KR20150122011A - Pharmaceutical Compositions for Preventing or Treating Spinal Muscular Atrophy - Google Patents

Abstract

The present invention provides a pharmaceutical composition for preventing or treating spinal muscular atrophy comprising PTB-associated splicing factor (PSF) or polynucleotide coding the same as an active ingredient. According to the present invention, the PSF increases mRNA formation comprising exon 7 when splicing SMN2 pre-mRNA. Therefore, the composition of the present invention can prevent or treat spinal muscular atrophy by controlling splicing of SMN2 pre-mRNA.

Description

Technical Field [0001] The present invention relates to a pharmaceutical composition for preventing or treating spinal muscular atrophy,

The present invention relates to a pharmaceutical composition for preventing or treating spinal muscular atrophy.

Spinal muscular atrophy (SMA) is an autosomal recessive genetic disorder that occurs at a rate of one per 11,000 newborns. In Type I SMA patients, motor neurons in the spinal cord's forehead are severely damaged and do not support the respiratory system, usually before the age of two. As a result, SMA is one of the leading causes of infant mortality. The genetic cause of SMA is the deletion or mutation of the SMN1 gene (1). There are two SMN genes in humans, and SMN1 genes are defective in SMA patients. The SMN1 gene encodes a full-length functional SMN protein. Functional SMN proteins regulate the assembly and degradation of the U snRNP complex and may also control the transport of -actin mRNA in neurons (2-9). On the other hand, the SMN2 gene remains intact and intact in SMA patients, but contains several different nucleotides from the SMN1 gene (10-12). Because of this, the SMN2 pre-mRNA is spliced into an mRNA variant encoding the SMN7 protein that lacks the exon 7 essential for the full function of the SMA protein. Since SMN7 protein does not form oligomers well, it breaks down rapidly and is almost nonexistent in cells (13). In SMA patients, most SMN7 proteins are produced that do not function from the SMN2 gene, and only a small amount of the full-length functional SMN protein is produced (14, 15). Therefore, when SMN2 mRNA containing exon 7 is produced in SMA patients, the problem caused by deletion or damage of SMN1 gene can be compensated to some extent. Based on these results, many studies have been made to treat SMA by enhancing the inclusion of exon 7 into SMN2 mRNA, thereby producing more full-length functional SMN protein.

Exon 7 splicing of SMN2 pre-mRNA is regulated by complex positive and negative factors. hnRNP A1 inhibits the inclusion of exon 7 into SMN mRNA, while the C-T mutation of SMN2 pre-mRNA promotes skipping of exon 7 by providing a binding site for hnRNP A1 (16, 17). On the other hand, SRSF1 interacts with the C-T mutation and promotes the inclusion of exon 7 (18). In addition, tra2, SRp30, hnRNP Q, and Sam68 are known as regulators that promote the inclusion of exon 7 (19-22). Several RNA sequences on exon 7, intron 6 and intron 7 have also been identified as enhancers or inhibitors for exon 7 splicing of SMN2 pre-mRNA (23-25). However, in order to develop better therapeutic agents targeting the splicing of exon 7, it is necessary to understand the splicing mechanism of exon 7 in more detail.

The PTB-associated splicing factor (PSF) was first discovered as a protein complex with PTB (27, 28). Since PTB-PSF complexes are necessary at the beginning of spliceosome formation and are essential in the catalytic step II (27, 29, 30), PSF plays an important role in the regulation of pre-mRNA splicing It has been thought to do. For example, PSF is known to regulate alternative splicing of tau and CD45 pre-mRNA (31, 32), and is associated with a purine rich sequence at the 3 'end of U5 snRNA Can mediate the stimulation of transcriptional activator-dependent pre-mRNA processing (33). In addition to its role in pre-mRNA splicing, PSF plays an important role in cancer formation and cancer cell proliferation as well as in transcription and translation (34-40).

The present inventors have found that PSF is a novel regulator of SMN2 pre-mRNA splicing. That is, it was confirmed that PSF interacted with GAAGGA, an enhancer sequence of exon 7, to improve the inclusion of exon 7 into SMN2 mRNA. These findings provide clues to further understand how splicing of SMN2 pre-mRNAs is regulated and open up the possibility of developing new therapeutic agents.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have made efforts to develop a method for preventing or treating spinal muscular atrophy by controlling the splicing of SMN2 pre-mRNA. As a result, it was found that mRNA production including exon 7 was significantly increased during SMN2 pre-mRNA splicing by overexpression of PSF in spinal muscular atrophy patients, and by using this, Thereby completing the present invention.

It is therefore an object of the present invention to provide a pharmaceutical composition for the prevention or treatment of spinal muscular atrophy.

It is yet another object of the present invention to provide a method for screening for a therapeutic agent for spinal muscular atrophy.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating spinal muscular atrophy comprising, as an active ingredient, a PTB-associated splicing factor (PSF) or a polynucleotide encoding the same.

The present inventors have made efforts to develop a method for preventing or treating spinal muscular atrophy by controlling the splicing of SMN2 pre-mRNA. As a result, it was confirmed that mRNA production of exon 7-containing type was significantly increased in SMN2 pre-mRNA splicing by overexpression of PSF in patients with spinal muscular atrophy, and the prevention or treatment of the disease was confirmed by using this.

Spinal muscular atrophy is known to be a genetic disorder caused by lack of functional SMN protein production due to deletion or mutation of SMN1 gene. According to the present invention, the PSF increases the production of exon 7-containing mRNA by selective splicing of SMN2 pre-mRNA, thereby increasing the production of functional SMN protein containing exon 7, Spinal muscular atrophy caused by massive production of SMN protein can be prevented or treated.

As used herein, the term exon 7-containing mRNA refers to SMN1 Or an exon 1 through exon 8 formed by splicing in pre-mRNA transcribed from SMN2 gene. The exon 7-containing mRNA encodes a functional SMN protein.

According to some embodiments of the present invention, the effect of increasing the mRNA formation of the exon 7-containing form by the PSF is achieved by inserting the PSF into the GAAGGA in the purine-rich sequence of the SMN2 pre-mRNA exon 7 . The GAAGGA sequence is a binding sequence of the PSF newly identified by the present inventors, in which PSF binds to the pre-mRNA exon 7 for the splicing control of SMN2 pre-mRNA.

The therapeutic strategies of the present invention can be roughly divided into protein treatment and gene therapy.

The protein therapeutic strategy of the present invention uses a PSF protein. In order for the spinal muscular atrophy to be effectively prevented or treated, the PSF must penetrate into cells. To this end, the protein transduction domain (PTD) can be bound to the PSF directly or through a linker. That is, a fusion protein can be prepared by fusing a protein transport domain with the protein in order to introduce PSF, which is an effective component of the pharmaceutical composition of the present invention, into cells (permeable peptide transduction). The protein transport domain mainly contains basic amino acid residues such as lysine / arginine, and functions to permeate the proteins fused therewith through the cell membrane to penetrate into cells.

Protein delivery domains (PTDs) that can be used in the present invention include, but are not limited to, HIV-1 Tat protein, homeodomain of Drosophila Antennapedia, HSV VP22 transcriptional regulatory protein, vFGF-derived MTS peptide, penetralin, trans- And the like.

Further, in order to effectively prevent or treat spinal muscular atrophy according to the present invention, the PSF needs to be stably infiltrated into the nucleus. That is, since the pre-mRNA (for example, SMN2 pre-mRNA) targeted by the PSF is located in the nucleus, stable infiltration into the nucleus is required for the PSF to bind to the target pre-mRNA to control its splicing. will be.

As a method for this purpose, a Nuclear localization sequence (NLS) can be fused to the N- or C-terminus of the PSF, either directly or through a linker. NLS is well known in the art and is widely described in the literature, so any known functional NLS can be used. Actually experimentally determined and predicted or proposed NLS and strategies for identifying it are described in Lange et al., J. Biol. Chem . 2007, 282 (8), 5101-5105; Makkerh et al., Current Biology 1996, 6 (8), 1025-1027; Leslie et al., Methods 2006, 39, 291-308; And Lusk et al. Nature Reviews MCB 2007, 8, 414-420.

In the present invention, the content of the PSF with respect to cell and nuclear penetration can also be applied to the case of using a PSF-coding polynucleotide.

The gene therapeutic strategy of the present invention uses PSF-coding polynucleotide, and the composition of the present invention can be applied in vivo through various transportation methods commonly known in the field of gene therapy.

According to some embodiments of the present invention, the polynucleotide of the present invention is naked DNA or contained in a gene carrier. When a gene carrier containing a polynucleotide sequence encoding a PSF is used as an active ingredient, the PSF-encoding polynucleotide is preferably present in a suitable expression construct.

As used herein, the term gene carrier refers to a substance that helps to transfer a gene into a cell, and gene transfer has the same meaning as transduction of a gene into a cell. At the tissue level, the term gene transfer has the same meaning as the spread of a gene. Therefore, the gene delivery system of the present invention can be described as a gene penetration system and a gene diffusion system.

In such expression constructs, the PSF-coding polynucleotide may be operatively linked to a promoter. Refers to a functional linkage between a nucleic acid sequence that is operatively linked to the term nucleic acid expression control sequence (e.g., an array of promoter, signal sequence, or transcription factor binding site) and another nucleic acid sequence, Regulate the transcription and / or translation of the sequence. In the present invention, a promoter bound to a PSF-coding polynucleotide is one capable of regulating the transcription of a PSF-coding polynucleotide by working in an animal cell (for example, a mammalian cell), including a promoter derived from a mammalian virus, A cytomegalo virus (CMV) promoter, an adenovirus late promoter, a vaccinia virus 7.5K promoter, an SV40 promoter, an HSV tk promoter, an RSV promoter, an EF1 alpha promoter, a metallo Promoter of the human IL-2 gene, a promoter of the human IFN gene, a promoter of the human IL-4 gene, a promoter of the human lymphotoxin gene, and a promoter of the human GM-CSF gene. But is not limited thereto.

The expression constructs used in the present invention may comprise polyanenylation sequences (e. G. Bovine growth hormone terminator and SV40-derived polyadenylation sequences).

According to some embodiments of the present invention, the PSF-coding polynucleotide used in the present invention has the structure of a promoter-PSF-coding polynucleotide sequence-polyadenylation sequence.

The gene delivery system of the present invention can be constructed in various forms including (i) naked recombinant DNA molecules, (ii) plasmids, (iii) viral vectors, and (iv) the naked recombinant DNA molecules or plasmids It can be produced in the form of a liposome or a niosome.

PSF-coding polynucleotide sequences can be applied to all gene delivery systems used in conventional gene therapy, such as plasmids, adenoviruses (Lockett LJ, et al., Clin. Cancer Res. 3: 2075-2080 (1997) , Adeno-associated viruses (AAV, Lashford LS., Et al., Gene Therapy Technologies, Applications and Regulations Ed. A. Meager, 1999), retroviruses (Gunzburg WH, et al., Retroviral vectors. Gene Therapy Technologies, Applications and Regulations Ed. A. Meager, 1999), lentivirus (Wang G. et al., J. Clin. Invest. 104 (11): R55-62 (1999)), herpes simplex virus (Puhlmann M. et al., Human Gene Therapy 10: 649-657 (1999)), Liposomes (Methods in Molecular Biology, Vol. 199, SC Basu and M. Basu (Eds.), Human Press 2002) or niosomes.

According to some embodiments of the present invention, the gene delivery system of the present invention may be embodied as a plasmid.

a. Plasmid

Plasmids are widely used in the art as gene delivery systems that are very simple and easy to handle. In the present invention, the plasmid comprising the PSF-coding polynucleotide sequence is preferably capable of expressing PSF by working in animal cells, more preferably mammalian cells, and includes expression vectors well known in the art do. For example, pcDNA3, pcDNA3.1 series (such as pcDNA3.1D / V5-His-TOPO, pcDNA3.1 / mycHisC (-)), pcDNAI, pTracer-SV40, pCDM8, pCEP4, pRc / CMV, pREP9, pCDM8 , pCR2.1, pCR3.1, pCRIII, pSG5, pCMV4, pCMV / Nu / Myc, pGW1, pEGFP-C2, pC1-S65T, YIp5, YCpl9, pMSG, pAV009 / A +, pMAMneo-5, pSecTag2B or yT & But are not limited to,

b. Adenovirus

Adenoviruses are widely used as gene transfer vectors because of their moderate size, ease of manipulation, high titer, broad target cells and excellent infectivity. Both ends of the genome contain an inverted terminal repeat (ITR) of 100-200 bp, which is a sis element essential for DNA replication and packaging. The E1 regions of the genome (E1A and E1B) encode proteins that regulate transcription and transcription of host cell genes. The E2 regions (E2A and E2B) encode proteins involved in viral DNA replication. Of currently developed adenovirus vectors, non-replicable adenoviruses lacking the E1 region are widely used. On the other hand, the E3 region is removed from the conventional adenoviral vector to provide a site for insertion of a foreign gene (Thimmappaya, B. et al., Cell, 31: 543-551 (1982); and Riordan, JR et al. Science, 245: 1066-1073 (1989)). Thus, the PSF-coding polynucleotide of the present invention is preferably inserted into the deleted E1 region (E1A region and / or E1B region) or E3 region, more preferably inserted into the deleted E3 region. The term used herein in connection with the viral genome sequence, deletion, as used herein, is meant to include not only the complete deletion of the sequence but also the partial deletion.

In addition, since the adenovirus can pack up to about 105% of the wild type genome, about 2 kb can be additionally packaged (Ghosh-Choudhury et al., EMBO J., 6: 1733-1739 (1987)). Thus, PSF-encoding polynucleotides that are inserted into adenoviruses may additionally bind to the adenovirus genome.

The adenovirus has 42 different serotypes and a subgroup of A-F. Of these, adenovirus type 5 belonging to subgroup C is the most preferable starting material for obtaining the adenovirus vector of the present invention. Biochemical and genetic information on adenovirus type 5 is well known.

PSF-encoding polynucleotides carried by adenovirus are replicated in the same manner as episomes and have very low genetic toxicity to host cells. Therefore, in the present invention, gene therapy using an adenovirus gene delivery system is considered to be very safe.

c. Retrovirus

Retroviruses are widely used as gene transfer vectors because they can insert their genes into host genomes, carry large quantities of foreign genetic material, and have a broad spectrum of cells that can be infected.

In order to construct a retroviral vector, the PSF-coding polynucleotide is inserted into the retroviral genome in place of the sequence of the retrovirus to produce a non-replicating virus. In order to produce virions, a packaging cell line containing the gag, pol and env genes but without LTR (long terminal repeat) and sequence is constructed (Mann et al., Cell, 33: 153-159 (1983) ). Upon introduction of the recombinant plasmid containing the PSF-coding polynucleotide, LTR and sequence into the cell line, the sequence enables production of the RNA transcript of the recombinant plasmid, which transcript is packaged as a virus, (Nicolas and Rubinstein "Retroviral vectors," In: Vectors: A survey of molecular cloning vectors and their uses, Rodriguez and Denhardt (eds.), Stoneham: Butterworth, 494-513 (1988)). The medium containing the recombinant retrovirus is harvested and concentrated to use as a gene delivery system.

Genetic transfer using a second generation retroviral vector was announced. Kasahara et al. According to Science, 266: 1373-1376 (1994), variants of Molonimurina leukemia virus were prepared, in which an EPO (erythropoietin) sequence was inserted into the envelope region to produce chimeric proteins with novel binding properties . The gene delivery system of the present invention can also be produced according to the construction strategy of the second generation retroviral vector.

d. AAV vector

Adeno-associated virus (AAV) is suitable as a gene delivery system of the present invention because it can infect non-dividing cells and infect various types of cells. A detailed description of the preparation and use of AAV vectors is disclosed in detail in U.S. Patent Nos. 5,139,941 and 4,797,368.

A study of AAV as a gene delivery system is described in LaFace et al, Viology, 162: 483486 (1988), Zhou et al., Exp. Hematol. (NY), 21: 928-933 (1993), Walsh et al, J. Clin. Invest., 94: 1440-1448 (1994) and Flotte et al., Gene Therapy, 2: 29-37 (1995).

Typically, the AAV virus is a plasmid (McLaughlin et al., J. Virol., 62: 1963-1973 (1988); and Samulski et al. (McCarty et al., J. Virol., 65: 2936-2945 (1991)), which contains wild-type AAV coding sequences without terminal repeats (McCarty et al., J. Virol., 63: 3822-3828 )). ≪ / RTI >

e. Other viral vectors

Other viral vectors may also be used as the gene delivery system of the present invention. Rudgeway, "Mammalian expression vectors," In: Vectors: A survey of molecular cloning vectors and their uses, Rodriguez and Denhardt, eds. "Transgenic and stable expression of transferred genes," In: Kucherlapati R, ed Gene transfer New York: Plenum (1988); (1986) and Coupar et al., Gene, 68: 1-10 (1988)), lentivirus (Wang G. et al., J. Clin. Invest. 104 (11): R55-62 (1999)) or vectors derived from herpes simplex virus (Chamber R., et al., Proc Natl Acad Sci USA 92: 1411-1415 (1995)) also carry the PSF-coding polynucleotide into cells Lt; RTI ID = 0.0 > a < / RTI >

f. Liposome

Liposomes are formed automatically by phospholipids dispersed in the aqueous phase. Examples of successful delivery of foreign DNA molecules into cells by liposomes are described by Nicolau and Sene, Biochim. Biophys. Acta, 721: 185-190 (1982) and Nicolau et al., Methods Enzymol., 149: 157-176 (1987). On the other hand, Lipofectamine (Gibco BRL) is the most widely used reagent for transformation of animal cells using liposomes. The liposomes containing the PSF-encoding polynucleotide interact with the cells through a mechanism such as endocytosis, adsorption to the cell surface, or fusion with the plasma membrane, thereby transporting the PSF-encoding polynucleotide into the cell.

The method of introducing the gene carrier of the present invention into cells may be carried out through various methods known in the art.

In the present invention, when the gene carrier is prepared based on a viral vector, it is carried out according to a virus infection method known in the art. Infection of host cells with viral vectors is described in the above cited documents.

(Capecchi, MR, Cell, 22: 479 (1980); Harland and Weintraub, J. Cell Biol. 101: 1094-9), in the case where the gene delivery system is a naked recombinant DNA molecule or plasmid, 1099 (1985)), calcium phosphate precipitation method (Graham, FL et al., Virology, 52: 456 (1973), Chen and Okayama, Mol. Cell. Biol. 7: 2745-2752 (1986)), liposome-mediated transfection (see, for example, Neumann, E. et al., EMBO J. 1: 841 (1982); Tur-Kaspa et al., Mol. Cell Biol., 6: 716-718 (1982); Nicolau et al., Methods Enzymol., 149: 157-176 (1987); Wong, TK et al., Gene, 10:87 (1980); Nicolau Sene, Biochim. Biophys. Acta, 721: 185-190 ), DEAE-dextran treatment (Gopal, MoI. Cell Biol., 5: 1188-1190 (1985)) and genetic bombardment (Yang et al., Proc. Natl. Acad. Sci., 87: 9572 (1990)). ≪ / RTI >

Meanwhile, the amino acid or nucleotide sequence of the PSF that can be used in the present invention is interpreted to include an amino acid or nucleotide sequence showing substantial identity with the wild-type human PSF sequence (GenBank accession No. 6421). Said substantial identity is determined by aligning the amino acid or nucleotide sequence of the human PSF with any other sequence to the greatest extent of correspondence and analyzing the aligned sequence using algorithms commonly used in the art, Homologous, more preferably at least 90% homologous, most preferably at least 95% homologous to the amino acid sequence or nucleotide sequence. Alignment methods for sequence comparison are well known in the art. .

The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier in addition to a pharmaceutically effective amount of the active ingredient. The term, pharmaceutically effective amount means an amount sufficient to achieve efficacy or activity of the above-mentioned active ingredients.

The pharmaceutical composition of the present invention is preferably parenterally administered, and can be administered, for example, by intravenous administration, subcutaneous administration, or local administration.

The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as the formulation method, administration method, age, body weight, sex, severity of disease symptoms, food, administration time, administration route, excretion rate and responsiveness of the patient Ordinarily skilled physicians can easily determine and prescribe dosages effective for the desired treatment. Generally, the daily dosage of the pharmaceutical composition of the present invention is 0.0001-100 /.

The pharmaceutical composition of the present invention may be formulated into a unit dosage form by using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container.

According to another aspect of the present invention, the present invention provides a method for screening a substance for preventing or treating spinal muscular atrophy comprising the steps of:

(a) treating a test substance to be analyzed with a cell comprising a PSF protein or a PSF encoding nucleotide sequence; And

(b) analyzing the expression or activity of the PSF protein;

Wherein the test substance is considered to be a substance for preventing or treating spinal muscular atrophy when the expression or activity of the PSF protein is increased.

According to the method of the present invention, the test substance to be analyzed is first treated with cells containing PSF protein or PSF encoding nucleotide sequence.

As used herein, the term treatment refers to an administration process in which the test substance is administered to a cell to induce contact between the cell and the test substance to enable the assay to be assayed for its efficacy, They can be used interchangeably with contacts.

The cells containing the nucleotide sequence of the present invention are not particularly limited, and specifically are mammalian cells.

The term used in reference to the screening method of the present invention means an unknown substance used in screening to check whether the test substance affects the expression or activity of the PSF protein. Test substances that can be used in the present invention include chemicals, siRNA (small interference RNA), shRNA (small hairpin RNA or short hairpin RNA), miRNA (microRNA), ribozyme, DNAzyme, , Antisense oligonucleotides, peptides, antibodies, aptamers and natural extracts.

The sample analyzed by the screening method of the present invention is a single compound or a mixture of compounds (e.g., a natural extract or a cell or tissue culture). Samples can be obtained from a library of synthetic or natural compounds. Methods for obtaining libraries of such compounds are known in the art. Synthetic compound libraries are commercially available from Maybridge Chemical Co., Comgenex (USA), Brandon Associates (USA), Microsource (USA) and Sigma-Aldrich (USA) ) And MycoSearch (USA). Samples can be obtained by various combinatorial library methods known in the art and include, for example, biological libraries, spatially addressable parallel solid phase or solution phase libraries, 1-compound library method, and synthetic library method using affinity chromatography screening. Methods for synthesis of molecular libraries are described in DeWitt et al., Proc. Natl. Acad. Sci. USA 90, 6909, 1993; Erb et al. Proc. Natl. Acad. Sci. USA 91, 11422, 1994; Zuckermann et al., J. Med. Chem. 37, 2678, 1994; Cho et al., Science 261, 1303, 1993; Carell et al., Angew. Chem. Int. Ed. Engl. 33,2059,1994; Carell et al., Angew. Chem. Int. Ed. Engl. 33, 2061; Gallop et al., J. Med. Chem. 37, 1233, 1994, and the like.

Next, the expression or activity of the PSF protein is measured in the cells to which the test substance has been treated. If it is determined that the expression or activity of these proteins is up-regulated as a result of the measurement, the test substance can be determined as a substance for preventing or treating spinal muscular atrophy.

When the screening method of the present invention is carried out by analyzing the expression of PSF protein, the change in the amount of PSF protein can be performed according to various quantitative or qualitative immunoassay protocols known in the art. For example, immunoassay, radioimmunoassay, radioimmunoprecipitation, Western blotting, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), capture-ELISA, inhibition or competition analysis, sandwich assay, flow cytometry, But are not limited to, immunofluorescent staining and immunoaffinity purification.

Methods of immunoassay or immunostaining are described in Enzyme Immunoassay, E. T. Maggio, ed., CRC Press, Boca Raton, Florida, 1980; Gaastra, W., Enzyme-linked immunosorbent assay (ELISA), in Methods in Molecular Biology, Vol. 1, Walker, J.M. ed., Humana Press, NJ, 1984; And Ed Harlow and David Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999.

When the method of the present invention is carried out by an ELISA method, the present invention relates to a method for screening a solid substrate, comprising the steps of: (i) coating the surface of a solid substrate with an analyte of an unknown cell sample to be analyzed; (ii) reacting said cell lysate with an antibody to a target as a primary antibody; (iii) reacting the result of step (ii) with an enzyme-conjugated secondary antibody; And (iv) measuring the activity of the enzyme. The enzyme bound to the secondary antibody may include, but is not limited to, an enzyme catalyzing a chromogenic reaction, a fluorescent reaction, a luminescent reaction, or an infrared reaction.

When the method of the present invention is carried out in a Capture-ELISA fashion, certain embodiments of the present invention comprise (i) an antibody against a target of the invention (e. G., A PSF protein) as a capturing antibody on the surface of a solid substrate Coating; (ii) reacting the capture antibody with a cell sample; (iii) reacting the result of step (ii) with a detecting antibody which is labeled with a signal generating label and specifically reacts with PSF protein; And (iv) measuring a signal originating from the label. The detection antibody has a label that generates a detectable signal. The label may be a chemical (e.g. biotin), an enzyme (alkaline phosphatase, -galactosidase, horseradish peroxidase and cytochrome P ₄₅₀ ), a radioactive material (e.g. C ¹⁴ , I ¹²⁵ , P ³² And S ³⁵ ), fluorescent materials (e.g., fluorescein), luminescent materials, chemiluminescent materials, and fluorescence resonance energy transfer (FRET). Various labels and labeling methods are described in Ed Harlow and David Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,

In the ELISA method and the capture-ELISA method, measurement of the activity of the final enzyme or measurement of the signal can be performed according to various methods known in the art. The detection of such signals enables a qualitative or quantitative analysis of the target of the present invention. If biotin is used as a label, it can be easily detected by streptavidin. When luciferase is used, luciferin can easily detect a signal.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a pharmaceutical composition for preventing or treating spinal muscular atrophy comprising PSF or a polynucleotide encoding the same as an active ingredient.

(b) According to the present invention, PSF improves mRNA formation involving exon 7 upon splicing of SMN2 pre-mRNA.

(c) According to the present invention, PSF directly contacts GAAGGA, the enhancer sequence of SMN2 pre-mRNA exon 7, enhancing the inclusion of exon 7 into SMN2 mRNA.

(d) The composition of the present invention can prevent or treat spinal muscular atrophy by controlling the splicing of SMN2 pre-mRNA.

Figure 1 is an illustration showing that increased expression of PSF promotes the inclusion of exon 7 in SMN2 pre-mRNA.
1A is a diagram showing an overview of SMN2-L and SMN2-S mini genes. The exon was labeled as a box and the intron as a line. The primers used for RT-PCR analysis were indicated by arrows.
1B shows RT-PCR analysis of exon 7 splicing of the SMN2-L minigene gene in 293A, C33A and SH-SY5Y cells treated with either pcDNA 3.1 (+) plasmid or PSF expression plasmid, respectively It is a picture. GAPDH was used as a loading control. Quantitation results are shown in the lower panel. To confirm the expression of PSF, Western blotting was performed using an anti-Myc antibody and an anti-tubulin antibody was used as a control.
Figure 1c shows RT-PCR analysis of exon 7 splicing of the SMN2-S minigene gene in 293A, C33A and SH-SY5Y cells treated with pcDNA 3.1 (+) plasmid or PSF expression plasmid, respectively, It is a picture.
Figure 2 is a graph showing that the increased expression of PSF promotes the inclusion of exon 7 in SMNl pre-mRNA.
2a shows RT-PCR analysis of exon 7 splicing of SMN2-L minigene in 293A, C33A and SH-SY5Y cells treated with either pcDNA 3.1 (+) plasmid or PSF expression plasmid, respectively It is a picture. GAPDH was used as a loading control. Quantitation results are shown in the lower panel.
Figure 2b shows RT-PCR analysis of exon 7 splicing of the SMN2-S minigene gene in 293A, C33A and SH-SY5Y cells treated with either none or treated pcDNA 3.1 (+) plasmid or PSF expression plasmid, respectively It is a picture. GAPDH was used as a loading control. Quantitation results are shown in the lower panel.
Figure 3 is a graph showing that the decreased PSF expression promotes skipping of exon 7 in SMN2 and SMNl pre-mRNA.
Figure 3a shows the overall strategy for isolating RT-PCR products of SMN1 and SMN2 pre-mRNA. Unlike SMN1 pre-mRNA, the RT-PCR product of SMN2 pre-mRNA is cleaved by restriction enzyme Dde I in exon 8, resulting in shorter fragments of DNA fragments on agarose gel. Primers for PCR were indicated by arrows.
FIG. 3B shows the expression of endogenous SMN1 and SMN2 pre-mRNA in the SH-SY5Y cells treated with either non-silencing shRNA virus or PSF shRNA virus and the presence and exclusion of skeletal exon 7 by RT-PCR It is a picture. The decrease in expression of PSF was confirmed by RT-PCR and Western blotting analysis. GAPDH and -tubulin were used as the spinning controls. Quantitation results are shown in the panel below.
Figure 4 is a graph showing that PSF improves the inclusion of exon 7 in cells of SMA patients.
4A shows the expression of SMN2-L, SMN2-S, SMN1-L and SMN1-S minigene in exon 7 splice of these mini genes in cells of SMA patients transformed simultaneously with pcDNA3.1 (+) plasmid or PSF plasmid Figure 1 shows the results of RT-PCR analysis. GAPDH was used as a loading control. Quantitation results are shown in the lower panel.
FIG. 4b is an analysis of RT-PCR analysis of exon 7 splicing of endogenous SMN2 pre-mRNA in cells of SMA patient overexpressing PSF. Expression of PSF was confirmed by anti-myc antibody and -tubulin was used as a loading control. The results of quantitation of exon 7 splicing in endogenous SMN2 pre-mRNA are shown in the lower panel.
Figure 5 is an illustration showing that elimination of the enhancer in exon 7 eliminates the effect of PSF. 5A is a diagram showing the RNA sequences of wild-type exon 7, deletion mutants 7-1, 7-2 and 7-3. The deletion was marked with (-).
FIG. 5B shows the expression of wild-type exon 7, mutants 7-1, 7-2 and 7-3 SMN2-S by transforming PSF or pcDNA3.1 (+) plasmids into cells expressing exon 7 splicing and RT- PCR analysis. The quantified results are shown in the lower panel.
Figure 6 is a graph showing that the PSF improves the inclusion of exon 7 by contacting the GAAGGA sequence of exon 7;
6A is a diagram showing the RNA sequence of exon 7 of wild type and UUA mutation. The mutated parts were underlined. After transformation of PSU, Tra2 or pcDNA3.1 (+) vector plasmid into cells expressing UUA mutant SMN2-S, exon 7 splicing was analyzed by RT-PCR. The quantified results are shown in the lower panel.
Figure 6b shows the interaction between the PSF and exon 7 RNA sequences. The panel above shows the sequence of wild-type and UAA mutant RNA labeled with biotin. Biotin labeled RNA was incubated with nuclear extracts of HeLa cells. The RNA-protein complex was submerged in streptavidin beads. The presence of PSF was analyzed by Western blotting using anti-PSF antibody.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Materials and Experiments

Plasmid production

All primers used for plasmid production are summarized in Table 1. Wild type SMN1 and SMN2 minigene were obtained by PCR amplification using human genomic DNA as template. SMN1-S and SMN-2 were constructed by overlapping PCR reactions. SMN1-GFP and SMN2-GFP plasmids were used as template in the first PCR reaction and SMNE6 (B) .F and SMNI6 (D). R was used as a primer. In the second PCR reaction, human genomic DNA (Promega) was used as a template and SMNI6 (D) .F and SMNE8 (X) .R were used as primers. The third overlapping PCR product was cloned into the pcDNA3.1 (+) plasmid using restriction enzymes BamHI (TAKARA) and XhoI (TAKARA). Site-directed mutagenesis was performed using the following primers to generate SMN2-E7-1, SMN2-E7-2, SMN2-E7-3 and SMN2-E7-UCC mutant mini genes; Specific primer (forward direction: E7GA.F, reverse direction: E7GA.R), SMN2-E7-A specific primer (forward direction: SMNE6 (B) .F, reverse direction: SMNE8 (Forward: E7A.F, reverse: E7A.R) and SMN2-E7-UCC specific primers (forward: E7-UCC.F, reverse: E7-UCC.R). All minigene genes were identified by sequencing analysis (Cosmo Genetech).

Name Sequences SMNE6 (B) .F 5-TACTCGGATCCATAATTCCCCCACCACCTCC-3 SMNI6 (D) .R 5-GACCTCTAATCCCAGCTACGACAGGCGTGGTGGCAG-3 SMNI6 (D) .F 5-CTGCCACCACGCCTGTCGTAGCTGGGATTAGAGGTC-3 SMNE8 (X) .R 5-CTAACCTCGAGAACAGTACAATGAACAGCCATG-3 E7-1.F 5-ACAGGGTTTTAGACAAAAAGAAGGAAGG-3 E7-1.R 5-CCTTCCTTCTTTTTGTCTAAAACCCTGT-3 E7-2.F 5-TTTTAGACAAAATCGAAGGAAGGTGCTC-3 E7-2.R 5-GAGCACCTTCCTTCGATTTTGTCTAAAA-3 E7-3.F 5-GACAAAATCAAAAAAGGTGCTCACATTC-3 E7-3.R 5-GAATGTGAGCACCTTTTTTGATTTTGTC-3 E7-UUA.F 5-GACAAAATCAAAAAGAATTAAGGTGCTCACATTC-3 E7-UUA.R 5-GAATGTGAGCACCTTAATTCTTTTTGATTTTGTC-3 Ex5.F 5-CTATCATGCTGGCTGCCT-3 Ex8.R 5-CTACAACACCCTTCTCACAG-3 pcI.F 5-GCTAACGCAGTCAGTGCTTC-3 GFP-220AS.R 5-ATAATTCCCCCACCACCTCC-3 SMNE6.F 5-CTGAAGCACTGCACGCCGTAC-3 pcDNA.R 5-CTAGAAGGCACAGTCGAGGCT-3 PSF.F 5-AAGGCAAAGGATTCGGATTT-3 PSF.R 5-TGGCTAAAGGCTTCTTCCAA-3 GAPDH.F 5-ACCACAGTCCATGCCATCA-3 GAPDH.R 5-TCCACCACCCTGTTGCTGTA-3

Cell culture

C33A, 293A, neuroblastoma SH-SY5Y cells were cultured in DMEM (Hyclone) supplemented with 10% fetal bovine serum (FBS, Hyclone) and antibiotics (100 U / penicillin G and 100 / streptomycin). Human SMA type I fibroblast GM03813 (Coriell Repositories) was cultured in DMEM (Hyclone) medium containing 10% fetal bovine serum. HCT 116 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum. All cells were cultured at 37, 5% CO ₂ .

RT-PCR

RT-PCR analysis of exon 7 splicing of SMN2 and SMNl pre-mRNA was performed according to the method previously described (41). Total RNA was extracted from animal cells using RiboEx reagent (Geneall) and ethanol precipitation.

The reverse transcription reaction was performed in a total volume of 20, and 1 RNA, 0.5 oligo-dT, dNTP mix (0.5 mM each dNTP) and 6 mM MgCl ₂ , 4 were added to 5 ImProm- -II < / RTI > TM reverse transcriptase (Promega).

PCR was performed using pcI.F and GFP-220AS.R primers to detect spliced forms from SMN1-L and SMN2-L minigene. SMN.F and pcDNA.R. were used as PCR primers for SMN2-E7-1, SMN2-E7-2, SMN2-E7-3 and SMN2-E7-UUA mutant mini genes. PSF and control GAPDHRT were primers of PSF.F, PSF.R, GAPDH.F and GAPDH.R.

The PCR product was analyzed using 2% agarose gels containing 0.5% ethidium bromide solution. To analyze the exon 7 splicing of endogenous SMN1 and SMN2 by RT-PCR, primers located at exons 5 and 8 were used. The PCR product was digested with DdeI (NEB) and analyzed using 5% native polyacrylamide gels.

Plasmid transformation

Transient transfection of cells was performed using polyethyleneimide (PEI) reagent. Cells were aliquoted 24 hours before transfection so that the cell concentration reached 50% on the day of transfection. A mixture containing 0.5 SMN1 or SMN2 mini gene plasmid and 0.5 PSF plasmid was placed in a medium containing PEI and then applied to the cells. After 4 hours, the medium was replaced with fresh medium. Total RNA was extracted 48 hours after transformation.

shRNA treatment

To produce shRNA lentivirus for the PSF gene, 0.5 lentivirus pLKO.1 plasmid (openbiosystem) was transfected simultaneously with PSPAX2 and PMD2G helper plasmids of 0.3 using 293T cells with PEI reagent Respectively. After 12 hours of incubation, the growth medium was replaced with fresh medium. Again 24 hours later, supernatants containing lentivirus were collected. To inhibit PSF expression, the cells were cultured for 24 hours in a medium containing lentivirus and cultured for 48 hours in a fresh medium.

RNA pulldown analysis

RNA pulldown analysis was performed according to the method previously described (42). Streptavidin agarose beads (Upstate) were first incubated with NETN buffer (100 mM NaCl, 200 mM Tris-Cl (pH 8.0), 1 mM EDTA, 0.5% NP) supplemented with 200 mM PMSF and 0.5 mM DTT -40], followed by incubation with 5 'of biotinylated RNA (Bioneer) for 4 to 60 min. The beads were then washed with NETN buffer to remove unbound RNA. Streptavidin beads conjugated with biotin-labeled RNA were incubated with nuclear extract of HeLa cells for 4 hours at 4 ° C. After 7 washes with NETN buffer again, SDS-PAGE loading buffer was added to dissolve the RNA-bound protein. Samples were analyzed by western blotting using anti-PSF and anti-myc antibodies.

Experiment result

Increased expression of PSF promotes the inclusion of exon 7 into SMN2 mRNA

Since exon 7 of SMN2 pre-mRNA contains a GAAGGA sequence, and since this sequence is similar to the PSF binding site, we investigated the possibility that PSF is involved in SMN2 splicing. In order to confirm whether PSF affects exon 7 splicing of SMN2 pre-mRNA, 293A cells were transfected with SMN2 minigene and PSF gene alone or simultaneously. The first mini-gene tested was SMN2-L (41) as previously described (41), SMN2-L contains exons 6-8, a substantial portion of exon 8 has been removed and a reporter for exon 7 (Fig. 1A). After 48 hours of transformation, total RNA was extracted and the exon 7 splicing of the RNA transcript from this minigene was analyzed by RT-PCR. The primers used were tailored to the pCI-neo vector and GFP coding region as shown by the arrows in Fig. When the SMN2-L gene alone was transformed, SMN2-L minigene produced a mutant lacking the exon 7 (98%) (Fig. 1a), consistent with our previous findings, The mini-gene was confirmed to be very similar to the endogenous SMN2 gene in SMA patients (41). Figure 1b shows that the increased expression of PSF significantly promotes the inclusion of exon 7 in SMN2 mRNA in 293A cells transfected with SMN2-L compared to the control (34%). PSF plasmids were expressed in C33A and SH-SY5Y cells to determine if the effect of PSF was specific to 293A cells. These two types of cells, as well as 293 cells, usually express a type that contains exon 7 and most of them have a feature that expresses no exon 7. After the expression of PSF in C33A and SH-SY5Y cells, the exon 7-containing forms increased to 7% and 9% of total RNA, respectively. This shows that the PSF has a universal effect on the splicing of SMN2-L transcripts. Next, we examined the effect of PSF on splicing of transcripts made from SMN2-S minigene with shorter intron 6 and full-length exon 8 (Fig. 1A). PSF enhanced exon 7 in SMN2 mRNA by 62%, 66% and 60%, respectively, in 293A, C33A and SH-SY5Y cells (Fig. Taken together, these results suggest that PSF enhances the inclusion of exon 7 into SMN2 mRNA, and although the quantitative values of the effect are different in the two minigene genes, the PSF effect is mediated by the intermediate part of intron 6 and the exon 8, which is independent of the downstream part.

Increased expression of PSF promotes the inclusion of exon 7 into SMN1 pre-mRNA

The effect of PSF on SMM1-L and SMN1-S, two minigene genes of SMN1, was investigated in order to confirm whether the effect of PSF is related to the CT base pair difference between SMN2 gene and SMN1 gene. These SMM1-L and SMN1-S minigene genes were designed similar to the SMN2-L and SMN2-S minigene genes (Fig. 1a). The SMN1-L minigene produces a form containing mostly exon 7 in 293A, C33A and SH-SY5Y cells (Fig. 2a), similar to exon 7 splicing of endogenous SMNl pre-mRNA . In 293A, C33A, and SH-SY5Y cells, PSF significantly reduced exon 7 missing form and increased the wildtypes to 22%, 49%, and 16% of total RNA levels, respectively. SMN1-S produced only molds containing exon 7 and SMN7 was rarely found (Fig. 2a). Figure 2b shows that PSF expression only produces a form containing exon 7. From the results of FIGS. 1 and 2, it can be concluded that the effect of PSF on exon 7 splicing is not related to the point mutation in exon 7 of SMN2 pre-mRNA.

PSF vesicular reduction improves exon 7 skipping of SMN2 and SMNl pre-mRNA

To further investigate the effect of PSF on SMN splicing, we reduced the expression of PSF by infecting cells with a virus containing PSF shRNA. Since exon 8 of SMN2 has a cleavage site of DdeI restriction enzyme but SMN1 does not have it (Fig. 3a), RT-PCR products of SMN1 and SMN2 mRNA are digested with DdeI and then differentiated by different sizes Could. In the neuroblastoma SH-SY5Y cells, SMN1 mRNA was mostly wild-type containing exon 7, whereas SMN2 mRNA was mostly skipped exon 7 (Fig. 3B). (Fig. 3b, lane 3), SMN1 increased the SMN1 exon 7 skipped type from 32% to 32% of the total SMN1 RNA level (Fig. 3b, lane 3) , The type in which exon 7 was skipped increased by 8% of the total SMN2 mRNA level. These results indicate that PSF improves the inclusion of exon 7 in both SMN1 and SMN2 mRNA.

PSF promotes the inclusion of exon 7 in cells of SMA patients

In order to confirm whether PSF improves the inclusion of exon 7 in fibroblasts of SMA patients, the PSF was used in combination with SMN2-L, SMN2-S, SMN1-L or SMN1-S minigene, Overexpression. In patients with SMN2-L and SMN2-S minigene genes, PSF enhanced exon 7-containing forms by 39% and 62%, respectively, of the total SMN2 RNA level (Fig. 4a) , Which was consistent with the result of Fig. As in normal cells, the SMN1-L and SMN1-S minigene genes in the SMA patient cells produced only molds containing exon 7 (Fig. 4A, lanes 7 and 10) Making it difficult to observe the increase. Consistent with the results of the mini-gene, overexpression of PSF increased the exon 7-containing form by 14% at the endogenous SMN2 mRNA level (FIG. 4b). These results have led to the conclusion that PSF also promotes the inclusion of exon 7 in SMA patient cells.

Elimination of the enhancer in exon 7 eliminates the effect of PSF

It has previously been reported that PSF promotes or represses splicing by direct contact with RNA sequences (31, 33, 43). For example, it has been shown that PSF contacts the UGGAGAGGAAC sequence to promote splicing (33). We have found by sequencing that there is a sequence (GAAGGA) similar to the PSF contact site in the middle of exon 7, which was previously known as an enhancer that can be targeted to tra2. To confirm whether PSF promotes splicing of exon 7 with the GAAGGA sequence of exon 7, GAAGGA sequence was removed from exon 7 (7-3) and two other deletion mutants were generated as controls , Which were either those in which the upstream AAAAUC sequence was removed (7-1) or the AAAAA sequence was deleted (7-2) (Fig. 5A). 7-3 mutant only produced a form in which exon 7 was skipped, and there was almost no increase in mRNA containing exon 7 even in the presence of PSF (Fig. 5B, lane 12). By contrast, splicing of exon 7 in the 7-1 mutation was still well controlled by the PSF (Fig. 5b, lane 6). On the other hand, control plasmids without PSF did not improve the splicing of exon 7 in 7-3 or 7-1 (lane 5). Taken together, these results suggest that the GAAGGA sequence may be a potential target for PSF in exon 7 splicing of SMN2 pre-mRNA.

PSF contacts the GAAGGA sequence of exon 7 to enhance the inclusion of exon 7

To further elucidate the target sequence of PSF in exon 7, the GAAGGA sequence was mutated to GAAUUA (UUA). Tra2 also improved the inclusion of exon 7 in the UUA mutation (Figure 6a, lane 4), consistent with previous reports that GA2 alone in the GAAGGA sequence is sufficient to regulate the splicing of SMN exon 7 (44). In contrast, in the case of PSF, only the expression of the exon 7 skipped in the UUA mutant was expressed, even though the PSF was sufficiently expressed (lane 3). In the UAA mutation, the phenomenon that the PSF improves the inclusion of exon 7 has disappeared.

To confirm that PSF directly contacts the GAAGGA sequence to enhance the inclusion of exon 7, 5 'biotin labeled AA GAAGGA AG and AA GAAUUA AG RNA sequences were synthesized. The wild type (GAAGGA) sequence of the potential PSF binding site and the two ends of the UUA mutation (GAAUUA) sequence were surrounded by two nucleotides. The biotin-labeled RNA was then incubated with a nuclear extract of HeLa cells and the RNA-protein complex was submerged in streptavidin beads. The presence of PSF in the complex was examined by Western blotting using antibodies against PSF. Experimental results showed that PSF was in direct contact with wild-type RNA (lane 3) but not with UAA mutants (Fig. 6b). Therefore, we concluded that PSF promotes the inclusion of exon 7 into SMN2 mRNA by contacting the GAAGGA sequence of exon 7.

references

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Claims (6)

A pharmaceutical composition for preventing or treating Spinal Muscular Atrophy (SMA) comprising, as an active ingredient, a PTB-associated splicing factor (PSF) or a polynucleotide encoding the same.
The pharmaceutical composition according to claim 1, wherein the PSF increases mRNA formation of the exon 7 included form during splicing of the SMN2 pre-mRNA.
3. The pharmaceutical composition according to claim 2, wherein the PSF binds to GAAGGA, an enhancer of SMN2 pre-mRNA exon 7, to increase exon 7-containing mRNA formation.
2. The pharmaceutical composition according to claim 1, wherein the polynucleotide is contained in a gene carrier.
5. The pharmaceutical composition according to claim 4, wherein the gene carrier is a plasmid, a viral vector or a liposome.
A method for screening a substance for preventing or treating spinal muscular atrophy comprising the steps of:
(a) treating a test substance to be analyzed with a cell comprising a PSF protein or a PSF encoding nucleotide sequence; And
(b) analyzing the expression or activity of the PSF protein; Wherein the test substance is considered to be a substance for preventing or treating spinal muscular atrophy when the expression or activity of the PSF protein is increased.

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