Compositions and methods for editing RNA

Abstract

To provide methods for editing a sequence of an endogenous RNA in a cell, particularly in the nucleus of the cell.SOLUTION: A method for editing a target sequence of an endogenous RNA in a cell comprises delivering, to the cell, a) a nucleic acid molecule encoding a fusion protein comprising an RNA editing enzyme linked to an RNA binding domain and b) a nucleic acid molecule encoding a guide RNA. The fusion protein comprises a nuclear localization signal. The guide RNA comprises a sequence specifically recognized by the RNA binding domain. The guide RNA specifically hybridizes with the target sequence in the endogenous RNA and comprises a mismatch at a nucleotide to be edited.SELECTED DRAWING: Figure 1-1

Description

Technical Field

[0001] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 62 / 569,376, filed Oct. 6, 2017 . The foregoing application is hereby incorporated by reference in its entirety .

[0002] This invention was made with government support under Grant No. NS087726 awarded by the National Institutes of Health. The government has certain rights in the invention .

[0003] Description of Electronically Submitted Text File The content of the text file submitted electronically herewith is hereby incorporated by reference in its entirety: a computer-readable copy of the Sequence Listing (filename: SEQL IST.txt; date recorded: Oct. 9, 2018; file size: 44.6 KB ).

[0004] The present invention relates to the field of nucleic acid editing. Specifically, compositions and methods for the therapeutic editing of RNA, particularly endogenous nuclear RNA, are disclosed .

Background Art

[0005] To describe the prior art in the field to which the present invention pertains, several publications and patent documents are cited throughout this specification. Each of these citations is hereby incorporated by reference in its entirety as if fully set forth .

[0006] Strategies for editing or modifying genetic material, e.g., various gene editing technologies, have advanced . However, there remains a need for methodologies to correct disease-causing mutations, particularly in the nervous system .​​

[0007] Rett syndrome is caused by sporadic sudden mutations in the transcription factor methyl CpG-binding protein 2 (MECP2). It is a neurodevelopmental disorder caused by mutation (Amir, et al. (1999)). (Nat. Genet., 23:185-188). MECP2 is located on the X chromosome. Due to the mechanism of dosage compensation in mammals Women with Rett syndrome are mosaics, and their cells are a mix of wild-type and mutant cells. The ratio is approximately 50:50. Women with the MECP2 mutation have speech and intentional They experience a setback in early developmental milestones, such as hand movements, followed by severe respiratory problems. They acquire motor abnormalities and die by an average age of 40 (Neul, et al., (201 0)Ann.Neurol.,68:944-950;Percy,et al.(20 10) Ann. Neurol., 68:951-955). It has a mutation in MECP2. Males with only one X chromosome develop even more serious illnesses and usually die before the age of two. (Schule, et al. (2008) Clin. Genet., 74:116 (-126). There is no cure for Rett syndrome. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Amir, et al. (1999) Nat. Genet., 23:185-188 [Non-Patent Document 2] Neul,et al.,(2010)Ann.Neurol.,68:944-950 [Non-Patent Document 3] Percy,et al.(2010)Ann.Neurol.,68:951-955 [Non-Patent Document 4] Schule,et al.(2008)Clin.Genet.,74:116-126 [Overview of the Initiative] [Means for solving the problem]

[0009] According to one aspect of the present invention, a method for editing the sequence of endogenous RNA within a cell, particularly within the nucleus of a cell. A method is provided. In certain embodiments, this method involves i) nuclear localization signaling and RNA binding. A nucleic acid molecule encoding a fusion protein containing an RNA editing enzyme linked to a domain, and ii) Steps to deliver one or more nucleic acid molecules encoding guide RNA to cells It contains a guide RNA, which contains a sequence that is specifically recognized by the RNA-binding domain. The guide RNA also specifically hybridizes with the target sequence in the endogenous RNA and edits it. The nucleotides include mismatches. In certain embodiments, RNA editing enzymes are used to edit RNA Adenosine deaminases (ADARs) that act on it, such as ADRAR1 or ADAR2 In certain embodiments, the RNA-binding domain is a λN peptide, and the RNA-binding domain The sequence specifically recognized by the main is the BoxB sequence. In certain embodiments, The causative RNA is methyl CpG-binding protein 2 (MECP2)RNA. Morphologically, the nuclear localization signal is the SV40 large T antigen nuclear localization signal. The nucleic acid molecule in this method is a viral vector (for example, an adeno-associated virus (AAV) vector). It may be contained within a single vector such as -).

[0010] According to another aspect of the present invention, the sequence of endogenous RNA within a cell, particularly within the nucleus of a cell, is edited. Additional methods are provided. In certain embodiments, the method involves one or more guide RNs. The process includes the step of delivering a nucleic acid molecule encoding A to a cell, and the guide RNA is used to make RNA The specificity depends on the endogenous deaminase used, such as human adenosine deaminase (ADAR). It contains (one or more) sequences and / or structures that are specifically recognized. Guide RNA also It specifically hybridizes with the target sequence in endogenous RNA and edits the nucleotide with a mistake. Includes matching. In certain embodiments, ADAR is ADRA1 or ADAR2. In one embodiment, endogenous RNA is methyl CpG-binding protein 2 (MECP2)RN A. In certain embodiments, the nucleic acid molecule is a viral vector (e.g., an AAV vector). It is included within ().

[0011] According to another aspect of the present invention, the sequence of endogenous RNA within a cell, particularly within the nucleus of a cell, is edited. An additional method is provided. In certain embodiments, the method involves a nucleus encoding a guide RNA. The process includes the step of delivering an acid molecule (e.g., within an AAV) to a cell, and the guide RNA is RNA It is specifically recognized by endogenous human adenosine deaminase (ADAR), which acts on it. Includes (one or more) sequences and / or structures. Therefore, in various aspects, this editorial The method involves the absence of recombinant RNA editing enzymes (for example, the absence of recombinant ADARs). ) can be done. Therefore, in the embodiments, this method may have an effect on the editing described herein. It is responsible for the endogenous ADAR activity necessary to exert its effects.

[0012] According to another aspect of the present invention, the treatment, suppression and / or prevention of Rett syndrome in a target area A method is provided. In certain embodiments, the method uses the RNA editing method of the present invention. Includes a step. For example, this method includes an RNA editing enzyme linked to an RNA-binding domain. The target is nucleic acid molecules encoding fusion proteins and nucleic acid molecules encoding guide RNA. The procedure may include a step of administering the fusion protein, which contains a nuclear localization signal. In this case, the method includes a step of administering a nucleic acid molecule encoding guide RNA to the target. Guide RNA acts on RNA, such as endogenous adenosine deaminase (ADAR). (1 or more) sequences and / or (1) that are specifically recognized by starfish aminase The structure includes (or multiple) structures, and the guide RNA is methyl CpG-binding protein 2 (MECP2). It specifically hybridizes with RNA and is a mutant nucleotide of endogenous MECP2 RNA. This includes mismatches. [Brief explanation of the drawing]

[0013] [Figure 1]This demonstrates that editing efficiency is sequence-dependent. Figure 1A is a schematic diagram showing the locations of three G>A mutations in the methyl DNA-binding domain (MBD), transcriptional repressor domain (TRD), and NCoR interaction domain (NID) in MeCP2. Figure 1B is a schematic diagram of the core components of site-directed RNA editing. The hybrid editase contains an RNA-binding domain derived from bacteriophage λ (λN) and a catalytic domain (deaminase domain) of human adenosine deaminase (hADAR2) that acts on RNA 2. Three copies of the nuclear localization signal (NLS) and two copies of the human influenza hemagglutinin (HA) epitope tag are also included but not shown. The guide RNA is complementary to Mecp2 mRNA and contains a hairpin (stem-loop) recognized by the λN RNA-binding domain. For target A, C is introduced into the guide to enhance editing efficiency. Figure 1C is a sequencing chromatogram of Mecp2W104X cDNA after transfection of N2A neuroblastoma cells with or without guide editase. Figure 1D shows the edit percentage (mean ± SD; n=3) from A to I, including the data from Figure 1C, quantified using direct sequencing of Mecp2 cDNA. Light gray bars: cells transfected with editase only; dark gray bars: cells transfected with both editase and guide. ***P<0.001, ****P<0.0001, one-way ANOVA with Bonferroni post-hoc test. ns: not significant. [Figure 2]This study demonstrates that using site-specific AG mismatch guidance for Mecp2 mRNA can reduce off-target editing by more efficient editases. Figure 2A shows the percentage of A-to-I editing (mean ± SD; n=3, including data from Figure 2B) at the R106Q site after transfection of N2A cells with guide RNA and editase WT or editase E488Q. Figure 2B is a representative chromatogram of Mecp2R106Q cDNA edited with editase WT (top) or editase E488Q (bottom). Figure 2C shows Mecp2 mRNA against two different guide RNAs. The standard guide (top) contains an AC mismatch (R106Q) at target A (bold) to enhance editing. The modified guide (bottom) contains an AG mismatch at off-target A, marked with an asterisk, to inhibit editing at this site. The provided target sequence is Sequence ID No. 52. Figure 2D is a chromatogram of Mecp2 cDNA after transfection of N2A cells with an editase E488Q and a guide containing mismatches only at the target site (top) or a modified guide containing both on-target AC mismatches and off-target AG mismatches (bottom). Figure 2E shows that the guide containing AG mismatches significantly reduces off-target editing (mean ± SD; n=3, including data from Figure 2D). Figure 2F shows that the presence of off-target AG mismatches does not affect editing at the R106Q site (mean ± SD; n=3, including data from Figure 2D). Light gray bars: cells transfected with editase only; dark gray bars: cells transfected with editase and guide; black bars: cells transfected with editase and guide containing AG mismatches. **P<0.01, ***P<0.001, ****P<0.0001, one-way ANOVA with Bonferroni post-hoc test. ns: not significant. [Figure 3]This figure shows sequence analysis of endogenous MeCP2 mRNA after AAV1 / 2 transduction of primary neurons. Figure 3A shows the quantification of editing (mean ± SD, n=3) by sequence analysis of cDNA isolated from Mecp2R106Q / y hippocampal neurons (DIV14) 7 days after transduction with AAV1 / 2 virus. +Guide refers to AAV1 / 2 containing six copies of the guide, which are expressed under the control of the synapsin I promoter and the U6 promoter, respectively. The guide includes a C mismatch at target A of R106Q and a G mismatch at off-target A T105T. The control virus encodes the editase under the control of the synapsin I promoter but lacks both guide sequences (-Guide). ****P<0.0001 by unpaired two-sided t-test. Figure 3B shows the Mecp2 mRNA (SEQ ID NO: 52) and primary amino acid sequence (SEQ ID NO: 53) relative to the guide RNA region. Target A is shown in bold, and asterisks indicate off-target edited A residues. Guide hairpins represent the locations of BoxB sequences recognized by the λN peptide. The graph provides quantification of off-target editing within Mecp2 mRNA (mean ± SD; n=3). Residue N126S is outside the guide region. [Figure 4] This figure shows that site-directed RNA editing increases MeCP2 protein levels, thereby indicating functional recovery of the endogenous disease-causing protein after editing. Representative Western blots of whole-cell solubilites from Mecp2R106Q / y or wild-type (WT, Mecp2+ / y) sibling hippocampal neurons (DIV14) transduced 7 days prior to editing with either editase alone or with editase and guide expressing AAV1 / 2. The guide includes C mismatch at the R106Q site and off-target A, G mismatch at T105T. The graph provides quantification (mean ± SD, n=3) of Western blots normalized to β-actin for each condition. Light gray bars: cells transduced with editase alone. Dark gray bars: cells transduced with editase and guide. ***P<0.001 by unpaired two-sided t-test. [Figure 5]Site-directed RNA editing has been shown to restore MeCP2's ability to bind to heterochromatin, demonstrating the recovery of the endogenous protein's function after editing. Representative confocal images of hippocampal neurons (DIV14) immunolabeled for editase (HA) and MeCP2 are shown. DAPI staining outlines the nuclear contour and reveals heterochromatic foci. Insets define cell boundaries imaged at higher magnification and gain in adjacent panels. Figure 5A shows a culture of wild-type (Mecp2+ / y) neurons. Figure 5B shows a culture of Mecp2R106Q / y neurons transduced with AAV1 / 2 virus expressing only editase (without guide). These neurons never showed MeCP2 enrichment in heterochromatin. Figure 5C shows a culture of Mecp2R106Q / y neurons transduced with AAV1 / 2 virus expressing editase and a guide containing a C mismatch at target A. Figure 5D shows a culture of Mecp2R106Q / y neurons transduced with AAV1 / 2 virus expressing editase and a guide containing a C mismatch at target A. In Figure 5D, + and - indicate nuclei with and without MeCP2 enrichment in heterochromatin, respectively. Scale bar, 10 μm. Figures 5E-5G: Each histogram represents the quantification of cells from three fields in each of three slides (mean ± SD) (editase only, n=134; editase and guide, n=137). Figure 5E shows the percentage of editase+ cells identified by HA nuclear staining after thresholding signals from uninfected cells. Percentages are relative to the total number of DAPI+ cells. Figure 5F shows the percentage of editase+ cells with MeCP2 enrichment in heterochromatin (structure). Figure 5G shows the percentage of all cells with MeCP2 enrichment in heterochromatin (structure). ns: not significant. [Figure 6]This graph shows the MeCP2 intensity in dentate neuronal heterochromatin of the brains of wild-type mice or Mecp2317G>A(Mecp2R106Q) mice treated with editase alone or with an AAV vector encoding editase and guide RNA. [Figure 7] Schematic diagrams of various guide RNAs and graphs of the edited percentage of Mecp231 7G>A(Mecp2R106Q) in HEK cells treated with untreated or guide RNAs containing two BoxB stem loops, guide RNAs containing an R / G binding site from GluA2, or guide RNAs containing an internal loop. HEK cells were also transfected with full-length native ADAR2 cDNA under the control of the CMV promoter. [Modes for carrying out the invention]

[0014] This invention, in part, utilizes site-directed RNA editing to process RNA at the RNA level (e.g., mRN). A) The point mutation that causes the disease, for example, the methyl CpG mutation that underlies Rett syndrome. guanosine to adenosine in the DNA-binding domain gene of the binding protein 2 (MECP2) This is based on the remarkable discovery that the (G>A) mutation can be repaired. Furthermore, this site-directed RNA editing repairs endogenous RNA and improves protein function. It is useful for recovering site-specific RNA. Therefore, in embodiments, the present invention is useful for site-specific RNA editing. This relates to compositions and methods for collection.

[0015] Sudden Mecp2 mutation that causes Rett syndrome in humans, limited to germline or nerve cells. Mice manipulated to carry the mutation exhibit growth abnormalities, anxiety, and other symptoms similar to those seen in patients with Rett syndrome. It shows motor impairment (Guy, et al. (2001) Nat. Genet., 27:3 22-326;Lioy,et al.(2011)Nature 475:497-5 00;Chen,et al.(2001)Nat.Genet.,27:327-33 1). Studies in mice have shown that the most severe Rett syndrome phenotype is neurological, involving neurons. This has shown that it affects both glial cells (Lioy, et al. (2011)N ature 475:497-500;Luikenhuis,et al.(2004 (Proc.Natl.Acad.Sci.,101:6033-6038) many Other organizations may also be affected (Ross, et al. (2016) Hum.M (ol. Genet., 25:4389-4404). Similar to humans, male redmouths are female. It is more severe than in mice. For example, female red mice live a normal lifespan, but male mice do not. They die at 3-4 months of age (Guy, et al. (2001) Nat. Genet., 27:322-326;Chen,et al.(2001)Nat.Genet.,2 7:327-331). At the cellular level, the nerve cells of male and female Rett mice are cells The body and nucleus are small, and the complexity of the processes is reduced (Belichenko, et al. (2009)Neurobiol.Dis.,34:71-77;Belichenk o,et al.(2009)J.Comp.Neurol.,514:240-258 ;Fukuda,et al.(2005)J.Neuropathol.Exp.Ne urol.,64:537-544;Kishi,et al.(2004)Mol.C ell.Neurosci.,27:306-321;Robinson,et al. (2012)Brain 135:2699-2710;Tropea,et al.( 2009)Proc.Natl.Acad.Sci.,106:2029-2034;S tuss, et al. (2012) PLoS One 7:e31896), affected It is reminiscent of human cells (Armstrong, et al. (1995) J. Neur opathol.Exp.Neurol.,54:195-201;Li,et al. (2013)Cell Stem Cell 13:446-458;Belichen ko,et al.(1994)Neuroreport.,5:1509-1513; Bauman, et al. (1995) Neurology 45:1581-158 6). Importantly, conditional Cre recombinase (Guy, et al. (2007) (Science 315:1143-1147) or gene therapy approach (Sin nett,et al.(2017)Mol.Ther.Methods Clin.D ev.,5:106-115;Gadalla,et al.(2017)Mol.Th er.Methods Clin.Dev.,5:180-190;Garg,et a l.(2013)J.Neurosci.,33:13612-13620;Gadal la, et al. (2013) Mol. Ther., 21:18-30) via Me The recovery of MeCP2 in cp2 null mice indicates that even in the later stages of the disease, Rett-like syndrome and Many cell defects are retrograde. Phenotypic reversal is seen in humans, particularly in Rett syndrome, which is a genetic replacement strategy. This shows that it can be treated (Robinson, et al. (2012) )Brain 135:2699-2710;Sinnett,et al.(2017 )Mol.Ther.Methods Clin.Dev.,5:106-115;Ga dalla,et al.(2017)Mol.Ther.Methods Clin. Dev.,5:180-190;Garg,et al.(2013)J.Neuros ci.,33:13612-13620;Gadalla,et al.(2013)M (ol.Ther.,21:18-30). However, the MECP2 gene in humans This duplication leads to MeCP2 overexpression and severe neurological impairment (Van Esch, et al. (2005) Am.J.Hum.Genet.,77:442-453). Furthermore, mouse MeCP2 is expressed at various levels in various neuronal cell types, and in mice... Loss of MeCP2 function leads to cell-specific changes in gene expression (Skene, et al. l.(2010)Mol.Cell.,37:457-468;Ballas,et a l.(2009)Nat.Neurosci.,12:311-317;Shahbaz ian,et al.(2002)Hum.Mol.Genet.,11:115-12 4;Sugino,et al.(2014)J.Neurosci.,34:1287 7-12883;Linhoff,et al.(2015)Cell 163:246 (-255). These findings indicate normal MeCP2 levels across diverse cell types of the nervous system. And the task of MECP2 gene replacement, which must be fine-tuned to restore cell physiology. The title is being emphasized.

[0016] In this specification, since RNA is repaired in the environment of a normal transcript, M at the RNA level Repairing ECP2 mutations can lead to problems with both MECP2 overexpression and cell type-specific regulation. It has been shown that this can be avoided. Guanosine to adenosine, which underlies Rett syndrome. (G>A) mutation to (Fyfe, et al. (2003) J.Child.Neu The target was RNA, a family of natural enzymes. (rol.,18:709-713) The adenosine deaminase (ADAR) that acts on endogenous mRNA converts A to inosine (I) Hydrolytic deamination (Bass, et al. (1988) Cell 55:1089-1098;Bass,et al.(1987)Cell 48:60 7-613;Melcher,et al.(1996)Nature 379:460 -464;O'Connell,et al.(1998)Methods 15:51 -62;Kim,et al.(1994)Proc.Natl.Acad.Sci., 91:11457-11461). The inosine base pairs with cytosine (C) and ribosol Translated as G by M (Basilio, et al. (1962) Proc. (Natl.Acad.Sci.,48:613-616). Member of the ADAR family. ADAR2, one of these, is highly expressed in the brain and, after transcription, de-acidifies the primary transcript. Through ionization, protein functions such as ion channel permeability are altered (Bhall a,et al.(2004)Nat.Struct.Mol.Biol.,11:95 0-956;Sommer,et al.(1991)Cell 67:11-19;B (1997) Nature 387:303-308). ADA In addition to its catalytic activity, R2-mediated natural editing appropriately targets A into exons for editing. Recognition of the double-stranded RNA structure mediated by the introns of the pre-mRNA to be placed is necessary. (Bhalla, et al. (2004) Nat. Struct. Mol. Bio) l.,11:950-956;Dawson,et al.(2004)J.Biol. Chem.,279:4941-4951;Higuchi,et al.(1993) Cell 75:1361-1370;Maas, et al. (1996) J.Bio l.Chem.,271:12221-12226;Lomeli,et al.(19 94)Science 266:1709-1713;Yang,et al.(199 7) Proc. Natl. Acad. Sci., 94:4354-4359). Human AD The cloning catalytic domain of AR2 (hADAR2) is typically a stop codon, and heterologously To target G>A repair of expressed mRNA, it has been used in various configurations (H answillemenke,et al.(2015)J.Am.Chem.Soc. ,137:15875-15881;Vogel,et al.(2014)ChemM edChem 9:2021-2025;Vogel,et al.(2014)Ang ew Chem.Int.Ed.Engl.,53:6267-6271;Schnei der,et al.(2014)Nucleic Acids Res.,42:e8 7;Montiel-Gonzalez,et al.(2016) Nucleic A cids Res.,44:e157;Montiel-Gonzalez,et al (2013)Proc.Natl.Acad.Sci.,110:18285-182 90). One approach involves the native RNA-binding domain of ADAR2 being used at nanomolar concentrations. Binds to specific short RNA hairpins with affinity (Austin, et al. (2002) (J.Am.Chem.Soc., 124:10966-10967) Bacteriophage Diramuda (λN; Montiel-Gonzalez, et al. (2013) Pro (c.Natl.Acad.Sci.,110:18285-18290) RNA string derived from RNA string It was replaced with a synthetic peptide. Subsequently, targeted editing of heterologous mRNA occurred in the λN-recognizing stem cell. Hybrid ADAR2 with an RNA guide containing regions complementary to the target mRNA and target mRNA. This is achieved by expressing proteins (Montiel-Gonzalez, et al. (2016) Nucleic Acids Res.,44:e157;M ontiel-Gonzalez,et al.(2013)Proc.Natl.Ac ad.Sci.,110:18285-18290).

[0017] Previously, both endogenous RNA and mRNA were repaired in a way that produced functional proteins. Regarding this, it has not been repaired by site-directed RNA editing. However, This approach to G>A mutations in endogenous Mecp2 is described herein for the first time. This has been demonstrated. Mutations in Mecp2 affect the domain that encodes a well-established function. Yes. Furthermore, the fidelity of repair can be measured using sequence analysis, Western blotting, and single-cell analysis. It can be monitored by immunochemistry at the level. Here, endogenous Mecp2 transcription To effectively repair G>A mutations within a substance, recombinant hADAR2-λN protein ( This specification uses an editase (also called an editase). Transfected mouse neuroblasts After determining the parameters for Mecp2 editing in cytoplasmic tumor (N2A) cells, adeno-associated virus Using (AAV), Rett disease with severe human G>A mutations in the DNA-binding domain. Primary neuronal cultures were transduced from a candidate mouse model (MeCP2 317G>A ; MeCP2 R106Q This mutation leads to a decrease in MeCP2 protein levels. Binding to heterochromatin is significantly reduced. The editing efficiency of mutant RNA is first improved. Quantification was performed using Ron, and then the MeCP2 protein levels were rescued by editing. Heterochromacy is an important characteristic of MeCP2 in cells, including ron, glial, and non-neuronal cell types. We tested whether this would lead to a concentration of bonds in the loratin structure. The results show that site-directed RNA editing is effective for Rett syndrome and other conditions that can be treated with gene therapy. This demonstrates that the MECP2 mutation, which is the underlying cause of many diseases, can be therapeutically repaired. They are doing it.

[0018] According to the present invention, a method for editing nucleic acid molecules within cells is provided. In a particular embodiment, The nucleic acid molecule being edited is an RNA molecule, especially a nuclear RNA molecule (e.g., a primary transcript). It is pre-mRNA, or mRNA (for example, mRNA before transport from the nucleus). In the embodiment, the nucleic acid molecule to be edited is an endogenous and / or nuclear transcript. CRI In gene editing techniques such as SPR, off-target mutations in the genome are permanent. In other words, intracellular RNA turnover is a transient process when off-target mutations occur within RNA. This means that... Furthermore, RNA editing, unlike CRISPR genome editing, is step-by-step. It can be hierarchical. As a result, off-target mutations are not necessarily edited to 100%. That's not the case.

[0019] In embodiments, the present invention provides nucleic acids that offer fine-tuning of protein expression and / or function. The present invention provides a method for RNA editing molecules. For example, the method of the present invention is used to edit an unedited state. Compared to this, it allows for the restoration of normal levels of protein expression and / or function. In the context of the treatment described in the details, the method of the present invention, compared to the untreated state, is normal. Restoration of protein expression and / or function to levels or at least close to normal levels is possible. Make it Noh.

[0020] In embodiments, the present invention, for example, in the context of the described therapy, (for example, via medication) This provides a method for RNA editing nucleic acid molecules that offers adjustable transient editing. The invention enables reversible editing of target RNA.

[0021] In certain embodiments, the cells to be edited are non-dividing cells. In certain embodiments, editing The cells being treated are neurons and / or glial cells. In certain embodiments, edited The cells being edited are non-neuronal cells. In certain embodiments, the cells being edited are neurons. Cells (e.g., neurons) are found in the central nervous system (e.g., brain, spinal cord) and / or peripheral nervous systems. They can be found in the nervous system. Cells can be present in the target being treated (for example, in an in vivo procedure). The cells may be treated in vitro and then administered to a subject (e.g., EVA). Sovivo treatment method).

[0022] In certain embodiments of the present invention, the method involves 1) linking or fusing to an RNA-binding domain 1) A nucleic acid molecule encoding an RNA editing enzyme and 2) guide RNA or guide RNA The process includes the step of delivering the encoding nucleic acid molecule to the cell. In certain embodiments, RNA binding is involved. The domain is ligated to the N-terminus of the RNA editing enzyme. In the embodiment, the RNA editing enzyme and A fusion containing an RNA-binding domain produces at least one nuclear localization signal (NLS). Not included. In the embodiment, the fusion containing the RNA editing enzyme and RNA binding domain is small It also contains at least one additional nuclear localization signal (NLS). For example, RNA editing enzymes and The fusion containing the RNA-binding domain contains 1, 2, 3, 4, 5 or more NLSs. It also includes. When multiple NLSs are used, each NLS may be directly connected to each other. Alternatively, they may be separated by amino acid linkers of 1 to approximately 5 amino acids. In this embodiment, the NLS is located at the N-terminus of the fusion protein. An example of an NLS is Kosug i et al. (J.Biol.Chem.(2009)284:478-485; Provided in this specification (as incorporated herein). In certain embodiments, NLS is Consensus array K(K / R)X(K / R)(Sequence number 58)(For example, monosegmental type (mon Includes (oppartite)NLS). In certain embodiments, NLS is a consensus array (K / R)(K / R)X 10~12 (K / R) 3 / 5 (Sequence number 59) (In the formula, (K / R ) 3 / 5 This means that at least three of the five amino acids are either lysine or arginine. This includes (representing that it is). In certain embodiments, the NLS includes the c-myc NLS. In a particular embodiment, the c-myc NLS is sequence PAAKRVKLD (sequence number 54) Includes. In certain embodiments, NLS is nucleoplasmin NLS. In this state, nucleoplasmin NLS has the sequence KRPAATKKAGQAKKKK (sequence number Includes (No. 60). In certain embodiments, the NLS comprises the SV40 large T antigen NLS. In a specific embodiment, the SV40 large T antigen NLS is sequence PKKKRKV (SEQ ID NO: 4 7) Includes. In certain embodiments, the fusion is made up of three SV40 large T antigens NLS (e.g. If, then, include the sequence DPKKKRKVDPKKKRKVDPKKKRKV (sequence number 67) In various embodiments, the NLS is the above sequence (for example, sequence numbers 58, 59, 60, 4 7, 54 or 67) In which these sequences are replaced, added or deleted by one or more substitutions, additions or deletions (e.g.) For example, about 1, or about 2, or about 3, or about 4, or about 5, or about 10, or Approximately 15 substitutions, additions, or omissions (including approximately 1-5, or approximately 1-10, or approximately 1-15). Mutations / mutations may include, for example, the loss of lysine in the NLS. In certain embodiments, lysine in the NLS may include, The amino acids may be substituted with arginine amino acids, and / or within the NLS The ginine amino acid may be substituted with the lysine amino acid. Fusion proteins are fusion proteins. The protein may further contain a purified tag (e.g., an HA tag) at its N-terminus.

[0023] The nucleic acid molecules of the present invention may be contained within a single vector or in separate vectors. It may be included in the RNA editing domain, for example, RNA editing linked or fused to the RNA-binding domain. Nucleic acid molecules encoding enzymes and nucleic acid molecules encoding guide RNA are contained within a single vector. It is included in. Nucleic acid molecules are sequential (before or after) and / or simultaneously (at the same time). It can be delivered to cells. Nucleic acid molecules can be delivered in the same composition or in a separate composition (for example, a separate composition). (If contained in a vector) it can be delivered. In certain embodiments, nucleic acid molecules can be delivered in a single vector. They are delivered, in particular, by viral vectors such as AAV vectors.

[0024] In certain embodiments, the RNA editing enzyme is human. In certain embodiments, RNA editing The enzyme is a deaminase. Examples of deaminases include, but are not limited to, those that act on RNA. Adenosine deaminase (ADAR), apolipoprotein Bm mRNA editing enzyme, Catalyst polypeptide-like (APOBEC (e.g., APOBEC1, APOBEC3A, AP) OBEC3G)), and activation-inducible cytidine deaminase (AICDA or AID; Examples include C:G being converted to T:A). In certain embodiments, RNA editing enzymes are used. ADARs such as ADAR1, ADAR2, or ADAR3. In certain embodiments, The RNA editing enzyme is ADAR1 (e.g., Gene ID:103 and GenBa nk deposit numbers NM_001111.5 and NP_001102.3 and these (See isoform). In certain embodiments, the RNA editing enzyme is ADAR2. The editing enzyme may be less than the full length. In certain embodiments, the RNA editing enzyme is of its natural It lacks the RNA-binding domain. For example, an RNA editing enzyme lacks the catalytic domain of the enzyme. It may contain, or it may consist of the catalytic domain of the enzyme.

[0025] An example of the amino acid sequence of human ADAR1 is: MNPRQGYSLS GYYTHPFQGY EHRQLRYQQP GPGSSP SSFL LKQIEFLKGQ LPEAPVIGKQ TPSLPPSLPG LR PRFPVLLA SSTRGRQVDI RGVPRGVHLR SQGLQRGFQ H PSPRGRSLPQ RGVDCLSSHF QELSIYQDQE QRILK FLEEL GEGKATTAHD LSGKLGTPKK EINRVLYSLA K KGKLQKEAG TPPLWKIAVS TQAWNQHSGV VRPDGHSQ GA PNSDPSLEPE DRNSTSVSED LLEPFIAVSA QAWN QHSGVV RPDSHSQGSP NSDPGLEPED SNSTSALEDP LEFLDMAEIK EKICDYLFNV SDSSALNLAK NIGLTKA RDI NAVLIDMERQ GDVYRQGTTP PIWHLTDKKR ERM QIKRNTN SVPETAPAAI PETKRNAEFL TCNIPTSNAS NNMVTTEKVE NGQEPVIKLE NRQEARPEPA RLKPPV HYNG PSKAGYVDFE NGQWATDDIP DDLNSIRAAP GE FRAIMEMP SFYSHGLPRC SPYKKLTECQ LKNPISGLL E YAQFASQTCE FNMIEQSGPP HEPRFKFQVV INGRE FPPAE AGSKKVAKQD AAMKAMTILL EEAKAKDSGK S EESSHYSTE KESEKTAESQ TPTPSATSFF SGKSPVTT LL ECMHKLGNSC EFRLLSKEGP AHEPKFQYCV AVGA QTFPSV SAPSKKVAKQ MAAEEAMKAL HGEATNSMAS DNQPEGMISE SLDNLESMMP NKVRKIGELV RYLNTNP VGG LLEYARSHGF AAEFKLVDQS GPPHEPKFVY QAK VGGRWFP AVCAHSKKQG KQEAADAALR VLIGENEKAE RMGFTEVTPV TGASLRRTML LLSRSPEAQP KTLLPLT GSTF HDQIAMLSHR CFNTLTNSFQ PSLLGRKILA AI IMKKDSED MGVVVSLGTG NRCVKGDSLS LKGETVNDC H AEIISRRGFI RFLYSELMKY NSQTAKDSIF EPAKG GEKLQ IKKTVSFHLY ISTAPCGDGA LFDKSCSDRA M ESTESRHYP VFENPKQGKL RTKVENGEGT IPVESSDI VP TWDGIRLGER LRTMSCSDKI LRWNVLGLQG ALLT HFLQPI YLKSVTLGYL FSQGHLTRAI CCRVTRDGSA FEDGLRHPFI VNHPKVGRVS IYDSKRQSGK TKETSVN WCL ADGYDLEILD GTRGTVDGPR NELSRVSKKN IFL LFKKLCS FRYRRDLLRL SYGEAKKAAR DYETAKNYFK KGLKDMGYGN WISKPQEEKN FYLCPV (Sequence ID 72) .

[0026] In certain embodiments, the deaminase domain of ADAR1 is the amino acid of SEQ ID NO: 72 Includes 839-1222. In certain embodiments, the RNA editing enzyme is its deaminase. At least 80%, 85%, 90%, 95%, 97%, and 99% of the main Array No. 72. Or 100% homology or identity, in particular at least 95%, 97%, 99%, or 1 Contains sequences with 00% homology or identity.

[0027] In certain embodiments, the RNA editing enzyme contains the deaminase domain of human ADAR2. In certain embodiments, the deaminase domain of human ADAR2 is used in GenBank Contains amino acids 299-701 of accession number U82120. In certain embodiments, ADAR2 or its deaminase domain contains the E488Q mutation (Montiel-Go nzalez, et al. (2016) Nucleic Acids Res.,44 :e157). In certain embodiments, the deaminase domain of human ADAR2 is LHLDQTPSRQPIPSEGLQLHLPQVLADAVSRLVLGKFGD LTDNFSSPHAR RKVLAGVVMTTGTDVKDAKVISVSTGTKCINGEYMSDRG LALNDCHAEII SRRSLLRFLYTQLELYLNNKDDQKRSIFQKSERGGFRLK ENVQFHLYIST SPCGDARIFSPHEPILEEPADRHPNRKARGQLRTKIESG E GTIPVRSNAS IQTWDGVLQGERLLTMSCSDKIARWNVVGIQGSLLSIFV EPIYFSSIILG SLYHGDHLSRAMYQRISNIEDLPPLYTLNKPLLSGISNA EARQPGKAPNF SVNWTVGDSAIEVINATTGKDELGRASRLCKHALYCRWM RVHGKVPSHLL RSKITKPNVYHESKLAAKEYQAAKARLFTAFIKAGLGAW VEKPTEQDQFS Includes LTP (sequence number 55; E488 is indicated).

[0028] In certain embodiments, the deaminase domain of human ADAR2 is LHLDQTPSRQPIPSEGLQLHLPQVLADAVSRLVLGKFGD LTDNFSSPHAR RKVLAGVVMTTGTDVKDAKVISVSTGTKCINGEYMSDRG LALNDCHAEII SRRSLLRFLYTQLELYLNNKDDQKRSIFQKSERGGFRLK ENVQFHLYIST SPCGDARIFSPHEPILEEPADRHPNRKARGQLRTKIESG Q GTIPVRSNAS IQTWDGVLQGERLLTMSCSDKIARWNVVGIQGSLLSIFV EPIYFSSIILG SLYHGDHLSRAMYQRISNIEDLPPLYTLNKPLLSGISNA EARQPGKAPNF SVNWTVGDSAIEVINATTGKDELGRASRLCKHALYCRWM RVHGKVPSHLL RSKITKPNVYHESKLAAKEYQAAKARLFTAFIKAGLGAW VEKPTEQDQFS Includes LTP (sequence number 71; E488Q is indicated).

[0029] In certain embodiments, the RNA editing enzyme is at least 80 with SEQ ID NO: 55 or 71 Homologie or identity of %, 85%, 90%, 95%, 97%, 99%, or 100%, special Sequences having at least 95%, 97%, 99%, or 100% homology or identity Includes.

[0030] As described above, RNA editing enzymes are linked to the RNA-binding domain. The element may be directly linked to the RNA-binding domain (i.e., without a linker sequence). Alternatively, they may be linked via a polypeptide linker. For example, a polypeptide linker - 1 to approximately 50 amino acids, 1 to approximately 25 amino acids, 1 to approximately 20 amino acids, 1 It may contain approximately 15 amino acids, 1 to approximately 10 amino acids, or 1 to approximately 5 amino acids. In certain embodiments, the linker is the array (GGGGS) n (Sequence number 44) In the formula, n includes 1 to approximately 10, especially 1 to approximately 5. For example, the linker sequence is GG It may be GGSGGGGSGGGGS (Sequence No. 45). In various embodiments, the linker - However, in the above sequence (for example, sequence number 44 or 45), one of these sequences or Multiple substitutions, additions, or deletions (e.g., about 1, or about 2, or about 3, or about 4, etc.) or approximately 5, or approximately 10, or approximately 15 (approximately 1-5, or approximately 1-10, or approximately 1-1 Mutations / variations may include substitutions, additions, or deletions of (including 5).

[0031] The RNA-binding domain binds to specific RNA sequences and / or RNA structures (e.g., hairpins). It may be any polypeptide that specifically recognizes RNA. In certain embodiments, RNA binding The binding domain is an artificial RNA-binding domain, and in particular, has a high affinity for RNA. Yes. In certain embodiments, the RNA-binding domain is a phage RNA-binding domain. (For example, Keryer-Bibens, et al., Biol. Cell (2008 (See 100:125-138). For example, the RNA-binding domain is a λN peptide (for example) (See Cilley, et al., RNA (1997) 3:57-67) or MS2 coat protein (e.g., Johansson, et al. Sem. Vi) (See rol. (1997) 8:176-185). In certain embodiments, λN pept Tide has the amino acid sequence: MNARTRRRERRAEKQAQWKAAN (SEQ ID NO: 46) ) includes. In certain embodiments, the λN peptide is at least 80% of the SEQ ID NO: 46. 85%, 90%, 95%, 97%, 99%, or 100% homology or identity, especially less It contains sequences with at least 95%, 97%, 99%, or 100% homology or identity. nothing.

[0032] In this embodiment, the fusion protein is de-aminobutyric to human ADAR2 via an amino acid linker. λN peptide linked to the amino terminus of the enzyme domain (e.g., SEQ ID NO: 55 or 71) Includes cido (sequence number 46). In certain embodiments, the linker is sequence (GGGGS) n (Sequence code 44; where n is 1 to approximately 10 or 1 to approximately 5) or sequence GGGGSG Contains GGGSGGGGS (SEQ ID NO: 45). In certain embodiments, the fusion protein is array: MNARTRRRERRAEKQAQWKAANGGGGSGGGGGSGGGGSLH LDQTPSRQPIP SEGLQLHLPQVLADAVSRLVLGKFGDLTDNFSSSPHARRK VLAGVVMTTGT DVKDAKVISVSTGTKCINGEYMSDRGLALNDCHAEIISR RSLLRFLYTQL ELYLNNKDDQKRSIFQKSERGGFRLKENVQFHLYISTSP CGDARIFSPHE PILEEPADRHPNRKARGQLRTKIESGEGTIPVRSNASIQ TWDGVLQGERL LTMSCSDKIARWNVVGIQGSLLSIFVEPIYFSSIILGSL YHGDHLSRAMY QRISNIEDLPPLYTLNKPLLSGISNAEARQPGKAPNFSV NWTVGDSAIEV INATTGKDELGRASRLCKHALYCRWMRVHGKVPSHLLRS KITKPNVYHES KLAAKEYQAAKARLFTAFIKAGLGAWVEKPTEQDQFSLT Includes P (sequence number 68).

[0033] In certain embodiments, the fusion protein further comprises at least one NLS at its N-terminus. In certain embodiments, the NLS is the SV40 large T antigen NLS (e.g., SEQ ID NO: 4). 7) Includes. In certain embodiments, the fusion contains three SV40 large T antigen NLS (e.g. For example, it includes sequence number 67). In certain embodiments, the fusion protein is sequence: DPKKKRKVDPKKKRKVDPKKKRKVMNARTRRRERRAEKQ AQWKAANGGGG SGGGGSGGGGSLHLDQTPSRQPIPSEGLQLHLPQVLADA VSRLVLGKFGD LTDNFSSPHARRKVLAGVVMTTGTDVKDAKVISVSTGTK CINGEYMSDRG LALNDCHAEIISRRSLLRFLYTQLELYLNNKDDQKRSIF QKSERGGFRLK ENVQFHLYISTSPCGDARIFSPHEPILEEPADRHPNRKA RGQLRTKIESG EGTIPVRSNASIQTWDGVLQGERLLTMSCSDKIARWNVV GIQGSLLSIFV EPIYFSSIILGSLYHGDHLSRAMYQRISNIEDLPPLYTL NKPLLSGISNA EARQPGKAPNFSVNWTVGDSAIEVINATTGKDELGRASR LCKHALYCRWM RVHGKVPSHLLRSKITKPNVYHESKLAAKEYQAAKARLF TAFIKAGLGAW Includes VEKPTEQDQFSLTP (sequence number 69).

[0034] In certain embodiments, the fusion protein is at least 80 with SEQ ID NO: 68 or 69. Homologie or identity of %, 85%, 90%, 95%, 97%, 99%, or 100%, special Sequences having at least 95%, 97%, 99%, or 100% homology or identity Includes.

[0035] The guide RNA of the present invention targets or specifically hybridizes a target sequence. The sequence that performs the action (e.g., the complementary sequence), and the sequence recognized by the RNA-binding domain Includes. Guide RNA is directed towards the nucleotide (e.g., adenosine) that is being modified or edited. Includes mismatch with the target sequence. When used herein, "specifically hive The term "redize" refers to the requirement that nucleic acid molecules be 100% complementary to the target sequence. This does not mean that the (one or more) mismatched nucleotides that are changed / edited are Sequences that are not included are those that are at least 80%, 85%, 90%, 95%, 97%, and 99% of the target sequence. % or 100% complementary, especially at least 95%, 97%, 99%, or 100% complementary. It is possible. However, as described herein, the sequence is off-target editing. To reduce this, additional mismatches (e.g., G-mismatches) may be included. Embodiments So, the array consists of approximately 1, 2, 3, 4, or 5 elements, Approximately 10 or 15 (approximately 1-5, or approximately 1-10, or approximately 1-15) This may include mismatches in areas of complementarity (e.g., guide). In certain embodiments, this may include mismatches in areas of complementarity (e.g., guide). The gap between RNA and the target sequence is at least approximately 10, at least approximately 12, at least approximately 15, at least about 17, at least about 20, at least about 25, at least about 30, at least about 35, or more nucleotides. In certain embodiments The complementary region (for example, between the guide RNA and the target sequence) has approximately 15 to 30 nucleotides. Cleotide, approximately 15-25 nucleotides, approximately 20-30 nucleotides, approximately 2 0 to approximately 25 nucleotides, or approximately 20, 21, 22, 23, 24, and It consists of 25 nucleotides. Typically, a mismatch occurs between the guide RNA and the target sequence. The center of the complementary region between the guide RNA and the target sequence (for example, the guide RNA sequence) It is directed towards the central 50%. In certain embodiments, the target sequence includes sequence number 52. nothing.

[0036] Guide RNA contains the correct or desired editing of RNA within the cell. Using A, any mutation, including missense mutations and nonsense mutations, In particular, point mutations can be corrected. For example, in Rett syndrome, the common G>A abruption can be corrected. Mutations exist. These mutations result in amino acid changes R106Q(CAA), W10 4X(UAG) and R306H(CAC) are obtained. In the case of nonsense mutations, These are C-to-T mutations, where A is at the 3' position and forms a stop codon, or in the middle. It is in the central position (for example, UAG to UGG). Edit the nonsense mutation and the stop code The 'n' can be removed. In certain embodiments, the guide RNA is (for example, ADAR (By) A can be deaminated, including C which matches A in these mutants. .

[0037] The guide RNA of the present invention is recognized by an RNA-binding domain and contains one or more RNAs Includes a column. In certain embodiments, the guide RNA is recognized by the RNA-binding domain. It contains two sequences. In certain embodiments, two sequences recognized by the RNA-binding domain The sequence may be on both sides of a sequence that is a mismatch or specifically hybridizes with the target sequence. For example, one sequence recognized by the RNA-binding domain (e.g., BoxB) The target mutation is located at approximately 15-20 or 16-18 nucleotides 5' of the RNA. The second sequence recognized by the syntactic domain (e.g., BoxB) is approximately 8% of the target mutation. It is located at ~12 or approximately 10 nucleotides 3'. In certain embodiments, RNA-binding domain The sequence recognized by the guide RNA is not at the end of the guide RNA (i.e., the end of the guide RNA) (The terminal sequence may be complementary to the target sequence). Recognized by the RNA-binding domain. If there are more than one sequence, those sequences may be the same or different, but The same RNA-binding domain is recognized. In certain embodiments, RNA binding The sequence recognized by the domain is the BoxB sequence. In a particular embodiment, BoxB The sequence is GCCCUGAAAAAGGGC (sequence number 48) or GGCCUGAAA Includes AAGGGCC (sequence number 49). In certain embodiments, the BoxB sequence is sequence number At least 80%, 85%, 90%, 95%, 97%, 99% with number 48 or 49, and This refers to 100% homology or identity, particularly at least 95%, 97%, 99%, or 100%. It has a percentage of homology or identity.

[0038] In certain embodiments, the guide RNA is the sequence shown in Table 1 (RNA bar of DNA molecule). Targets or includes sequences (including John). In certain embodiments, the guide RNA is , at least 80%, 85%, 90%, 95%, 97%, and 99% of the sequences shown in Table 1. Or 100% homology or identity, in particular at least 95%, 97%, 99%, or 1 Targets or contains sequences that have 00% homology or identity. Specific implementation In this state, the guide RNA is sequence numbers 15-22, especially sequence numbers 15, 17, 19 or 2 Includes one of 1, or sequence numbers 15-22, especially sequence numbers 15, 17, 19 or 2 At least 80%, 85%, 90%, 95%, 97%, 99%, or 100% of one of the 1 % homology or identity, in particular homology of at least 95%, 97%, 99%, or 100%. Contains sequences that have sex or identity.

[0039] A nucleic acid molecule containing a nucleic acid sequence that codes for guide RNA is a nucleic acid that codes for guide RNA. It may contain multiple copies of the sequence. For example, each nucleic acid molecule may be under the control of a promoter. Nucleic acid sequences 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 that code for a guide RNA , or more copies.

[0040] In certain embodiments, the nucleic acid molecule of the present invention is used as a vector (e.g., plasmid), particularly in the case of Delivered to cells via an Virus Vector (e.g., infection, transfection, electricity) It is expressed (via perforation, etc.). The expression vector of the present invention has a strong promoter, constitutive Promoter, tissue or cell-specific promoter, ubiquitous promoter, and / or a control promoter can be used. In certain embodiments, RNA-bound The promoter of the nucleic acid molecule encoding the RNA editing enzyme, which is linked or fused to the main, It is a tissue or cell-specific promoter. In certain embodiments, the promoter is a new It is a ron-specific promoter or a ubiquitous promoter. An example of a promoter is... It is well known in the technical field, and not limited to it, but synaptic promoters, especially synapsins Includes I promoter, CAG promoter, and MECP2 promoter. Guide RN Regarding A, examples of RNA promoters are well-known in this field, and are not limited to them. RNA polymerase III promoters (e.g., U6 and H1; e.g., Mysli nski et al. (2001) Nucl. Acids Res.,29:2502 -09) or other promoters known to express short RNAs. In this embodiment, the promoter is a human U6 promoter. Examples of expression vectors include, but are not limited to, plasmids and viral vectors. For example, adeno-associated viruses (AAVs), adenoviruses, retroviruses, and lentiviruses. Examples include viruses. In certain embodiments, the vector may be AAV (e.g., AAV-1 ~AAV-12 and other serotypes and hybrid AAV vectors; e.g., AAV1 , or AAV2, or AAV3, or AAV4, or AAV5, or AAV6 , or AAV7, or AAV8, or AAV9, or AAV10, or AAV 11, or AAV12). In certain embodiments, the vector is a neuron and / or it can infect glial cells.

[0041] In another embodiment, the present invention includes RNA editing and therapeutic methods, as described above. The step includes delivering guide RNA or nucleic acid encoding guide RNA to a cell, Uses nucleic acid molecules encoding RNA editing enzymes linked to or fused to an RNA-binding domain. No. ADARs (e.g., ADAR 1-3) are expressed at high levels in the nervous system. Therefore, the guide RNA of the present invention is an ADAR enzyme, particularly ADAR1 or ADAR2, etc. The endogenous deaminase enzyme can be attracted to the endogenous MECP2 RNA. In the embodiments, the present invention enables the involvement of endogenous ADAR enzymes and recombinant ADAR enzymes It does not require any raw materials. By using only guide RNA, non-mammalian RNA binding is possible. The potential immune response to the main target is avoided. Furthermore, this method allows for highly repetitive It is now possible to include guide sequences in the AAV vector, reducing off-target editing. In a particular embodiment of this aspect of the present invention, the guide targets or targets the target sequence. Sequences that specifically hybridize to (e.g., complementary sequences), and deaminases, particularly Includes sequences recognized by ADAR (e.g., ADAR1 or ADAR2). The RNA is not limited to, but includes RNA hairpins (e.g., based on the natural targets of ADARs). , mismatches that create a double-stranded "bulge" recognized by ADAR, and / or It may contain any other sequences that are typically required for targeted editing using endogenous ADARs. In the application morphology, the guide RNA contains one, two, or more BoxB sequences. In this embodiment, the guide RNA includes an R / G binding site from GluR2 (Wette ngel,et al.(2017) Nucleic Acids Res.,45(5 ):2797-2808;Fukuda,et al.(2017)Sci.Rep., 7:41478; For example, GUGGAAUAGUAUAACAA-UAUGCUAAAU GUUGUUAUAGUAUCCCAC (SEQ ID NO: 70), or at least 80%, 85%, 90%, 95%, 97%, 99%, or 100% homology or identity, especially less It contains sequences with at least 95%, 97%, 99%, or 100% homology or identity. (m). In certain embodiments, the guide RNA includes having an internal loop (Lehm ann,et al.(1999)J.Mol.Biol.,291(1):1-13; For example, loops containing 4, 6, 8, 10, or more nucleotides. In a specific embodiment, the guide RNA is a region complementary to the MECP2 RNA, with editing errors. Includes matches (e.g., A:C) and A:G mismatches for off-target editing. In certain embodiments, the nucleic acid molecule encoding the guide RNA is described herein. It is contained within the vector. In certain embodiments, the guide RNA is the U6 promoter. Alternatively, it may be expressed by other promoters that express small RNA molecules.

[0042] According to the present invention, a method for treating, suppressing and / or preventing hereditary disorders of the central nervous system is Provided. According to the present invention, progressive neurodevelopmental disorders are treated, suppressed and / or prevented. A method is provided. For example, the present invention, in an embodiment, is characterized by a mutation of the target RNA. This provides treatment, suppression, and / or prevention of hereditary central nervous system disorders. In embodiments, this The invention directly or By indirectly recovering, for example by editing the read RNA as G This provides methods for treating, suppressing, and / or preventing progressive neurodevelopmental disorders.

[0043] According to the present invention, hereditary central nervous system disorders, including progressive neurodevelopmental disorders such as Rett syndrome, are available. Methods for treating, suppressing and / or preventing the disease are effective in vivo or ex vivo. For example, in the case of the in vivo method, (one or more) nucleic acid molecules (e.g., the fusion described) Administered to targets that code for proteins (e.g., those coding for the described guide RNA). In the exovivo method, cells (syngeneic or homogeneous) are subjected to this nucleic acid molecule (e.g., as described). It is brought into contact with a compound protein (for example, one that codes for the described guide RNA). The drug is introduced into the target. Embodiments relating to the treatment are equally applicable to in vivo and ex vivo methods. It is used.

[0044] According to the present invention, hereditary diseases related to the MECP2 gene can be treated, suppressed and / or A method for prevention is provided. In embodiments, the present invention relates to the functional MECP2 protein To restore Bell to the level of a non-disease or healthy subject, for example, at the RNA level, It provides gene editing. Mecp2 can be human or mouse, especially human. Implementation In this context, the present invention increases the level of functional MECP2 protein in response to a disease state. For example, it provides gene editing at the RNA level. In an embodiment, MECP2 Genetic disorders associated with this condition include neonatal encephalopathy, microcephaly, X-linked intellectual disability, and PPM-X syndrome. (Manic-depressive illness, pyramidal tract signs, Parkinsonian syndrome and megatesticle), bipolar disorder, Parkinson Son's syndrome, increased muscle tone, grandiose reflex, and megatesticle syndrome, or a combination of these. In this embodiment, a genetic disorder related to the MECP2 gene affects either a male or female subject. It has an effect.

[0045] According to the present invention, a method for treating, suppressing, and / or preventing Rett syndrome is provided. In certain embodiments, Rett syndrome is characterized by a G>A mutation in MeCP2. For example, Rett syndrome is characterized by MECP2 mutations R106Q, W104X, or R306H. This can be considered a characteristic. Exemplary amino acid and nucleotide sequences of human MECP2 are shown in GenBa nk Gene ID 4204 and GenBank deposit number NM_004992.3 and provided in NP_004983.1 (GenBank deposit number NM_00111) 0792.1, NP_001104262.1, NM_001316337.1 and N (See also the isoform P_001303266.1). In certain embodiments, Rett syndrome The candidate group includes the R106Q mutation in MeCP2. In certain embodiments, this method is used for RN Nucleic acid molecules encoding RNA editing enzymes linked to or fused to the A-binding domain, and The procedure includes the step of administering a nucleic acid molecule encoding an id RNA or guide RNA. In the embodiment, the method administers guide RNA or a nucleic acid molecule encoding guide RNA. The procedure includes the following steps: The nucleic acid molecule may be administered directly to the target or delivered to cells. And this can be administered to the target.

[0046] In certain embodiments, the amino acid sequence of MECP2 is MVAGMLGLRE EKSEDQDLQG LKDKPLKFKK VKKDKK EEKE GKHEPVQPSA HHSAEPAEAG KAETSEGSGS APAVPEASAS PKQRRS IIRD RGPMYDDPTL PEG W T R KLKQ RKSGRSAGKY DVYLINPQGK AFRSKV ELIA YFEKVGDTSL DPNDFDFTVT GRGSPSRREQ KP PKKPKSPK APGTGRGRGR PKGSGTTRPK AATSEGVQVK RVLEKSPGKL LVKMPFQTSP GGKAEG GGAT TSTQVMVIKR PGRKRKAEAD PQAIPKKRGR KPGSVVAAAA AEAKKK AVKE SSIRSVQETV LPIKK R KTRE TVSIEVKEVV KPLLVSTLGE KSGKGL KTCK SPGRKSKESS PKGRSSSASS PPKKEHHHHH HH SESPKAPV PLLPPLPPPP PEPESSEDPT SPPEPQDLS S SVCKEEKMPR GGSLESDGCP KEPAKTQPAV ATAAT AAEKY KHRGEGERKD IVSSSMPRPN REEPVDSRTP V This is TERVS (sequence number 56).

[0047] R106, W104, and R306 are shown above with an underline.

[0048] In a particular embodiment, the nucleic acid encoding MECP2 is atgg tagctgggat gttagggctc agggaagaaa a gtcagaaga ccaggacctc cagggcctca aggacaaa cc cctcaagttt aaaaaggtga agaaagataa gaaa gaagag aaagagggca agcatgagcc cgtgcagcca tcagcccacc actctgctga gcccgcagag gcaggca aag cagagacatc agaagggtca ggctccgccc cggctgtgcc ggaagc ttct gcctccccca aacagcggcg ctccatcatc cg tgaccggg gacccatgta tgatgacccc acctgcct g aaggctggac acggaagctt aagcaaagga aatct ggccg ctctgctggg aagtatgatg tgtatttgat c aatccccag ggaaaagcct ttcgctctaa agtggagt tg attgcgtact tcgaaaaggt aggcgacaca tccc tggacc ctaatgattt tgacttcacg gtaactggga gagggagccc ctcccggcga gagcagaaac cacctaa gaa gcccaaatct cccaaagctc caggaactgg cag aggccgg ggacgcccca aagggagcgg caccacgaga cccaaggcgg ccacgtcaga gggtgtgcag gtgaaa aggg tcctggagaa aagtcctggg aagctccttg tc aagatgcc ttttcaaact tcgccagggg gcaaggctg a ggggggtggg gccaccacat ccacccaggt catgg tgatc aaacgccccg gcaggaagcg aaaagctgag g ccgaccctc aggccattcc caagaaacgg ggccgaaa gc cggggagtgt ggtggcagcc gctgccgcg aggc caaaaa gaaagccgtg aaggagtctt ctatccgatc tgtgcaggag accgtactcc ccatcaagaa gcgcaag acc cgggagacgg tcagcatcga ggtcaaggaa gtg gtgaagc cctgctggt gtccaccctc ggtgagaaga gcgggaaagg actgaagacc tgtaagagcc ctgggc ggaa aagcaaggag agcagcccca aggggcgcag ca gcagcgcc tcctcacccc ccaagaagga gcaccacca c catcaccacc actcagagtc cccaaaggcc cccgt gccac tgctcccacc cctgccccca cctccacctg a gccgagag ctccgaggac cccaccagcc cccctgag cc ccaggacttg agcagcagcg tctgcaaaga ggag aagatg cccagaggag gctcactgga gagcgacggc tgccccaagg agccagctaa gactcagccc gcggttg cca ccgccgccac ggccgcagaa aagtacaaac acc gagggga gggagagcgc aaagacattg tttcatcctc catgccaagg ccaaacagag aggagcctgt ggacag ccgg acgcccgtga ccgagagagt tagctga (SEQ ID NO: 5 7).

[0049] In embodiments, the present invention provides a method for treating, suppressing and / or preventing Rett syndrome, including classical Rett syndrome and variant Rett syndrome (also known as non-typical Rett syndrome). In embodiments, the Rett syndrome is the Zappella variant, Hanefeld variant, Rolando variant and / or "incomplete" variant. In embodiments, the present invention provides, but is not limited to, ataxia, uncontrolled hand movements (e.g., hand wringing or squeezing, clapping, rubbing, washing, or movement from hand to mouth), acquired microcephaly, autistic-like behaviors, respiratory irregularities, feeding and swallowing difficulties, growth retardation, hypotension, panic attacks,

[0050] In embodiments, the present invention provides a reduction, improvement and / or suppression of one or more symptoms of Rett syndrome, including, but not limited to, ataxia, uncontrolled hand movements (e.g., hand wringing or squeezing, clapping, rubbing, washing, or movement from hand to mouth), acquired microcephaly, autistic-like behaviors, respiratory irregularities, feeding and swallowing difficulties, growth retardation, hypotension, panic attacks, teeth grinding (Bruxism), tremors, apraxia, cardiac irregularities (e.g., QT interval and / or T-wave abnormalities), and seizures. In embodiments, the present composition can be used in combination with any of the following in the method for treating, suppressing and / or preventing, for example, Rett syndrome: tridecanoic acid, fingolimod (e.g., GILENYA), ketamine, EPI-743 (batquinone), sarizotan ( EMD-128, 130), statins (e.g., lovastatin), tricyclic antidepressants (TC

[0051] In embodiments, the present composition can be used in combination with any of the following in the method for treating, suppressing and / or preventing, for example, Rett syndrome: tridecanoic acid, fingolimod (e.g., GILENYA), ketamine, EPI-743 (batquinone), sarizotan ( (e.g., EMD-128, 130), statins (e.g., lovastatin), tricyclic antidepressants (TC (e.g., EMD-128, 130), statins (e.g., lovastatin), tricyclic antidepressants (TC A, for example, desipramine), glatiramer acetate (e.g., COPAXONE), dextro lometorfan, and / or an oral cholesterol 24-hydroxylase (CH24 H) inhibitor (e.g., TAK-935 / OV935).

[0052] As described above, the present invention provides nucleic acid molecules, vectors, and compositions and methods for suppressing, treating, and / or preventing Rett syndrome. Compositions containing at least one nucleic acid described herein are also included in the present invention. In certain embodiments, the composition comprises at least one guide RNA or a nucleic acid molecule encoding a guide RNA (e.g., an expression vector ter) and at least one pharmaceutically acceptable carrier. The composition may further comprise a nucleic acid molecule encoding an RNA editing enzyme that is mainly linked or fused to an RNA binding domain . In certain embodiments, all of the nucleic acid molecules are encoded within a single expression vector (e.g., a viral vector er (e.g., AAV)). Alternatively, the nucleic acid molecules may be included within separate compositions containing at least one pharmaceutically acceptable carrier. The present invention also encompasses a kit comprising a first composition comprising at least one guide RNA or a nucleic acid molecule encoding a guide RNA (e.g., an expression vector ) and a second composition comprising at least one nucleic acid molecule encoding an RNA editing enzyme linked or fused to an RNA binding domain . The first and second compositions may further comprise at least one pharmaceutically acceptable carrier . In certain embodiments, the kit of the present invention comprises at least one guide RNA or a nucleic acid molecule encoding a guide RNA (e.g., an expression vector) and / or an RNA binding domain . The first and second compositions may further comprise at least one pharmaceutically acceptable carrier . In certain embodiments, the kit of the present invention comprises at least one guide RNA or a nucleic acid molecule encoding a guide RNA (e.g., an expression vector) and / or an RNA binding domain RNA (e.g., an expression vector) and / or an RNA binding domain A first composition comprising a nucleic acid molecule encoding an RNA editing enzyme linked to or fused to an RNA The first and second compositions further comprise at least one pharmaceutically acceptable carrier. It is visible.

[0053] As described above, the compositions of the present invention are useful for treating Rett syndrome. A therapeutically effective amount of the composition may be administered to the subject in need. Dosage, method and The administration time can be readily determined by those skilled in the art, given the teachings provided herein. be.

[0054] The components described herein are generally administered to patients as formulations. The terms “patient” or “subject” used in this invention refer to human or animal subjects. Minutes may be used therapeutically under the guidance of a physician to treat the indicated disease or disorder. ru.

[0055] A formulation containing the components of the present invention is water, buffered saline, ethanol, polyol (for example, Glycerol, propylene glycol, liquid polyethylene glycol, etc.), dimethyl Lufoxide (DMSO), oil, surfactant, suspending agent, or suitable mixture thereof, etc. Formulated for convenient administration using an acceptable medium (e.g., a pharmaceutically acceptable carrier). The concentration of the drug in the selected medium may vary, and the medium may be the desired route of administration of the formulation. Selection may be made based on the following criteria, unless the conventional medium or drug is incompatible with the drug being administered. And its use in pharmaceutical formulations is intended.

[0056] The selection of an appropriate formulation depends on the chosen method of administration. For example, the components of the present invention are optional. It can be administered by direct injection into the desired tissue (e.g., the brain) or the surrounding area. In this case, the formulation contains components dispersed in a medium compatible with the blood or the target tissue. In this case, the formulation contains components dispersed in a medium compatible with the blood or the target tissue.

[0057] The treatment can be administered, for example, parenterally, by injection into the bloodstream (e.g., intravenously), or subcutaneously, intramuscularly or intraperitoneally. In certain embodiments, the treatment is administered by direct injection (e.g., into the tissue to be treated). Injectable formulations are known in the art. When injection is selected as the method of administering the treatment, measures must be taken to ensure that a sufficient amount of the molecule reaches the target cells to exert a biological effect.

[0058] A pharmaceutical composition containing the compound of the present invention as an active ingredient, intimately mixed with a pharmaceutical carrier, can be prepared by conventional pharmaceutical compounding techniques. The carrier can take a variety of forms depending on the desired form of preparation for administration, such as intravenous, oral or parenteral, etc. In the preparation of oral dosage forms, for example, in the case of oral liquid preparations (e.g., suspensions, elixirs and solutions, etc.), conventional pharmaceutical media such as water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, etc.; or in the case of oral solid preparations (e.g., powders, capsules and tablets, etc.), any of the carriers such as starch, sugar, diluents, granulating agents, lubricants, binders, disintegrants, etc. can be used. Injectable suspensions can be prepared, in which case

[0059] The formulations of the present invention are formulated in dosage unit form for ease of administration The dosage unit forms used herein are those of formulations suitable for the patient receiving treatment. It refers to a distinct unit in theory. Each dosage, along with the selected drug carrier, produces the desired effect. It should contain the calculated amount of active ingredient. Procedure for determining the appropriate dosage unit. This is well known to those skilled in the art. The dosage unit increases or decreases proportionally based on the patient's weight. To obtain. The appropriate concentration for alleviating a particular pathological condition is known in the art. This can be determined by calculating the drug concentration curve.

[0060] The method of the present invention provides for the treatment of a target disease or disorder after administration of one or more compositions of the present invention. The procedure may further include monitoring to check the effectiveness of the method. For example, if the subject has Rett disease. The characteristics of the suspected group can be monitored.

[0061] definition The following definitions are provided to facilitate understanding of the present invention: The singular forms "a," "an," and "the" are different depending on the context. Unless it is clear that it has the meaning of a step, it may include multiple referents.

[0062] "Pharmacologically acceptable" means that the use in animals, especially humans, is approved by the federal or state government. Approval by the prefectural regulatory authority, or the United States Pharmacopeia or other generally recognized drugs This indicates that it is listed in the Pharmacopoeia.

[0063] "Carrier" refers to, for example, diluents, adjuvants, and preservatives (e.g., thymelsol (Thi Mersol, benzyl alcohol, antioxidants (e.g., ascorbic acid, metabisulfite) Sodium sulfate), solubilizer (e.g., Tween® 80, polysorbate 8) 0) Emulsifier, buffer (e.g., Tris-HCl, acetate, phosphate), antimicrobial agent, bulking agent A substance (e.g., lactose, mannitol), an excipient, an adjuvant, or the activator of the present invention may be used in combination with the substance (e.g., lactose, mannitol), an excipient, an adjuvant, or the activator of the present invention. This refers to the vehicle to which the drug is administered. Pharmaceutically acceptable carriers are water and oil (petroleum, animal, plant). It may be a sterile liquid such as water or saline solution, including substances or synthetic sources. In addition, aqueous solutions of dextrose and glycerol are preferred as carriers, especially for injection solutions. It is used. Suitable drug carriers are available at Remington: The Science and Practice of Pharmacy,(Lippincott,Willia ms and Wilkins);Liberman,et al.,Eds.,Pha rmaceutical Dosage Forms,Marcel Decker,N New York, NY; and Rowe, et al., Eds., Handboo k of Pharmaceutical Excipients,Pharmaceu It is listed in tical Pr.

[0064] As used herein, the term “treat” means (for example, to treat one or more symptoms) (In this case) providing benefits to patients suffering from a disease, including improvement of the patient's condition and delay of the progression of the condition. It refers to all kinds of procedures.

[0065] As used herein, the term “prevent” means the probability that an object will develop a condition. This refers to preventative measures for individuals at risk of developing a condition that can lead to a decline in health.

[0066] The "therapeutic effective amount" of a compound or pharmaceutical composition is defined as the amount of a specific disorder or disease and / or This refers to the amount that is effective in preventing, suppressing, or treating the symptoms.

[0067] As used herein, the term “subject” means animals, in particular mammals, in particular humans. To point.

[0068] The term "isolation" refers to the separation of a compound from other components present during its manufacture. "Isolation" refers to the artificial or synthetic mixture of a compound or material with other compounds or materials, or the basic activity of a compound. Although it does not substantially interfere, impurities that may exist due to, for example, incomplete purification or the addition of stabilizers may be present. It is not intended to exclude the existence of pure substances.

[0069] The terms "linker," "linker domain," and "linking" refer to covalent or minor connections. At the very least, two compounds, such as an RNA editing enzyme and an RNA-binding domain, can covalently bind to each other. It refers to the chemical part containing a chain of atoms. A linker is an amino acid sequence (for example, 1 to 50 atoms). 1 amino acid, 1-25 amino acids, 1-20 amino acids, 1-15 amino acids, 1 It can consist of up to 10 amino acids, or 1 to 5 amino acids.

[0070] As used herein, the term “olivone complex” refers to two or more, preferably three. It contains nucleic acid molecules composed of one or more ribonucleotides and / or deoxyribonucleotides. The precise size of an oligonucleotide depends on various factors, as well as the specific oligonucleotide. It depends on the application and use of [the system / technology].

[0071] As used herein, "nucleic acid" or "nucleic acid molecule" refers to either single-stranded or double-stranded nucleic acids. This refers to DNA or RNA molecules, and in the case of single-stranded DNA, their complementary sequences in a linear or circular form. This refers to the molecule. In discussions of nucleic acid molecules, the sequence or structure of a particular nucleic acid molecule is defined as starting from 5'. Nucleic acids of the present invention may be described herein in accordance with the usual practice of providing the sequence in the 3' direction. In this regard, the term "isolated nucleic acid" is sometimes used. This term applies to DNA. In that case, it is isolated from directly adjacent sequences in the naturally occurring genome of the organism from which it originates. It refers to isolated DNA molecules. For example, "isolated nucleic acid" refers to plasmids or viral vectors. Which vector was inserted, or which is the genomic DNA of a prokaryotic or eukaryotic cell or host organism? It may contain DNA molecules incorporated into it.

[0072] A "vector" is a gene such as a plasmid, cosmid, bacmid, phage, or virus. It is a genetic element, to which another gene sequence or element (either DNA or RNA) is present. They can be combined. To result in a combined array or a copy of an element, the vector is a replica. The vector may be either RNA or DNA, and may be single-stranded or double-stranded. It can be a chain. The vector is not limited to, but can also be a promoter, enhancer, or translation initiation chain. Polyadenylation signals, terminators, and other polynes of host cells or organisms Transcriptional and translational regulatory sequences that promote the expression of creotide or polypeptide coding sequences, etc. It may include an expression operon or element.

[0073] "Expression operons" include promoters, enhancers, and translation initiation signals (e.g., AT G or AUG codons, polyadenylation signals, terminators, etc., in the host cell or transcriptional and translational regulatory sequences that promote the expression of nucleic acid or polypeptide coding sequences in organisms. This refers to a nucleic acid segment that may contain [a specific gene / expression vector]. "Expression vector" refers to a nucleus in a host cell or organism. It is a vector that promotes the expression of acid or polypeptide coding sequences.

[0074] As used herein, a "nuclear localization signal" (NLS) refers to a molecule or polypeptide that promotes the movement of an associated polypeptide into the nucleus of a cell. In certain embodiments, the nuclear localization signal is a peptide that directs a protein to the nucleus. Typically, an NLS consists mainly of basic, positively charged amino acids (notably lysine and arginine). An NLS can be monopartite, bipartite or multipartite. An NLS is typically a short peptide (e.g., less than about 20 amino acids, about less than 15 amino acids, or less than about 10 amino acids). Examples of NLSs are provided by Kosugi et al. (J. Biol. Chem. (2009) 284:478 - 485; incorporated herein by reference ). In certain embodiments, the NLS comprises the consensus sequence K(K / R)X(K / R) (SEQ ID NO: 58) (e.g., a monopartite NLS). In certain embodiments, the NLS comprises the consensus sequence ( (K / R)(K / R)X 10~12 (K / R) 3 / 5 (SEQ ID NO: 59), where (K / R) 3 / 5 indicates that at least 3 of 5 amino acids are either lysine or arginine 3 / 5 (SEQ ID NO: 59). In certain embodiments, the NLS is the SV40 large T antigen NLS 3 / 5 (e.g., PKKKRKV (SEQ ID NO: 47)). In certain embodiments, the c - myc NLS comprises the sequence PAAKRVKLD (SEQ ID NO: 54). In certain embodiments, the N LS is the nucleoplasmin NLS KRPAATKKAGQAKKKK (SEQ ID NO: 60 ) With respect to the provided sequence, the lysine and arginine amino acids are interchangeable. .

[0075] In various embodiments, by including NLS, editing in the absence of NLS is possible, for example. Conversely, it reduces or suppresses off-target editing. For example, in various embodiments, This gene editing method, including NLS, causes off-target editing of approximately 10% or 20%. Or about 30%, or about 40%, or about 50%, or about 60%, or about 70% Or reduce by approximately 80%, 90%, or 100%. In various embodiments, This gene editing method, including NLS, increases off-target editing by approximately 2x, or 3x, or It reduces it by approximately 5 times, 10 times, or 30 times.

[0076] The following examples are provided to illustrate various embodiments of the present invention. Therefore, this invention is by no means intended to limit itself.

[0077] [Example 1] material and method Plasmid construction pcDNA 3 encoding a λN peptide fused to the wild-type ADAR2 catalytic domain. I obtained a .1+ plasmid (Thermo Fisher Scientific). Montiel-Gonzalez, et al. (2016) Nucleic Aci ds Res.,44:e157;Montiel-Gonzalez,et al.( 2013)Proc.Natl.Acad.Sci.,110:18285-18290 ). Editor E488Q cDNA is analyzed using overlapping PCR with wild-type editase. It was created by and cloned into pcDNA3.1+. Two copies of the HA epitope - And three copies of the SV40 NLS are placed in the frame of the hybrid editor. By inserting into the frame and at the N-terminus, both versions of the editor can be used with pcD. Modified in NA3.1+ (pGM1090, wild type; pGM1091, E488Q). The Mecp2-BoxB guide describes three different Mecp2 G>A mutations. Synthetic oligonucleotides and their antisense sequences are used with Bsa1 overhand. It anneals and pENTR / U6 polylinker [pGM1099(W104X), pGM1181(306H), pGM1085(R106Q)](Thermo Fis Cloned in her Scientific. Off-target AG mismatch The Mecp2-BoxB guide (pGM11089), which includes this, is also available in pENTR / U6. Regarding ecp2 editing substrates, mouse Mecp2 E1 isoform cDNA (Gen The EcoRI-KpnI fragment with bank deposit number NP_001075448.1) is used in pE It was cloned into a multi-cloning site of GFP-N3 (Clontech). Individual G>A mutations in ecp2 can be overwritten using the same restriction enzyme site overhang. - Prepared by wrap PCR, pEGFP-N3 (Thermo Fisher Sc Cloned in frame as a fusion protein with eGFP (enthific) All subclonings were validated by sequence analysis. Plasmid construction and PCR Table 1 shows the primer sequences used for amplification. [Table 1] TIFF2023145597000003.tif171156

[0078] Plasmid construct Contains a fusion cDNA (editase) of λN peptide and wild-type ADAR2 catalytic domain. The first construction is described (Montiel-Gonzalez, et al. (2 016) Nucleic Acids Res.,44:e157;Montiel-G onzalez,et al.(2013)Proc.Natl.Acad.Sci., 110:18285-18290). This is two copies of the HA epitope tag, pull Next, the in-frame and N-terminal SVs are sent to the hybrid editor (pGM1090). Modified to include three copies of 40 NLS. To do this, HA epitope Two copies of the sequence and two single-stranded oligonucleotides encoding the Kozak sequence, Anneal with EcoRI and BspeI overhangs and 5' λN domain sequence They were linked together. Three copies of SV40 NLS were connected to pECFP-Nuc(Clontec h) Amplified by PCR and the HA epitope is used with BspEI overhang The tag was added between the tag and the λN domain. The ADAR2 catalytic domain contains the E488Q mutation. Plasmid pGM1091 ​​was prepared using the same steps.

[0079] The plasmid pGM1258 used in the AAV transduction experiment is a synapsin I promoter. Editase cDNA under the control of the human U6 promoter, respectively. Mecp2 with off-target AG mismatch located in R106Q Guide DNA 6 Includes two copies. U6-Mecp2 R106QTo introduce a guide region, human U6 Promoter and plasmid pX552(60958;Addgene;Swiech ,et al.(2015)Nat.Biotechnol.,33:102-106) The CRISPR sgRNA sequence is removed by restriction digestion (NdeI / ApaI). , 6 U6-Mecp2 R106Q The guide array is placed in two steps between these sites. Inserted. In the first step, U6-Mecp2 R106Q Three copies of the guide area The following restricted areas: NdeI / MfeI, MfeI / SpeI, and SpeI / NheI 4 PCR amplicons (pGM1108 template) containing +ApaI(pGM1257) It was cloned to pX552 by directional ligation. In the second step, U6- Mecp2 R106Q Three additional copies of the guide region are placed in the following restricted area: NheI / Use primers that add SacI, SacI / AfIII, and AfIII / ApaI. The final plasmid pGM12 was prepared by PCR amplification from pGM1108. 58 is limited to pGM1257 digested with NheI / ApaI, and three U6-Me cp2 R106Q The samples were prepared by four-directional ligation using PCR amplicons. To introduce the editase cDNA into pGM1258, the editase ORF is matched. The sequence was amplified from plasmid pGM1091, and NcoI and EcoRI overhaul Using a ping, pGM1257 was added downstream of the synapsin I promoter.

[0080] AAV vectors and virus preparations AAV1 / 2 backbone vector containing human synapsin I promoter, pX552 This was obtained from Addgene (plasmid 60958; Swiech, et al.). (2015) Nat. Biotechnol., 33:102-106). Guide cDN A(pGM1186, editase only; pGM1258, editase and R106Q) Using and without the six copies of the id, the eGFP-KASH coding sequence was modified for HA pX552 was modified by replacing it with a tagged NLS editor cDNA. Before creating the virus, the editorase and guide sequences were validated by sequence analysis.

[0081] Each AAV1 / 2 chimeric vector was transfected using the adenovirus-free transfection method (M atsushita,et al.(1998)Gene Ther.,5:938-9 45;Earley,et al.(2017)J.Virol.,91:e01980 -16) Three 225cm per vector 2 On a flask scale, human embryo Prepared using infant-derived kidney 293 (HEK293) cells. Each flask contained approximately 2 × 10⁶ cells. 7 H EK293 cells were mixed with polyethyleneimine (PEI) in a DNA:PEI weight ratio of 1:2. The following four plasmid DNAs were transfected with a total of 45 μg: Plasmid D The NA mixture contains 15 μg of pHelper (Agilent) and 7.5 μg of each of the following: It has HLP19-1 and pHLP19-2, as well as two terminal inversion sequences (ITRs). AAV vector editor recombinant plasmid containing the AAV vector genome sequence It contained (15 μg). pHLP19-1 contains AAV2 Rep protein and AA This is an AAV1 helper plasmid that supplies the V1 VP protein, pHLP19-2 AAV2 Hell, which supplies AAV2 Rep protein and AAV2 VP protein. It is a perplasmid (Grimm, et al. (2003) Blood 102:2 412-2419). Cells were collected three days after transfection. Then, AAV Vector particles are recovered from cells by cell lysis and stored in a HiTrap® heparin column. (GE Healthcare; Desterro, et al. (2003) J.Ce Purification was performed using (ll Sci., 116:1805-1818). Each viral titer was measured. Quantitative dot blotting using probes prepared against editor-zecode sequences. This was determined by assay.

[0082] cell culture Neuro2A cells (ATCC CCL-131) are placed in a 5% CO2 humidified incubator. - At 37°C, 10% FBS (Lot No. AAC20-0955; HyClone) in D Maintained by MEM (Thermo Fisher Technologies). First generation The ureon was created by targeted homologous recombination (Janelia Farms), and the gene Mecp2 characterized by type determination R106Q Induced from mouse strains. All movements The experiment was approved by the Oregon Health & Science University Animal Experimentation Committee. The offspring (P0) was isolated. Killed by the head, the brain is chilled in an ice-cold Hanks basal salt solution (HBSS) containing 25 mM Hepes. Dissection was performed in a pH of 7.4. Individual hippocampi were excised without meninges and pooled according to genotype. The tissue was then subjected to 1% trypsin and 0.01% DNas in HBSS for 10 minutes at 37°C. The tissue was treated with e I. The tissue sample was rinsed three times in HBSS at room temperature, and then treated with 25 mM glucose and 1% PE. Nishilin / Streptomycin, 1% Equine Serum (Lot No. B02307-7021; H They were isolated in yClone and minimal essential medium (Gibco) containing 1% FBS. The fluorocarbon was separated by filtering through a 0.4 μm filter, and Neuroba sal-A (Thermo Fisher Scientific), 1×Glutam ax (Thermo Fisher Scientific), 2%B27 (Therm (Fischer Scientific), and penicillin / streptomycin In a neuronal proliferation medium, one well of a 12-well dish was placed in a poly-L-lysine coated dish. 5 x 10 per unit 5 Individual cells, or 5 × 10⁶ cells in a 96-well glass chamber. 4 at individual density The seeds were sown. After 24 hours, the neurons were given a complete culture medium change to remove cell debris. 2 Half of the culture medium was replaced every 3 days. The cells were maintained at 37°C in 5% CO2.

[0083] Mecp2 R106Q Mouse generation and genotyping Mecp2 R106Q The targeting vector for creating mice is the Mecp2 exon. 3. Subsequently, the neomycin cassette adjacent to the flippase recognition target (frt) of intron 3... The first 1.2kb of Mecp2 exon 4, and phosphoglycerate kinase pro It consisted of a neomycin resistance gene expressed from the motor (PGK). Linear construct Electroporation was performed into mouse embryonic stem cells (mESCs), and the correctly targeted clone was identified as G418. Identified by susceptibility testing and sequencing. Knock-in Mecp2 R106Q Alleles Neomycin-resistant mice were generated from mESCs using a standard procedure. Mecp2 R106Q Mice and flipper jelly from the Rosa 26 gene locus A mouse expressing combinase (stock number 009086; Jackson Labs) The cells were removed by crossbreeding. The removal of the cassette was confirmed by sequencing.

[0084] The following primers amplify the third intron region of the Mecp2 gene: Mecp2 -R106Q Fwd(5′ ggacctatgtatgatgaccc 3′(array) Number 50)) and Mecp2-R106Q Rev(5′ ggtcattgggct Using agactgaa 3′ (SEQ ID NO: 51), Mecp2R106Q mice Genotyping was performed. Mecp2 R106Q The amplicons of knock-in animals are neo Since it contains the remaining frt portion used to remove the mycin cassette, PC The R product is 93 base pairs larger than in the wild type (392 bp vs. 299 bp).

[0085] RNA editing To analyze N2A cells, 1.3 × 10⁶ cells were used per well in a 12-well plate. 3 pieces Cells were seeded at a cell density. After 24 hours, Opti-MEM (trademark) low serum medium (Th (Fisher Scientific) Lipofectami in a 2:1 ratio n(trademark)2000 (Thermo Fisher Scientific) and DNA Use to select cells in wild-type or E488Q editase (pGM1090 and 1091 ), 1 copy of the guide (pGM1099, pGM1181 or pGM1108), and Mecp2-egfp cDNA (pGM1174, pGM1172 or pGM11 Transfected with plasmid D containing 73). Plasmid D added per well The amounts of NA were 125 ng of target, 250 ng of editase, and 2.5 μg of guide. Yes. After 72 hours, collect the cells and follow the manufacturer's instructions for Purelink (registered trademark). Total RNA was isolated using the RNA Mini Kit (Ambion). TURB Using the O DNA-Free (Trademark) Kit (Ambion), residual plasmid DNA is removed. Removed. All RNA was removed using SuperScript(registered trademark) III First-St. rand Synthesis System(Life Technologies) Reverse transcription was performed using [method name] and primed with oligo dT. Transfect Mec p2-egfp cDNA is used as the 5' primer for the CMV promoter of pEGFP-N3. and for sequence analysis, PCR using reverse primers for the egfp gene. The width was reduced. For editing analysis of primary neurons, DIV7 was used, 5 × 10 5 Individual Kaiba First Generation New ¹ 4 AAV1 / 2 based on the infection multiplicity of individual viral genomes Transfection was performed. The viral load did not exceed 5% of the total culture medium volume. After one week, collect the cells and edit them as described for transfected N2A cells. We analyzed the rates.

[0086] Reverse transcription PCR (RT-PCR) and direct sequencing of PCR products can be used to sequence A to I. The efficiency of editing was determined. Quantification of sequence peak height from antisense strands was performed using Bioed. IT software package (mbio.ncsu.edu / BioEdit / bio) edit.html;File > Batch Export of Raw Seq Using the Trace Data, we will process the four-color trace sequence. Therefore, it was determined. Next, the amount of editing in each part was determined by T (unedited) and C ( (Edited) The percentage of edited cDNA was calculated and determined using the maximum peak height {1 00% × [C height / (T height + C height)]}. 5% editing detection limit, ratio decreases R1 GA peak height was measured for a mixture containing the 06Q mutant and the wild-type Mecp2 plasmid. This was determined by using the A / G peak height of the sense strand, which is more accurate than using the A / G peak height of the sense strand. Therefore, the C / T peak height of the antisense chain was quantified (Eggington, et al. (2011) Nat.Commun., 2:319). However, in order to clarify All chromatograms are shown in reverse complementary strands.

[0087] Western blotting Primary hippocampal neurons transduced using AAV1 / 2 were transduced using 100 μL of whole cell lysis buffer. (25mM Tris, pH 7.6, 150mM NaCl, 1% Igepal CA-6) 30; Sigma), 1% deoxycholic acid, 0.1% SDS, protease inhibitor (complete) Completely EDTA-free; Roche), 1 mM β-mercaptoethanol, and 250 units Dissolved in benzonase (Sigma-Aldrich) at 4°C. The solubilized product was then incubated at 4°C. The soluble fraction was isolated by centrifugation at 9300 × g for 10 minutes. BCA protein assay Using Ikit (Pierce Biotechnology), the protein concentration The measurement was performed using Mops-SDS electrophoresis buffer (Thermo). Equal amounts of protein solubilized material were added. Fisher Scientific NuPage (registered trademark) 4-12% Bis-T Separation was performed using a lithogel (Thermo Fisher Scientific), and the protein was separated. to nitrocellulose membrane (GE Healthcare Life Sciences) Blotting was performed. The membrane was 1x TBST (0.05% Tween(registered trademark) 20 Blocking with 3% BSA (including TBS) for 1 hour, then, rabbit anti-mMeCP2(C) Either ovance or rabbit anti-β-actin (8227; Abcam) at 4°C Incubated overnight. After washing three times with 1x TBST, the blot was removed using anti-rabbit IgG. DyLight (registered trademark) 680 (1:10000 dilution; Thermo Science) Incubated with tific for 1 hour. Odyssey® Imaging Quantitative blot analysis was performed using the System (LI-COR Biosciences). did.

[0088] immunostaining Primary hippocampal neurons were fixed in 4% paraformaldehyde in PBS for 20 minutes at room temperature. The fixed cells were then subjected to 1×PBSG (0.1M glycine in 1×PBS) for 10 minutes at room temperature. The cells were washed twice. Then the cells were blocked and permeabilized at 4°C for 1 hour [0.5 %Igepal CA-630, Sigma; 3% BSA in 1x PBS (source), added MeCP2 (Rabbit mAb D4F3; Cell Sign) was stored overnight at 4°C in a humid chamber. Aling) and HA (rat mAb 3F10; Roche) produced The cells were incubated with the next antibody. The cells were washed three times with 1×PBS containing 0.5% Igepal. Furthermore, secondary antibodies Alexa 488 and Alexa 568 (Thermo Fishe Incubated with (r Scientific) for 1 hour. Contains 0.5% Igepal. After further washing with 1×PBS, incubate the cells with 300nM DAPI for 5 minutes. Next, the cells were washed again with 1×PBS. Using a color-preventing reagent (Thermo Fisher Scientific), overnight I took all images using a 40x immersion objective lens with a Zeiss 710 confocal lens. Acquired as z-stacks of 0.5 μm optical sections using a microscope. HA and MeCP2 fluorescence Images were acquired using the same settings across all samples. Total cell count or antibody-positive cells. The number of cells is measured using the ImageJ cell counter plugin (National Institutes of Health, imagej.ni Determined by h.gov / ij, version 1.60_65 (32-bit).

[0089] statistical analysis All statistics are calculated using GraphPad version 6.0 software (Prism). The procedure was carried out using one-way ANOVA to determine the percentage of A-to-I editing in N2A cells. The analysis was performed using the Bonferroni post-hoc test. Mecp2 R10 6Q / y Level of A-to-I editing in transduced neurons, MeCP2 protein level Western blots comparing the structures and MeCP2 enrichment in heterochromatin structures are shown. The number of neurons was analyzed using independent t-tests. All experimental results Expressed as mean ± standard deviation.

[0090] result Mecp2 mRNA is targeted by an editase that targets heterologously expressed Mecp2 mRNA. G>A mutations can be repaired. Human MECP2 has at least three G>A mutations that cause classical Rett syndrome. There is MeCP2. R106Q and MeCP2 W104X The two mutations are in methyl DNA It resides within the Joint Domain (MBD) and contains one MeCP2 R306H This is the NCoR interaction (Fyfe, et al. (2003) J.Child.) Neurol.,18:709-713;Lyst,et al.(2013)Nat. Neurosci., 16:898-902) (Figure 1A). Editase is used to treat these sudden To determine whether the mutation can be repaired, editase, guide RNA and Mec Editing was tested after transient transfection of p2 cDNA into N2A cells. To distinguish species-specific MeCP2 proteins from endogenous ones, heterologous expression is used. The C-terminal eGFP was tagged to the MeCP2. Editorase and Mecp2-g fp cDNA enhances the cytomegalovirus (CMV) early gene promoter. The sensor is expressed, and the guide is expressed from the human U6 nuclear small RNA gene promoter. ADAR2 edits endogenous mRNA in the nucleus as its primary transcript, so λN-P In addition to ptido, there are three Simian virus 40 large T antigen nuclear localization signals (NLS). A copy of this was added to the editor (Desterro, et al. (2003) J (Cell.Sci.,116:1805-1818). Each guide RNA is λN peptide It contains two stem loops (BoxB) that represent the array recognized by the code. The Box B stem loop is located at 16-18 bases 5' of target A, and the second is at 3' of target A. It is located at 10 bases (Figure 1B). The number and location of stem loops relative to target A are studied. Based on (Montiel-Gonzalez, et al. (2016) Nuclei c Acids Res.,44:e157;Montiel-Gonzalez,et al.(2013)Proc.Natl.Acad.Sci.,110:18285- (18290) Mecp2 was empirically determined by transfection analysis. This is optimal for the C mismatch at that site of the complementary guide (Schneider, et al.). al. (2014) Nucleic Acids Res.,42:e87;Wong ,et al.(2001)RNA 7:846-858;Kallman,et al. (2003) Nucleic Acids Res.,31:4874-4881), All Mecp2 guide mRNAs contain this mismatch.

[0091] N2A cells are subjected to an editase, a separate plasmid encoding MeCP2-GFP, The plasmid was cotransfected with a third plasmid containing or lacking the guide sequence. Three days later, cDN synthesized from the target region of Mecp2-gfp mRNA using the Sanger method. A was analyzed (Figures 1C and 1D). The relative peak height at target A was determined. Therefore, editing efficiency was measured. All three Mecp2 mutations were edited in a guide-dependent manner. This was consistent with ADAR2-mediated editing requiring double-stranded RNA (Figure 1C and Figure 1D). The editing percentage of target A is similar to the sequence preference of the ADAR2 catalytic domain, as is the 5' nucleotide. It changed depending on the environment (Eggington, et al. (2011) Nat. Co mmun.,2:319;Lehmann,et al.(2000)Biochemi (stry 39:12875-12884). Specifically, in in vitro screening Based on this, the optimal 5' nucleotide hierarchy for A deamination by the ADAR2 catalytic domain is U>A>C>G, and the most optimal 3' nucleotide is C~G~A>U. W104 X(UAG) was edited most efficiently (76±10%), R306H(CAC, 34±3 %) and R106Q (CAA, 25±2%) follow, but in the case of Mecp2, statistically There was no difference (Figure 1D). Editor Zess for repairing Mecp2 G>A mutations To further optimize the stem, the W104X mutation is more common in human patients. To cause a more severe form of Rett syndrome than R306H, focus on R106Q. (Fyfe, et al. (2003) J.Child. Neurol., 18: 709-713;Cuddapah,et al.(2014)J.Med.Genet .,51:152-158).

[0092] Deaminase domain mutation, E488Q, improves the editing efficiency of hybrid editases. increase The hADAR2 catalytic domain containing the E488Q mutation exhibits a catalytic rate (Montiel- Gonzalez,et al.(2016)Nucleic Acids Res., 44:e157;Kuttan,et al.(2012)Proc.Natl.Aca d.Sci.,109:E3295-E3304) and the catalytic domain for substrate RNA Affinity (Lehmann, et al. (2000) Biochemistry 39: By increasing both 12875-12884), A>I editing efficiency is increased. This characteristic makes the E488Q mutation more suitable for unfavorable 5' and 3' environments. It becomes possible to achieve a high level of editing (Montiel-Gonzalez, et al. (2016) Nucleic Acids Res.,44:e157;Kutt an,et al.(2012)Proc.Natl.Acad.Sci.,109:E 3295-E3304). Editase E488Q However, Mec has an optimal temperature of 5'C. p2 R106Q To test whether it increases the editing efficiency of target A, N2A cells to Mecp2 R106Q -egfp and editor E488Q Cotran in cDNA Sfected. Sequence analysis revealed that guide expression is required for editing, and Mecp2 mR NA editing % compared to wild-type editor E488Q It increased by approximately 2 times. This was shown to be the case (51±11% vs. 22±5%, n=3, P<0.01) (Figure 2A).

[0093] Hybrid editor enzyme with wild-type hADAR2 or hADAR2 E488Q Catalyst Use the main to transfect Mecp2 R106Q -egfp cDNA One off-target editing area was detected within the region (Figure 2B). Editing at this area... Silent codon changes, such as T105T (ACA>ACG), occur in off-target sites. G mismatch reduces off-target editing in the transfect substrate. This is possible (Vogel, et al. (2014) Angew Chem. Int. Ed. (Engl., 53:6267-6271). G mismatch is an O of Mecp2 mRNA. To decide whether to also reduce target editing, Mecp2 R106Q -eg fp cDNA, editor E488Q , and G miss at nearby off-target A Editing efficiency in N2A cells transfected with plasmids encoding a guide containing . This was analyzed (Figure 2C). When the editorase was targeted with a guide containing AG mismatch... The amount of off-target editing was significantly reduced (4.9±0% mismatch, 33±5%). No mismatch, n=3, P<0.0001; Figures 2D and 2E) Editing at target A There was no significant effect (Figures 2D and 2F). In all editing events, guided RN The presence of A was necessary (Figures 2E and 2F).

[0094] Site-directed RNA editing repairs endogenous mutations that cause Ret, and protein remission. Restore Bell and MeCP2 functionality Next, editor E488Q (i) Endogenous Mecp2 mRNA R106Q miss (ii) Repair sense mutations, (ii) Restore protein levels, (iii) In mice It binds to heterochromatin, a characteristic functional feature necessary for reversing reticular symptoms. We tested whether MeCP2's capabilities could be restored (Garg, et al.). (2013) J. Neurosci., 33:13612-13620). These tests Next, we isolated neurons from mice and introduced the R106Q mutation into the endogenous Mecp2 gene. The cells were manipulated to include one of two AAVs (AAV1 / 2). Quality was introduced. Both viruses are controlled by the human synapsin I promoter and editaseE48 8Q It expresses (Swiech, et al. (2015) Nat. Biotechnol .,33:102-106), each virus is controlled by the human U6 promoter Further including the 6 copies of the guide below (Off-Target Mismatch Guide; Figure 2C) It was there. Other viruses served as controls and lacked all guide sequences. Hippocampal neuron P0 Mecp2 R106Q / y Created from mice and tested in vitro for 7 days (DIV7). Shaped with a guide containing an AAV1 / 2 hybrid capsid or a control AAV vector. Quality was introduced. After further 7 days of virus expression, Mecp2 cDNA was added to the experimental culture. Both the eddie and guide were prepared from the control cultures and analyzed by Sanger assay. In cultures expressing this gene, 72±5% of Mecp2 mRNA is repaired (Figure 3A). No editing was detected in neurons transduced with a control virus lacking a guide. In addition to editing at 106Q, sequence analysis revealed several offshoots within the Mecp2 cDNA. The GET editing sites were also identified (Figure 3B). Off-target sites are complementary to the guide RNA. Most events occurred within the region, but one event occurred outside the guide (N126S).

[0095] Western blotting reveals MeCP2 protein in AAV1 / 2 transduced cultures. The functional outcomes of RNA editing were tested by measuring the quantity and quality. Other sudden changes in MBD Similar to mutations (Goffin, et al. (2011) Nat. Neurosci., 15:274-283;Brown,et al.(2016)Hum.Mol.Gen et., 25:558-570), MeCP2 R106QProtein levels are at wild-type levels. It is lower compared to the previous level (Figure 4). The decrease in the level of mutant MeCP2 protein indicates that This could be caused by instability (Goffin, et al. (2011) Nat. Neurosci., 15:274-283). Eddy in mutant primary neurons The expression of tase and guide compared to the expression of editase alone, which is beneficial for MeCP2 protein expression. The quality level was increased by approximately three times (Figure 4; 12.9±1% compared to no guidance, 35.3± (2% guided, n=3, P<0.001). This is the cause of the endogenous disease after editing. This is the first demonstration of functional restoration of proteins.

[0096] MeCP2 binds to methyl-CpG with high affinity both in vitro and in vivo. Skene,et al.(2010)Mol.Cell.,37:457-468;L agger,et al.(2017)PLoS Genet.,13:e100679 3) This is an important characteristic for normal function. In mouse cells, MeCP2 MB The D mutation leads to the formation of heterochromatin containing mCG-rich amplified satellite sequences. The ratio decreases (Brown, et al. (2016) Hum. Mol. Genet., 25:558-570;Heckman,et al.(2014)eLife 3:e 02676). MeCP2, an MBD mutation. R106Q Also, in vitro methyl-C It shows a decrease in binding to pG (Yang, et al. (2016) ACS Chem. Bi (ol.,11:2706-2715). MeCP2 R106Q This similarly reduces intracellular binding. Whether or not the amount was reduced, and the editing of G>A mutant Mecp2 RNA in heterochromatin To determine whether to restore enrichment, the nucleus is used as a guide or control. Me introduced using AAV1 / 2, which encodes an HA-tagged editor that does not contain the 'd' gene. cp2 R106Q / y Immunolabeling was performed on neuronal cultures (Figure 5). DNA rich in AT. DAPI(4',6-diamidino-2-phenyl) is a fluorescent indicator that binds strongly to the region. We identified the nucleus and heterochromatin using (Me) dendr. cp2 + / y In cultures from ), the nuclei reflect functional MBD, and DAPI staining hetamine The chloromatin (structure) showed classical MeCP2 enrichment (Figure 5A). In contrast, the guide distribution Mecp2 transduced with an editorase virus lacking rows. R106Q / y Prepared from siblings In the cultured samples, MeCP2 immunofluorescence indicated that mutations in MBD that interfere with DNA binding were predicted. As expected, they were scattered and distributed throughout the nucleus (Goffin, et al. (201 1) Nat. Neurosci., 15:274-283; Heckman, et al. (2014) eLife 3:e02676) (Figure 5B). The staining intensity is also greater than that of wild-type nuclei. Low, likely reflecting an unstable MeCP2 protein. In contrast, editase Mecp2 expresses both the γ and the guide RNA. R106Q Neurons are similar to wild-type nuclei. Clear increase in MeCP2 immunofluorescence up to the level, and MeCP2 in heterochromatin structure The enrichment of two proteins demonstrated functional recovery of MBD (Figures 5C and 5D). To quantify the fluorescence results, editors were used at the same proportion of cells, regardless of the presence of guides. We were the first to determine in three experiments that ze is expressed (editase only 67±7%, ede 67±10% of cells contained nitrase and guide; n=134 and 137 cells, respectively; Figure 5E). Next, in cultures transduced with eddieze and guide, eddieze is produced 74±11% of the cells exhibiting heterochromatic chromosomes (Figure 5F) and 49±8% of all cells were heterochromatic. It was determined that the structure shows enrichment of MeCP2 (Figure 5G). MeCP2 enrichment This is Mecp2 transduced with a virus lacking a guide. R106Q Nuclear heterochromatin The sequencing results were consistent with the fact that no editing was detected within the structure, indicating that the editing depends on the presence of guides. They are doing it.

[0097] ADAR repairs exogenous mRNA G>A mutations in African clawed frog oocytes. It has been used for this purpose (Woolf, et al. (1995) Proc. Natl. Acad.Sci.,92:8298-8302). The data presented herein is based on the operation Site-directed RNA editing using the generated hADAR2 catalytic domain is caused by an endogenous mutation m The ability to repair RNA and reverse cellular defects caused by mutations. This demonstrates that.

[0098] In mice and humans, three genes encode the ADAR protein, but AD Only AR1 and ADAR2 show catalytic activity from A to I (Nishikura, K. (2 010)Annu.Rev.Biochem.,79:321-349). natural ADAR Electron-mediated editing is crucial for post-transcriptional regulation of brain protein function, and iontochemistry is also important. First demonstrated in channels and receptors (Bhalla, et al. (2004)N at.Struct.Mol.Biol.,11:950-956;Sommer,et al.(1991)Cell 67:11-19;Burns,et al.(199 7) Nature 387:303-308) shows that many other proteins and non-coding proteins are involved. It is now known that it also affects RNA (Chen, et al. (2012) Curr .Top.Microbiol.Immunol.,353:111-121;Nish ikura, K. (2016) Nat.Rev.Mol.Cell Biol.,17: 83-96). The ADAR2 catalytic domain is used for its ability to edit heterologous mRNA (V ogel,et al.(2014)Angew Chem.Int.Ed.Engl. ,53:6267-6271;Schneider,et al.(2014)Nucl eic Acids Res.,42:e87;Montiel-Gonzalez,e t al. (2016) Nucleic Acids Res.,44:e157;Mo ntiel-Gonzalez,et al.(2013)Proc.Natl.Aca d.Sci.,110:18285-18290;Wong,et al.(2001) RNA 7:846-858) and its well-characterized editing mechanism (Kutt an,et al.(2012)Proc.Natl.Acad.Sci.,109:E 3295-E3304;Matthews,et al.(2016) The focus is on Nat. Struct. Mol. Biol, 23:426-433. In fact, when the editase contains the E488Q mutation within its catalytic domain, Mec Improved editing efficiency of p2 mRNA was observed (Montiel-Gonzalez, et al.) al. (2016) Nucleic Acids Res.,44:e157;Kut tan,et al.(2012)Proc.Natl.Acad.Sci.,109: E3295-E3304;Phelps,et al.(2015)Nucleic A cids Res., 43:1123-1132). hAD complexed into double-stranded RNA. Elucidation of the structure of the AR2 catalytic domain (Matthews, et al. (2016) Nat (Struct.Mol.Biol.,23:426-433) MeCP2 and others To further optimize the editing efficiency and specificity of the mutations, create other mutations. Provides valuable resources for manufacturing (Wang, et al. (2016) Nucle (ic Acids Res., 44:9872-9880). In contrast to previous approaches. ADARs typically edit primary transcripts within the nucleus, so all of the constructs here are special. It contained NLS that increased the editing efficiency of endogenous mRNA (Wong, et al.). (2001) RNA 7:846-858).

[0099] In transfected cells, the editase at target A E488Q Using a higher braid Due to improved collection efficiency, off-target editing in a single area within the guide region has also been enhanced. The number of target editing areas was reduced by using GA mismatch (Schn eider,et al.(2014)Nucleic Acids Res.,42: e87). In particular, five cDNs that represent highly expressed mRNA other than the target mRNA. The sequencing of A did not show off-target editing (Montiel-Gonzale z,et al.(2016)Nucleic Acids Res.,44:e157 ). However, surprisingly, in this study using neurons, off-target editing was observed. The site is between transfected Mecp2 mRNA and endogenous Mecp2 mRNA. They were different. Specifically, in endogenous repaired Mecp2 mRNA, the cDNA was different. Several of the Mecp2 mRNA expressed from within and outside the guide region are not present in the expressed Mecp2 mRNA. Additional off-target editing sites were observed (Figure 3B). Transfected Mecp2 m The difference in off-target editing sites between RNA and endogenous Mecp2 mRNA is that RNA Reflects array differences that may affect folding and other downstream processing events. It is possible that this is happening. Importantly, the off-target region of endogenous Mecp2 mRNA. None of these have been reported to cause Rett syndrome (Fyfe, et al. (2003) J. Child Neurol., 18:709-713). Rett syndrome Further using a group mouse model, we investigated the recovery of wild-type MeCP2 in symptomatic mice. Furthermore, it can be shown that behavioral symptoms can be reversed (Guy, et al. (2007)). Science 315:1143-1147;Sinnett,et al.(201 7)Mol.Ther.Methods Clin.Dev.,5:106-115;G adalla,et al.(2017)Mol.Ther.Methods Clin .Dev.,5:180-190;Garg,et al.(2013)J.Neuro sci.,33:13612-13620;Gadalla,et al.(2013) (Mol. Ther., 21:18-30).

[0100] [Example 2] Mecp2 mutation Mecp2317G>A Using mice that have this feature, in vivo this The method was tested. Mecp2 317G>A The mutation has an R106Q amino acid change. This results in MeCP2. In short, it's a Mecp2 mutation. 317G>A to have The mice used to perform the procedure were either with or without the use of 6 copies of guide RNA, according to the present invention. The nerves were treated with an AAV vector encoding an editase containing an 8Q mutation. Due to the characteristics of sex, an AAV vector containing the PHP.B capsid (AAV9 variant) was used. (Hordeaux et al. (2018) Mol. Ther., 26(3)) :664-668). 1.1 × 10 10 AAV of the viral genome equivalent (vge) The virus was injected stereotactically into the hippocampus of the mouse. Three to four weeks after direct viral injection, the mice were killed, and Me CP2 function was detected in the brain. As shown in Table 2, efficient targeting RNA in vivo. Editing and recovery of brain MeCP2 function were observed. Furthermore, based on RNA sequence analysis, The editing efficiency from A to I in dentate granule neurons was determined to be 39%, and CA1 neurons The editing efficiency from A to I in Ron was determined to be 64%. As can be seen in Figure 6, The MeCP2 intensity of dentate heterochromatin was measured by injection of AAV without guide RNA. Compared to mice, those injected with AAV containing guide RNA showed higher levels. The results indicate the rescue of MeCP2 DNA binding ability. [Table 2]

[0101] Table 2: Quantification of the number of cells expressing editase enzyme and exhibiting functional MeCP2 in vivo. Mecp2 encodes AAV PHP.B, which is an editor and guide RNA. 317 G>A The substance was injected into the hippocampus of mice. Three weeks after the injection, the mice were treated for immunohistochemistry. After thresholding the signal from uninfected cells, HA immunostaining of brain sections from injected mice was performed. Editase+ cells were identified by color. Percentages represent cells identified by DAPI. This is relative to the total number. Eddy showing MeCP2 enrichment within heterochromatin (structure) The percentage of tase-positive cells indicates the recovery of MeCP2 protein function. n=864 cells.

[0102] [Example 3] Plasmid construction The sequence encoding the full-length human ADAR2, including the amino-terminal Flag tag, was used in yeast expression. From the cadaver, pcDNA 3.1+ (Thermo Fisher Scientific) ) was subcloned into Mecp2, which was designed to mobilize the full-length ADAR2. To express the gene, synthetic oligonucleotides are used in the Bsa1 overhang and annealing. PENTR / U6 Polylinker [pGM1099 (2xBoxB Guide W104X ), pGM1192 (internal loop guide W104X), pGM1310 (GluA2 ste [Muloop W104X] (Thermo Fisher Scientific) I did it. Mecp2 311G>A Mecp2 editing substrates containing (W104X) mutations This is described in Example 1. All subclonings were verified by sequence analysis. Table 3 shows the primer sequences used for rasmid construction and PCR amplification.

[0103] cell culture HEK293T cells (ATCC CRL-3216) were incubated in a 5% CO2 humidified incubation environment. At 37°C inside the thermostat, 10% FBS contains DMEM (Thermo Fisher Techno (maintained in logies)

[0104] RNA editing To analyze editing using full-length ADAR2, one well in a 12-well plate 1.3 × 10 3 HEK293T cells were seeded at individual cell density. After 24 hours, Opt i-MEM™ Low Serum Medium (Thermo Fisher Scientific) Lipofectamin (trademark) 2000 (Thermo Fisher) in a 2:1 ratio. Using Scientific and DNA, cells were cellularized to the full length of human ADAR2 (pGM1). 155) One copy of the guide (pGM1099, pGM1192, or pGM1310) , and Mecp2 311G>A -egfp plasmid encoding cDNA Sfected. The amount of plasmid DNA added per well was 125 ng of target. The guides were 250 ng of human ADAR2 and 2.5 μg. After 72 hours, cells were collected. Collect the RNA and follow the manufacturer's instructions for the Purelink® RNA Mini Kit (A Total RNA was isolated using mbion. TURBO DNA-free (trademark) kit. Residual plasmid DNA was removed using Ambion. Total RNA was then processed using Sup erScript(registered trademark) III First-Strand Synthesis Reverse transcription is performed using the System (Life Technologies), and oligo-d Priming was performed using T. Transfected Mecp2. 311G>A -egfp cDNA was used with the 5' primer of the CMV promoter of pEGFP-N3 and the egfp gene. The genes were amplified for sequence analysis by PCR using reverse primers. [Table 3]

[0105] result Human fetal kidney (HEK) cells were subjected to a cytomegalovirus (CMV) promoter. Under controlled conditions, full-length human ADAR2 and Mecp2 317G>A It was transfected. Human ADAR2 was a full-length, naturally occurring ADAR2 molecule that mimicked endogenous ADAR2. ecp2 317G>A The mutation is an R106Q amino acid change (Mecp2 R106Q )of It brings about the following: Next, the cell is guided by RN, which has the two BoxB stem loops mentioned above. A (see, for example, Example 1), 2) Guide RNA containing an R / G binding site from GluA2 (Wettengel, et al. (2017) Nucleic Acids Res .,45(5):2797-2808;Fukuda,et al.(2017)Sci .Rep.,7:41478), or 3) Guide RNA with an internal loop (Lehm ann, et al. (1999) J.Mol.Biol.,291(1):1-13) The treatment was performed as shown in Figure 7, using a guide RNA containing two BoxB stem loops. The included guide RNA mobilizes full-length ADAR2 to transfect HEK cells with Me We were able to edit cp2 RNA. These results, in addition to the target RNA sequence, Full-length ADAR to target RNA may include sequences not normally present in the target RNA. This demonstrates the effectiveness of mobilization.

[0106] Although some preferred embodiments of the present invention have been described and specifically illustrated above, the present invention However, this is not intended to be limited to such embodiments. The following claims are shown. Various modifications can be made without departing from the scope and spirit of the present invention.

Claims

1. A method for editing a target sequence of endogenous RNA within a cell, a) A nucleic acid molecule encoding a fusion protein containing an RNA editing enzyme linked to an RNA-binding domain, and b) Nucleic acid molecules encoding guide RNA The step includes delivering to the cells, Here, the fusion protein includes a nuclear localization signal, Here, the guide RNA includes a sequence that is specifically recognized by the RNA-binding domain, and here, The method wherein the guide RNA specifically hybridizes with the target sequence of the endogenous RNA and contains a mismatch at the nucleotide to be edited.

2. The method according to claim 1, wherein the endogenous RNA is located in the nucleus of the cell.

3. The method according to claim 1 or 2, wherein the RNA editing enzyme is an adenosine deaminase (ADAR) enzyme that acts on RNA.

4. The RNA-binding domain is a λN peptide or a variant thereof. The sequence specifically recognized by the RNA-binding domain is the BoxB sequence, and / or The method according to any one of claims 1 to 3, wherein the nuclear localization signal is an SV40 large T antigen nuclear localization signal or a variant thereof.

5. The method according to any one of claims 1 to 4, wherein the endogenous RNA is RNA expressed in central nervous system cells, and preferably the endogenous RNA is methyl CpG-binding protein 2 (MECP2) RNA.

6. The method according to any one of claims 1 to 5, wherein the guide RNA further comprises one or more mismatches upstream or downstream of the nucleotide to be edited.

7. The method according to any one of claims 1 to 6, wherein the nucleic acid molecules of a) and b) are contained in a single vector, preferably the vector is a viral vector, and more preferably the viral vector is an adeno-associated virus (AAV).

8. The method according to any one of claims 1 to 7, wherein the guide RNA can repair a nonsense mutation from C to T by deamination of A at the 3' position.

9. For the treatment, suppression and / or prevention of hereditary disorders of the central nervous system in the subject, a) A nucleic acid molecule encoding a fusion protein containing an RNA editing enzyme linked to an RNA-binding domain, and b) A combination of nucleic acid molecules that encode guide RNA, Here, its use involves editing the target sequence of endogenous RNA within the cell. Here, the fusion protein includes a nuclear localization signal, Here, the guide RNA comprises a sequence specifically recognized by the RNA-binding domain, and the guide RNA is a combination that specifically hybridizes with the target sequence of the endogenous RNA, including a mismatch in the nucleotide to be edited.

10. The combination according to claim 9, wherein the RNA editing enzyme is an adenosine deaminase (ADAR) enzyme that acts on RNA.

11. The combination according to claim 9 or 10, wherein the endogenous RNA is located in the nucleus of the cell.

12. The RNA-binding domain is a λN peptide or a variant thereof. The sequence specifically recognized by the RNA-binding domain is the BoxB sequence, and / or The combination according to any one of claims 9 to 11, wherein the nuclear localization signal is the SV40 large T antigen nuclear localization signal or a variant thereof.

13. The method according to any one of claims 9 to 12, wherein the endogenous RNA is RNA expressed in central nervous system cells.

14. The combination according to claim 13, wherein the endogenous RNA is methyl CpG-binding protein 2 (MECP2) RNA.

15. The combination according to any one of claims 9 to 14, wherein the guide RNA further comprises one or more mismatches upstream or downstream of the nucleotide to be edited.

16. The combination according to any one of claims 9 to 15, wherein the nucleic acid molecules of a) and b) are contained within a single vector.

17. The combination according to claim 16, wherein the vector is a viral vector.

18. The combination according to claim 17, wherein the viral vector is adeno-associated virus (AAV).

19. The combination according to any one of claims 9 to 18, wherein the guide RNA can repair a nonsense mutation from C to T by deamination of A at the 3' position.

20. The combination according to any one of claims 9 to 19, wherein the hereditary disorder of the central nervous system is neonatal encephalopathy, microcephaly, X-linked intellectual disability, PPM-X syndrome, bipolar disorder, Parkinson's syndrome, hyponia, grandiosity, and megatesticle, or a combination thereof.

21. The combination according to any one of claims 9 to 19, wherein the hereditary disorder of the central nervous system is Rett syndrome.

22. A vector comprising a nucleic acid molecule encoding a fusion protein containing an RNA editing enzyme, ligated to an RNA-binding domain, wherein the fusion protein contains a nuclear localization signal.

23. The vector according to claim 22, wherein the RNA editing enzyme is an adenosine deaminase (ADAR) enzyme that acts on RNA.

24. A vector comprising a nucleic acid molecule encoding a guide RNA, wherein the guide RNA comprises a sequence specifically recognized by an RNA-binding domain.

25. a) A nucleic acid molecule encoding a fusion protein containing an RNA editing enzyme linked to an RNA-binding domain, and b) A vector comprising a nucleic acid molecule encoding guide RNA, Here, the fusion protein includes a nuclear localization signal, Herein, the guide RNA comprises a sequence specifically recognized by the RNA-binding domain, and therein, the guide RNA comprises a vector containing a nucleotide mismatch that specifically hybridizes with the target sequence of the endogenous RNA and is edited.

26. The vector according to claim 25, wherein the RNA editing enzyme is an adenosine deaminase (ADAR) enzyme that acts on RNA.

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