JP4463608B2 - SiRNA that specifically suppresses expression of mutant MJD gene - Google Patents

Description

  The present invention relates to siRNA (short interfering RNA) capable of specifically suppressing the expression of a mutant allele of the Macado-Joseph disease gene, its use, and the like.

  Macado-Joseph disease (MJD) is an autosomal dominant inherited neurodegenerative disease characterized clinically by cerebellar ataxia, cone and extrapyramidal signs, peripheral neuropathy and ocular muscle paralysis, 10-15 Bedridden in about a year, treatment is currently only symptomatic treatment. The number of 3 base sequence (CAG) repeat sequences (CAG repeats) in the MJD1 gene, which is the causative gene, is 13 to 36 in normal individuals, while it extends to 62 to 82 in MJD patients. [1]. The etiology of MJD is thought to be “acquisition of toxic functions” by abnormally elongated polypolyglutamine of mutant MJD protein [2,3].

  Therefore, the most effective and simple treatment for MJD is to reduce this abnormal mutant protein. Furthermore, the wild-type MJD1 gene product (ataxin-3) is thought to play an important role in cell survival such as endoplasmic reticulum regulation [4] and DNA repair [5]. Only the mutant ataxin-3 needs to be selectively reduced without affecting this wild-type protein.

  RNA interference (RNAi) is a sequence-specific post-transcriptional gene silencing process initiated by double-stranded RNA (dsRNA). This has a multi-step process where a siRNA of 21-23 nucleotides is generated, thereby causing degradation of the homologous RNA. This siRNA needs to be long enough to mediate gene-specific repression, while being short enough to avoid the negative effects of dsRNA in mammalian cells.

  In recent years, using the very high ability to suppress the expression of a target gene due to RNA interference, application to the medical field is expected as a powerful tool for gene therapy of diseases.

An RNA interference method for the MJD1 gene has also been reported. This method focuses on the single nucleotide polymorphism of guanine / cytosine located directly under the CAG repeat, and converts the abnormal gene and normal gene into their primary sequence. It is identified from the difference (Non-Patent Document 1). The siRNA described in this report suppresses the expression of a sequence having Q ₁₆₆ C (base sequence in which “C” is bound immediately below 166 CAG repeats), while Q ₂₈ G (28 CAG repeats). The expression of a sequence having a base sequence (with a “G” bound immediately below) had a slight effect. However, Q ₂₈ C has not been investigated.

Japanese translation of PCT publication No. 2002-516062 Special table 2003-529374 gazette Special table 2003-535583 gazette Miller VM, Xia H, MarrGL, et al. Allele-specific silencing of dominant disease genes.Proc. Natl. Acad. Sci., USA 2003; 100: 7195-7200

With regard to the guanine / cytosine single nucleotide polymorphism located directly under the CAG repeat in the MJD1 gene, it has been found that there is an extreme bias between the mutant and the normal MJD1 allele. That is, mutant alleles have exclusively (CAG) nC, whereas normal alleles have both (CAG) nC and (CAG) nG at the same frequency [ 7,8] (Fig. 1A)
.

  Therefore, in the RNA interference method described in Non-Patent Document 1 based on simple single nucleotide polymorphism (difference in primary sequence), normal and abnormal genes cannot be distinguished in about half of MJD patients. However, since there is a possibility that the expression of a normal allele of (CAG) nC type may be suppressed with a probability of about 50%, it is expected to be a major obstacle in terms of clinical application.

  In order to solve the above problems, the present invention provides siRNA capable of specifically distinguishing RNAs derived from normal alleles and abnormal (mutant) alleles in the MJD1 gene.

That is, the present invention relates to siRNA that can distinguish between wild-type alleles and mutant alleles of the Macado-Joseph disease gene.
Furthermore, the present invention relates to an siRNA capable of specifically binding to mRNA derived from Macada-Joseph disease mutant allele, independent of the primary sequence of RNA.

  The present invention also relates to a carrier containing the siRNA as an active ingredient and a DNA vector that expresses the siRNA.

  The present invention also relates to a composition comprising any of the above siRNA, carrier, or DNA expression vector. This composition can be used, for example, for the treatment of Macad-Joseph disease for pharmaceutical use.

  Furthermore, the present invention provides a method for specifically inhibiting the expression of a mutant allele of a Macado-Joseph disease gene by transfection of a cell with the siRNA, and the action of the siRNA incorporated into the cell, and the siRNA The present invention relates to a method of transfecting a cell and reducing the cytotoxicity caused by the mutant allele product of the Macado-Joseph disease gene by the action of siRNA incorporated into the cell. Although there is no restriction | limiting in particular in the kind of this cell, For example, mammalian cells, such as a human as used in the Example of this specification, can be mentioned.

  The siRNA of the present invention makes it possible to specifically suppress only the expression of an abnormal mutant gene without impairing the expression of the normal gene of the patient in the genotypes of all patients with Macad-Joseph disease. It was.

The siRNA of the present invention can be prepared by any method known to those skilled in the art. For example, as shown in the following examples, it can be easily chemically synthesized by methods well known to those skilled in the art. As a preferred specific example of the siRNA of the present invention, siRNA containing the base sequence: 5′- g cag cag cag cgg gac cua-3 ′ (SEQ ID NO: 1) can be mentioned. This base sequence is the sequence described in Non-Patent Document 1 (5′-CAG CAG CAG
CGG GAC CTA TC-
Compared with 3 ', three bases are different (underlined portion in each sequence above).

  In addition, in order for RNA interference to occur in an animal cell, siRNA needs to have about 21 to 23 bases and 2 bases protrude from each 3 'side. Therefore, more specifically, the siRNA of the present invention having the above-mentioned sense strand is, for example, shown as siRNA MJD3 in FIG. 1 of the present specification.

  For example, when the siRNA of the present invention is used for the treatment of Macad-Joseph disease, any drug delivery system known in the art can be used. RNA itself is an unstable substance. For example, when it is injected into blood, it is degraded by an RNA degrading enzyme. Therefore, siRNA is modified by encapsulating siRNA in a carrier such as various functional liposomes or polymer micelles, or by binding a target induction molecule such as an adhesion factor or antibody molecule or PEG to both ends of the siRNA. However, it is possible to increase the stability in blood or cells.

  As another method for effectively expressing and acting on siRNA in the body, there is an expression vector for the siRNA of the present invention, that is, an expression DNA vector obtained by incorporating a DNA corresponding to the siRNA as a foreign DNA fragment. As such an expression vector, an appropriate vector system known to those skilled in the art can be appropriately selected and prepared by an appropriate method known in the art. Such a DNA fragment corresponding to the siRNA of the present invention can also be easily prepared by any method known to those skilled in the art.

  In such an expression vector, a “hairpin type” or “stem” in which a DNA sequence corresponding to the sense strand of siRNA and a DNA sequence corresponding to the antisense strand are encoded in one DNA downstream of the promoter. There is a type called "loop type". This is transcribed in the body into hairpin (stem loop) RNA, and then undergoes an enzymatic reaction to produce siRNA.

  Specific examples of such expression vectors include various virus vectors such as adenovirus vectors and adeno-associated virus vectors. Adenovirus vectors can introduce genes into a wide range of cells including cells with low division rate such as nerve cells, and the in vivo conductivity is almost 100%. Transgenes usually reside outside the host cell chromosome and are rarely integrated into the chromosome. Therefore, the expression of the transgene is transient and usually 1 to 4 weeks. In addition, adeno-associated virus vectors are highly safe and can be introduced into non-dividing cells such as nerve cells. Furthermore, since the transgene is integrated into the host chromosome, the transgene can be introduced for more than one year. It is a system capable of long-term expression.

  A carrier containing the above siRNA as an active ingredient and an siRNA expression vector can be prepared by methods known to those skilled in the art. Moreover, the composition containing these contains arbitrary components known to those skilled in the art, such as buffers and adjuvants, depending on the purpose, components and the like, and further, depending on the use and administration method thereof. It can be in any form such as a solution, suspension or emulsion.

  In the method of the present invention, transfection of cells can be performed by any method known to those skilled in the art. For example, transfection can be performed in vivo by administering the above-described composition of the present invention into an animal body including a human by an appropriate route using an appropriate means such as injection. In such a case, the method of the present invention will function as a gene therapy method for McDough Joseph patients. Alternatively, as shown in the examples below, transfection can be performed in vitro on various cultured cell lines.

  EXAMPLES Hereinafter, although this invention is concretely demonstrated according to an Example, the technical scope of this invention is not restrict | limited at all by these description.

Plasmid construction and siRNA design
The ataxin-3 expression plasmid was provided by Dr. Akira Kakizuka [4]. MJDI in the plasmid
cDNA is 22 (normal, pCMX HA-Q22C) or 79 (extended pCMX
HA-Q79C) CAG repeats and a binding fragment in which hemagglutinin (HA) was fused to the N-terminus thereof. pCMX
The cytosine immediately after CAG repeat in HA-Q22C is a QuikChange site-directed mutagenesis system (Stratagene, La
Jolla (CA)) and changed to guanine (pCMX HA-Q22G). Furthermore, these MJD cDNAs were sub-rotated into pIRES-hrGFP-la (Stratagene) to obtain pGFP-Q22G, pGFPQ22C and pGFP-Q79C, respectively.

Four different siRNAs were designed to target the CAG repeat and the binding site at its 3 ′ end (FIG. 1B), and these RNAs were chemically synthesized by methods known to those skilled in the art. In other words, the nucleic acid synthesizer (ABI)
After adding dT with Expedite, Inc., the siRNA was provided with single-base changes in the C / G polymorphism at positions 5, 8, 11 and 11 from the 5 ′ end of the sense strand in siRNA, respectively.
MJD1, siRNA MJD2, siRNA MJD3, and siRNA MJD4 were synthesized and purified using HPLC.

Transfection and Western blot
Humans in which each siRNA (10 or 25 nM) and ataxin-3 expression plasmid (4 μg) are cultured in a 6-well culture plate for the purpose of examining the effect of the siRNA prepared above on MJD RNA in the expression of ataxin-3 Fetal kidney cell line 293T (purchased from ATCC) was cotransfected using Lipofectamine Plus reagent (Life Technologies, Rockville, MD). siRNA was annealed and then transfected according to the method described in Ref. [9]

24 hours after transfection, TNG buffer (50m) containing a protease inhibitor cocktail (Roche)
Cells were harvested with Tris-HCl, 150 mM NaCl, 1% Triton X-100), separated on a 15% SDS polyacrylamide gel, and transferred onto polyvinylidene difluoride thin film (Bio-Rad). Mouse monoclonal anti-HA antibody (Roche) reaction, enhanced chemiluminescence detection kit (ECL;
Amerham Pharmacia Biotech).

As a result, mutant ataxin-3 (Q79C) was most effectively suppressed by 25 nM siRNA MJD3. In addition, the signal intensity on the Western blot decreased by 96.0% compared to the control (FIG. 2A). In contrast, the expression of wild type ataxin-3 (Q22G) is
Although moderately suppressed by MJD4, it was clearly not affected by siRNA MJDl, siRNA MJD2, and siRNA MJD3. From the above results, siRNA which is an example of the siRNA of the present invention
MJD3 best recognized one nucleotide change between C and G, had little effect on Q22G expression, and specifically suppressed Q79C expression. SiRNA for Q79C expression
The effect of MJD3 was dose-dependent, whereas the suppression of Q22G was similar with 10 nM and 25 nM siRNA. That is, 25nM siRNA MJD3 hardly suppresses Q22G (5.9%), and only slightly suppresses Q22C (22.5%), whereas Q79C is 96.0%
Was also suppressed. Unexpectedly, despite the identical target sequences in (CAG) 79C and (CAG) 22C, siRNA MJD3 did not significantly reduce the expression of another wild type allele (Q22C) ( FIG. 2B).

  Furthermore, the above trend in suppression results shows that DsRed fluorescence is used as a control for transfection efficiency, and the fluorescence of GFP expressed by the internal ribosome entry site with ataxin-3 (pGFP-Q22C or pGFP-Q79C) decreases. (FIG. 2C).

  From the above results, it was confirmed that the siRNA of the present invention can distinguish between the wild-type allele and the mutant allele of the Macado-Joseph disease gene. Furthermore, it was confirmed that the siRNA of the present invention specifically binds to mRNA derived from Macad-Joseph disease mutant allele independently of the primary sequence of RNA.

Evaluation of cell death To assess the effect of siRNA on cell death caused by the expression of variant-3, Neuro2a cells (purchased from ATCC) in 24-well culture plates were transformed into pCMXQ79C (1 μg / well) and siRNA.
Co-transfected with MJD3 (100 nM / well). Forty-eight hours after transfection, Neuro2a cell death was determined by trypan blue exclusion and Cytotox.
It was determined by analyzing both cytoplasmic lactate dehydrogenase (LDH) activity measurements by 96 non-radioactive cytotoxicity assay (Promega).

As a result, 48 hours after transfection, the expression of mutant MJD (Q79C) was toxic to Neuro2a. However, the expression of wild type ataxin-3 (Q22G and Q22C) did not affect cell survival. SiRNA
MJD3 significantly suppressed the toxicity of the mutant ataxin-3, thereby reducing cell death by 62.8% (LDH test) and 75.9% (trypan blue exclusion method), respectively.
Statistical analysis consists of Student t-test and single factor ANOVA, followed by Fisher's
This was done using the protected 1east-significant differenc post hoc test.

  The present invention is not limited by the following theoretical considerations, but the siRNA of the present invention can specifically distinguish RNAs derived from normal alleles and abnormal (mutant) alleles in the MJD1 gene. A possible reason is that not all RNA sequences are equally accessible to siRNA. That is, certain target sequences may be embedded within their secondary structure, especially if the target RNA is highly folded [10]. Furthermore, the optimal target site of siRNA for highly folded RNA is almost the same as for ribozyme, and its cleavage efficiency is highly influenced by the secondary structure of the target RNA [9].

The target site of the C / G polymorphism of the MJD gene is just downstream of the CAG repeat that shows a tight stem morphology in secondary structure on computer prediction. Therefore, MJD due to large difference in GAG repeat length
It is possible that changes in the secondary structure of RNA may affect the effects of siRNA MJD3.

Alternatively, another possibility exists that there is an RNA binding protein that binds preferentially to (CAG) 22C over (CAG) 79C, and thus siRNA
This means that MJD3 is prevented from approaching (CAG) 22C. Although an RNA binding protein for MJD mRNA has not yet been discovered, CUG repeats in the myotonic dystrophy protein kinase gene have been found to bind to CUG binding proteins in a repeat length-dependent manner [11]. Furthermore, the existence of an RNA binding protein that adheres to CAG repeats in Huntington's disease has been suggested [12].

[Cited document]
1. Kawaguchi Y, Okamoto T, Taniwaki M,
et al. CAG expansions in a novel gene for
Machado-Joseph disease at chromosome
14q32.1. Nat Genet 1994; 8: 221-228.
2. Ross CA. When more is less:
pathogenesis of glutamine repeat neurodegenerative
diseases. Neuron 1995; 15: 493-496:
3. Ikeda H, Yamaguchi M, Satoshi 5, Aze
Y, Narumiya 5, Kakizuka A. Expanded
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p97 / valosin-containing protein (VCP) is
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HHR23A and HHR23B. Hum Mol Genet
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6. Elbashir 5, Harborth J, Lendeckel W,
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Futamura N, Hirano M, Ueno S.
Relationship of (CAG) nC configuration to
repeat instability of the Machado-Joseph
disease gene. Hum Genet 1996; 98: 643-645.
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  The siRNA-based nucleic acid drug of the present invention provides the possibility of gene therapy for McDough Joseph patients.

In the figure, A (upper) shows the G / G polymorphism immediately downstream of the CAG repeat in the human MJD1 gene. A (bottom) shows the distribution of G / G polymorphism and CAG repeat in 56 MJD patients. All extended alleles have (CAG) nC, while normal alleles have (CAG) nG and (CAG) nG at the same frequency (0.46 and 0.54). Arrows indicate CAG repeats in the ataxin3-expression vector construct used in the Examples herein. B shows the sequence of siRNA targeting (CAG) nG in MJD1 mRNA. Capital letters mean DNA. 2 is an electrophoresis photograph showing the specific inhibitory effect of the siRNA of the present invention on the expression of mutant MJD1. Each value in the A and B bar graphs represents the mean and SEM. C is a photograph showing a GFP image (x200) of 293T cells. The detection result by the LDH assay (*** p <0.0001) and trypan blue exclusion method (** p <0.001) which show the influence of siRNAMJD3 with respect to the toxicity by the variant Ataxin 3 in a mammalian cell is shown. Cytotoxicity was not shown by overexpression of Q22C or Q22G. The pcDNA3.1 plasmid (Invitrogen) was used as a mock transfection. Each value in the bar graph represents the average and SEM.

Claims (3)

SiRNA containing the base sequence: 5′-g cag cag cag cgg gac cua-3 ′ (SEQ ID NO: 1) in the sense strand, or the sense strand has the base sequence: 5′-g cag cag cag cgg gac cua tt-3 ′ A method in which a cell is transfected with an siRNA comprising , and the expression of a mutant allele of the Macado-Joseph disease gene is specifically suppressed by the action of the siRNA incorporated into the cell, except for in vivo in humans. SiRNA containing the base sequence: 5′-g cag cag cag cgg gac cua-3 ′ (SEQ ID NO: 1) in the sense strand, or the sense strand has the base sequence: 5′-g cag cag cag cgg gac cua tt-3 ′ A method of transfecting a cell with an siRNA consisting of and reducing cytotoxicity due to a mutant allele product of the Macado-Joseph disease gene by the action of the siRNA incorporated into the cell, except in vivo in humans. SiRNA containing the base sequence: 5′-g cag cag cag cgg gac cua-3 ′ (SEQ ID NO: 1) in the sense strand, or the sense strand has the base sequence: 5′-g cag cag cag cgg gac cua tt-3 ′ 3. Transfection is performed in vivo by administering a composition comprising either a siRNA comprising: a carrier containing the siRNA as an active ingredient; or a DNA vector expressing the siRNA. The method described.

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