Mecp2 transcript-targeting trans-splicing ribozyme, and use thereof

Abstract

The present invention relates to a MeCP2 gene-specific trans-splicing ribozyme, and a use thereof. The trans-splicing ribozyme according to the present invention replaces the RNA of a mutant MeCP2 gene, which causes Rett syndrome, with a wild type MeCP2 RNA, so as to reduce the expression of the mutant MeCP2 protein and increase the expression of the wild type MeCP2 protein, and enables regulated expression so that wild type MeCP2 expression can be prevented from exceeding physiological levels, and thus can be effectively used for preventing or treating Rett syndrome.

Description

MECP2 transcript-specific trans-splicing ribozyme and uses thereof

[0001] The present invention relates to a MeCP2 gene-specific trans-splicing ribozyme and uses thereof.

[0002]

[0003] It has been reported that a group I intronic ribozyme from Tetrahymena thermophilla can induce a trans-splicing reaction in vitro, whereby the exon RNA attached to the 3' end of the ribozyme can be targeted to the 5' exon in trans, thereby linking it to a separate transcript. This suggests that the trans-splicing reaction of group I ribozymes could also be used to correct transcripts of genes associated with various genetic diseases. Indeed, it was recently shown that a β-globin gene transcript could be converted to an anti-sickling, γ-globin gene transcript in erythrocyte precursor cells from patients with sickle cell disease by a trans-splicing ribozyme.

[0004] As a gene therapy method, trans-splicing ribozymes can correct mutant genetic RNA with normal RNA. Similarly, they can simultaneously reduce the expression of specific genes while simultaneously activating target gene expression in host cells. Because ribozymes operate at the RNA level, they have the advantage of avoiding undesirable immune responses or safety concerns due to gene insertion at random locations within the host cell genome.

[0005] Meanwhile, Rett syndrome (RTT) is a neurodevelopmental disorder with a relatively high incidence of 1 in 10,000 to 15,000 births. Rett syndrome is an X-dominant inherited disorder that is characterized by relatively normal development from 6 to 18 months of age, followed by a decrease in head circumference, loss of acquired cognitive and motor skills, loss of language function, and characteristic hand stereotypy, which involves repetitive hand-washing movements. Familial history is rare and it occurs only in girls, while males with the disorder are known to die at birth.

[0006] It is known that 96-98% of patients diagnosed with Rett syndrome are caused by mutations in the MeCP2 gene, which encodes the MeCP2 protein (Methyl-CpG binding protein 2) on the X chromosome (Xq28). The MeCP2 protein is important for the development of the nervous system, especially the brain. Mutations in the MeCP2 gene cause reduced or incomplete production of the MeCP2 protein, resulting in failure to develop normally in certain brain regions responsible for sensory, emotional, motor, and autonomic functions. It is known that there are eight main MeCP2 mutations in Rett syndrome, and the location and type of mutation affect the onset and severity of the syndrome symptoms, of which the MeCP2 T158M mutation and MeCP2 R106W mutation are the most common. In addition, MeCP2 duplication syndrome is caused by overexpression of MeCP2 due to duplication of the MeCP2 gene, and causes abnormalities in cognitive function and neurodevelopment. MecP2 duplication syndrome occurs in boys and is mainly inherited from the mother, but it is also known to occur sporadically in rare cases.

[0007] That is, while expression of WT MeCP2 capable of performing its unique function is important for treating Rett syndrome, its excessive expression must be controlled. Accordingly, the present inventors have conducted extensive research into a method for treating Rett syndrome without inducing excessive WT MeCP2 expression in cells using a trans-splicing ribozyme targeting MeCP2, and have completed the present invention.

[0008] The technical problem to be achieved by the present invention is to provide a MeCP2 transcript-specific trans-splicing ribozyme that is excellent in safety and expression efficiency of normal MeCP2 RNA and can control the expression amount, and its use in treating Rett syndrome.

[0009] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below.

[0010] To solve the above problem, the present invention provides a trans-splicing ribozyme targeting MeCP2 RNA.

[0011] As one embodiment of the present invention, the trans-splicing ribozyme may have a structure of 5'- IGS (internal guide sequence)-Ribozyme*-3'.

[0012] As another embodiment of the present invention, the trans-splicing ribozyme may have a structure of 5'- IGS (internal guide sequence)-Ribozyme*-exon-3'.

[0013] As another embodiment of the present invention, the IGS region may be composed of a base sequence of 2 to 10 nt in length that can specifically bind to MeCP2 RNA and form a G / U wobble base pair, and preferably, may be composed of a base sequence of 2 to 10 nt in length that can complementarily bind to a region including positions +18, +31, +33, +36, +45, +54, +127, +145, +148, +162, +167, +181, +188, +211, +294, +289, +296, +304, +314 or +357 of MeCP2 RNA and form a G / U wobble base pair.

[0014] In another embodiment of the present invention, the IGS region may comprise or consist of a base sequence of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, or 61.

[0015] Specifically, when the reaction site of the ribozyme is +18 of MeCP2 RNA, the IGS region may include or consist of the base sequence of SEQ ID NO: 3, when +31, the IGS region may include or consist of the base sequence of SEQ ID NO: 61, when +33, the IGS region may include or consist of the base sequence of SEQ ID NO: 5, when +36, the IGS region may include or consist of the base sequence of SEQ ID NO: 7, when +45, the IGS region may include or consist of the base sequence of SEQ ID NO: 9, when +54, the IGS region may include or consist of the base sequence of SEQ ID NO: 11, when +127, the IGS region may include or consist of the base sequence of SEQ ID NO: 13, when +145, the IGS region may include or consist of the base sequence of SEQ ID NO: 15, and when +148 The IGS region may comprise or consist of the base sequence of SEQ ID NO: 17, and in the case of +162, the IGS region may comprise or consist of the base sequence of SEQ ID NO: 19, in the case of +167, the IGS region may comprise or consist of the base sequence of SEQ ID NO: 21, in the case of +181, the IGS region may comprise or consist of the base sequence of SEQ ID NO: 23, in the case of +188, the IGS region may comprise or consist of the base sequence of SEQ ID NO: 25, in the case of +211, the IGS region may comprise or consist of the base sequence of SEQ ID NO: 27, in the case of +289, the IGS region may comprise or consist of the base sequence of SEQ ID NO: 29, and in the case of +294, the IGS region may comprise or consist of the base sequence of SEQ ID NO: 31,In the case of +296, the IGS region may include or consist of the base sequence of SEQ ID NO: 33, in the case of +304, the IGS region may include or consist of the base sequence of SEQ ID NO: 35, in the case of +314, the IGS region may include or consist of the base sequence of SEQ ID NO: 37, and in the case of +357, the IGS region may include or consist of the base sequence of SEQ ID NO: 39.

[0016] As another embodiment of the present invention, the trans-splicing ribozyme may additionally include an AS (antisense sequence) region capable of complementarily binding to a portion of MeCP2 RNA upstream of the IGS region.

[0017] As another embodiment of the present invention, the length of the AS region may be 10 nt to 300 nt.

[0018] As another embodiment of the present invention, the trans-splicing ribozyme may further include a base sequence extended in the 5' direction of the IGS region, and a part of the extended base sequence may be composed of a base sequence capable of complementarily binding to MeCP2 RNA together with the IGS region to form a P1 helix, and a part of the extended base sequence may be composed of a base sequence capable of complementarily binding to a base sequence in the 5' direction of the exon region to form a P10 helix.

[0019] The present invention confirms high trans-splicing efficiency by trans-splicing ribozymes targeting +33, +45, or +304, randomly selects a trans-splicing ribozyme targeting +45 as a representative, designs various P10 structures in the trans-splicing ribozyme targeting +45, and confirms the superiority of ribozymes v1 and v2 in SH-SY5Y cells, a neuroblastoma cell line.

[0020] Accordingly, a portion of the extended base sequence forming P10 in the ribozyme of the present invention may include or consist of the base sequence of SEQ ID NO: 43, 45, 47, or 49.

[0021] In addition, the present invention provides a trans-splicing ribozyme expression vector comprising the base sequence of the trans-splicing ribozyme and / or a base sequence complementary thereto.

[0022] In one embodiment of the present invention, the expression vector may further comprise a promoter operably linked to the ribozyme gene, wherein the promoter may be a neuron-specific promoter, and the neuron includes neurons, astrocytes, and oligodendrocytes. The promoter is not limited thereto and may be a viral promoter, a constitutively active promoter, a CAG promoter, or the like.

[0023] In addition, the present invention provides a system for delivering the trans-splicing ribozyme or a gene delivery system comprising a trans-splicing ribozyme expression vector.

[0024] In addition, the present invention provides a pharmaceutical composition for preventing or treating Rett syndrome, comprising as an active ingredient at least one selected from the group consisting of the trans-splicing ribozyme, a ribozyme delivery system, a vector capable of expressing the ribozyme, and a gene delivery system comprising the vector.

[0025] In addition, the present invention provides a method for preventing or treating Rett syndrome, comprising administering to a subject at least one selected from the group consisting of the trans-splicing ribozyme, the trans-splicing ribozyme expression cassette, the vector capable of expressing the trans-splicing ribozyme, and the gene delivery system comprising the trans-splicing ribozyme, the expression cassette, or the vector.

[0026] As one embodiment of the present invention, the gene delivery system may be a viral or non-viral gene delivery system.

[0027] In addition, the present invention provides a use of the trans-splicing ribozyme, the ribozyme expression cassette, the vector capable of expressing the ribozyme, or the gene delivery system for the manufacture of a drug for preventing or treating Rett syndrome.

[0028] The trans-splicing ribozyme according to the present invention can be usefully used for the prevention or treatment of Rett syndrome by replacing the RNA of the MeCP2 mutant gene that causes Rett syndrome with wild type MeCP2 RNA, thereby lowering the expression of the mutant MeCP2 protein, increasing the expression of the wild type MeCP2 protein, and regulating the expression of WT MeCP2 so that it does not exceed the physiological level.

[0029] Figure 1 is a schematic diagram of the mechanism of action of a trans-splicing ribozyme targeting MeCP2 RNA.

[0030] Figure 2 is the structure of a vector for expressing a trans-splicing ribozyme targeting MeCP2 RNA.

[0031] Figure 3 is a schematic diagram showing the experimental process for selecting a target site of MeCP2 RNA through mapping.

[0032] Figure 4 is a schematic diagram of the reaction site mapping experiment of MeCP2 RNA in vitro and in cells.

[0033] Figure 5a shows the results of reaction site mapping in vitro and in cells.

[0034] Figure 5b shows the IGS of the reaction site target selected as a result of mapping and its target site sequence.

[0035] Figure 6 is a schematic diagram of various variants of MeCP2 RNA transcripts. The red block represents the 5'UTR region, the blue block represents the ORF, and the blue arrow indicates the location of the initiation codon (ATG).

[0036] Figure 7 shows the results of performing an in vitro trans-splicing reaction by producing +33, +45, or +304 target ribozymes.

[0037] Figure 8 is a ribozyme design for the +45 target site of various P10 structures.

[0038] Figures 9a and 9b show the results of the trans-splicing reaction of MeCP2 mRNA by a +45 target ribozyme with various P10 structures in SH-SY5Y cells. Figure 9b shows the results of confirming whether a trans-splicing product was generated, and Figure 9c shows the results of confirming the reaction site where trans-splicing occurred in the trans-splicing product.

[0039] Figure 10 shows the results of confirming the ribozyme activity on the +45 target site of various P10 structures in 293A cells.

[0040] Figures 11a to 11d show the results of co-transfecting SH-SY5Y cells with MeCP2 E1 expression vectors and +45 target ribozyme expression vectors of various P10 structures, and confirming the levels of MeCp2 E1, MeCP2 E2, total MeCp2, ribozyme, and TSP (Trans Splicing Product, substituted RNA) and confirming the trans splicing reaction site by ribozyme.

[0041] Figures 12a to 12d show the results of co-transfecting SH-SY5Y cells with MeCP2 E2 expression vectors and +45 target ribozyme expression vectors of various P10 structures, and confirming the levels of MeCp2 E1, MeCP2 E2, total MeCp2, ribozyme, and TSP, and confirming the trans-splicing reaction site by ribozyme.

[0042] Figure 13 shows the results of confirming the action of +45 target ribozymes of various P10 structures in iPSC-derived neurons.

[0043] Figure 14 is a schematic diagram of an example of the trans-splicing reaction region of a +18, +33, +45 or +54 target ribozyme and a target MeCP2 transcript.

[0044] The present inventors have completed the present invention by conducting extensive research on a gene therapy method for preventing and treating Rett syndrome targeting MeCP2 mutations based on statistical results showing that MeCP2 mutations are prevalent among Rett syndrome patients.

[0045] Specifically, the present inventors identified bases +18, +31, +33, +36, +45, +54, +127, +145, +148, +162, +167, +181, +188, +211, +294, +289, +296, +304, +314, and +357 as reaction sites through sequence analysis of trans-splicing products generated by mixing MeCP2 RNA and random library ribozymes to secure a target region capable of effectively inducing trans-splicing in a ribozyme targeting MeCP2 RNA.

[0046] Meanwhile, MeCP2 has multiple variants. Therefore, the inventors selected target sites that could cover all variants, based on the results obtained through in vitro mapping and the trans-splicing effect of the selected ribozyme.

[0047] The present inventors produced ribozymes specific for the +33, +45, or +304 targets among the target sites identified through the mapping process and performed in vitro trans-splicing reactions. As a result, it was confirmed that the ribozymes for each reaction site target trans-spliced ​​with MeCP2 RNA to produce reaction products.

[0048] Furthermore, each ribozyme expression vector was transfected into SH-SY5Y cells, which are MeCP2-positive cells, and the levels of E1 MeCP2, E2 MeCP2, and E1 / E2 total MeCP2 were measured. As a result, it was confirmed that the expression vectors transfected into the cells expressed ribozymes, and the expressed ribozymes reacted precisely with MeCP2 RNA at the target site. In this specification, the results for the ribozyme targeting +45 are presented as a representative example.

[0049] Meanwhile, the inventors of the present invention designed various P10 structures to improve the efficiency of the trans-splicing reaction with the target and confirmed the reaction by transfecting ribozyme expression vectors of the various P10 structures in MeCP2-positive cells. As a result, the P10 structures showed differences in efficiency depending on the endo MeCP2 or exo MeCP2 experiment and depending on the cell line. Specifically, in 293A cells, high levels of trans-splicing products were confirmed with ribozymes v2 and v4 when targeting endo MeCP2, and high levels of trans-splicing products were confirmed with ribozymes v1 and v2 when targeting exo MeCP2 in SH-SY5Y cells. In summary, it was confirmed that v1, v2, and v4 showed excellent trans-splicing efficiency.

[0050] The present inventors provide a trans-splicing ribozyme for reducing the expression of MeCP2 mutant protein for the treatment of Rett syndrome, wherein the ribozyme may have a structure of 5'- IGS (internal guide sequence)-Ribozyme*-3', and may have a structure of 5'- IGS (internal guide sequence)-Ribozyme*-exon-3' in which a target gene is linked in the 3' direction. In addition, the ribozyme may additionally include an AS (antisense sequence) region of a sequence complementary to MeCP2 RNA in the 5' direction of the IGS region to increase trans-splicing efficiency.

[0051] The ribozyme of the present invention can form a P1 helix and / or a P10 helix region by including nucleotides extended in the leading edge (5' direction) of the IGS. The P1 helix is ​​a secondary structure formed by complementary binding of the IGS region of a group I intron ribozyme and three nucleotides extended upstream thereof to the target MeCP2 RNA, and can increase the binding specificity and binding strength of the ribozyme to the target. The P10 helix is ​​a secondary structure formed by complementary binding of a 5-10 nt long base sequence extended upstream of the IGS region of the ribozyme and a 5' base sequence of the exon region included in the ribozyme, and can increase the trans-splicing efficiency of the ribozyme.

[0052] In this specification, the base sequence forming the P1 helix is ​​referred to as the P1 region or P1 helix region, and similarly, the base sequence forming the P10 helix is ​​referred to as the P10 region or P10 helix region.

[0053] The term "ribozyme" as used herein refers to a molecule composed of an RNA molecule or a protein containing such an RNA molecule, which acts like an enzyme. Also called RNA enzyme or catalytic RNA, it refers to an RNA molecule with a distinct tertiary structure that performs chemical reactions and possesses catalytic or autocatalytic properties. Some ribozymes are known to cleave their own or other RNA molecules, thereby inhibiting their activity, while others catalyze the aminotransferase activity of the ribosome. These ribozymes may include hammerhead ribozymes, VS ribozymes, and hairpin ribozymes.

[0054] As used herein, “trans-splicing” means joining RNAs from different genes together.

[0055] In this specification, the ribozyme refers to a trans-splicing ribozyme that cleaves MeCP2 RNA and replaces it with its own RNA (particularly, the 3' exon region of the ribozyme) to inhibit the expression of the MeCP2 mutant protein in the host cell and induce the expression of the gene encoded in the exon region (Fig. 1). In the present invention, the ribozyme is a 5'-IGS (internal guide sequence)-Ribozyme. * - Exon-3' or 5'-AS (antisense) - IGS - Ribozyme * - It can have the structure of exon -3'. Ribozyme within the above structure * The region refers to the region that performs the function of cleavage and / or substitution of MeCP2 RNA into an exon region within a trans-splicing ribozyme. Unless otherwise specified herein, a ribozyme is a region that performs a substantial function, Ribozyme * Refers to a trans-splicing ribozyme including the entire structure including the region.

[0056] In the present invention, the gene encoded in the 3' exon region of the ribozyme may be a wild-type MeCP2 gene and may not encode a protein. Since the substitution action of the trans-splicing ribozyme is dependent on the level of target RNA, the gene encoded in the exon region of the ribozyme is expressed in a manner dependent on the level of target RNA.

[0057] In addition, the present invention provides a vector expressing a trans-splicing ribozyme targeting the MeCP2 transcript. The vector comprises a trans-splicing ribozyme expression cassette targeting the MeCP2 transcript, and the expression cassette may include a splicing donor / splicing acceptor sequence (SD / SA sequence) linked in the 5' direction of the trans-splicing ribozyme coding region to increase the expression efficiency of the ribozyme, and may include a Woodchuck hepatitis virus Posttranscriptional Regulatory Element (WPRE) linked in the 3' direction of the trans-splicing ribozyme coding region (Fig. 2).

[0058] As used herein, “vector” refers to a gene construct that is an expression vector capable of expressing a trans-splicing ribozyme in a suitable host cell, and that includes essential regulatory elements operably linked to enable expression of a ribozyme gene insert contained within the vector.

[0059] As used herein, “operably linked” means that a nucleic acid expression control sequence that performs a general function and a nucleic acid sequence encoding a gene of interest are functionally linked (functional linkage).

[0060] For example, when a ribozyme coding sequence is operably linked to a promoter, the expression of the ribozyme coding sequence is under the influence or control of the promoter. Two nucleic acid sequences (a ribozyme coding sequence and a promoter region sequence at the 5' end of the sequence) are operably linked when the promoter action is induced to transcribe the ribozyme coding sequence, and the linkage characteristics between the two sequences do not induce a frameshift mutation, and the expression control sequence does not inhibit the expression of the ribozyme. The operably linked sequence with the recombinant vector can be prepared using genetic recombination techniques well known in the art, and the site-specific DNA cleavage and ligation can be performed using enzymes generally known in the art.

[0061] The vector according to the present invention comprises, in addition to expression control elements such as a promoter, operator, initiation codon, termination codon, polyadenylation signal, and enhancer, a signal sequence or leader sequence for membrane targeting or secretion, and can be manufactured in various ways depending on the purpose. The promoter of the vector may be constitutive or inducible. In addition, the expression vector may comprise a selectable marker for selecting a host cell containing the vector, and, if it is a replicable expression vector, may comprise an origin of replication. The vector may be self-replicating or integrated into the host DNA.

[0062] The vector according to the present invention may preferably be a plasmid vector, a cosmid vector, a viral vector, or the like, and most preferably a viral vector. The viral vector may preferably be a vector derived from a retrovirus, for example, human immunodeficiency virus (HIV), murine leukemia virus (MLV), avian sarcoma / leucosis virus (ASLV), spleen necrosis virus (SNV), Rous sarcoma virus (RSV), mouse mammary tumor virus (MMTV), adenovirus, adeno-associated virus (AAV), or herpes simplex virus (HSV), but is not limited thereto. The recombinant vector according to the present invention may most preferably be a recombinant adeno-associated virus vector.

[0063] The term "promoter" as used herein refers to a portion of DNA involved in the binding of RNA polymerase to initiate transcription. It is typically located adjacent to and upstream of a target gene, and is a site where RNA polymerase or a transcription factor, a protein that induces RNA polymerase, binds, thereby directing the enzyme or protein to the correct transcription start site. Specifically, it is located at the 5' end of the gene to be transcribed on the sense strand, and induces RNA polymerase to bind to that site directly or through a transcription factor, thereby initiating mRNA synthesis for the target gene. It has a specific genetic sequence.

[0064] The promoter according to the present invention may be a viral promoter, a constitutively active promoter, a CAG promoter, or a neuronal cell type-specific promoter from the viewpoint of increasing gene expression in a neuronal cell, wherein the neuronal cell includes a neuron, an astrocyte, and an oligodendrocyte.

[0065] In this specification, “gene delivery system” means a system that can increase expression efficiency by increasing the delivery efficiency of ribozyme RNA or ribozyme gene and / or nucleic acid sequence into cells, and can be classified into a viral-mediated system and a non-viral system.

[0066] Viral vector systems use viral vectors such as retrovirus vectors, adenovirus vectors, adeno-associated virus vectors, and herpes simplex virus, and are known to have relatively high intracellular gene transfer efficiency compared to non-viral systems because they utilize the unique intracellular penetration mechanism of viruses that cause infection in human cells. In addition, non-viral vectors have the problem of genes being degraded in endolysosomes after endosomes fuse with lysosomes after entering the cell, whereas viral vectors have the advantage of high gene transfer efficiency due to small gene loss due to a mechanism that delivers genes into the nucleus without passing through lysosomes.

[0067] The viral vector that can be used in the present invention functions as a gene delivery vehicle and may be a vector derived from a retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, etc., as described above for the recombinant vector. Such a viral vector can be assembled into a viral particle and then introduced into a cell by a transduction method such as infection.

[0068] The above non-viral system is a method that uses a cationic lipid carrier or cationic polymer carrier as a delivery medium for nucleic acids and / or genes, or uses LNP or electroporation.

[0069] Cationic lipid delivery systems utilize the positive charge of nanometer-sized liposomes or lipid-based nanoparticles, which are mainly composed of cationic lipids, to form a complex with negatively charged ribozyme RNA or genes, expression vectors containing genes, or nucleic acids, and then deliver this complex into cells through phagocytosis. The complex delivered into the cell is first transported from the endosome to the lysosome and then released into the cytoplasm for expression. Cationic polymer delivery systems deliver ribozyme RNA or genes in a similar manner to cationic lipid delivery systems, except that they use polymers instead of lipids. Representative cationic polymers include polyethyleneimine, poly-L-lysine, and chitosan.

[0070] Therefore, a complex formed by combining the recombinant vector of the present invention with a cationic lipid carrier or cationic polymer carrier can be used as a gene delivery vehicle.

[0071] In the present invention, the gene delivery system includes the recombinant vector described above, and both a viral-mediated system and a non-viral system can be used, but it is preferable to use a viral-mediated system.

[0072] Meanwhile, the trans-splicing ribozyme of the present invention can target MeCP2 RNA in nerve cells and inhibit the expression of MeCP2 mutant protein, and MeCP2 mutation is known to be a major risk factor for Rett syndrome. Therefore, the trans-splicing ribozyme of the present invention, the ribozyme delivery system, the ribozyme expression vector, and the gene delivery system including the vector can be used in a pharmaceutical composition for preventing or treating Rett syndrome.

[0073] The term “prevention” as used in the present invention means any act of suppressing or delaying the onset of Rett syndrome by administering a pharmaceutical composition according to the present invention.

[0074] The term "treatment" as used in the present invention means any action that improves Rett syndrome or beneficially changes its symptoms by administering a pharmaceutical composition according to the present invention.

[0075] The pharmaceutical composition according to the present invention may further comprise a pharmaceutically acceptable carrier, excipient or diluent. Examples of pharmaceutically acceptable carriers, excipients and diluents that can be used in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, calcium carbonate, cellulose, methyl cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxy benzoate, talc, magnesium stearate, mineral oil and the like.

[0076] The pharmaceutical composition of the present invention can be administered orally or parenterally depending on the intended method, but parenterally administration is preferred.

[0077] According to one embodiment of the present invention, the pharmaceutical composition according to the present invention can be administered intravenously, intraarterially, intracerebrally, subcutaneously, or by intracisternal injection or intrathecal injection. The injection according to the present invention can be in a form dispersed in a sterile medium so that it can be used as is when administered to a patient, and can also be in a form in which distilled water for injection is added to disperse it at an appropriate concentration and then administered. In addition, when manufactured as an injection, it can be mixed with buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, etc., and can be manufactured in the form of a unit dosage ampoule or multiple dosage.

[0078] The dosage of the pharmaceutical composition of the present invention may vary depending on the patient's condition and weight, the severity of the disease, the drug form, the route of administration, and the time of administration, but can be appropriately selected by those skilled in the art. Meanwhile, the pharmaceutical composition of the present invention may be used alone or in combination with a therapeutic agent or treatment method for Tarrett syndrome.

[0079] Hereinafter, embodiments are described in detail with reference to the attached drawings. However, the embodiments may be modified in various ways, and the scope of the patent application is not limited or restricted by these embodiments. It should be understood that all modifications, equivalents, or alternatives to the embodiments are included within the scope of the patent application.

[0080]

[0081] The terms used in the examples are for illustrative purposes only and should not be construed as limiting. Singular expressions include plural expressions unless the context clearly dictates otherwise. In this specification, terms such as "comprise" or "have" are intended to indicate the presence of a feature, number, step, operation, component, part, or combination thereof described in the specification, but should be understood to not preclude the presence or addition of one or more other features, numbers, steps, operations, components, parts, or combinations thereof.

[0082]

[0083] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person of ordinary skill in the art to which the embodiments pertain. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and shall not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

[0084]

[0085] In addition, when describing with reference to the attached drawings, identical components will be assigned the same reference numerals regardless of the drawing numbers, and redundant descriptions thereof will be omitted. When describing embodiments, if a detailed description of a related known technology is judged to unnecessarily obscure the gist of the embodiment, the detailed description will be omitted.

[0086]

[0087] [Example]

[0088] Example 1. In vitro mapping

[0089] To secure various tools for removing MeCP2 mutations, MeCP2 RNA and random library ribozyme were mixed in a test tube, reacted, RT-PCR was performed, and the MeCP2 RNA site where trans-splicing occurred was confirmed through sequence analysis of the PCR DNA band expected to be a trans-splicing product (Fig. 3).

[0090] Specifically, in vitro mapping and intracellular mapping were performed using E1 MeCP2 RNA or E2 MeCP2 RNA and a ribozyme RNA library. For in vitro mapping, MeCP2 RNA or intracellular mRNA and a random library ribozyme were mixed and reacted in a test tube, and RT-PCR was performed. The PCR DNA band expected to be a trans-splicing product was sequenced to identify the MeCP2 RNA region where trans-splicing occurred. More specifically, MeCP2 RNA was prepared by synthesizing it through an in vitro transcription process or by extracting mRNA from MeCP2-expressing cells, and a trans-splicing ribozyme RNA library having a random sequence (GNNNNN) at the 5' end was synthesized through an in vitro transcription process. 0.1 pmole of MeCP2 RNA was mixed with 1X in vitro trans-splicing reaction buffer (50 mM Hepes (pH 7.0), 150 mM NaCl, 5 mM MgCl2) to make a total of 10 uL, and 1 pmole of ribozyme library RNA was mixed with 1X in vitro trans-splicing reaction buffer to make a total of 9 uL, and 1 uL of 1 mM GTP was added to make a final concentration of 0.1 mM GTP. These were reacted at 95°C for 1 minute and 37°C for 3 minutes, respectively, and the two solutions were mixed and reacted at 37°C for 3 hours. RNA was recovered from the reaction solution through phenol extraction / EtOH precipitation, and then RT-PCR was performed.

[0091] Next, ribozyme specific RT primer (5'-ATGTGCTGCAAGGCGATT-3', SEQ ID NO: 56) was added, and cDNA was synthesized by reverse transcription in 20uL volume conditions. Using 2uL of synthesized cDNA, 10 pmole each of MeCP2 mRNA specific 5' primer #1 (5'-GCGCGCGCTCCCTCCTCT-3', SEQ ID NO: 57) or 5' primer #2 (5'-GCGCGCGCTCCCTCCTCTCGGAGAG-3', SEQ ID NO: 58), and ribozyme specific 3' primer (5'- TGTAAAACGACGGCCAGTG-3', SEQ ID NO: 59) were used to perform 30 cycles of PCR under the conditions of 98℃ for 30 sec, 58℃ for 30 sec, and 72℃ for 30 sec, and the PCR band was confirmed on a 2% TAE agarose gel. Among the confirmed PCR bands, bands showing differences compared to the MeCP2 only RNA sample were eluted through gel extraction, and the region of the MeCP2 RNA where the trans-splicing reaction occurred was confirmed through base sequence analysis (Figs. 5a and 5b). Among the target sites confirmed through mapping, considering the target with the best mapping result or the utility in cells, +33, +45, and +304 are considered to have the best target efficiency.

[0092]

[0093] Example 2. Selection of the optimal target site among various MeCP2 variants.

[0094] Meanwhile, MeCP2 mRNA has various variants. Therefore, we aimed to select the optimal target region for the production of ribozymes capable of effective trans-splicing across various MeCP2 mRNA variants. Figure 6 is a schematic diagram showing the untranslated region (UTR) and coding sequence (CDS) regions of 12 MeCP mRNA variants.

[0095] Bases +18, +31, +33, +36, and +45 are located in Exon 1, so ribozymes targeting bases +18, +31, +33, +36, and +45 can target variants 1, 2, 3, 4, 5, 6, 8, 9, and 10.

[0096] Bases +127, +145, +148, +162, +167, +181, +188, and +211 are located in Exon 2, so ribozymes targeting bases +127, +145, +148, +162, +167, +181, and +211 can target variants 1, 3, 4, 5, 8, 9, and X1.

[0097] Bases +289, +294, +296, +304, +314, and +357 are located in Exon 3, so ribozymes targeting bases +294, +298, +304, +314, and +357 can target variants 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, X1, and X3.

[0098]

[0099] Example 3. Confirmation of the trans-splicing effect of ribozymes targeting each reaction site.

[0100] Among the target sites identified through mapping, ribozymes targeting sites +33, +45, and +304 were produced based on the best mapping results or considering their utility in cells, and an in vitro trans-splicing reaction was performed to confirm whether trans-splicing occurred.

[0101] More specifically, 0.1 pmole of MeCP2 E1 or E2 RNA was mixed with 1 pmole of each trans-splicing ribozyme library RNA to induce a trans-splicing reaction. RNA was recovered from each reaction solution, RT-PCR was performed, and the produced cDNA was PCR amplified using MeCP2 RNA specific 5' primer #1 (5'- GCGCGCGCTCCCTCCTCT-3', SEQ ID NO: 57) and ribozyme specific 3' primer (5'- TGTAAAACGACGGCCAGTG-3', SEQ ID NO: 59) (95 ℃ 30 sec, 58 ℃ 30 sec, 72 ℃ 30 sec, 35 cycles), and each amplification product was confirmed by electrophoresis on an agarose gel.

[0102] As a result, trans-splicing by ribozymes targeting the +33, +45, and +304 sites of MeCP2 RNA was confirmed (Fig. 7).

[0103]

[0104] Example 4. Design and construction of a MeCP2 RNA +45 targeting trans-splicing ribozyme.

[0105] Among the target sites where the in vitro trans-splicing reaction was confirmed, various forms of P10 were designed to confirm whether the ribozyme acts on the +45 target site (Fig. 8).

[0106]

[0107] Example 5. Confirmation of the trans-splicing effect of the MeCP2 RNA-targeting ribozyme.

[0108] To confirm the effect of the +45 targeting ribozyme produced in Example 4, SH-SY5Y cells, which are MeCP2 positive cells, were used. The MeCP2 targeting ribozyme was transfected with an expression vector, and RNA was extracted and RT-PCR was performed to confirm the trans-splicing effect.

[0109] Specifically, cells were seeded at 4 x 10^5 cells per 6-well plate and cultured in a 37°C, 5% CO2 incubator. 3 μg of MeCP2 target ribozyme expression vector and 100 μl of Opti-MEM were mixed in a 1.5 ml tube, 9 μl of Fugene HD and 100 μl of Opti-MEM were mixed in another 1.5 ml tube, and the contents of the two tubes were mixed and stored at room temperature for 15 minutes to allow the complex to form. After 15 minutes, the tubes were centrifuged for 10 seconds and then sprayed onto each cell for transfection, and after 4 hours, the medium was replaced with fresh medium.

[0110] After culturing for 48 hours in a 37℃ 5% CO2 incubator, cells were washed with 1X PBS and treated with 500 ul of trizol to extract RNA. 5 µg of the extracted RNA was treated with 1 µl of DNase I to remove gDNA, and 1 µg of this was reverse transcribed to synthesize cDNA. Using the synthesized cDNA, the trans-splicing product was confirmed by PCR (Fig. 9a), and the precise trans-splicing reaction was confirmed by base sequence analysis (Fig. 9b).

[0111] In addition, another MeCP2 positive cell, 293A cells, were transfected with a MeCP2 target ribozyme expression vector, and RNA was extracted to confirm the trans-splicing effect, and RT-PCR was performed.

[0112] Specifically, cells were seeded at 4 x 10^5 cells per 6-well plate and cultured in a 37°C, 5% CO2 incubator. 3 μg of MeCP2 targeting ribozyme expression vector and 100 μl of Opti-MEM were mixed in a 1.5 ml tube, and 6 μl of lipofectamine P3000 was dispensed each. 4.5 μl of lipofectamine 3000 and 100 μl of Opti-MEM were mixed in another 1.5 ml tube, and the contents of the two tubes were mixed and stored at room temperature for 15 minutes to form liposome-shaped complexes. After 15 minutes, the tubes were centrifuged for 10 seconds and then sprayed onto each cell for transfection, and after 4 hours, the medium was replaced with fresh medium.

[0113] After culturing for 48 hours in a 37℃ 5% CO2 incubator, cells were washed with 1X PBS and treated with 500 ul of trizol to extract RNA. 5 ㎍ of the extracted RNA was treated with 1 ㎕ of DNase I to remove gDNA, and 1 ㎍ of this was reverse transcribed to synthesize cDNA. The expression levels of E1 MeCP2, E2 MeCP2, and E1 / E2 total MeCP2 were confirmed through qPCR using the synthesized cDNA, and all ribozymes were confirmed to have a reducing effect on endogenous E1 and E2 (Fig. 10A). Ribozyme expression did not show a significant difference by design, and as a result of checking the trans-splicing products, it was confirmed that trans-splicing products were produced for all designs, although the degree of production differed depending on the ribozyme design. Among the compared constructs, it was determined that the efficiency by v2 and v4 was relatively good (Fig. 10B and C).

[0114]

[0115] Example 6. Confirmation of the trans-splicing effect of the MeCP2 RNA-targeting ribozyme on E1 and E2.

[0116] To confirm the effect of ribozymes on MeCP2 E1 and MeCP2 E2, SH-SY5Y cells were co-transfected with MeCP2-targeting ribozymes and MeCP2 E1 or E2 expression vectors. Specifically, 3 μg of MeCP2-targeting ribozyme expression vector and 0.5 μg of MeCP2 expression vector were mixed in 100 μl of Opti-MEM in a 1.5 ml tube, and 10.5 μl of fugene HD and 100 μl of Opti-MEM were added to another 1.5 ml tube and mixed. The contents of the two tubes were mixed and left at room temperature for 15 minutes. After transfection, RT-qPCR was performed using the same method as above.

[0117] As a result, it was confirmed that when co-transfected with the E1 MeCP2 expression vector, the expressed E1 MeCP2 was reduced (Fig. 11a). In addition, although there were differences in ribozyme expression depending on the construct, it was expressed normally (Fig. 11b), and although there were differences in the production amount, it was confirmed that trans-splicing products were produced (Fig. 11c). The trans-splicing products confirmed through qPCR were sequenced, and the results confirmed that trans-splicing occurred precisely at the target site, confirming that MeCP2 E1 was accurately replaced with the desired RNA (Fig. 11d).

[0118] When co-transfected with the E2 MeCP2 expression vector in the same manner as the E1 MeCP2 control described above, it was confirmed that the expressed E2 MeCP2 was reduced (Figs. 12a to 12d).

[0119] Therefore, it can be seen that the trans-splicing ribozyme targeting MeCP2 RNA according to the present invention can not only correct specific MeCP2 variants, but also replace mutation sites regardless of the variant.

[0120]

[0121] Example 7. Confirmation of the effect of MeCP2 RNA-targeting ribozyme in neurons.

[0122] Rett syndrome is a neurodevelopmental disorder in which mutations in single proteins are known to affect neurons. Therefore, iPSC-derived neuronal cells were used to confirm the effects of ribozymes on actual neurons. Two MeCP2-targeting ribozyme expression vectors were selected and transfected into neurons.

[0123] Specifically, differentiated neurons were seeded at 1.5 x 10^5 in a 6-well plate and cultured in a 37°C, 5% CO2 incubator. After maturation for 3 weeks, the MeCP2 target ribozyme expression vector was transfected using Lipofectamine™ Stem Transfection Reagent. 1.25 μg of the MeCP2 target ribozyme expression vector and 100 μl of Opti-MEM were mixed in a 1.5 ml tube, and 5 μl of Lipofectamine™ Stem Transfection Reagent and 100 μl of Opti-MEM were mixed in another 1.5 ml tube. The contents of the two tubes were mixed and left at room temperature for 10 minutes. After transfection, RT-qPCR was performed using the same method as above.

[0124] As a result, it was confirmed that the expression of neuronal MeCP2 was reduced, although the effect of reducing MeCP2 was not large, and it was confirmed that trans-splicing products were generated (Fig. 13).

[0125] Therefore, the effect on MeCP2 expressed in neurons by the produced MeCP2-targeting ribozyme was also confirmed.

[0126]

[0127] Although the embodiments described above have been described with limited drawings, those skilled in the art will appreciate that various technical modifications and variations can be applied based on the above. For example, appropriate results can still be achieved even if the described techniques are performed in a different order than described, and / or components of the described systems, structures, devices, circuits, etc. are combined or combined in a different manner than described, or are replaced or substituted with other components or equivalents.

[0128] Therefore, other implementations, other embodiments, and equivalents to the claims also fall within the scope of the claims described below.

Claims

1. A trans-splicing ribozyme targeting the MeCP2 transcript.

2. In paragraph 1, The above trans-splicing ribozyme is a 5'-IGS (internal guide sequence)-Ribozyme * - A trans-splicing ribozyme characterized by having an exon-3' structure.

3. In paragraph 2, A trans-splicing ribozyme characterized in that the above IGS region is composed of a base sequence of 2 to 10 nt in length that can complementarily bind to a region comprising bases +18, +31, +33, +36, +45, +54, +127, +145, +148, +162, +167, +181, +188, +211, +294, +289, +296, +304, +314, or +357 of the MeCP2 transcript represented by sequence number 1.

4. In paragraph 2, A trans-splicing ribozyme, wherein the IGS region comprises a base sequence of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, or 61.

5. In paragraph 2, A trans-splicing ribozyme, characterized in that it additionally comprises an AS (antisense sequence) region capable of complementarily binding to a part of the MeCP2 transcript in the 5'-terminal direction of the trans-splicing ribozyme.

6. In paragraph 2, A trans-splicing ribozyme, wherein the exon region comprises a polynucleotide sequence encoding a normal MeCP2 protein or a base sequence encoding a reporter protein.

7. In paragraph 2, The above trans-splicing ribozyme additionally comprises a base sequence extending in the 5' direction of the IGS region, A portion of the above extended base sequence may be composed of a base sequence that can complementarily bind to MeCP2 RNA together with the IGS region, thereby forming a P1 helix. A trans-splicing ribozyme, wherein a portion of the extended base sequence is composed of a base sequence capable of complementary binding to a base sequence in the 5' direction of the exon region and capable of forming a P10 helix.

8. In paragraph 1, The MeCP2 transcript is a trans-splicing ribozyme comprising a MeCP2 mutation.

9. In paragraph 8, The above MeCP2 mutation is a trans-splicing ribozyme in which at least one base is substituted, deleted, or added between bases 1 to 10467 of the MeCP2 transcript represented by SEQ ID NO:

1.

10. A trans-splicing ribozyme expression cassette encoding the trans-splicing ribozyme of any one of claims 1 to 9.

11. In paragraph 10, A trans-splicing ribozyme expression cassette comprising a promoter sequence operatively linked to the trans-splicing ribozyme expression cassette.

12. A trans-splicing ribozyme expression vector comprising a trans-splicing ribozyme expression cassette of any one of claims 10 to 11.

13. A recombinant virus comprising a trans-splicing ribozyme expression cassette of any one of claims 10 to 11.

14. In paragraph 13, The above virus is a recombinant virus, any one selected from the group consisting of adenovirus, adeno-associated viruses (AAV), retrovirus, lentivirus, herpes simplex virus, or vaccinia virus.

15. A nonviral gene vector comprising a trans-splicing ribozyme expression cassette of any one of claims 10 to 11.

16. A pharmaceutical composition for preventing or treating Rett syndrome, comprising as an active ingredient any one selected from the group consisting of the trans-splicing ribozyme of claims 1 to 9; the trans-splicing ribozyme expression cassette of claims 10 to 11; the expression vector of claim 12; and a gene delivery system comprising the expression vector.

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