KR101356469B1 - Pharmaceutical composition for prevention or treatment of a disease caused by motorneuronal cell death - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the prevention or treatment of diseases caused by motor neuron death. The pharmaceutical composition for preventing or treating diseases caused by motor neuronal cell death according to the present invention stabilizes the SMN protein by inhibiting polyubiquitination involved in the degradation of SMN protein, thereby preventing disease caused by motor neuronal cell death. Or has an effect on treatment.

Description

Pharmaceutical composition for the prevention or treatment of diseases caused by motor neuron death {PHARMACEUTICAL COMPOSITION FOR PREVENTION OR TREATMENT OF A DISEASE CAUSED BY MOTORNEURONAL CELL DEATH}

The present invention relates to a pharmaceutical composition for the prevention or treatment of diseases caused by motor neuron death.

Human survival motor neuron (SMN) genes are composed of two types, SMN1 and SMN2, on the telomere and centromere of autosomal 5q13.

Decreased SMN protein concentration due to deficiency of the SMN gene can cause the death of motor neurons and various diseases. Therefore, studies have been conducted to artificially increase the amount of SMN protein in cells (see Non-Patent Documents 1 and 2).

However, even if artificially increasing the intracellular SMN protein, it is inevitable that the SMN protein is degraded by the proteosome through polyubiquitination, which shortens the duration of the drug for increasing the SMN protein. There is a limit to the effect.

In addition, since the chemical composition is used in the process of artificially increasing the SMN, it may cause various side effects.

In the present invention, in order to overcome such limitations, attention has been paid to substances capable of inhibiting the degradation by polyubiquitination of SMN proteins.

In a previous study on the ASK1 protein, a novel function was revealed in the present invention, apoptosis due to stress caused by free radicals, cytokines that promote inflammatory reactions such as TNF-α, and deficiency of nerve growth factors such as NGF ( apoptosis has been concentrated on involvement in apoptosis.

However, the present inventors completed the present invention for the first time by discovering that a new function of the ASK1 protein stabilizes the SMN protein by inhibiting polyubiquitination, which is involved in the degradation of the SMN protein through binding to the SMN protein. .

 Le TT at al., 2005 Hum. Mol. Genet., 14: 845-857  Jarecki et al., 2005 Hum. Mol. Genet., 14: 2003-2018;

It is an object of the present invention to provide a pharmaceutical composition for the prevention or treatment of diseases caused by motor neuron death.

Another object of the present invention is to provide a kit for diagnosing a disease caused by motor neuron death.

In order to achieve the object of the present invention,

The present invention provides ASK1 (apoptosis signal-regulating kinase 1) protein or fragment thereof,

The gene of the ASK1 protein or fragment thereof, and

As ASK1 Protein Overexpression Vector

It provides a pharmaceutical composition for the prevention or treatment of diseases caused by motor neuron death, comprising one or more selected from the group consisting of.

The present invention also provides a kit for diagnosing a disease caused by motor neuron cell death comprising at least one siRNA selected from the group consisting of siRNA (small-interfering RNA) for ASK1 protein and siRNA for SMN protein.

The pharmaceutical composition for preventing or treating diseases caused by motor neuronal cell death according to the present invention stabilizes the SMN protein by inhibiting polyubiquitination involved in the degradation of SMN protein, thereby preventing disease caused by motor neuronal cell death. Or has an effect on treatment.

FIG. 1 is a diagram analyzing the mutual binding between ASK1 protein and SMN protein overexpressed in 293T cells by immunoprecipitation followed by Western blot.
Figure 2 is a diagram of the analysis of the interaction between the ASK1 protein and SMN present in HeLa cells at the cellular level through immunoprecipitation followed by Western blot.
Figure 3 is an analysis of the cross-linking between ASK1 protein and ASK1 (K709R) protein and SMN protein overexpressed in 293T cells via immunoprecipitation followed by Western blot.
Figure 4 is a diagram showing the binding of the ASK1 protein fragments and SMN protein.
5 is a diagram showing a relationship between the concentration of ASK1 protein or ASK1 (K709R) and the concentration of SMN protein.
6 is a diagram showing the effect of reducing the ASK1 protein on the SMN protein and its mRNA concentration change.
7 is a diagram showing the effect of ASK1 protein on the regulation of intracellular concentration of SMN protein.
8 is a diagram showing the effect of ASK1 protein on SMN1 and SMN2 proteins made from human SMN1 and SMN2 genes.
9 is a diagram showing changes in the amount of SMN protein and mRNA according to the reduction of ASK1 protein.
Fig. 10 shows the relationship between the concentration of ASK1 protein and the stability of SMN protein.
Figure 11 is a diagram of the correlation between the reduction of ASK1 protein and polyubiquitination of SMN protein.
12 is a diagram showing the correlation between kinase activity of ASK1 protein and polyubiquitination of SMN protein.
Figure 13 is a diagram showing the quantitative correlation between the ASK1 protein and SMN protein and the effect of the two proteins on neuronal differentiation.
FIG. 14 is a graph illustrating the length of cells having nerve segments and nerve segments in FIG. 13.
15 is a graph showing the relationship between the decrease of ASK1 protein and SMN protein and the amount of β-Actin, which is an indicator of neuronal segment differentiation.

Hereinafter, the present invention will be described in detail.

According to the present invention,

ASK1 (apoptosis signal-regulating kinase 1) protein or fragment thereof,

The gene of the ASK1 protein or fragment thereof, and

As ASK1 Protein Overexpression Vector

It provides a pharmaceutical composition for the prevention or treatment of diseases caused by motor neuron death, comprising one or more selected from the group consisting of.

The ASK1 protein of the present invention is one of the upper MAPK kinase kinase (MAPKKKs) of the mitogen-activated protein kinase (MAPK) signaling pathway, and is a cascade method of SEK1 / MKK7, MKK3 / MKK6 It is a protein that finally activates MAPK such as JNK, p38 through lower MAPK kinase (MAPKKs).

The amino acid sequence of the ASK1 protein of the present invention is represented by SEQ ID NO: 1, the nucleotide sequence of the ASK1 protein gene is represented by SEQ ID NO: 3.

According to one embodiment according to the invention, the ASK1 protein binds to the SMN protein (see FIGS. 1, 2 and 3), wherein the binding of the ASK1 protein to the SMN protein occurs particularly strongly at certain sites (FIG. 4). Reference).

Since only some fragments of the ASK1 protein bind to the SMN protein, some fragments of the ASK1 protein may be used for the prevention and treatment of diseases caused by motor neuronal cell death according to the present invention.

A fragment of the ASK1 protein according to the present invention preferably has an amino acid sequence of SEQ ID NO: 2, the gene of the fragment of the ASK1 protein preferably has a nucleotide sequence of SEQ ID NO: 4.

In addition, the ASK1 protein overexpression vector of the present invention refers to a recombinant vector capable of overexpressing ASK1 protein in a cell.

The vector may be any vector used in the art for transformation, transduction or transfection of cells, but preferably a recombinant plasmid vector, more preferably a vector having a nucleotide sequence of SEQ ID NO: 5 good.

According to one embodiment according to the invention, it can be seen that the ASK1 protein binds to the SMN protein (see FIGS. 1, 2 and 3).

According to another embodiment according to the present invention, SMN protein was maintained in the intracellular concentration dependent on the concentration of ASK1 protein (see FIGS. 5 and 9). That is, the concentration of SMN protein could be controlled by ASK1 protein. Regulation of the SMN protein was regulated at the protein level, not at the mRNA level (see FIGS. 6 and 9).

It was proved that the ASK1 protein according to the present invention stabilizes the SMN protein by inhibiting polyubiquitination of the SMN protein (see FIGS. 11 and 12).

According to another embodiment according to the present invention, it was confirmed that the reduction of ASK1 protein affects the differentiation of nerve segments (see FIGS. 13, 14 and 15).

Taken together, it can be easily seen that the ASK1 protein helps to stabilize the SMN protein and thus can help to differentiate the neuronal segment.

In particular, when the ASK1 protein decreases, the amount of β-Actin, which is an indicator of neuronal differentiation, decreases with the amount of SMN, the pharmaceutical composition according to the present invention is not only for spinal muscular atrophy (SMA) but also for Parkinson's disease ( It is evident that it will be effective against Parkinson's disease, Alzheimer's disease and atrophic axon sclerosis (ALS).

Disease caused by motor neuron cell death in the present invention means a disease caused by the deficiency of the SMN protein in the cell, preferably, spinal muscular atrophy (SMA), Parkinson's disease (Parkinson's disease), Alzheimer's disease (Alzheimer's disease) and amyotrophic axonal sclerosis (ALS).

The pharmaceutical composition for preventing or treating diseases caused by motor neuronal cell death according to the present invention stabilizes the SMN protein by inhibiting polyubiquitination, which is involved in the degradation of the SMN protein, so that the SMN protein in the cell is not reduced. It can prevent or treat diseases caused by motor neuron death.

According to one embodiment according to the invention, for cells with reduced ASK1 protein, polyubiquitination of the SMN protein proceeded (see FIG. 11), which was not related to the kinase activity known as the conventional function of the ASK1 protein (FIG. 11). 12).

Further, according to the present invention,

Provided is a kit for diagnosing a disease resulting from motor neuron cell death comprising at least one siRNA selected from the group consisting of siRNA (small-interfering RNA) for ASK1 protein and siRNA for SMN protein.

The siRNA for the ASK1 protein according to the present invention preferably has an oligonucleotide sequence of SEQ ID NO: 6 or SEQ ID NO: 7, and the siRNA for an SMN protein preferably has an oligonucleotide sequence of SEQ ID NO: 8.

Diseases caused by motor neuronal cell death that can be diagnosed in the diagnostic kit according to the present invention include spinal muscular atrophy (SMA), Parkinson's disease, Alzheimer's disease and atrophic axonal sclerosis (ALS). It means one or more selected from the group consisting of.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Example 1 Cross-linking Analysis of ASK1 Protein and SMN Protein

The present inventors transfected ASK1-Flag and SMN1-Myc into human kidney cell line 293T to confirm whether ASK1 protein and SMN protein can bind to each other in cells. After 48 hours, the transfected cells were washed with PBS (phosphate-buffered saline) solution, and then NETN buffer solution containing protease inhibitors (0.5% Nonidet P-40 (NP-40), 1 mM EDTA, 50). Cells were hemolysed with mM Tris-HCl (pH 7.5), 120 mM NaCl, 1 mM phenylmethylsulfonyl fluoride (PMSF), 2 μg / ml leupeptine, 2 μg / ml aprotinin, 2 mM Na ₃ VO ₄ ), and then to 12,000 g. Centrifuge at 4 ° C. for 15 minutes. After centrifugation, the precipitate was removed and an anti-Flag antibody was added to the obtained lysate to perform immunoprecipitation. Protein-G sepharose was added to the reaction solution and mixed at 4 ° for 1 hour. The immunocomplex obtained therefrom was subjected to SDS-polyacrylamide gel electrophoresis to perform western blot using anti-Myc antibody. As a result, it was found that the ASK1 protein and the SMN protein bind to each other in the cell (see FIG. 1).

We first found yeast 2-hybrid screening using the human HeLa cDNA library and found that ASK1 protein binds to SMN protein, so that cellular level concentrations in HeLa cells The binding between the two proteins present in the cells was analyzed using immunoprecipitation as in 293T cells. As a result, it was found that the two proteins are mutually bound even at the cellular level (FIG. 2).

In order to investigate whether the binding of ASK1 protein and SMN protein is affected by the kinase activity of ASK1 protein, ASK1-Flag with kinase activity and ASK1 (K709R) -Flag without kinase activity together with SMN1-Myc 293T cells After transfection, immunoprecipitation and Western blot were performed by the method as described in FIG. 1. As a result, it was found that the kinase activity of the ASK1 protein did not affect the mutual binding of the ASK1 protein and the SMN protein (see FIG. 3). The amino acid sequence of the ASK1 (K709R) protein is shown in SEQ ID NO: 9.

The inventors constructed an ASK1 protein deletion mutant to investigate where the SMN protein binds to the ASK1 protein. Specifically, the ASK1 protein N-terminus 1 to amino acid ASK1 protein containing amino acids 936-ΔC, the ASK1 protein N-terminal ASK1 protein-ΔN, ASK1 protein comprising amino acids from 649 to C- terminal 1375 The nucleotide sequence region of the ASK1 protein encoding the ASK1 protein-NT containing amino acids N-terminals 1 to 656 was inserted into the pcDNA3-HA vector. The ASK1 protein fragment prepared above is shown schematically in FIG. 4, above (FIG. 4, above).

HA-ASK1 protein, HA-ASK1 protein-ΔC, HA-ASK1 protein-ΔN, HA-ASK1 protein-NT and SMN1-Myc were transfected with 293T, and then anti-Myc as in <Fig. 1>. Immunoprecipitation was carried out using antibodies and Western blots were performed using anti-HA antibodies. As a result, it was found that all fragments of ASK1 protein and SMN1 were mutually bound. In particular, HA-ASK1 protein-ΔN from which the N-terminus of ASK1 protein, which is known to inhibit kinase activity of ASK1 protein, was removed, showed stronger binding with SMN protein than other deletion mutants of ASK1 protein, and anti-Myc. As can be seen from the Western blot using the antibody, the expression of HA-ASK1 protein-ΔN alone can increase the intracellular concentration of SMN protein (FIG. 4, middle).

In vitro binding experiments were performed to determine whether ASK1 protein and SMN protein can be directly bound. HA-ASK1 protein, HA-ASK1 protein-ΔC, HA-ASK1 protein-ΔN, HA-ASK1 protein-NT were converted into TNT reticulocyte lysate system (Promega) and ³⁵ S-mesay methionine ⁽³⁵ S-methionine) using transferred from the in vitro (transcription) and translated (translation) occurs by the ³⁵ S- methoxy Im methionine ⁽³⁵ S-methionine) labeling the protein with the ASK1 was obtained ASK1 protein deletion mutants (Figure 4, down-right). The ³⁵ S-methionine labeled ASK1 protein and ASK1 protein fragments were treated with GST-SMN1 and GST-buffered buffer A (50 mM Tris-HCl, pH 7.5), 150 mM NaCl, 2 mM EDTA, 1 mM dithiothreitol, 0.1% NP-40, 5 mg / ml BSA) and reacted at 4 ° for 1 hour. Glutathione-agarose beads were added to allow GST and GST-SMN1 protein to bind, followed by buffer B (50 mM Hepes (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol). , 0.1% Tween 20) three times, washed with GST-SMN1 combined ³⁵ S- methionine methoxy Im (by ³⁵ S-methionine) is a labeled ASK1 protein eluted ASK1 protein deletion mutants SDS- polyacrylamide gel electrophoresis After electrophoresis (SDS-polyacrylamide gel electrophoresis), autoradiograms were analyzed on BAS 2500 (Fuji). As a result, as in the immunoprecipitation method of <Fig. 4, middle>, all fragments of the ASK1 protein and the SMN1 protein can be seen to directly bind. In particular, it was found that ASK1 protein-ΔN including the ASK1 protein amino acid sequence 649 to 1375 site directly binds to SMN (FIG. 4, bottom).

Example 2- Whether the Effect of ASK1 Proteins on Increasing the Concentration of SMN Proteins Acts at RNA Level or Protein Level

The inventors of the present invention, when the expression of ASK1-Flag and SMN1-Myc at the same time as shown in the Western blot performed using the anti-Myc antibody of Figure 1 when the protein amount of SMN1-Myc express only SMN1-Myc The phenomenon was found to increase significantly.

Therefore, in order to determine whether the effect of increasing the intracellular concentration of ASK1-Flag in SMN1-Myc is proportional to the concentration of ASK1-Flag expressed in the cell or ASK1 (K709R) -Flag without kinase activity, In the case of sequentially increasing the concentrations of SMN1-Myc and ASK1-Flag or ASK1 (K709R) -Flag, the intracellular concentrations of SMN1-Myc were also proportional to the concentration of ASK1-Flag and ASK1 (K709R) -Flag. It was found to increase (see FIG. 5).

In order to test the stabilizing effect of SMN protein by ASK1 protein, ASK1 protein-siRNA was prepared by specifically binding to ASK1 protein mRNA and degrading ASK1 protein mRNA to reduce the concentration of ASK1 protein in the cell, and then the control siRNA (control-siRNA). 293T cells were transfected in parallel.

As shown in lanes 1 and 2 of FIG. 6, when the concentration of ASK1 protein in cells was decreased by ASK1 protein-siRNA, the amount of SMN protein also decreased.

Next, after treating cycloheximide (CHX, 100 μM), an inhibitor that inhibits the synthesis of new proteins, the effect of ASK1 protein on the stability of the existing SMN protein was examined. As shown in Fig. 4, the concentration of SMN mRNA was not affected by ASK1 protein (under FIG. 6, RT-PCR results), whereas the intracellular concentration of ASK1 protein was reduced by siRNA (293T / ASK1 protein-). siRNA) It was found that the stability of the SMN protein was reduced (see FIG. 6, Western blot results).

To demonstrate that ASK1 protein directly affects the regulation of intracellular concentrations of SMN proteins, ASK1 is used in 293T cells (293T / ASK1 protein-siRNA) that artificially reduced the amount of intracellular ASK1 protein using ASK1 protein-siRNA. -Flag or ASK1 (K709R) -Flag was transfected. As shown in the results of the Western blot using the anti-SMN antibody of FIG. 7, the amount of SMN protein in the cells was increased by the introduction of the ASK1 protein (see FIG. 7).

The effect of stabilizing the ASK1 protein SMN protein was found in both SMN1 and SMN2 (or SMNΔ7) protein (see Fig. 8).

Example 3 A mechanism for ASK1 protein stabilization of SMN protein

In order to investigate the SMN protein stabilization effect of ASK1 protein in motor neurons, the intracellular concentration of ASK1 protein was artificially reduced by using ASK1 protein-siRNA in NSC34 cells. The NSC34 cells thus prepared were named NSC34 / ASK1 protein-siRNA, and when produced as control si-RNA, NSC34 / Control1-siRNA was named.

In this case, similar to the results in 293T cells described above, the amount of SMN protein also decreased as the amount of ASK1 protein decreased (see FIG. 9, Western blot results).

In contrast, the amount of SMN mRNA was not affected by changes in the concentration of ASK1 protein (see Figure 9 below, RT-PCR results). The stability of SMN protein in NSC34 / ASK1 protein-siRNA cells was compared with the results of treatment with cycloheximide (CHX, 100 μM) for 1 hour, 2 hours, and 4 hours, respectively, and then to NSC34 / Control1-siRNA cells. .

Western blot results using the anti-SMN antibody shown in FIG. 10 show that, when the intracellular concentration of ASK1 protein is reduced in motility neurons, the stability of SMN protein is significantly reduced compared to NSC34 / Control1-siRNA cells. Could know. The density of the protein band on the western blot was quantified and shown as a graph as shown above in FIG. 10 (see FIG. 10).

SMN proteins are known to be degraded through the ubiquitin / proteasome pathway. One of the characteristics of proteins that are degraded through proteasomes is preceded by polyubiquitination.

Therefore, we compared the degree of polyubiquitination of SMN protein in NSC34 / ASK1 protein-siRNA cells and NSC34 / Control1-siRNA cells. Lysates of two cells were obtained as described in Example 1 above, followed by immunoprecipitation with anti-SMN antibody and Western blot with anti-ubiquitin (Ub) antibody. .

As a result, it was observed that polyubiquitination of SMN protein was significantly increased compared to control cells when the amount of ASK1 protein in cells was reduced (see FIG. 11).

Therefore, it was found that ASK1 protein has an effect of stabilizing SMN protein by inhibiting polyubiquitination of SMN protein and consequently, degradation of SMN protein through proteasome.

To investigate whether the inhibition of polyubiquitination of the ASK1 protein against SMN protein is affected by the kinase activity of ASK1 protein, 293T cells were transfected with SMN1-Myc, HA-Ub, ASK1-Flag or ASK1 (K709R) -Flag. After infection, the effect of ASK1-Flag or ASK1 (K709R) -Flag on polyubiquitination of SMN1-Myc was analyzed. The amino acid sequence of the human ASK (K709R) protein was as SEQ ID NO: 9.

In order to observe polyubiquitination of SMN1-Myc, proteasome inhibitor MG132 (10 μM) was treated for 6 hours before hemolysis of the transfected cells.

The transfected cells were washed with PBS and buffered (1% Nonidet P-40 (NP-40), 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 10 mM Na ₂ P _2). Cells were hemolyzed with O ₇ , 10 mM NaF, 2 mM Na ₃ VO ₄ , 1 mM PMSF, 2 μg / ml leupeptin, 2 μg / ml aprotinin), and then centrifuged at 12,000 g for 15 minutes at 4 ° C.

After centrifugation, the precipitate was removed, an anti-Myc antibody was added to the obtained lysate, and immunoprecipitation was performed, followed by Western blot with an anti-HA antibody.

As a result, ASK1 protein inhibited polyubiquitination of SMN regardless of its kinase activity (see FIG. 12).

Example 4 Effects of ASK1 and SMN Protein Reduction on Neuronal Differentiation in Motor Neurons

In the above embodiments of the present invention, first, when ASK1 protein and SMN protein were overexpressed at the same time, ASK1-Flag was shown to increase the intracellular concentration of SMN-Myc, and secondly, human kidney cell line 293T. In human motility neuronal cell line NSC34, when ASK1 protein-siRNA was used to artificially decrease the concentration of ASK1 protein in cells, the concentration of SMN protein in cells decreased in proportion to ASK1 protein concentration. We then investigated whether overexpressing ASK1-Flag and ASK1 (K709R) -Flag could increase the amount of SMN protein present in cells.

As shown in FIG. 13, A, overexpression of ASK1-Flag and ASK1 (K709R) -Flag showed that the concentration of SMN protein was increased.

13, B and C showed that the mutual binding between the ASK1 protein and the SMN protein is closely related to the differentiation of motor neurons.

Differentiation-induced media was obtained by transfecting NSC34 cells with ASK1 protein-siRNA (NSC34 / ASK1 protein-siRNA) and SMN-siRNA (NSC34 / SMN-siRNA) with control siRNA. Neuronal outgrowth was induced in DMEM / F-12 [1: 1], 1% FBS, and nonessential amino acid.

Here, the NSC34 / Conrol1-siRNA and NSC34 / Conrol2-siRNA transfected with the control siRNA are double-stranded oligonucleotides encoding partial sequences of the green fluorescent protein (GFP). PSuper retro vector (Oligoengine), which is the same vector as is inserted, and pSilencer 4.1-CMV vector (Ambion), which is the same vector where SMN-siRNA is inserted.

Differentiation-inducing medium induced nerve segment differentiation for 6 days by changing the medium every two days. To stain β-Actin as a marker of neural segment differentiation, the cells were fixed with 3.7% paraformaldehyde for 1 hour and then perforated for 5 minutes with 0.5% Triton X-100. permeabilization).

Blocking with 3% bovine serum albumin (BSA) for 30 minutes, staining β-Actin with rhodamine-phalloidin and observed with Zeiss Axioplan 2 microscope (Carl Zeiss Inc.) 13, B and C).

The length of the nerve segment (Neurite) was measured at 200 times magnification 12 or more using the Metamorph image analysis program (Universal Imaging). At this time, the length of the nerve segment was excluded 20μm or less.

After inducing neuronal differentiation of the motility neurons, neuron-bearing cells and neuron lengths were compared and measured, and the graphs are shown in FIG. 14. In addition, the changes of ASK1 protein, SMN protein, and β-Actin protein were changed to anti-ASK1 protein, anti-SMN, anti-Actin antibody and GAPDH, respectively, during the 6 days of induction of neuronal differentiation. Western blot was used to investigate.

As a result, it was found that β-Actin was markedly reduced in NSC34 / ASK1 protein-siRNA and NSC34 / SMN-siRNA cells, and that neuronal differentiation was significantly impaired compared to the control group (see FIG. 15).

In summary, the ASK1 protein is a protein having a novel function of binding to the SMN protein, and the ASK1 protein undergoes polyubiquitination, which is involved in the degradation of the SMN protein, regardless of its kinase activity through the interaction with the SMN protein. It can be seen that there is a function to stabilize the SMN protein by inhibiting.

In addition, when the ASK1 protein decreases, the amount of β-Actin, which is an indicator of neuronal differentiation, decreases along with the amount of SMN protein. It is evident that it will be effective for Parkinson's disease, Alzheimer's disease and atrophic axon sclerosis (ALS).

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Claims (9)

delete delete delete delete delete delete Spinal muscular atrophy (SMA) comprising at least one siRNA selected from the group consisting of siRNA (small-interfering RNA) for ASK1 protein represented by SEQ ID NO: 6 or SEQ ID NO: 7 and siRNA for SMN protein represented by SEQ ID NO: 8 ) Diagnostic kit. delete delete

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