gene expression levels were measured by quantitative real-time PCR using TaqMan® Gene Expression Assays (Thermo Fisher Scientific). In real-time PCR, the amount of PPIA mRNA of the housekeeping gene was also quantified at the same time, and the amount of CALM2 mRNA relative to the amount of PPIA mRNA was evaluated as the expression level of CALM2. The results are shown in FIG. In addition, ASO (-) shows the case where P19710002 was not added.

A method of screening for antisense oligonucleotides that can prevent, treat, and / or improve carmodulinopathy is provided. In addition, by using the screening method, it is possible to provide a novel drug for the prevention, treatment and / or improvement of carmodulinopathy.

This application is based on Japanese Patent Application No. 2020-036716 filed in Japan, the contents of which are all included in the present specification.

Claims (17)

1. A method of screening for therapeutic, prophylactic and / or ameliorating agents of carmodulinopathy containing antisense oligonucleotides, wherein:
   (1) A step of contacting the antisense oligonucleotide with cardiomyocytes derived from pluripotent stem cells having a mutation in the calmodulin gene.
   The step of measuring the action potential duration of the contacted cardiomyocytes, and the case where the action potential duration measured in the step (3) step (2) is not contacted with the antisense oligonucleotide. A method comprising the step of comparing the action potential duration of cardiomyocytes.
2. A method of screening for therapeutic, prophylactic and / or ameliorating agents of carmodulinopathy containing antisense oligonucleotides, wherein:
   (1) A step of contacting the antisense oligonucleotide with cardiomyocytes derived from pluripotent stem cells having a mutation in the calmodulin gene.
   (2) The step of measuring the expression level of the calmodulin gene in the contacted myocardial cells, and (3) the case where the expression level of the calmodulin gene measured in the step (2) was not contacted with the antisense oligonucleotide. A method comprising the step of comparing the expression level of the calmodulin gene in the myocardial cells.
3. A method of screening for therapeutic, prophylactic and / or ameliorating agents of carmodulinopathy containing antisense oligonucleotides, wherein:
   (1) A step of contacting the antisense oligonucleotide with cardiomyocytes derived from pluripotent stem cells having a mutation in the calmodulin gene.
   (2) The step of measuring the calcium current amount in the contacted cardiomyocyte, and (3) the cardiomyocyte when the calcium current amount measured in step (2) is not contacted with the antisense oligonucleotide. A method comprising a step of comparing with the amount of calcium current in.
4. The method according to any one of claims 1 to 3, wherein the pluripotent stem cell is an iPS cell derived from a carmodulinopathy patient.
5. A method of screening for therapeutic, prophylactic and / or ameliorating agents of carmodulinopathy containing antisense oligonucleotides, wherein:
   (1) A step of contacting the antisense oligonucleotide with mammalian cells,
   (2) The step of measuring the expression level of the carmodulin gene in the contacted cells, and (3) the case where the expression level of the carmodulin gene measured in the step (2) was not contacted with the antisense oligonucleotide. A method comprising the step of comparing the expression level of the carmodulin gene in the cell.
6. The method according to claim 5, wherein the mammalian cell is a human liver cancer-derived cell or a mouse fibroblast.
7. The method according to any one of claims 1 to 6, wherein the calmodulin gene is at least one selected from the group consisting of CALM1, CALM2, and CALM3.
8. The method according to any one of claims 1 to 7, wherein the antisense oligonucleotide is a CALM2 antisense oligonucleotide.
9. The method according to any one of claims 1 to 8, wherein the antisense oligonucleotide inhibits the expression of the calmodulin gene.
10. A therapeutic, prophylactic and / or ameliorating agent for calmodulinopathy, which comprises an antisense oligonucleotide that inhibits the expression of the calmodulin gene.
11. The therapeutic, prophylactic and / or ameliorating agent according to claim 10, wherein the calmodulin gene is CALM2.
12. The therapeutic, prophylactic and / or ameliorating agent according to claim 10 or 11, wherein the antisense oligonucleotide inhibits the expression of the calmodulin gene in cardiomyocytes.
13. The therapeutic, prophylactic and / or ameliorating agent according to any one of claims 10 to 12, wherein the antisense oligonucleotide shortens the duration of action potential in cardiomyocytes.
14. The therapeutic, prophylactic and / or ameliorating agent according to any one of claims 10 to 13, wherein the antisense oligonucleotide suppresses the amount of calcium current in cardiomyocytes.
15. A method for treating, preventing and / or ameliorating calmodulinopathy, which comprises the step of administering to a patient with calmodulinopathy an effective amount of an antisense oligonucleotide that inhibits the expression of the calmodulin gene.
16. A compound that is an antisense oligonucleotide that inhibits the expression of the calmodulin gene for use in the treatment, prevention and / or amelioration of calmodulinopathy.
17. Use of antisense oligonucleotides that inhibit the expression of the calmodulin gene in the manufacture of drugs to treat, prevent and / or improve calmodulinopathy.

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