Compositions and methods for editing RNA

Abstract

Compositions and methods for editing endogenous RNA molecules are provided. [Selected Figure] Figure 1A

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 disclosure of which is incorporated herein by reference in its entirety.

[0002] This invention was made with government support under grant number NS087726 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] Description of Electronically Submitted Text File The content of the text file submitted electronically herewith is incorporated herein by reference in its entirety: a computer-readable form copy of the Sequence Listing (file name: SEQLIST.txt; data record date: 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 therapeutically editing RNA, particularly endogenous RNA in the nucleus, 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 incorporated herein by reference as if fully set forth.

[0006] Strategies for editing or modifying genetic material, such as 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 a neurodevelopmental disorder caused by sporadic mutations in the transcription factor methyl-CpG-binding protein 2 (MECP2) (Amir, et al. (1999) Nat. Genet., 23:185-188). MECP2 is located on the X chromosome. Due to the gene dosage compensation mechanism in mammals, females affected by Rett syndrome are mosaics, approximately divided 50:50 between wild-type cells and mutant cells. Females with MECP2 mutations experience regression of early developmental milestones such as speech and intentional hand movements, then acquire severe motor abnormalities including breathing, and die by an average age of 40 years (Neul, et al., (2010) Ann. Neurol., 68:944-950; Percy, et al. (2010) Ann. Neurol., 68:951-955). Males with a mutation in MECP2 and only one X chromosome develop an even more severe disease and usually die before the age of 2 years (Schule, et al. (2008) Clin. Genet., 74:116-126). There is no treatment for Rett syndrome.

Prior Art Documents

Non-Patent Documents

[0008]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Summary of the Invention

Means for Solving the Problems

[0009] According to one aspect of the present invention, a method is provided for editing the sequence of endogenous RNA within a cell, particularly within the nucleus of a cell. In certain embodiments, the method includes the step of delivering to a cell a nucleic acid molecule encoding a fusion protein comprising a nuclear localization signal and an RNA editing enzyme linked to an RNA-binding domain, and ii) a nucleic acid molecule encoding one or more guide RNAs. The guide RNA comprises a sequence specifically recognized by the RNA-binding domain. The guide RNA also comprises a nucleotide mismatch that specifically hybridizes with the target sequence in the endogenous RNA and is edited. In certain embodiments, the RNA editing enzyme is an adenosine deaminase (ADAR) acting on RNA, e.g., ADRAR1 or ADAR2. In certain embodiments, the RNA-binding domain is a λN peptide, and the sequence specifically recognized by the RNA-binding domain is a BoxB sequence. In certain embodiments, the endogenous RNA is methyl CpG-binding protein 2 (MECP2) RNA. In certain embodiments, the nuclear localization signal is an SV40 large T antigen nuclear localization signal. The nucleic acid molecules in these methods may be contained within a single vector, such as a viral vector (e.g., an adeno-associated virus (AAV) vector).

[0010] Another aspect of the present invention provides an additional method for editing the sequence of endogenous RNA within a cell, particularly within the nucleus of a cell. In certain embodiments, the method comprises the step of delivering a nucleic acid molecule encoding one or more guide RNAs to a cell, the guide RNA comprising one or more sequences and / or structures that are specifically recognized by an endogenous deaminase, such as human adenosine deaminase (ADAR), which acts on RNA. The guide RNA also comprises a mismatch at the nucleotide to be edited, which specifically hybridizes with the target sequence in the endogenous RNA. In certain embodiments, the ADAR is ADRA1 or ADAR2. In certain embodiments, the endogenous RNA is methyl CpG-binding protein 2 (MECP2) RNA. In certain embodiments, the nucleic acid molecule is contained within a viral vector (e.g., an AAV vector).

[0011] Another aspect of the present invention provides an additional method for editing the sequence of endogenous RNA within a cell, particularly within the nucleus of a cell. In certain embodiments, the method comprises the step of delivering a nucleic acid molecule encoding a guide RNA (e.g., within an AAV) to a cell, wherein the guide RNA comprises (one or more) sequences and / or structures that are specifically recognized by an endogenous human adenosine deaminase (ADAR) acting on RNA. Thus, in various embodiments, the editing method can be carried out in the absence of recombinant RNA editing enzymes (e.g., in the absence of recombinant ADAR). Thus, in embodiments, the method assumes the endogenous ADAR activity necessary to influence the editing described herein.

[0012] According to another aspect of the present invention, a method for treating, suppressing and / or preventing Rett syndrome in a target is provided. In certain embodiments, the method includes a step of using the RNA editing method of the present invention. For example, the method may include a step of administering to a target a nucleic acid molecule encoding a fusion protein containing an RNA editing enzyme linked to an RNA-binding domain and a nucleic acid molecule encoding a guide RNA, wherein the fusion protein contains a nuclear localization signal. In certain embodiments, the method includes a step of administering to a target a nucleic acid molecule encoding a guide RNA, wherein the guide RNA includes (one or more) sequences and / or (one or more) structures that are specifically recognized by an endogenous human deaminase such as adenosine deaminase (ADAR) that acts on RNA, and the guide RNA specifically hybridizes with methyl CpG-binding protein 2 (MECP2) RNA and includes a mismatch in mutant nucleotides of the endogenous MECP2 RNA. [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 of 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 Mecp2317G>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 is partly based on the remarkable discovery that, using site-directed RNA editing, it is possible to repair disease-causing point mutations at the RNA level (e.g., mRNA), such as the guanosine-to-adenosine (G>A) mutation in the methyl CpG-binding protein 2 (MECP2) DNA-binding domain gene, which underlies Rett syndrome. Importantly, this site-directed RNA editing is particularly useful for repairing endogenous RNA and restoring protein function. Accordingly, in embodiments, this invention relates to compositions and methods for site-directed RNA editing.

[0015] Mice engineered to carry a mutation in Mecp2 localized to the germline or nerve cells that causes Rett syndrome in humans exhibit growth abnormalities, anxiety, and motor impairments similar to those seen in patients with Rett syndrome (Guy, et al. (2001) Nat. Genet., 27:322-326; Lioy, et al. (2011) Nature 475:497-500; Chen, et al. (2001) Nat. Genet., 27:327-331). Studies in mice have shown that the most severe Rett syndrome phenotype is neurological, affecting both neurons and glial cells (Lioy, et al. (2011) Nature 475:497-500; Luikenhuis, et al. (2004) Proc. Natl. Acad. Sci., 101:6033-6038), but many other tissues may also be affected (Ross, et al. (2016) Hum. Mol. Genet., 25:4389-4404). Similar to humans, male Rett mice are more severely affected than female mice. For example, female Rett mice live a normal lifespan, while male mice die at 3-4 months of age (Guy, et al. (2001) Nat. Genet., 27:322-326; Chen, et al. (2001) Nat. Genet., 27:327-331).At the cellular level, the neurons of male and female Rett mice have smaller cell bodies and nuclei, and reduced process complexity (Belichenko, et al. (2009) Neurobiol. Dis., 34:71-77; Belichenko, et al. (2009) J. Comp. Neurol., 514:240-258; Fukuda, et al. (2005) J. Neuropathol. Exp. Neurol., 64:537-544; Kishi, et al. (2004) Mol. Cell. Neurosci., 27:306-321; Robinson, et al. (2012) Brain 135:2699-2710; Tropea, et al. (2009) Proc. Natl. Acad. Sci., 106:2029-2034; Stuss, et al. (2012) PLoS One 7:e31896), it is reminiscent of infected human cells (Armstrong, et al. (1995) J. Neuropathol. Exp. Neurol., 54:195-201; Li, et al. (2013) Cell Stem Cell 13:446-458; Belichenko, et al. (1994) Neuroreport., 5:1509-1513; Bauman, et al. (1995) Neurology 45:1581-1586). Importantly, restoration of MeCP2 in Mecp2 null mice via conditional Cre recombinase (Guy, et al. (2007) Science 315:1143-1147) or gene therapy approaches (Sinnett, et al. (2017) Mol.Ther.Methods Clin.Dev., 5:106-115; Gadalla, et al. (2017) Mol.Ther.Methods Clin.Dev., 5:180-190; Garg, et al. (2013) J.Neurosci., 33:13612-13620; Gadalla, et al. (2013) Mol.Ther., 21:18-30) reverses many cases of Rett-like syndrome and cellular defects, even in the later stages of the disease.Phenotypic reversal demonstrates that Rett syndrome can be treated in humans with gene replacement strategies (Robinson, et al. (2012) Brain 135:2699-2710; Sinnett, et al. (2017) Mol.Ther.Methods Clin.Dev., 5:106-115; Gadalla, et al. (2017) Mol.Ther.Methods Clin.Dev., 5:180-190; Garg, et al. (2013) J.Neurosci., 33:13612-13620; Gadalla, et al. (2013) Mol.Ther., 21:18-30). However, duplication of the MECP2 gene in humans leads to MeCP2 overexpression and severe neurological disorders (Van Esch, et al. (2005) Am.J.Hum.Genet., 77:442-453). Furthermore, mouse MeCP2 is expressed at varying levels across different neuronal cell types, and loss of MeCP2 function in mice results in cell-specific changes in gene expression (Skene, et al. (2010) Mol. Cell., 37:457-468; Ballas, et al. (2009) Nat. Neurosci., 12:311-317; Shahbazian, et al. (2002) Hum. Mol. Genet., 11:115-124; Sugino, et al. (2014) J. Neurosci., 34:12877-12883; Linhoff, et al. (2015) Cell 163:246-255). These findings highlight the challenge of MECP2 gene substitution, which must be fine-tuned to restore normal MeCP2 levels and cellular physiology across diverse cell types of the nervous system.

[0016] This specification demonstrates that, because RNA is repaired in the environment of a normal transcript, repairing MECP2 mutations at the RNA level avoids problems with both MECP2 overexpression and cell type-specific regulation. The target was the guanosine-to-adenosine (G>A) mutation underlying Rett syndrome (Fyfe, et al. (2003) J.Child. Neurol., 18:709-713). Adenosine deaminases (ADARs), a family of natural enzymes that act on RNA, hydrolytically deaminate A to inosine (I) in endogenous mRNA (Bass, et al. (1988) Cell 55:1089-1098; Bass, et al. (1987) Cell 48:607-613; 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). Inosine bases pair with cytosine (C) and are translated as G by ribosomes (Basilio, et al. (1962) Proc. Natl. Acad. Sci., 48:613-616). ADAR2, a member of the ADAR family, is highly expressed in the brain and, after transcription, alters protein functions such as ion channel permeability through deamination of the primary transcript (Bhalla, et al. (2004) Nat. Struct. Mol. Biol., 11:950-956; Sommer, et al. (1991) Cell 67:11-19; Burns, et al. (1997) Nature 387:303-308).Innate editing by ADAR2 requires not only catalytic activity but also recognition of the double-stranded RNA structure mediated by premRNA introns that appropriately position target A in the exon for editing (Bhalla, et al. (2004) Nat. Struct. Mol. Biol., 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. Biol. Chem., 271:12221-12226; Lomeli, et al. (1994) Science 266:1709-1713; Yang, et al. al. (1997) Proc. Natl. Acad. Sci., 94:4354-4359). The cloning catalytic domain of human ADAR2 (hADAR2) is typically a stop codon and has been used in various configurations to target G>A repair in heterologously expressed mRNA (Hanswillemenke, et al. (2015) J.Am.Chem.Soc., 137:15875-15881; Vogel, et al. (2014) ChemMedChem 9:2021-2025; Vogel, et al. (2014) Angew Chem.Int.Ed.Engl., 53:6267-6271; Schneider, et al. (2014) Nucleic Acids Res., 42:e87; Montiel-Gonzalez, et al. (2016) Nucleic Acids Res., 44:e157; Montiel-Gonzalez, et al. al. (2013) Proc. Natl. Acad. Sci., 110:18285-18290). In one approach, the native RNA-binding domain of ADAR2 was replaced with an RNA-binding peptide derived from bacteriophage lambda (λN; Montiel-Gonzalez, et al. (2013) Proc. Natl. Acad. Sci., 110:18285-18290) that binds to specific short RNA hairpins at nanomolar affinity (Austin, et al. (2002) J. Am. Chem. Soc., 124:10966-10967).Subsequently, targeted editing of heterologous mRNA is achieved by expressing a hybrid ADAR2 protein along with an RNA guide containing a λN-recognizing stem-loop and a region complementary to the target mRNA (Montiel-Gonzalez, et al. (2016) Nucleic Acids Res., 44:e157; Montiel-Gonzalez, et al. (2013) Proc. Natl. Acad. Sci., 110:18285-18290).

[0017] Previously, neither endogenous RNA nor mRNA had been repaired by site-directed RNA editing, particularly in terms of repair to produce functional proteins. However, this approach to G>A mutations in endogenous Mecp2 is demonstrated for the first time herein. The mutation in Mecp2 is located in a domain that encodes a well-established function. Furthermore, the fidelity of the repair can be monitored by sequence analysis, Western blotting, and immunochemistry at the single-cell level. Here, recombinant hADAR2-λN protein (also referred to herein as editase) was used to effectively repair G>A mutations in endogenous Mecp2 transcripts. After determining the parameters of Mecp2 editing in transfected mouse neuroblastoma (N2A) cells, primary neuronal cultures from a Rett syndrome mouse model containing a severe human G>A mutation in the DNA-binding domain were transduced using adeno-associated virus (AAV) (Mecp2). 317G>A ;MeCP2 R106Q This mutation leads to a decrease in MeCP2 protein levels and a significant reduction in its binding to heterochromatin. We first quantified the editing efficiency of the mutant RNA in neurons and then tested whether editing rescued MeCP2 protein levels, leading to enrichment of MeCP2's binding to heterochromatin structures, a key characteristic of MeCP2 within cells including neurons, glial, and non-neuronal cell types. The results presented herein demonstrate that site-directed RNA editing can therapeutically repair the MECP2 mutations that underlie Rett syndrome and other neurological disorders for which gene therapy is available.

[0018] The present invention provides a method for editing nucleic acid molecules within a cell. In certain embodiments, the nucleic acid molecule to be edited is an RNA molecule, particularly a nuclear RNA molecule (e.g., a primary transcript, pre-mRNA, or mRNA (e.g., mRNA before transport from the nucleus)). In certain embodiments, the nucleic acid molecule to be edited is an endogenous and / or nuclear transcript. Gene editing, such as CRISPR technology, results in permanent off-target mutations in the genome. In contrast, RNA turnover within a cell means that off-target mutations in the RNA are transient. Furthermore, RNA editing, unlike CRISPR genome editing, can be stepwise. As a result, off-target mutations are not necessarily edited to 100%.

[0019] In embodiments, the present invention provides a method for RNA editing of nucleic acid molecules that provides fine-tuning of protein expression and / or function. For example, the method of the present invention allows for the restoration of normal levels of protein expression and / or function compared to the unedited state. In the context of the treatments described herein, the method of the present invention allows for the restoration of normal levels or at least near-normal levels of protein expression and / or function compared to the untreated state.

[0020] In embodiments, the present invention provides a method for RNA editing of nucleic acid molecules that provides adjustable transient editing (e.g., via drug administration) in the context of the described therapy. For example, the present invention enables reversible editing of target RNA.

[0021] In certain embodiments, the cells being edited are non-dividing cells. In certain embodiments, the cells being edited are neurons and / or glial cells. In certain embodiments, the cells being edited are non-neuronal cells. In certain embodiments, the cells being edited are neurons. Cells (e.g., neurons) may be present in the central nervous system (e.g., brain, spinal cord) and / or the peripheral nervous system. Cells may be present in a subject being treated (e.g., by an in vivo treatment method), or cells may be treated in vitro and then administered to a subject (e.g., by an ex vivo treatment method).

[0022] In certain embodiments of the present invention, the method includes the steps of 1) delivering a nucleic acid molecule encoding an RNA editing enzyme linked to or fused to an RNA-binding domain and 2) a guide RNA or a nucleic acid molecule encoding a guide RNA to a cell. In certain embodiments, the RNA-binding domain is linked to the N-terminus of the RNA editing enzyme. In embodiments, the fusion comprising the RNA editing enzyme and the RNA-binding domain does not include at least one nuclear localization signal (NLS). In embodiments, the fusion comprising the RNA editing enzyme and the RNA-binding domain further includes at least one nuclear localization signal (NLS). For example, the fusion comprising the RNA editing enzyme and the RNA-binding domain further includes 1, 2, 3, 4, 5 or more NLS. When multiple NLS are used, each NLS may be directly linked to one another or separated by an amino acid linker of 1 to about 5 amino acids. In certain embodiments, the NLS is located at the N-terminus of the fusion protein. Examples of NLS are provided in 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., monopartite NLS). In certain embodiments, the NLS comprises the consensus sequence (K / R)(K / R)X 10~12 (K / R) 3 / 5 (Sequence ID 59) (In the formula, (K / R) 3 / 5(This indicates that at least three of the five amino acids are either lysine or arginine). In certain embodiments, the NLS comprises c-myc NLS. In certain embodiments, the c-myc NLS comprises the sequence PAAKRVKLD (SEQ ID NO: 54). In certain embodiments, the NLS is a nucleoplasmin NLS. In certain embodiments, the nucleoplasmin NLS comprises the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 60). In certain embodiments, the NLS comprises an SV40 large T antigen NLS. In certain embodiments, the SV40 large T antigen NLS comprises the sequence PKKKRKV (SEQ ID NO: 47). In certain embodiments, the fusion comprises three SV40 large T antigen NLSs (e.g., sequences DPKKKRKVDPKKKRKVDPKKKRKV (SEQ ID NO: 67)). In various embodiments, the NLS may include mutations / mutations such that the above sequences (e.g., SEQ ID NOs. 58, 59, 60, 47, 54, or 67) contain one or more substitutions, additions, or deletions (e.g., about 1, or about 2, or about 3, or about 4, or about 5, or about 10, or about 15 (including about 1-5, or about 1-10, or about 1-15) substitutions, additions, or deletions). In certain embodiments, lysine amino acids in the NLS may be substituted with arginine amino acids, and / or arginine amino acids in the NLS may be substituted with lysine amino acids. The fusion protein may further include a purified tag (e.g., an HA tag) at the N-terminus of the fusion protein.

[0023] The nucleic acid molecules of the present invention may be contained within a single vector or within separate vectors. For example, a nucleic acid molecule encoding an RNA editing enzyme linked to or fused to an RNA-binding domain and a nucleic acid molecule encoding a guide RNA may be contained within a single vector. The nucleic acid molecules may be delivered to cells sequentially (before or after) and / or simultaneously. The nucleic acid molecules may be delivered in the same composition or in separate compositions (for example, if contained in separate vectors). In certain embodiments, the nucleic acid molecules are delivered in a single vector, particularly a viral vector such as an AAV vector.

[0024] In certain embodiments, the RNA editing enzyme is human. In certain embodiments, the RNA editing enzyme is a deaminase. Examples of deaminases include, but are not limited to, adenosine deaminases acting on RNA (ADAR), apolipoprotein B mRNA editing enzymes, catalytic polypeptide-like enzymes (APOBEC (e.g., APOBEC1, APOBEC3A, APOBEC3G)), and activation-inducible cytidine deaminases (AICDA or AID; C:G is converted to T:A). In certain embodiments, the RNA editing enzyme is an ADAR such as ADAR1, ADAR2, or ADAR3. In certain embodiments, the RNA editing enzyme is ADAR1 (e.g., Gene ID:103 and GenBank deposit numbers NM_001111.5 and NP_001102.3 and their isoforms). In certain embodiments, the RNA editing enzyme is ADAR2. The RNA editing enzyme may be less than full length. In certain embodiments, the RNA editing enzyme lacks its native RNA-binding domain. For example, an RNA editing enzyme may contain or consist of a catalytic domain.

[0025] An example of the amino acid sequence of human ADAR1 is: MNPRQGYSLS GYYTHPFQGY EHRQLRYQQP GPGSSPSSFL LKQIEFLKGQ LPEAPVIGKQ TPSLPPSLPG LRPRFPVLLA SSTRGRQVDI RGVPRGVHLR SQGLQRGFQH PSPRGRSLPQ RGVDCLSSHF QELSIYQDQE QRILKFLEEL GEGKATTAHD LSGKLGTPKK EINRVLYSLA KKGKLQKEAG TPPLWKIAVS TQAWNQHSGV VRPDGHSQGA PNSDPSLEPE DRNSTSVSED LLEPFIAVSA QAWNQHSGVV RPDSHSQGSP NSDPGLEPED SNSTSALEDP LEFLDMAEIK EKICDYLFNV SDSSALNLAK NIGLTKARDI NAVLIDMERQ GDVYRQGTTP PIWHLTDKKR ERMQIKRNTN SVPETAPAAI PETKRNAEFL TCNIPTSNAS NNMVTTEKVE NGQEPVIKLE NRQEARPEPA RLKPPVHYNG PSKAGYVDFE NGQWATDDIP DDLNSIRAAP GEFRAIMEMP SFYSHGLPRC SPYKKLTECQ LKNPISGLLE YAQFASQTCE FNMIEQSGPP HEPRFKFQVV INGREFPPAE AGSKKVAKQD AAMKAMTILL EEAKAKDSGK SEESSHYSTE KESEKTAESQ TPTPSATSFF SGKSPVTTLL ECMHKLGNSC EFRLLSKEGP AHEPKFQYCV AVGAQTFPSV SAPSKKVAKQ MAAEEAMKAL HGEATNSMAS DNQPEGMISE SLDNLESMMP NKVRKIGELV RYLNTNPVGG LLEYARSHGF AAEFKLVDQS GPPHEPKFVY QAKVGGRWFP AVCAHSKKQG KQEAADAALR VLIGENEKAE RMGFTEVTPV TGASLRRTML LLSRSPEAQP KTLPLTGSTF HDQIAMLSHR CFNTLTNSFQ PSLLGRKILA AIIMKKDSED MGVVVSLGTG NRCVKGDSLS LKGETVNDCHAEIISRRGFI RFLYSELMKY NSQTAKDSIF EPAKGGEKLQ IKKTVSFHLY ISTAPCGDGA LFDKSCSDRA MESTESRHYP VFENPKQGKL RTKVENGEGT IPVESSDIVP TWDGIRLGER LRTMSCSDKI LRWNVLGLQG ALLTHFLQPI YLKSVTLGYL FSQGHLTRAI CCRVTRDGSA FEDGLRHPFI VNHPKVGRVS IYDSKRQSGK TKETSVNWCL ADGYDLEILD GTRGTVDGPR NELSRVSKKN IFLLFKKLCS FRYRRDLLRL SYGEAKKAAR DYETAKNYFK KGLKDMGYGN WISKPQEEKN FYLCPV (Sequence ID 72).

[0026] In certain embodiments, the deaminase domain of ADAR1 contains amino acids 839-1222 of SEQ ID NO: 72. In certain embodiments, the RNA editing enzyme contains a sequence having at least 80%, 85%, 90%, 95%, 97%, 99%, or 100% homology or identity, particularly at least 95%, 97%, 99%, or 100% homology or identity, with SEQ ID NO: 72 of its deaminase domain.

[0027] In certain embodiments, the RNA editing enzyme contains the deaminase domain of human ADAR2. In certain embodiments, the deaminase domain of human ADAR2 contains amino acids 299-701 of GenBank deposit number U82120. In certain embodiments, ADAR2 or its deaminase domain contains the E488Q mutation (Montiel-Gonzalez, et al. (2016) Nucleic Acids Res., 44:e157). In certain embodiments, the deaminase domain of human ADAR2, LHLDQTPSRQPIPSEGLQLHLPQVLADAVSRLVLGKFGDLTDNFSSPHAR RKVLAGVVMTTGTDVKDAKVISVSTGTKCINGEYMSDRGLALNDCHAEII SRRSLLRFLYTQLELYLNNKDDQKRSIFQKSERGGFRLKENVQFHLYIST SPCGDARIFSPHEPILEEPADRHPNRKARGQLRTKIESG E GTIPVRSNAS IQTWDGVLQGERLLTMSCSDKIARWNVVGIQGSLLSIFVEPIYFSSIILG SLYHGDHLSRAMYQRISNIEDLPPLYTLNKPLLSGISNAEARQPGKAPNF SVNWTVGDSAIEVINATTGKDELGRASRLCKHALYCRWMRVHGKVPSHLL RSKITKPNVYHESKLAAKEYQAAKARLFTAFIKAGLGAWVEKPTEQDQFS Includes LTP (sequence number 55; E488 is indicated).

[0028] In certain embodiments, the deaminase domain of human ADAR2 is LHLDQTPSRQPIPSEGLQLHLPQVLADAVSRLVLGKFGDLTDNFSSPHAR RKVLAGVVMTTGTDVKDAKVISVSTGTKCINGEYMSDRGLALNDCHAEII SRRSLLRFLYTQLELYLNNKDDQKRSIFQKSERGGFRLKENVQFHLYIST SPCGDARIFSPHEPILEEPADRHPNRKARGQLRTKIESG Q GTIPVRSNAS IQTWDGVLQGERLLTMSCSDKIARWNVVGIQGSLLSIFVEPIYFSSIILG SLYHGDHLSRAMYQRISNIEDLPPLYTLNKPLLSGISNAEARQPGKAPNF SVNWTVGDSAIEVINATTGKDELGRASRLCKHALYCRWMRVHGKVPSHLL RSKITKPNVYHESKLAAKEYQAAKARLFTAFIKAGLGAWVEKPTEQDQFS Includes LTP (sequence number 71; E488Q is indicated).

[0029] In certain embodiments, the RNA editing enzyme includes a sequence having at least 80%, 85%, 90%, 95%, 97%, 99%, or 100% homology or identity with SEQ ID NO: 55 or 71, particularly at least 95%, 97%, 99%, or 100% homology or identity.

[0030] As described above, the RNA editing enzyme is linked to the RNA-binding domain. The RNA editing enzyme may be linked directly to the RNA-binding domain (i.e., without a linker sequence) or via a polypeptide linker. For example, the polypeptide linker may contain 1 to about 50 amino acids, 1 to about 25 amino acids, 1 to about 20 amino acids, 1 to about 15 amino acids, 1 to about 10 amino acids, or 1 to about 5 amino acids. In certain embodiments, the linker is a sequence (GGGGS) n The linker sequence may include (Sequence ID 44) (wherein n is 1 to about 10, particularly 1 to about 5). For example, the linker sequence may be GGGGSGGGGSGGGGS (Sequence ID 45). In various embodiments, the linker may include mutations / variations such that the above sequences (e.g., Sequence ID 44 or 45) contain one or more substitutions, additions or deletions (e.g., about 1, or about 2, or about 3, or about 4, or about 5, or about 10, or about 15 (including about 1 to 5, or about 1 to 10, or about 1 to 15)).

[0031] The RNA-binding domain can be any polypeptide that specifically recognizes a particular RNA sequence and / or RNA structure (e.g., a hairpin). In certain embodiments, the RNA-binding domain is an artificial RNA-binding domain, particularly one with high affinity for RNA. In certain embodiments, the RNA-binding domain is a phage RNA-binding domain (see, e.g., Keryer-Bibens, et al., Biol. Cell (2008) 100:125-138). For example, the RNA-binding domain is a λN peptide (see, e.g., Cilley, et al., RNA (1997) 3:57-67) or a phage MS2 coat protein (see, e.g., Johansson, et al., Sem. Virol. (1997) 8:176-185). In certain embodiments, the λN peptide contains the amino acid sequence:MNARTRRRERRAEKQAQWKAAN (SEQ ID NO: 46). In certain embodiments, the λN peptide comprises a sequence having at least 80%, 85%, 90%, 95%, 97%, 99%, or 100% homology or identity with SEQ ID NO: 46, particularly at least 95%, 97%, 99%, or 100% homology or identity.

[0032] In some embodiments, the fusion protein comprises a λN peptide (SEQ ID NO: 46) linked to the amino terminus of the deaminase domain of human ADAR2 (e.g., SEQ ID NO: 55 or 71) via an amino acid linker. In certain embodiments, the linker is sequence (GGGGS) n (Sequence ID 44; where n is 1 to about 10 or 1 to about 5) or the sequence GGGGSGGGGSGGGGS (Sequence ID 45). In certain embodiments, the fusion protein has the sequence: MNARTRRRERRAEKQAQWKAANGGGGSGGGGGSGGGGSLHLDQTPSRQPIP SEGLQLHLPQVLADAVSRLVLGKFGDLTDNFSSSPHARRKVLAGVVMTTGT DVKDAKVISVSTGTKCINGEYMSDRGLALNDCHAEIISRRSLLRFLYTQL ELYLNNKDDQKRSIFQKSERGGFRLKENVQFHLYISTSPCGDARIFSPHE PILEEPADRHPNRKARGQLRTKIESGEGTIPVRSNASIQTWDGVLQGERL LTMSCSDKIARWNVVGIQGSLLSIFVEPIYFSSIILGSLYHGDHLSRAMY QRISNIEDLPPLYTLNKPLLSGISNAEARQPGKAPNFSVNWTVGDSAIEV INATTGKDELGRASRLCKHALYCRWMRVHGKVPSHLLRSKITKPNVYHES Includes KLAAKEYQAAKARLFTAFIKAGLGAWVEKPTEQDQFSLTP (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 comprises an SV40 large T antigen NLS (e.g., SEQ ID NO: 47). In certain embodiments, the fusion comprises three SV40 large T antigen NLSs (e.g., SEQ ID NO: 67). In certain embodiments, the fusion protein comprises a sequence: DPKKKRKVDPKKKRKVDPKKKRKVMNARTRRRERRAEKQAQWKAANGGGG SGGGGSGGGGSLHLDQTPSRQPIPSEGLQLHLPQVLADAVSRLVLGKFGD LTDNFSSPHARRKVLAGVVMTTGTDVKDAKVISVSTGTKCINGEYMSDRG LALNDCHAEIISRRSLLRFLYTQLELYLNNKDDQKRSIFQKSERGGFRLK ENVQFHLYISTSPCGDARIFSPHEPILEEPADRHPNRKARGQLRTKIESG EGTIPVRSNASIQTWDGVLQGERLLTMSCSDKIARWNVVGIQGSLLSIFV EPIYFSSIILGSLYHGDHLSRAMYQRISNIEDLPPLYTLNKPLLSGISNA EARQPGKAPNFSVNWTVGDSAIEVINATTGKDELGRASRLCKHALYCRWM RVHGKVPSHLLRSKITKPNVYHESKLAAKEYQAAKARLFTAFIKAGLGAW Includes VEKPTEQDQFSLTP (sequence number 69).

[0034] In certain embodiments, the fusion protein includes a sequence having at least 80%, 85%, 90%, 95%, 97%, 99%, or 100% homology or identity with SEQ ID NO: 68 or 69, particularly at least 95%, 97%, 99%, or 100% homology or identity.

[0035] The guide RNA of the present invention comprises a sequence that targets or specifically hybridizes with a target sequence (e.g., a complementary sequence) and a sequence recognized by an RNA-binding domain. The guide RNA includes a mismatch with the target sequence directed to the nucleotide (e.g., adenosine) to be modified or edited. As used herein, the term “specifically hybridizes” does not mean that the nucleic acid molecule must be 100% complementary to the target sequence. Rather, a sequence that does not contain one or more mismatched nucleotides to be modified / edited may be at least 80%, 85%, 90%, 95%, 97%, 99%, or 100% complementary to the target sequence, and in particular at least 95%, 97%, 99%, or 100% complementary. However, as described herein, the sequence may include additional mismatches (e.g., G mismatches) to reduce off-target editing. In embodiments, the sequence may contain about 1, or about 2, or about 3, or about 4, or about 5, or about 10, or about 15 mismatches (including about 1 to 5, or about 1 to 10, or about 1 to 15). In particular embodiments, the complementary region (e.g., between the guide RNA and the target sequence) is at least about 10, at least about 12, at least about 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 particular embodiments, the complementary region (e.g., between the guide RNA and the target sequence) is about 15 to about 30 nucleotides, about 15 to about 25 nucleotides, about 20 to about 30 nucleotides, about 20 to about 25 nucleotides, or about 20, 21, 22, 23, 24, or 25 nucleotides. Typically, the mismatch between the guide RNA and the target sequence is directed towards the center of the complementarity region between the guide RNA and the target sequence (e.g., within the central 50% of the guide RNA sequence). In certain embodiments, the target sequence includes sequence number 52.

[0036] Guide RNA contains the correct or desired edits to RNA within the cell. Using guide RNA, any mutation, particularly point mutations, including missense and nonsense mutations, can be corrected. For example, in Rett syndrome, a common G>A mutation exists. These mutations result in the amino acid changes R106Q(CAA), W104X(UAG), and R306H(CAC). In the case of nonsense mutations, these are C-to-T mutations where A is at the 3' position forming a stop codon, or at the central position (e.g., UAG to UGG). Nonsense mutations can be edited to remove the stop codon. In certain embodiments, the guide RNA contains a C that matches the A in these mutants, so that A can be deaminated (e.g., by ADAR).

[0037] The guide RNA of the present invention comprises one or more sequences recognized by an RNA-binding domain. In certain embodiments, the guide RNA comprises two sequences recognized by an RNA-binding domain. In certain embodiments, the two sequences recognized by the RNA-binding domain may be on either side of a sequence that is mismatched or specifically hybridizes with the target sequence. For example, one sequence recognized by the RNA-binding domain (e.g., BoxB) is located at approximately 15-20 or approximately 16-18 nucleotides 5' of the target mutation, and a second sequence recognized by the RNA-binding domain (e.g., BoxB) is located at approximately 8-12 or approximately 10 nucleotides 3' of the target mutation. In certain embodiments, the sequences recognized by the RNA-binding domain are not at the end of the guide RNA (i.e., the sequence at the end of the guide RNA may be complementary to the target sequence). If there are two or more sequences recognized by the RNA-binding domain, those sequences may be the same or different, but preferably recognized by the same RNA-binding domain. In certain embodiments, the sequence recognized by the RNA-binding domain is the BoxB sequence. In certain embodiments, the BoxB sequence includes GCCCUGAAAAAGGGC (sequence number 48) or GGCCCUGAAAAAGGGCC (sequence number 49). In certain embodiments, the BoxB sequence has at least 80%, 85%, 90%, 95%, 97%, 99%, or 100% homology or identity with sequence number 48 or 49, and in particular at least 95%, 97%, 99%, or 100% homology or identity.

[0038] In certain embodiments, the guide RNA targets or includes sequences (including RNA versions of DNA molecules) shown in Table 1. In certain embodiments, the guide RNA targets or includes sequences having at least 80%, 85%, 90%, 95%, 97%, 99%, or 100% homology or identity with sequences shown in Table 1, particularly at least 95%, 97%, 99%, or 100% homology or identity. In certain embodiments, the guide RNA includes one of sequence numbers 15-22, particularly sequence numbers 15, 17, 19, or 21, or includes sequences having at least 80%, 85%, 90%, 95%, 97%, 99%, or 100% homology or identity with one of sequence numbers 15-22, particularly sequence numbers 15, 17, 19, or 21, particularly at least 95%, 97%, 99%, or 100% homology or identity.

[0039] A nucleic acid molecule containing a nucleic acid sequence encoding guide RNA may contain multiple copies of the guide RNA encoding nucleic acid sequence. For example, a nucleic acid molecule may contain one, two, three, four, five, six, seven, eight, nine, ten, or more copies of the guide RNA encoding nucleic acid sequence, each under the control of a promoter.

[0040] In certain embodiments, the nucleic acid molecule of the present invention is delivered to a cell (e.g., via infection, transfection, electroporation, etc.) and expressed via a vector (e.g., plasmid), particularly a viral vector. The expression vector of the present invention may use a strong promoter, a constitutive promoter, a tissue or cell-specific promoter, a ubiquitous promoter, and / or a regulatory promoter. In certain embodiments, the promoter of the nucleic acid molecule encoding an RNA editing enzyme linked to or fused to an RNA-binding domain is a tissue or cell-specific promoter. In certain embodiments, the promoter is a neuron-specific promoter or a ubiquitous promoter. Examples of promoters are well known in the art, but include synaptic promoters, particularly the synapsin I promoter, the CAG promoter, and the MECP2 promoter. With respect to guide RNA, examples of RNA promoters are well known in the art, but include RNA polymerase III promoters (e.g., U6 and H1; e.g., Myslinski et al. (2001) Nucl. Acids Res., 29:2502-09) or other promoters known to express short RNAs. In certain embodiments, the promoter is the human U6 promoter. Examples of expression vectors for expressing the molecules of the present invention include, but are not limited to, plasmids and viral vectors (e.g., adeno-associated viruses (AAVs), adenoviruses, retroviruses, and lentiviruses). In certain embodiments, the vector is an AAV (e.g., AAV-1 to 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 AAV11, or AAV12). In certain embodiments, the vector can infect neurons and / or glial cells.

[0041] In another embodiment, a method of the present invention, including RNA editing and therapeutic methods, includes the step of delivering a guide RNA or nucleic acid encoding the guide RNA to a cell, as described above, but without using a nucleic acid molecule encoding an RNA editing enzyme ligated or fused to an RNA-binding domain. ADARs (e.g., ADAR 1-3) are expressed to high levels in the nervous system. Thus, the guide RNA of the present invention can attract endogenous deaminase enzymes, particularly ADAR1 or ADAR2, to endogenous MECP2 RNA. Thus, in embodiments, the present invention enables the involvement of endogenous ADAR enzymes and does not require recombinant ADAR enzymes. By using only guide RNA, potential immune responses to non-mammalian RNA-binding domains are avoided. Furthermore, this method makes it possible to include highly repetitive guide sequences in the AAV vector, reducing off-target editing. In certain embodiments of this aspect of the present invention, the guide includes a sequence that targets or specifically hybridizes with a target sequence (e.g., a complementary sequence) and a sequence recognized by a deaminase, particularly ADAR (e.g., ADAR1 or ADAR2). The guide RNA may include, but is not limited to, RNA hairpins (e.g., based on the natural target of ADAR), mismatches that create a double-stranded "bulge" recognized by ADAR, and / or any other sequences typically required for targeted editing by endogenous ADAR. In certain embodiments, the guide RNA may include one, two, or more BoxB sequences. In certain embodiments, the guide RNA includes an R / G binding site from GluR2 (Wettengel, et al. (2017) Nucleic Acids Res., 45(5):2797-2808; Fukuda, et al. (2017) Sci.Rep., 7:41478; for example, GUGGAAUAGUAUAACAA-UAUGCUAAAUGUUGUUAUAGUAUCCCAC (SEQ ID NO: 70), or a sequence having at least 80%, 85%, 90%, 95%, 97%, 99%, or 100% homology or identity, particularly at least 95%, 97%, 99%, or 100% homology or identity).In certain embodiments, the guide RNA includes having an internal loop (Lehmann, et al. (1999) J. Mol. Biol., 291(1):1-13; e.g., a loop containing 4, 6, 8, 10, or more nucleotides). In certain embodiments, the guide RNA may include a region complementary to the MECP2 RNA, an editing mismatch (e.g., A:C), and an A:G mismatch for off-target editing. In certain embodiments, the nucleic acid molecule encoding the guide RNA is contained within the vector described herein. In certain embodiments, the guide RNA is expressed from a U6 promoter or another promoter expressing a small RNA.

[0042] The present invention provides methods for treating, suppressing, and / or preventing hereditary central nervous system disorders. The present invention also provides methods for treating, suppressing, and / or preventing progressive neurodevelopmental disorders. For example, in embodiments, the present invention provides methods for treating, suppressing, and / or preventing hereditary central nervous system disorders characterized by mutations in target RNA. In embodiments, the present invention provides methods for treating, suppressing, and / or preventing progressive neurodevelopmental disorders by directly or indirectly restoring the translation of RNA to a normal protein by restoring an abnormal G mutation, for example, by editing the RNA to be read as G.

[0043] According to the present invention, methods for treating, suppressing, and / or preventing genetic disorders of the central nervous system, including progressive neurodevelopmental disorders including Rett syndrome, are effective in vivo or ex vivo. For example, in the in vivo method, (one or more) nucleic acid molecules (e.g., encoding the described fusion protein, e.g., encoding the described guide RNA) are administered to the subject. In the ex vivo method, cells (syngeneic or homogeneous) are brought into contact with the nucleic acid molecules (e.g., encoding the described fusion protein, e.g., encoding the described guide RNA) and introduced into the subject. Embodiments relating to treatment are equally applicable to the in vivo and ex vivo methods.

[0044] The present invention provides methods for treating, suppressing, and / or preventing hereditary disorders associated with the MECP2 gene. In embodiments, the present invention provides gene editing, for example at the RNA level, to restore the level of functional MECP2 protein to the level of a non-disease or healthy subject. Mecp2 may be human or mouse, particularly human. In embodiments, the present invention provides gene editing, for example at the RNA level, to increase the level of functional MECP2 protein for a disease state. In embodiments, hereditary disorders associated with the MECP2 gene are neonatal encephalopathy, microcephaly, X-linked intellectual disability, PPM-X syndrome (manic-depressive illness, pyramidal tract signs, Parkinsonian syndrome, and megatesticle), bipolar disorder, Parkinsonian syndrome, hyponia, grandiosity, and megatesticle, or a combination thereof. In embodiments, the hereditary disorders associated with the MECP2 gene affect male or female subjects.

[0045] The present invention provides methods for treating, suppressing, and / or preventing Rett syndrome. In certain embodiments, Rett syndrome is characterized by a G>A mutation in MeCP2. For example, Rett syndrome may be characterized by R106Q, W104X, or R306H mutations in MECP2. Exemplary amino acid and nucleotide sequences of human MECP2 are provided in GenBank Gene ID 4204 and GenBank deposit numbers NM_004992.3 and NP_004983.1 (see also isoforms in GenBank deposit numbers NM_001110792.1, NP_001104262.1, NM_001316337.1 and NP_001303266.1). In certain embodiments, Rett syndrome includes an R106Q mutation in MeCP2. In certain embodiments, the method includes the step of administering a nucleic acid molecule encoding an RNA editing enzyme linked to or fused to an RNA-binding domain, and a guide RNA or a nucleic acid molecule encoding a guide RNA. In certain embodiments, the method includes the step of administering a guide RNA or a nucleic acid molecule encoding a guide RNA. The nucleic acid molecule may be administered directly to a subject, or it may be delivered to cells and then administered to the subject.

[0046] In certain embodiments, the amino acid sequence of MECP2 is MVAGMLGLRE EKSEDQDLQG LKDKPLKFKK VKKDKKEEKE GKHEPVQPSA HHSAEPAEAG KAETSEGSGS APAVPEASAS PKQRRSIIRD RGPMYDDPTL PEG W T R KLKQ RKSGRSAGKY DVYLINPQGK AFRSKVELIA YFEKVGDTSL DPNDFDFTVT GRGSPSRREQ KPPKKPKSPK APGTGRGRGR PKGSGTTRPK AATSEGVQVK RVLEKSPGKL LVKMPFQTSP GGKAEGGGAT TSTQVMVIKR PGRKRKAEAD PQAIPKKRGR KPGSVVAAAA AEAKKKAVKE SSIRSVQETV LPIKK R KTRE TVSIEVKEVV KPLLVSTLGE KSGKGLKTCK SPGRKSKESS PKGRSSSASS PPKKEHHHHH HHSESPKAPV PLLPPLPPPP PEPESSEDPT SPPEPQDLSS SVCKEEKMPR GGSLESDGCP KEPAKTQPAV ATAATAAEKY KHRGEGERKD IVSSSMPRPN REEPVDSRTP VTERVS (Sequence ID 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 agggaaa agtcagaaga ccaggacctc cagggcctca aggacaaacc cctcaagttt aaaaaggtga agaaagataa gaagaagag aaagagggca agcatgagcc cgtgcagcca tcagcccacc actctgctga gcccgcagag gcaggcaaag cagagacatc agaagggtca ggctccgcccc cggctgtgcc ggaagcttct gcctccccca aacagcggcg ctccatcatc cgtgaccggg gacccatgta tgatgacccc accctgcctg aaggctggac acggaagctt aagcaaagga aatctggccg ctctgctggg aagtatgatg tgtatttgat caatccccag ggaaaagcct ttcgctctaa agtggagttg attgcg tcgaaaaggt aggctactacaca tccctggacc ctaatgattt tgacttcacg gtaactggga gagggagccc ctcccgggga gaggagaaac cacctaagaa gcccaaatct cccaaagctc caggaactgg cagaggccgg ggacgcccca aagggagcgg caccacgaga cccaaggcgg ccacgtcaga gggtgtgcag gtgaaaaggg tcctggagaa aagtcctggg aagctccttg tcaagatgcc ttttcaaact tcgccagggg gcaaggctga gggggtggg gccaccacat ccacccaggt catggtgatc aaacgccccg gcaggaagcg aaaagctgag gccgaccctc aggccattcc caagaaacgg ggccgaaagc cggggagtgt ggtggcagcc gctgccgccg aggccaaaaa gaaagccgtg aaggagtctt ctatccgatc tgtgcaggag accgtactcc ccatcaagaa gcgcaagacc cgggagacgg tcagcatcga ggtcaaggaa gtggtgaagc ccctgctggt gtccaccctc ggtgagaaga gcgggaaagg actgaagacc tgtaagagcc ctgggcggaa aagcaaggag agcagcccca aggggcgcag cagcagcgcc tcctcacccc ccaagaagga gcaccaccaccatcaccacc actcagagtc cccaaaggcc cccgtgccac tgctcccacc cctgccccca cctccacctg agcccgagag ctccgaggac cccaccagcc cccctgagcc ccaggacttg agcagcagcg tctgcaaaga ggagaagatg cccagaggag gctcactgga gagcgacggc tgccccaagg agccagctaa gactcagccc gcggttgcca ccgccgccac ggccgcagaa aagtacaaac accgagggga gggagagcgc aaagacattg tttcatcctc catgccaagg ccaaacagag aggagcctgt ggacagccgg acgcccgtga ccgagagagt tagctga (SEQ ID NO: 57).

[0049] In embodiments, the present invention provides methods for treating, suppressing, and / or preventing Rett syndrome, including classical Rett syndrome and variant Rett syndrome (also known as atypical Rett syndrome). In embodiments, Rett syndrome is a Zappella variant, a Hanefeld variant, a Rolando variant, and / or an "incomplete" variant.

[0050] In embodiments, the present invention provides, but is not limited to, reduction, improvement, and / or suppression of one or more symptoms of Rett syndrome, including ataxia, uncontrolled hand movements (e.g., hand-rubbing or wringing, clapping, rubbing, washing, or hand-to-mouth movements), acquired microcephaly, autism-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.

[0051] In embodiments, the composition may 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 (vatiquinone), salizotan (EMD-128, 130), statins (e.g., lovastatin), tricyclic antidepressants (TCAs, e.g., desipramine), glatiramer acetate (e.g., COPAXONE), dextromethorphan, and / or oral cholesterol 24-hydroxylase (CH24H) inhibitors (e.g., TAK-935 / OV935).

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

[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 a subject in need. The dosage, method, and timing of administration can be readily determined by those skilled in the art, given the teachings provided herein.

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

[0055] Formulations containing the components of the present invention can be conveniently formulated for administration using acceptable media (e.g., pharmaceutically acceptable carriers) such as water, buffered saline, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), dimethyl sulfoxide (DMSO), oil, surfactants, suspending agents, or suitable mixtures thereof. The concentration of the drug in the selected media may vary, and the media may be selected based on the desired route of administration of the formulation. Use of conventional media or drugs in formulations is intended unless they are incompatible with the drug to be administered.

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

[0057] Therapy may be administered, for example, parenterally by injection into the bloodstream (e.g., intravenously), or by subcutaneous, intramuscular, or intraperitoneal injection. In certain embodiments, the therapy is administered by direct injection (e.g., into the tissue to be treated). Formulations for injection are known in the art. If injection is chosen as the method of administering the therapy, measures must be taken to ensure that a sufficient amount of molecules to exert a biological effect reaches the target cells.

[0058] A pharmaceutical composition containing the compound of the present invention as an active ingredient, tightly mixed with a pharmaceutical carrier, can be prepared by conventional pharmaceutical formulation techniques. The carrier can take a wide variety of forms depending on the preferred form of administration, such as intravenous, oral, or parenteral administration. In the preparation of oral dosage forms, for example, in the case of oral liquid preparations (e.g., suspensions, elixirs, and liquids), a common pharmaceutical medium such as water, glycol, oil, alcohol, flavoring agents, preservatives, and coloring agents can be used; or in the case of oral solid preparations (e.g., powders, capsules, and tablets), any carrier such as starch, sugar, diluent, granulator, lubricant, binder, or disintegrant can be used. An injectable suspension can be prepared, in which case a suitable liquid carrier, suspending agent, etc., can be used.

[0059] The formulations of the present invention may be formulated in dosage unit form for ease of administration and uniformity of dosage. As used herein, the dosage unit form refers to physically distinct units of the formulation appropriate for the patient receiving treatment. Each dosage should contain an amount of active ingredient calculated to produce the desired effect together with a selected pharmaceutical carrier. Procedures for determining appropriate dosage units are well known to those skilled in the art. Dosage units may be increased or decreased proportionally based on the patient's body weight. Appropriate concentrations for alleviating specific pathological conditions may be determined by dosage concentration curve calculations, as known in the art.

[0060] The method of the present invention may further include the step of monitoring the effectiveness of the method by monitoring the disease or disorder of a subject after administration of one or more compositions of the present invention. For example, the subject may be monitored for features of Rett syndrome.

[0061] definition The following definitions are provided to facilitate understanding of the present invention: The singular forms "a," "an," and "the" refer to multiple objects unless the context clearly indicates otherwise.

[0062] "Pharmacologically acceptable" means that it has been approved by a federal or state regulatory authority for use in animals, particularly humans, or that it is listed in the United States Pharmacopeia or other generally accepted pharmacopoeias.

[0063] "Carrier" refers to a vehicle administered together with, for example, diluents, adjuvants, preservatives (e.g., thymersol, benzyl alcohol), antioxidants (e.g., ascorbic acid, sodium metabisulfite), solubilizers (e.g., Tween® 80, polysorbate 80), emulsifiers, buffers (e.g., Tris-HCl, acetate, phosphate), antimicrobial agents, extenders (e.g., lactose, mannitol), excipients, adjuvants, or the activator of the present invention. Pharmaceutically acceptable carriers may be sterile liquids such as water and oil (including those of petroleum, animal, plant, or synthetic origin). Water or saline solution, as well as aqueous solutions of dextrose and glycerol, are particularly preferred as carriers for injection solutions. Appropriate drug carriers are described in Remington: The Science and Practice of Pharmacy, (Lippincott, Williams and Wilkins); Liberman, et al., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY; and Rowe, et al., Eds., Handbook of Pharmaceutical Excipients, Pharmaceutical Pr.

[0064] As used herein, the term “to treat” refers to any treatment that benefits a patient suffering from a disease, including, for example, improving the patient’s condition, slowing the progression of the condition, etc.

[0065] As used herein, the term “prevent” refers to a preventive measure taken by an object at risk of developing a condition, which results in a reduction of the object’s probability of developing the condition.

[0066] The “therapeutic dose” of a compound or pharmaceutical composition refers to the amount effective in preventing, suppressing, or treating a particular disorder or disease and / or its symptoms.

[0067] As used herein, the term “subject” refers to animals, in particular mammals, and in particular humans.

[0068] The term "isolation" refers to the separation of a compound from other components present during its manufacture. "Isolation" is not intended to exclude the presence of artificial or synthetic mixtures with other compounds or materials, or impurities that may be present, for example, due to incomplete purification or the addition of stabilizers, but which do not substantially interfere with the basic activity.

[0069] The terms “linker,” “linker domain,” and “linking” refer to a chemical portion containing a chain of atoms that covalently connects at least two compounds, such as an RNA editing enzyme and an RNA-binding domain. The linker may be an amino acid sequence (e.g., 1 to 50 amino acids, 1 to 25 amino acids, 1 to 20 amino acids, 1 to 15 amino acids, 1 to 10 amino acids, or 1 to 5 amino acids).

[0070] As used herein, the term “oligonucleotide” includes nucleic acid molecules composed of two or more, preferably three or more, ribo- and / or deoxyribonucleotides. The exact size of an oligonucleotide depends on various factors, as well as the specific application and use of the oligonucleotide.

[0071] As used herein, “nucleic acid” or “nucleic acid molecule” refers to a single-stranded or double-stranded DNA or RNA molecule, and in the case of a single-stranded molecule, refers to the molecule of its complementary sequence in a linear or circular form. In discussion of nucleic acid molecules, the sequence or structure of a particular nucleic acid molecule may be described herein in accordance with the usual convention of providing the sequence in the 5' to 3' direction. In relation to the nucleic acids of the present invention, the term “isolated nucleic acid” is sometimes used. When applied to DNA, this term refers to a DNA molecule isolated from a directly adjacent sequence in the naturally occurring genome of the organism from which it originates. For example, “isolated nucleic acid” may include a DNA molecule inserted into a vector such as a plasmid or viral vector, or incorporated into the genomic DNA of a prokaryotic or eukaryotic cell or host organism.

[0072] A "vector" is a genetic element such as a plasmid, cosmid, bacmid, phage, or virus to which another gene sequence or element (either DNA or RNA) may be bound. A vector may be a replicon to result in replication of the bound sequence or element. A vector can be either RNA or DNA and can be single-stranded or double-stranded. A vector may contain, but is not limited to, expression operons or elements such as promoters, enhancers, translation initiation signals, polyadenylation signals, or terminators—transcriptional and translational regulatory sequences that promote the expression of polynucleotide or polypeptide coding sequences in a host cell or organism.

[0073] An "expression operon" refers to a nucleic acid segment that may contain transcriptional and translational regulatory sequences that promote the expression of nucleic acid or polypeptide coding sequences in a host cell or organism, such as promoters, enhancers, translation initiation signals (e.g., ATG or AUG codons), polyadenylation signals, and terminators. An "expression vector" is a vector that promotes the expression of nucleic acid or polypeptide coding sequences in a host cell or organism.

[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 contains predominantly 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, less than about 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 contains the consensus sequence K(K / R)X(K / R) (SEQ ID NO: 58) (e.g., a monopartite NLS). In certain embodiments, the NLS contains the consensus sequence (K / R)(K / R)X 10~12 (K / R) 3 / 5 (SEQ ID NO: 59) wherein (K / R) 3 / 5 represents that at least three of five amino acids are either lysine or arginine). In certain embodiments, the NLS is the SV40 large T antigen NLS (e.g., PKKKRKV (SEQ ID NO: 47)). In certain embodiments, the c-myc NLS contains the sequence PAAKRVKLD (SEQ ID NO: 54). In certain embodiments, the NLS is the nucleoplasmin NLS KRPAATKKAGQAKKKK (SEQ ID NO: 60). With respect to the sequences provided, lysine and arginine amino acids are exchangeable.

[0075] In various embodiments, the inclusion of NLS reduces or suppresses off-target editing compared to editing in the absence of NLS, for example. For example, in various embodiments, the gene editing method including NLS reduces off-target editing by about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 100%. In various embodiments, the gene editing method including NLS reduces off-target editing by about 2, or about 3, or about 5, or about 10, or about 30 times.

[0076] The following examples are provided to illustrate various embodiments of the present invention. These examples are illustrative and are not intended to limit the present invention in any way.

[0077] [Example 1] material and method Plasmid construction We obtained a pcDNA 3.1+ plasmid (Thermo Fisher Scientific) encoding a λN peptide fused to the wild-type ADAR2 catalytic domain (Montiel-Gonzalez, et al. (2016) Nucleic Acids Res., 44:e157; Montiel-Gonzalez, et al. (2013) Proc. Natl. Acad. Sci., 110:18285-18290). E488QcDNA was constructed by overlap PCR with wild-type editase and cloned into pcDNA3.1+. Both versions of the editase were modified in pcDNA3.1+ by inserting two copies of the HA epitope and three copies of the SV40 NLS in-frame and N-terminus into the frame of the hybrid editase (pGM1090, wild-type; pGM1091, E488Q). For the Mecp2-BoxB guide, synthetic oligonucleotides representing three different Mecp2 G>A mutations, and their antisense sequences, were annealed with a Bsa1 overhang and cloned into the pENTR / U6 polylinker [pGM1099(W104X), pGM1181(306H), pGM1085(R106Q)] (Thermo Fisher Scientific). A Mecp2-BoxB guide containing an off-target AG mismatch (pGM11089) is also present in pENTR / U6. For the Mecp2 editing substrate, the EcoRI-KpnI fragment of mouse Mecp2 E1 isoform cDNA (GenBank deposit number NP_001075448.1) was cloned into the multi-cloning site of pEGFP-N3 (Clontech). Individual G>A mutations in Mecp2 were constructed by overlap PCR using the same restriction enzyme site overhang and cloned in-frame as fusion proteins with eGFP in pEGFP-N3 (Thermo Fisher Scientific). All subclonings were validated by sequence analysis. The primer sequences used for plasmid construction and PCR amplification are shown in Table 1. [Table 1] TIFF0007893585000002.tif171156

[0078] Plasmid construct The first constructs containing a fusion cDNA (editase) of the λN peptide and the wild-type ADAR2 catalytic domain have been described (Montiel-Gonzalez, et al. (2016) Nucleic Acids Res., 44:e157; Montiel-Gonzalez, et al. (2013) Proc. Natl. Acad. Sci., 110:18285-18290). This was modified to include two copies of the HA epitope tag, followed by three copies of the SV40 NLS in frame and at the N-terminus of the hybrid editase (pGM1090). To do this, two copies of the HA epitope tag and two single-stranded oligonucleotides encoding the Kozak sequence were annealed with EcoRI and BspeI overhangs and ligated at 5' to the λN domain sequence. Three copies of SV40 NLS were amplified by PCR from pECFP-Nuc (Clontech), and a BspEI overhang was added between the HA epitope tag and the λN domain. Plasmid pGM1091, containing the E488Q mutation in the ADAR2 catalytic domain, was constructed using the same steps.

[0079] The plasmid pGM1258 used in the AAV transduction experiment contained an editase cDNA under the control of the synapsin I promoter, and Mecp2 with an off-target AG mismatch, both under the control of the human U6 promoter. R106Q Contains six copies of guide DNA. U6-Mecp2 R106Q To introduce the guide region, the human U6 promoter and CRISPR sgRNA sequences from plasmid pX552 (60958; Addgene; Swiech, et al. (2015) Nat. Biotechnol., 33:102-106) were removed by restriction digestion (NdeI / ApaI), and six U6-Mecp2 R106Q The guide sequence was inserted between these sites in two steps. In the first step, U6-Mecp2 R106QThree copies of the guide region were cloned into pX552 by four-directional ligation of a PCR amplicon (pGM1108 template) having the following restriction sites: NdeI / MfeI, MfeI / SpeI, and SpeI / NheI+ApaI (pGM1257). In the second step, U6-Mecp2 R106Q Three additional copies of the guide region were generated by PCR amplification from pGM1108 using primers that added the following restriction sites: NheI / SacI, SacI / AfIII, and AfIII / ApaI. The final plasmid pGM1258 was then converted to the restriction site of pGM1257 digested with NheI / ApaI, and three U6-Mecp2 R106Q The amplicons were prepared by four-directional ligation using PCR amplicons. To introduce the editase cDNA into pGM1258, the sequence corresponding to the editase ORF was amplified from plasmid pGM1091 ​​and added downstream of the synapsin I promoter in pGM1257 using NcoI and EcoRI overhangs.

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

[0081] Each AAV1 / 2 chimeric vector was transfected using the adenovirus-free transfection method (Matsushita, et al. (1998) Gene Ther., 5:938-945; Earley, et al. (2017) J. Virol., 91:e01980-16), resulting in three 225 cm³ transfections per vector.2 The samples were prepared on a flask scale using human fetal kidney 293 (HEK293) cells. Each flask contained approximately 2 × 10⁶ cells. 7 HEK293 cells were transfected with a total of 45 μg of the following four plasmid DNAs mixed with polyethyleneimine (PEI) in a DNA:PEI weight ratio of 1:2. The plasmid DNA mixture contained 15 μg of pHelper (Agilent), 7.5 μg each of pHLP19-1 and pHLP19-2, and one of the AAV vector editorse recombinant plasmids (15 μg) containing an AAV vector genome sequence with two terminal inversion sequences (ITRs). pHLP19-1 is an AAV1 helper plasmid supplying AAV2 Rep protein and AAV1 VP protein, and pHLP19-2 is an AAV2 helper plasmid supplying AAV2 Rep protein and AAV2 VP protein (Grimm, et al. (2003) Blood 102:2412-2419). Cells were collected 3 days after transfection. Next, AAV vector particles were recovered from cells by cell lysis and purified using a HiTrap® heparin column (GE Healthcare; Desterro, et al. (2003) J. Cell Sci., 116:1805-1818). Each viral titer was determined by quantitative dot blot assay using probes prepared against editor-zecode sequences.

[0082] cell culture Neuro2A cells (ATCC CCL-131) were maintained in DMEM (Thermo Fisher Technologies) in 10% FBS (lot number AAC20-0955; HyClone) at 37°C in a 5% CO2 humidified incubator. Primary neurons were generated by targeted homologous recombination (Janelia Farms) and characterized by genotyping. R106QThe strain was derived from a mouse strain. All animal experiments were approved by the Animal Experiments Committee of Oregon Health & Science University. Offspring (P0) were killed by decapitation, and the brains were dissected in ice-cold Hanks basal salt solution (HBSS, pH 7.4) containing 25 mM Hepes. Individual hippocampi were excised without meninges and pooled by genotype. The tissues were treated with 1% trypsin and 0.01% DNase I in HBSS at 37°C for 10 minutes. The tissue pieces were rinsed three times in HBSS at room temperature and separated in minimal essential medium (Gibco) containing 25 mM glucose, 1% penicillin / streptomycin, 1% horse serum (lot number B02307-7021; HyClone), and 1% FBS. Neurons were separated by filtration through a 0.4 μm filter and placed in a neuronal growth medium consisting of Neurobasal-A (Thermo Fisher Scientific), 1 × Glutamax (Thermo Fisher Scientific), 2% B27 (Thermo Fisher Scientific), and penicillin / streptomycin in a poly-L-lysine coated dish, with 5 × 10 per well in a 12-well dish. 5 Individual cells, or 5 × 10⁶ cells in a 96-well glass chamber. 4 The cells were seeded at a density of [number] cells. After 24 hours, the neurons were given a complete culture medium change to remove cell debris. Half of the culture medium was changed every 2-3 days. The cells were maintained at 37°C in 5% CO2.

[0083] Mecp2 R106Q Mouse generation and genotyping Mecp2 R106Q The targeting vector for generating mice consisted of Mecp2 exon 3, followed by a neomycin cassette adjacent to the flippase recognition target (frt) in intron 3, the first 1.2 kb of Mecp2 exon 4, and a neomycin resistance gene expressed from the phosphoglycerate kinase promoter (PGK). Linear constructs were electroporated into mouse embryonic stem cells (mESCs), and correctly targeted clones were identified by G418 sensitivity and sequencing. Knock-in Mecp2 R106QMice expressing the allele were generated from mESCs using a standard procedure. Neomycin-resistant cassettes were created using Mecp2. R106Q The cassette was removed by crossing mice with mice expressing flipper jellycombinase from the Rosa 26 locus (stock number 009086; Jackson Labs). Cassette removal was confirmed by sequencing.

[0084] Genotyping of Mecp2R106Q mice was performed using the following primers that amplify the third intron region of the Mecp2 gene: Mecp2-R106Q Fwd(5′ ggacctatgtatgatgaccc 3′ (SEQ ID NO: 50)) and Mecp2-R106Q Rev(5′ ggtcattgggctagactgaa 3′ (SEQ ID NO: 51)). R106Q The amplicon of the knock-in animal contains the remaining frt region used to remove the neomycin cassette, so the PCR product is 93 base pairs larger than 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. 3Cells were seeded at individual cell density. After 24 hours, cells were transfected with plasmids containing wild-type or E488Q editase (pGM1090 and 1091), one copy of guide (pGM1099, pGM1181, or pGM1108), and Mecp2-egfp cDNA (pGM1174, pGM1172, or pGM1173) using Lipofectamin® 2000 (Thermo Fisher Scientific) in a 2:1 ratio in Opti-MEM® low serum medium (Thermo Fisher Scientific) and DNA. The amount of plasmid DNA added per well was 125 ng of target, 250 ng of editase, and 2.5 μg of guide. After 72 hours, cells were collected and total RNA was isolated using the Purelink® RNA Mini Kit (Ambion) according to the manufacturer's instructions. Residual plasmid DNA was removed using the TURBO DNA Free® Kit (Ambion). Total RNA was reverse transcribed using the SuperScript® III First-Strand Synthesis System (Life Technologies) and primed with oligo-dT. Transfected Mecp2-egfp cDNA was amplified for sequence analysis by PCR using a 5' primer for the CMV promoter of pEGFP-N3 and a reverse primer for the egfp gene. For editing analysis of primary neurons, 5 × 10⁶ samples were taken in DIV7. 5 Each primary hippocampal neuron is 3-6 × 10⁶ 4 Transduction was performed using AAV1 / 2 with individual viral genome infection multiplicity. Viral load did not exceed 5% of the total culture medium volume. Cells were collected one week after transfection and analyzed for editing efficiency as described for transfected N2A cells.

[0086] The efficiency of A-to-I editing was determined by reverse transcription PCR (RT-PCR) and direct sequencing of the PCR products. Quantification of sequence peak heights from the antisense strand was determined by processing the tetrachromic trace sequence using the Bioedit software package (mbio.ncsu.edu / BioEdit / bioedit.html; File > Batch Export of Raw Sequence Trace Data). The amount of editing at each site was then determined by calculating the percentage of edited cDNA using the maximum T (unedited) and C (edited) peak heights at a given site {100% × [C height / (T height + C height)]}. The 5% editing detection limit was determined by measuring the GA peak height of a mixture containing R106Q mutants and wild-type Mecp2 plasmids where the ratio decreases. C / T peak heights of the antisense strand were quantified because they are more accurate than using the A / G peak heights of the sense strand (Eggington, et al. (2011) Nat. Commun., 2:319). However, for clarity, all chromatograms are shown using inverse complementary strands.

[0087] Western blotting Primary hippocampal neurons transduced with AAV1 / 2 were dissolved in 100 μL of whole cell lysis buffer (25 mM Tris, pH 7.6, 150 mM NaCl, 1% Igepal CA-630; Sigma), 1% deoxycholic acid, 0.1% SDS, a protease inhibitor (completely EDTA-free; Roche), 1 mM β-mercaptoethanol, and 250 units / mL benzoase (Sigma-Aldrich). The solubilized product was centrifuged at 9300 × g for 10 minutes at 4°C, and the soluble fraction was isolated. Protein concentration was measured using a BCA protein assay kit (Pierce Biotechnology). Equal volumes of protein solubilized product were separated on NuPage® 4-12% bis-Tris gel (Thermo Fisher Scientific) in Mops-SDS electrophoresis buffer (Thermo Fisher Scientific), and the proteins were blotted onto nitrocellulose membrane (GE Healthcare Life Sciences). The membranes were blocked with 3% BSA in 1x TBST (TBS containing 0.05% Tween® 20) for 1 hour, and then incubated overnight at 4°C with either rabbit anti-mMeCP2 (Covance) or rabbit anti-β-actin (8227; Abcam). After washing three times with 1x TBST, the blots were incubated with anti-rabbit IgG DyLight® 680 (1:10000 dilution; Thermo Scientific) for 1 hour. The blots were quantified using the Odyssey® Imaging System (LI-COR Biosciences).

[0088] immunostaining Primary hippocampal neurons were fixed with 4% paraformaldehyde in PBS for 20 minutes at room temperature. The fixed cells were washed twice with 1×PBSG (0.1M glycine in 1×PBS) for 10 minutes at room temperature. The cells were then blocked and permeabilized at 4°C for 1 hour [0.5% Igepal CA-630, Sigma; 3% BSA (source) in 1×PBS], and incubated overnight at 4°C in a humidified chamber with primary antibodies produced against MeCP2 (rabbit mAb D4F3; Cell Signaling) and HA (rat mAb 3F10; Roche). The cells were washed three times with 1×PBS containing 0.5% Igepal and incubated with secondary antibodies Alexa 488 and Alexa 568 (Thermo Fisher Scientific) for 1 hour. After further washing with 1×PBS containing 0.5% Igepal, the cells were incubated with 300 nM DAPI for 5 minutes and then washed again with 1×PBS. Cells were mounted overnight using ProLong® Gold anti-bleeding reagent (Thermo Fisher Scientific). All images were acquired as z-stacks of 0.5 μm optical sections using a Zeiss 710 confocal microscope with a 40x water immersion objective lens. HA and MeCP2 fluorescence images were acquired across all samples using the same setup. Total cell count or antibody-positive cell count was determined by the ImageJ cell counter plugin (National Institutes of Health, imagej.nih.gov / ij, version 1.60_65 (32-bit)).

[0089] statistical analysis All statistics were performed using GraphPad version 6.0 software (Prism). The percentage of A-to-I editing in N2A cells was analyzed using one-way ANOVA, followed by Bonferroni post-hoc tests. Mecp2 R106Q / y Western blotting comparing the level of A-to-I editing in transduced neurons, MeCP2 protein levels, and the number of neurons showing MeCP2 enrichment in heterochromatin structures were analyzed using independent t-tests. All experimental results are expressed as mean ± SD.

[0090] result Mecp2 G>A mutations can be repaired by targeting editases that target heterologously expressed Mecp2 mRNA. Human MECP2 has at least three G>A mutations that cause classical Rett syndrome. R106Q and MeCP2 W104X The two mutations are located within the methyl DNA binding domain (MBD), one of which is MeCP2. R306HThe mutation is located in the NCoR interaction domain (NID) (Fyfe, et al. (2003) J. Child. Neurol., 18:709-713; Lyst, et al. (2013) Nat. Neurosci., 16:898-902) (Figure 1A). To determine whether the editase could repair these mutations, editing was tested after transient transfection of the editase, guide RNA, and Mecp2 cDNA into N2A cells. To distinguish heterologously expressed MeCP2 protein from endogenous MeCP2, the heterologously expressed MeCP2 was tagged with C-terminal eGFP. The editase and Mecp2-GFP cDNA were expressed from a cytomegalovirus (CMV) early gene promoter-enhancer, and the guide was expressed from a human U6 nuclear small RNA gene promoter. Since ADAR2 edits endogenous mRNA in the nucleus as its primary transcript, three copies of the Simian virus 40 large T antigen nuclear localization signal (NLS), in addition to the λN peptide, were added to the editase (Desterro, et al. (2003) J. Cell. Sci., 116:1805-1818). Each guide RNA contains two stem-loops (Box B) representing the sequence recognized by the λN peptide. One Box B stem-loop is located at 16-18 bases 5' of target A, and the second is located at 10 bases 3' of target A (Figure 1B). The number and location of stem-loops relative to target A were determined empirically for Mecp2 by transfection analysis, based on studies (Montiel-Gonzalez, et al. (2016) Nucleic Acids Res., 44:e157; Montiel-Gonzalez, et al. (2013) Proc. Natl. Acad. Sci., 110:18285-18290). Editing is optimal with a C mismatch at that site of the complementary guide (Schneider, et 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), and all Mecp2 guide mRNAs contain this mismatch.

[0091] N2A cells were cotransfected with an editase, a separate plasmid encoding MeCP2-GFP, and a third plasmid containing or lacking a guide sequence. Three days later, cDNA synthesized from the target region of Mecp2-GFP mRNA was analyzed using the Sanger method (Figure 1C and Figure 1D). Editing efficiency was measured by determining the relative peak height at target A. All three Mecp2 mutations were edited in a guide-dependent manner, consistent with ADAR2-mediated editing requiring double-stranded RNA (Figure 1C and Figure 1D). The editing percentage at target A varied depending on the 5' nucleotide environment, as well as the sequence preference of the ADAR2 catalytic domain (Eggington, et al. (2011) Nat. Commun., 2:319; Lehmann, et al. (2000) Biochemistry 39:12875-12884). Specifically, based on in vitro screening, the optimal 5' nucleotide hierarchy for A deamination by the ADAR2 catalytic domain is U>A>C>G, and the most optimal 3' nucleotide hierarchy is C~G~A>U. W104X(UAG) was edited most efficiently (76±10%), followed by R306H(CAC, 34±3%) and R106Q(CAA, 25±2%), but there was no statistically significant difference in the case of Mecp2 (Figure 1D). To further optimize the editase system for repairing Mecp2 G>A mutations, we focused on R106Q, which is more common than W104X mutations in human patients and causes a more severe form of Rett syndrome than R306H (Fyfe, et al. (2003) J.Child.Neurol., 18:709-713; Cuddapah, et al. (2014) J.Med.Genet., 51:152-158).

[0092] A mutation in the deaminase domain, E488Q, increases the editing efficiency of hybrid editases. The hADAR2 catalytic domain containing the E488Q mutation increases A>I editing efficiency by increasing both the catalytic rate (Montiel-Gonzalez, et al. (2016) Nucleic Acids Res., 44:e157; Kuttan, et al. (2012) Proc. Natl. Acad. Sci., 109:E3295-E3304) and the affinity of the catalytic domain to the substrate RNA (Lehmann, et al. (2000) Biochemistry 39:12875-12884). This characteristic allows the E488Q mutation to achieve a higher level of editing in the unfavorable 5' and 3' environments (Montiel-Gonzalez, et al. (2016) Nucleic Acids Res., 44:e157; Kuttan, et al. (2012) Proc. Natl. Acad. Sci., 109:E3295-E3304). Editase E488Q However, Mecp2 has a temperature below the optimal 5'C. R106Q To test whether it increases the editing efficiency of target A, N2A cells were treated with Mecp2 R106Q -egfp and editor E488Q Cotransfected with cDNA. Sequence analysis revealed that guide expression is required for editing, and the editing percentage of Mecp2 mRNA was compared to that of wild-type editase. E488Q It was shown that the value increased by approximately twofold (51±11% vs. 22±5%, n=3, P<0.01) (Figure 2A).

[0093] Hybrid editor enzyme with wild-type hADAR2 or hADAR2 E488Q Transfect Mecp2 using the catalytic domain. R106Q-A single off-target editing site was detected within the guide region of the egfp cDNA (Figure 2B). Editing at this site results in a silent codon change T105T (ACA>ACG). G mismatch at the off-target site can reduce off-target editing in the transfect substrate (Vogel, et al. (2014) Angew Chem. Int. Ed. Engl., 53:6267-6271). To determine whether G mismatch also reduces off-target editing in Mecp2 mRNA, we used Mecp2 R106Q -egfp cDNA, editor E488Q Editing efficiency was analyzed in N2A cells transfected with plasmids encoding a guide with a G mismatch at nearby off-target A (Figure 2C). Targeting the editase with a guide containing an AG mismatch significantly reduced the amount of off-target editing (4.9±0% with mismatch, 33±5% without mismatch, n=3, P<0.0001; Figures 2D and 2E), but there was no significant effect on editing at target A (Figures 2D and 2F). The presence of guide RNA was required for all editing events (Figures 2E and 2F).

[0094] Site-directed RNA editing repairs endogenous Ret-related mutations, restoring protein levels and MeCP2 function. Next, editor E488Q We tested whether it could (i) repair the R106Q missense mutation in endogenous Mecp2 mRNA, (ii) restore protein levels, and (iii) restore the ability of MeCP2 to bind to heterochromatin, a characteristic functional feature necessary to reverse Rett-like symptoms in mice (Garg, et al. (2013) J. Neurosci., 33:13612-13620). In these tests, neurons were isolated from mice and engineered to contain the R106Q mutation in the endogenous Mecp2 gene. Cultured neurons were transduced with one of two AAVs (AAV1 / 2). Both viruses were editase-controlled under the control of the human synapsin I promoter. E488QThe virus expressed (Swiech, et al. (2015) Nat. Biotechnol., 33:102-106), and each virus contained an additional 6 copies of guides (off-target mismatch guides; Figure 2C) under the control of the human U6 promoter. The other virus served as a control and lacked all guide sequences. Hippocampal neurons were subjected to P0 Mecp2 R106Q / y Mecp2 cDNA was generated from mice and transduced in vitro with guide-containing or control AAV vectors containing a 7-day (DIV7) AAV1 / 2 hybrid capsid. After expressing the virus for another 7 days, Mecp2 cDNA was prepared from experimental and control cultures and analyzed by Sanger assay. In cultures expressing both the editase and the guide, 72±5% of Mecp2 mRNA was repaired (Figure 3A), but no editing was detected in neurons transduced with the guide-deficient control virus. In addition to editing at R106Q, sequence analysis also identified several off-target editing sites within the Mecp2 cDNA (Figure 3B). Off-target sites mainly occurred within regions complementary to the guide RNA, although one event occurred outside the guide (N126S).

[0095] The functional outcomes of RNA editing were tested by measuring the amount of MeCP2 protein in AAV1 / 2 transduced cultures using Western blotting. Similar to other mutations in MBD (Goffin, et al. (2011) Nat. Neurosci., 15:274-283; Brown, et al. (2016) Hum. Mol. Genet., 25:558-570), MeCP2 R106QProtein levels were reduced compared to wild-type levels (Figure 4). The decrease in mutant MeCP2 protein levels may be due to destabilization (Goffin, et al. (2011) Nat. Neurosci., 15:274-283). Expression of both editase and guide in mutant primary neurons increased MeCP2 protein levels by approximately threefold compared to expression of editase alone (Figure 4; 35.3±2% with guide compared to 12.9±1% without guide, n=3, P<0.001). This is the first demonstration of functional recovery of the protein responsible for endogenous disease after editing.

[0096] MeCP2 binds to methyl-CpG with high affinity both in vitro and in vivo (Skene, et al. (2010) Mol. Cell., 37:457-468; Lagger, et al. (2017) PLoS Genet., 13:e1006793), a characteristic important for normal function. In mouse cells, MBD mutations in MeCP2 reduce binding to heterochromatin containing mCG-rich amplified satellite sequences (Brown, et al. (2016) Hum. Mol. Genet., 25:558-570; Heckman, et al. (2014) eLife 3:e02676). MBD mutations in MeCP2 R106Q However, it shows decreased binding to methyl-CpG in vitro (Yang, et al. (2016) ACS Chem. Biol., 11:2706-2715). MeCP2 R106Q To determine whether intracellular binding was similarly reduced and whether editing of G>A mutant Mecp2 RNA restores heterochromatin enrichment, nuclei were transduced with AAV1 / 2 encoding HA-tagged editases, with or without the guide as a control. R106Q / y Neuron cultures were immunolabeled (Figure 5). The nucleus and heterochromatin were identified using DAPI (4',6-diamidino-2-phenylindole), a fluorescent indicator that strongly binds to AT-rich regions of DNA. Wild-type neurons (Mecp2) + / yIn cultures from ), the nuclei showed classical MeCP2 enrichment in DAPI-stained heterochromatin (structure), reflecting functional MBD (Figure 5A). In contrast, Mecp2 transduced with an editase virus lacking a guide sequence. R106Q / y In cultures prepared from siblings, MeCP2 immunofluorescence was scattered throughout the nucleus, as would be expected from mutations in MBD that interfere with DNA binding (Goffin, et al. (2011) Nat. Neurosci., 15:274-283; Heckman, et al. (2014) eLife 3:e02676) (Figure 5B). The staining intensity was also lower than that of wild-type nuclei, likely reflecting an unstable MeCP2 protein. In contrast, MeCP2 expressing both editase and guide RNA... R106Q Neurons showed a clear increase in MeCP2 immunofluorescence to levels similar to wild-type nuclei, and enrichment of MeCP2 protein in heterochromatin structures, indicating functional recovery of MBD (Figures 5C and 5D). To quantify the immunofluorescence results, we first determined in three experiments that the same proportion of cells expressed eddiease regardless of the presence of the guide (67±7% of cells with eddiease alone, and 67±10% with eddiease and guide; n=134 and 137 cells, respectively; Figure 5E). Subsequently, in cultures transduced with eddiease and guide, it was determined that 74±11% of cells expressing eddiease (Figure 5F) and 49±8% of all cells showed enrichment of MeCP2 in heterochromatin structures (Figure 5G). MeCP2 enrichment was observed in MeCP2 transduced with the virus lacking the guide. R106Q The absence of detection within the nuclear heterochromatin structure is consistent with sequencing results indicating that editing depends on the presence of a guide.

[0097] ADARs have been used to repair G>A mutations in exogenous mRNA in African clawed frog oocytes (Woolf, et al. (1995) Proc. Natl. Acad. Sci., 92:8298-8302). The data presented herein demonstrate that site-directed RNA editing using an engineered hADAR2 catalytic domain can repair endogenous mutant mRNA and reverse mutation-induced cellular defects.

[0098] In mice and humans, three genes encode ADAR proteins, but only ADAR1 and ADAR2 exhibit catalytic activity from A to I (Nishikura, K. (2010) Annu. Rev. Biochem., 79:321-349). Natural ADAR-mediated editing is crucial for post-transcriptional regulation of brain protein function and was first demonstrated in ion channels and receptors (Bhalla, et al. (2004) Nat. Struct. Mol. Biol., 11:950-956; Sommer, et al. (1991) Cell 67:11-19; Burns, et al. (1997) Nature 387:303-308), but is now known to extend to many other proteins and non-coding RNAs (Chen, et al. (2012) Curr. Top. Microbiol. Immunol., 353:111-121; Nishikura, K. (2016) Nat. Rev. Mol. Cell Biol., 17:83-96). The ADAR2 catalytic domain is recognized for its ability to edit heterologous mRNA (Vogel, et al. (2014) Angew Chem. Int. Ed. Engl., 53:6267-6271; Schneider, et al. (2014) Nucleic Acids Res., 42:e87; Montiel-Gonzalez, et al. (2016) Nucleic Acids Res., 44:e157; Montiel-Gonzalez, et al. (2013) Proc. Natl. Acad. Sci., 110:18285-18290; Wong, et al. (2001) RNA 7:846-858) and for its well-characterized editing mechanism (Kuttan, et al. al.(2012)Proc.Natl.Acad.Sci.,109:E3295-E3304;Matthews,et al.(2016) The focus was on Nat. Struct. Mol. Biol, 23:426-433. In fact, improved editing efficiency of Mecp2 mRNA was observed when the editase contained the E488Q mutation in the catalytic domain (Montiel-Gonzalez, et al. (2016) Nucleic Acids Res., 44:e157; Kuttan, et al. (2012) Proc. Natl. Acad. Sci., 109:E3295-E3304; Phelps, et al. (2015) Nucleic Acids Res., 43:1123-1132). The structural analysis of the hADAR2 catalytic domain complexed onto double-stranded RNA (Matthews, et al. (2016) Nat. Struct. Mol. Biol., 23:426-433) provides a valuable resource for generating other mutations to further optimize the editing efficiency and specificity of MeCP2 and other mutations (Wang, et al. (2016) Nucleic Acids Res., 44:9872-9880). In contrast to previous efforts, since ADARs typically edit primary transcripts in the nucleus, all of the constructs here contained NLSs that particularly enhance the editing efficiency of endogenous mRNA (Wong, et al. (2001) RNA 7:846-858).

[0099] In transfected cells, the editase at target A E488QThe higher editing efficiency achieved using GA mismatch also resulted in increased off-target editing at a single site within the guide region. Single off-target editing sites were reduced by using GA mismatch (Schneider, et al. (2014) Nucleic Acids Res., 42:e87). In particular, sequencing of five cDNAs representing highly expressed mRNAs other than the target mRNA showed no off-target editing (Montiel-Gonzalez, et al. (2016) Nucleic Acids Res., 44:e157). However, also surprisingly, in this study using neurons, off-target editing sites differed between transfected Mecp2 mRNA and endogenous Mecp2 mRNA. Specifically, the endogenous repaired Mecp2 mRNA showed several additional off-target editing sites both inside and outside the guide region that were not present in the Mecp2 mRNA expressed from cDNA (Figure 3B). Differences in off-target editing sites between transfected Mecp2 mRNA and endogenous Mecp2 mRNA may reflect sequence differences that could affect RNA folding and other downstream processing events. Importantly, none of the off-target sites in endogenous Mecp2 mRNA have been reported to cause Rett syndrome (Fyfe, et al. (2003) J. Child Neurol., 18:709-713). Further use of the Rett syndrome mouse model can demonstrate that cellular and behavioral symptoms can be reversed by restoring wild-type MeCP2 in symptomatic mice (Guy, et al. (2007) Science 315:1143-1147; Sinnett, et al. (2017) Mol.Ther.Methods Clin.Dev., 5:106-115; Gadalla, et al. (2017) Mol.Ther.Methods Clin.Dev., 5:180-190; Garg, et al. (2013) J.Neurosci., 33:13612-13620; Gadalla, et al. (2013) Mol.Ther., 21:18-30).

[0100] [Example 2] Mecp2 mutation Mecp2 317G>A This method was tested in vivo using mice possessing Mecp2. 317G>A The mutation results in MeCP2 with an R106Q amino acid change. In short, Mecp2 mutation Mecp2 317G>A Mice possessing the E488Q mutation were treated with an AAV vector encoding an editase having the E488Q mutation of the present invention, with or without the use of six copies of guide RNA. Due to its neurotropic properties, an AAV vector with a PHP.B capsid (AAV9 variant) was used (Hordeaux et al. (2018) Mol.Ther., 26(3):664-668). 1.1 × 10 10 A viral genome equivalent (vge) AAV was stereotactically injected into the hippocampus of mice. Three to four weeks after direct viral injection, the mice were killed, and MeCP2 function was detected in the brain. As shown in Table 2, efficient targeted RNA editing and recovery of MeCP2 function in the brain were observed in vivo. Furthermore, based on RNA sequence analysis, the A-to-I editing efficiency in dentate granule neurons was determined to be 39%, and the A-to-I editing efficiency in CA1 neurons was determined to be 64%. As shown in Figure 6, the MeCP2 intensity in dentate heterochromatin was higher in mice injected with AAV containing guide RNA compared to mice injected with AAV without guide RNA. These results indicate the rescue of MeCP2 DNA binding ability. [Table 2]

[0101] Table 2: Quantification of the number of cells expressing the editase enzyme in vivo and exhibiting functional MeCP2. AAV PHP.B encoding the editase and guide RNA is used for MeCP2. 317G>AThe drug was injected into the hippocampus of mice. Three weeks after injection, the mice were treated for immunohistochemistry. After thresholding the signal from uninfected cells, ededitase+ cells were identified by HA immunostaining of brain sections from injected mice. Percentages are relative to the total number of cells identified by DAPI. Percentages of ededitase+ cells showing MeCP2 enrichment within heterochromatin (structure) indicate recovery of MeCP2 protein function. n=864 cells.

[0102] [Example 3] Plasmid construction The sequence encoding full-length human ADAR2, including the amino-terminal Flag tag, was subcloned from a yeast expression vector into pcDNA 3.1+ (Thermo Fisher Scientific). To express a Mecp2 guide designed to recruit full-length ADAR2, synthetic oligonucleotides were annealed to a Bsa1 overhang and cloned into pENTR / U6 polylinkers [pGM1099 (2x BoxB guide W104X), pGM1192 (internal loop guide W104X), pGM1310 (GluA2 stem-loop W104X)] (Thermo Fisher Scientific). 311G>A The Mecp2 editing substrate containing the (W104X) mutation is described in Example 1. All subclonings were validated by sequence analysis. The primer sequences used for plasmid construction and PCR amplification are shown in Table 3.

[0103] cell culture HEK293T cells (ATCC CRL-3216) were maintained in DMEM (Thermo Fisher Technologies) in 10% FBS at 37°C in a 5% CO2 humidified incubator.

[0104] RNA editing To analyze editing using full-length ADAR2, 1.3 × 10⁶ wells were used per well in a 12-well plate. 3HEK293T cells were seeded at individual cell density. After 24 hours, cells were labeled with full-length human ADAR2 (pGM1155), one-copy guide (pGM1099, pGM1192, or pGM1310), and Mecp2 using Lipofectamin® 2000 (Thermo Fisher Scientific) in a 2:1 ratio in Opti-MEM® low-serum medium (Thermo Fisher Scientific) and DNA. 311G>A -egfp cDNA-encoding plasmids were used for transfection. The amount of plasmid DNA added per well was 125 ng of target, 250 ng of human ADAR2, and 2.5 μg of guide. After 72 hours, cells were collected and total RNA was isolated using the Purelink® RNA Mini Kit (Ambion) according to the manufacturer's instructions. Residual plasmid DNA was removed using the TURBO DNA Free® Kit (Ambion). Total RNA was reverse transcribed using the SuperScript® III First-Strand Synthesis System (Life Technologies) and primed with oligo-dT. Transfected Mecp2 311G>A -egfp cDNA was amplified for sequence analysis by PCR using a 5' primer for the CMV promoter of pEGFP-N3 and a reverse primer for the egfp gene. [Table 3]

[0105] result Human fetal kidney (HEK) cells were subjected to full-length human ADAR2 and Mecp2 under the control of the cytomegalovirus (CMV) promoter. 317G>A It was transfected with Mecp2. Human ADAR2 was a full-length native ADAR2 molecule that mimicked endogenous ADAR2. 317G>A The mutation is an R106Q amino acid change (Mecp2 R106Q) results in the following: The cells were then treated with 1) guide RNA having the two BoxB stem loops described above (see, e.g., 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 having an internal loop (Lehmann, et al. (1999) J.Mol.Biol., 291(1):1-13). As seen in Figure 7, guide RNA containing two BoxB stem loops was able to recruit full-length ADAR2 to edit Mecp2 RNA in transfected HEK cells. These results demonstrate the recruitment of full-length ADAR to target RNA, which may include the presence of sequences not normally present in the target RNA, in addition to the target RNA sequence.

[0106] While some preferred embodiments of the present invention have been described and illustrated above, the present invention is not intended to be limited to such embodiments. Various modifications can be made without departing from the scope and spirit of the invention, as set forth in the following claims. In one embodiment, the present invention includes the following: [Item 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, The aforementioned fusion protein contains a nuclear localization signal, The guide RNA includes a sequence that is specifically recognized by the RNA-binding domain, The method wherein the guide RNA specifically hybridizes with the target sequence of the endogenous RNA and contains a mismatch in the nucleotide to be edited. [Item 2] The method according to item 1, wherein the endogenous RNA is located in the nucleus of the cell. [Item 3] The method according to item 1, wherein the RNA editing enzyme is a deaminase. [Item 4] The method according to item 1, wherein the RNA editing enzyme is an adenosine deaminase (ADAR) that acts on RNA. [Item 5] The method according to item 4, wherein the ADAR is selected from the group consisting of ADAR1, ADAR2, fragments thereof, and variants thereof. [Item 6] The method according to item 5, wherein the ADAR has at least 90% identity with sequence number 55. [Item 7] The method according to any one of items 1 to 6, wherein the RNA-binding domain is a λN peptide or a variant thereof. [Item 8] The method according to item 7, wherein the RNA-binding domain has at least 90% identity with SEQ ID NO: 46. [Item 9] The method according to any one of items 1 to 8, wherein the sequence specifically recognized by the RNA-binding domain is the BoxB sequence. [Item 10] The method according to any one of items 1 to 9, wherein the endogenous RNA is RNA expressed in central nervous system cells. [Item 11] The method according to item 10, wherein the endogenous RNA is methyl CpG-binding protein 2 (MECP2) RNA. [Item 12] The method according to any one of items 1 to 11, wherein the guide RNA further comprises one or more mismatches upstream or downstream of the nucleotide to be edited. [Item 13] The method according to any one of items 1 to 12, wherein the nuclear localization signal is the SV40 large T antigen nuclear localization signal or a variant thereof. [Item 14] The method according to item 13, wherein the nuclear localization signal includes SEQ ID NO: 47 or SEQ ID NO: 47 having one, two, or three substitutions, additions, or deletions. [Item 15] The method according to item 1, wherein the nuclear localization signal is an SV40 large T antigen nuclear localization signal, the RNA binding domain is a λN peptide, the RNA editing enzyme is an adenosine deaminase (ADAR) that acts on RNA, and the sequence specifically recognized by the RNA binding domain is a BoxB sequence. [Item 16] The method according to any one of items 1 to 15, wherein the nucleic acid molecules of a) and b) are contained within a single vector. [Item 17] The method according to item 16, wherein the vector is a viral vector. [Item 18] The method according to item 17, wherein the viral vector is adeno-associated virus (AAV). [Item 19] A method for editing a target sequence of endogenous RNA within a cell, comprising the step of delivering a nucleic acid molecule encoding a guide RNA to the cell, The aforementioned guide RNA includes a sequence that is specifically recognized by endogenous human adenosine deaminase (ADAR) that acts on RNA. The method wherein the guide RNA specifically hybridizes with the target sequence of the endogenous RNA and contains a mismatch in the nucleotide to be edited. [Item 20] The method according to item 19, wherein the endogenous RNA is located in the nucleus of the cell. [Item 21] The method according to item 19, wherein the ADAR is ADAR1 or ADAR2. [Item 22] The method according to item 21, wherein the ADAR has at least 90% identity with sequence number 55. [Item 23] The method according to item 19, wherein the endogenous RNA is methyl CpG-binding protein 2 (MECP2) RNA. [Item 24] The method according to any one of items 19 to 23, wherein the guide RNA further comprises one or more mismatches upstream or downstream of the nucleotide to be edited. [Item 25] The method according to any one of items 19 to 24, wherein the endogenous ADAR recognizes the guide RNA. [Item 26] The method according to any one of items 19 to 25, wherein the endogenous ADAR deaminates the bases of the nucleotides of the endogenous RNA. [Item 27] The method according to any one of items 19 to 26, wherein the editing of the target sequence alters the level and / or function of the protein encoded by the target sequence. [Item 28] The method according to any one of items 19 to 27, wherein the nucleic acid molecule is contained within a viral vector. [Item 29] The method according to item 28, wherein the viral vector is adeno-associated virus (AAV). [Item 30] A method for treating, suppressing and / or preventing a genetic disorder of the central nervous system, 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 administering the above to the subject, The aforementioned fusion protein contains a nuclear localization signal, The guide RNA includes a sequence that is specifically recognized by the RNA-binding domain, A method for correcting mismatch mutations associated with the genetic disorder of the central nervous system, using the RNA editing enzyme. [Item 31] The method according to item 30, wherein the aforementioned hereditary disorder of the central nervous system is Rett syndrome. [Item 32] The method according to item 31, wherein the guide RNA specifically hybridizes with methyl CpG-binding protein 2 (MECP2) RNA and includes a mismatch in the mutated nucleotide of the endogenous MECP2 RNA. [Item 33] The method according to any one of items 30 to 32, wherein the endogenous RNA is located in the nucleus of the cell. [Item 34] The method according to any one of items 30 to 33, wherein the RNA editing enzyme is an adenosine deaminase (ADAR) that acts on RNA. [Item 35] The method according to item 34, wherein the ADAR is selected from ADAR1, ADAR2, and fragments or variants thereof. [Item 36] The method according to item 35, wherein the ADAR has at least 90% identity with sequence number 55. [Item 37] The method according to any one of items 30 to 36, wherein the RNA-binding domain is a λN peptide or a variant thereof. [Item 38] The method according to item 37, wherein the RNA-binding domain has at least 90% identity with SEQ ID NO: 46. [Item 39] The method according to any one of items 30 to 38, wherein the sequence specifically recognized by the RNA-binding domain is a BoxB sequence. [Item 40] The method according to any one of items 30 to 39, wherein the nuclear localization signal is the SV40 large T antigen nuclear localization signal or a variant thereof. [Item 41] The method according to item 40, wherein the nuclear localization signal includes SEQ ID NO: 47 or SEQ ID NO: 47 having one, two, or three substitutions, additions, or deletions. [Item 42] The method according to item 30, wherein the nuclear localization signal is an SV40 large T antigen nuclear localization signal, the RNA binding domain is a λN peptide, the RNA editing enzyme is an adenosine deaminase (ADAR) that acts on RNA, and the sequence specifically recognized by the RNA binding domain is a BoxB sequence. [Item 43] The method according to any one of items 30 to 42, wherein the nucleic acid molecules of a) and b) are contained within a single vector. [Item 44] The method according to item 43, wherein the vector is a viral vector. [Item 45] The method according to item 44, wherein the viral vector is adeno-associated virus (AAV). [Item 46] A method for treating, suppressing and / or preventing a genetic disorder of the central nervous system of a subject, comprising the step of administering a nucleic acid molecule encoding a guide RNA to the subject, wherein the guide RNA comprises a sequence specifically recognized by an RNA-acting endogenous human adenosine deaminase (ADAR). [Item 47] The method according to item 46, wherein the aforementioned hereditary disorder of the central nervous system is Rett syndrome. [Item 48] The method according to item 47, wherein the guide RNA specifically hybridizes with methyl CpG-binding protein 2 (MECP2) RNA and includes a mismatch in the mutant nucleotide of the endogenous MECP2 RNA. [Item 49] The method according to any one of items 46 to 48, wherein the endogenous RNA is located in the nucleus of the cell. [Item 50] The method according to any one of items 46 to 49, wherein the ADAR is ADAR1 or ADAR2 or a variant thereof. [Item 51] The method according to item 50, wherein the ADAR has at least 90% identity with sequence number 55. [Item 52] The method according to any one of items 46 to 51, wherein the endogenous ADAR recognizes the guide RNA. [Item 53] The method according to any one of items 46 to 52, wherein endogenous ADAR deaminates the bases of the nucleotides of the endogenous RNA. [Item 54] The method according to any one of items 46 to 53, wherein the editing of the target sequence alters the level and / or function of the protein encoded by the target sequence. [Item 55] The method according to any one of items 46 to 54, wherein the nucleic acid molecule is contained within a viral vector. [Item 56] The method according to item 55, wherein the viral vector is adeno-associated virus (AAV).

Claims

1. A composition for editing the target sequence of endogenous RNA within a cell, The composition comprises a guide RNA or a nucleic acid molecule encoding a guide RNA. Here, the guide RNA can form a double-stranded RNA structure with the target sequence by having at least 80% complementarity with the target sequence, and the double-stranded RNA structure has an internal loop or one or more mismatches in addition to adenosine nucleotides to be edited, and is specifically recognized by endogenous human adenosine deaminase (ADAR) that acts on RNA. Here, the guide RNA contains a mismatch in the adenosine nucleotide to be edited, and here, The composition wherein the endogenous RNA is methyl CpG-binding protein 2 (MECP2) RNA.

2. The composition according to claim 1, wherein the endogenous human adenosine deaminase (ADAR) that acts on RNA is ADAR1 or ADAR2, and / or the endogenous RNA that is edited in the cell is located in the nucleus of the cell.

3. The nucleotide being edited is a point mutation and / or MECP2 RN The composition according to claim 1, wherein A is a point mutation that causes a disease.

4. The point mutation that causes the disease is a mutation from guanine to adenosine in MECP2 RNA. The composition according to claim 3, which is a mutation.

5. The composition according to claim 1, wherein the nucleotide to be edited is a nonsense mutation, and the editing deletes a stop codon, or the guide RNA can correct a nonsense mutation from C to T by deamination of A at the 3' position.

6. The composition according to any one of claims 1 to 5, wherein the editing of the target sequence alters the level and / or function of the protein encoded by the target sequence.

7. The composition according to any one of claims 1 to 6, wherein the nucleic acid molecule encoding the guide RNA is contained within the viral vector.

8. The composition according to any one of claims 1 to 7, wherein the viral vector is adeno-associated virus (AAV).

9. A composition for treating, suppressing and / or preventing a target genetic disorder of the central nervous system, comprising editing a target sequence of endogenous RNA in a cell, Here, the composition comprises a guide RNA or a nucleic acid molecule encoding the guide RNA, wherein the guide RNA is capable of forming a double-stranded RNA structure with the target sequence by having at least 80% complementarity with the target sequence, the double-stranded RNA structure having an internal loop or one or more mismatches in addition to mismatches at the adenosine nucleotide to be edited, and is specifically recognized by endogenous human adenosine deaminase (ADAR) acting on RNA. The aforementioned guide RNA contains a mismatch in the adenosine nucleotide to be edited, and here, The endogenous RNA is methyl CpG-binding protein 2 (MECP2) RNA. The aforementioned composition.

10. The composition according to claim 9, wherein the central nervous system disorder is Rett syndrome, and the endogenous human adenosine deaminase (ADAR) acting on RNA is ADAR1 or ADAR2, and / or the endogenous RNA edited within the cell is located in the nucleus of the cell.

11. The mismatch in the edited nucleotide of the endogenous MECP2 RNA suddenly The composition according to claim 9, wherein the guide RNA is a mutation and / or a disease-causing point mutation such as a nonsense mutation in which a stop codon is deleted by editing, or the guide RNA can modify a nonsense mutation from C to T by deamination of A at the 3' position.

12. The composition according to any one of claims 9 to 11, wherein the editing of the target sequence alters the level and / or function of the protein encoded by the target sequence.

13. The composition according to any one of claims 9 to 12, wherein the nucleic acid molecule encoding the guide RNA is contained within the viral vector.

14. The composition according to any one of claims 9 to 13, wherein the viral vector is adeno-associated virus (AAV).

15. A guide RNA or a nucleic acid molecule encoding a guide RNA, wherein the guide RNA has at least 80% complementarity with the target sequence, thereby enabling it to form a double-stranded RNA structure with a target sequence contained in endogenous RNA within the cell, and the double-stranded RNA structure has an internal loop or one or more mismatches in addition to a mismatch at the adenosine nucleotide to be edited, and is specifically recognized by endogenous human adenosine deaminase (ADAR) acting on RNA. The aforementioned guide RNA contains a mismatch in the adenosine nucleotide to be edited, and here, The endogenous RNA is methyl CpG-binding protein 2 (MECP2) RNA. Guide RNA or a nucleic acid molecule that encodes guide RNA.

16. The guide RNA or nucleic acid molecule encoding the guide RNA according to claim 15, wherein the mismatch between the nucleotide to be modified or edited and the target sequence is an A:C mismatch, and / or the endogenous deaminase is ADAR1 or ADAR2.

17. The nucleotide being edited is a point mutation and / or MECP2 RN A guide RNA or nucleic acid molecule encoding a guide RNA, which is a point mutation in A that causes a disease.

18. The point mutation that causes the disease is a mutation from guanine to adenosine in MECP2 RNA. A mutant, the guide RNA or nucleic acid molecule encoding the guide RNA as described in claim 17.

19. The nucleotide to be edited is a nonsense mutation that can be edited to delete a stop codon, or the guide RNA or nucleic acid molecule encoding the guide RNA according to claim 15, wherein the guide RNA can correct a nonsense mutation from C to T by deamination of A at the 3' position.

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