Compositions and methods for treating RETT syndrome

Abstract

The present disclosure relates to compositions and methods for treating Rett syndrome and related MECP2 disorders.

Description

[0001] COMPOSITIONS AND METHODS FOR TREATING RETT SYNDROME

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 661,438, filed June 18, 2024, the contents of which are incorporated herein by reference in their entirety.

[0004] BACKGROUND

[0005] The disruption of the methyl-CpG-binding protein 2 (MECP2), encoded on the X chromosome, is known to cause severe neurodevelopmental diseases referred to as the MECP2-related disorders. These disorders include classic Rett syndrome, variant Rett syndrome, MECP2 -related severe neonatal encephalopathy, nonsyndromic X-linked intellectual disability, PPM-X syndrome, CDKL5 disorder, and A- / / T7U duplication syndrome. Because classic Rett syndrome is an X-linked dominant disorder, it is usually observed in female heterozygotes, as males with this mutation on their only X chromosome typically fail to survive birth. Female patients with Rett syndrome present with short stature overall, but have a relatively more profound microcephaly phenotype, suggesting a prominent role for MECP2 in the brain. Accordingly, while MECP2 protein is present in all tissues, the expression of MECP2 is particularly high in all types of mature neurons of the CNS. MECP2 has been described as both a transcriptional stimulator and inhibitor, a regulator of RNA splicing, and a regulator of DNA methylation or reader of methylation. Moreover, loss of MECP2 has been shown to lead to a variety of cell physiological defects such as mitochondrial permeability, diminished dendritic branching, altered electrophysiological activity, and changes in nuclear and nucleolar size, etc. Despite these observations, it is still not clear which of these are relevant for human Rett syndrome patients, and which should serve as proxies for experimentally targeting and developing therapeutic strategies. To date, only one therapy, trofinetide, has been approved by the U.S. Food and Drug Administration for treating Rett Syndrome. However, this treatment has specific drawbacks, such as severe gastrointestinal side effects, including diarrhea and vomiting, which may lead to dehydration and seizures. In view of the foregoing, there is an unmet need to develop new treatments for Rett syndrome. SUMMARY

[0006] In some aspects, the present disclosure provides methods for treating a disease or condition characterized by a mutation of the MECP2 gene in a subject in need thereof, the method comprising administering an effective amount of nicotinamide adenine dinucleotide (NAD+) or a precursor or salt thereof to the subject.

[0007] In some aspects, the present disclosure provides methods for treating a disease or condition characterized by a mutation of the MCEP2 gene in a subject in need thereof, the method comprising administering an effective amount of an agent that promotes DNA damage repair to the subj ect.

[0008] In some aspects, the present disclosure provides methods of treating CDKL5 disorder in a subject in need thereof, the method comprising administering an effective amount of nicotinamide adenine dinucleotide (NAD+) or a precursor or salt thereof to the subject.

[0009] In some aspects, the present disclosure provides compositions comprising an agent that promotes DNA damage repair for treating a subject having a disease or condition characterized by a mutation of the MECP2 gene; optionally wherein the agent is NAD+ or a precursor or salt thereof.

[0010] In some aspects, the present disclosure provides compositions comprising an agent that promotes DNA damage repair for treating a subject having CDKL5 disorder; optionally wherein the agent is NAD+ or a precursor or salt thereof.

[0011] In some aspects, the present disclosure provides the use of an agent that promotes DNA damage repair for treating a subject having a disease or condition characterized by a mutation of the MECP2 gene; optionally wherein the agent is NAD+ or a precursor or salt thereof.

[0012] In some aspects, the present disclosure provides the use of an agent that promotes DNA damage repair for treating a subject having CDKL5 disorder; optionally wherein the agent is NAD+ or a precursor or salt thereof.

[0013] BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIGS. 1A-1F depict a method to isolate and molecularly profile MECP2-Wildtype and MECP2-Mutant neurons from Rett brain. FIG. 1A shows a diagram depicting the process of nuclei isolation that employs a sucrose gradient and several washes and centrifugations followed by immunostaining and FACS. FIG. IB shows immunostaining of the sorted MECP2hi and MECP21ow neuronal nuclei after cytospin from Rett patient brain. The immunostaining shows a decrease in MECP2expression in the MECP21ow fraction, images taken at 40x. It was previously shown that the hi and low populations represent wildtype MECP2 expressing versus mutant MECP2 expressing neurons. To the right of the immunostaining, the quantification of immunofluorescence signal is depicted. Statistical significance was checked using a paired t-test, where * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < o 0001. FIG. 1C shows Western blot analysis verifying a decrease in MECP2 expression in the MECP21ow fraction isolated by FACS. FIG. ID shows proportions of neuronal subtypes in MECP2hi, MECP21ow and WT nuclei from Greenberg data and three independent experiments. Mutant (MECP2-Mutant nuclei from Rett brain), WT (MECP2- wild type nuclei from Rett brain), Normal (wild type nuclei from wild type brain). FIG. IE shows UMAP plots of abundant neuronal subtypes as defined by the indicated markers. FIG. IF shows UMAP showing 19 clusters identified during the analysis of RNA-seq. MECP21ow (MUT) and MECP2hi (WT) nuclei contribute to multiple identified clusters, expressing excitatory and inhibitory neuronal subtypes.

[0015] FIGS. 2A-2F show a transcriptome analysis revealing alteration in synaptic and metabolic gene expression due to lack of MECP2. FIG. 2A shows a summary of the number of Differentially Expressed Genes (DEGs) in several of the main cell types. FIG. 2B provides a Venn diagram showing the number of genes that overlap between excitatory and inhibitory neuronal populations. FIG. 2C depicts a Venn diagram showing the number of overlapping genes in several excitatory and inhibitory neuronal populations between the instant data and a published data set from the Greenberg lab, validating the instant approach to isolate wildtype versus mutant neurons. FIG. 2D shows Gene ontology (GO) analysis of misregulated pathways indicate significant alterations in metabolic gene expression in inhibitory neurons (VIP) and synaptic genes in both excitatory (SLC17A7) and inhibitory neurons. FIG. 2E shows Gene Set Enrichment Analysis (GSEA) to evaluate specifically oxidative phosphorylation RNA signatures in 6 cell type clusters. FIG. 2F shows analysis of the transcription factor binding sites within the genes significantly altered due to lack of MECP2 in excitatory (SLC17A7) and inhibitory (VIP) neurons.

[0016] FIGS. 3A-3G show evidence of increased incidence of DNA damage present in human neurons with MECP2 mutations. FIG. 3A shows measurement of GLB 1 gene expression as an indicator of senescent cells in MECP2 mutant and wildtype neurons from Rett brain. FIG. 3B shows expression of a senescence signature in wildtype and MECP2 mutant neurons in 6 clusters by comparison of its differentially expressed genes established senescent transcriptomic signatures. FIG. 3C shows a Venn diagram showing the number of genes that overlap between excitatory mutant neurons from single-nuclei RNAseq experiment and from previously published dataset of our in vitro Rett syndrome model (3i). Statistical significance was checked using a hypergeometric distribution test, where * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. FIG. 3D shows immunostaining of the sorted MECP2high and MECP21ow neuronal nuclei after cytospin from Rett patient brain. The immunostaining shows an increase in gamma-H2AX punch as quantified in the right panel. FIG. 3E shows the single cell gel electrophoresis comet assay used to measure extent of DNA damage in sorted MECP2high and MECP21ow neuronal nuclei from Rett cortical samples, images taken at 10X. Quantification of tail moment of 3 individual Rett patient samples shown on the right. FIG. 3F shows the comet assay used to measure the extent of DNA damage in in vitro neurons with or without MECP2 mutant allele, images taken at 10X. Quantification of tail moment of neurons (Control is R18 (z.e., Rett line 18), Mutant 1 is R17, Mutant 2 is R15), neural progenitor cells (NPCs) (Control is R16, Mutant is R17), and human induced pluripotent stem cells (hiPSCs) (Control is R16, Mutant is R17) shown on the right. FIG. 3G shows immunostaining neurons for phospho-ATR (pATR) or gamma-H2AX, DNA damage response proteins, and neuronal marker MAP2, images taken at 40X (Control is R18, Mutant is R15). Quantification of mean fluorescence intensity for pATR and punch per cell for gamma-H2AX is shown on the right. Statistical significance for comet and immunostaining experiments was calculated using student’s t test, (* p < 0.05, ** p < 0.01, *** p < 0.001).

[0017] FIGS. 4A-4J show that Rett neurons exhibit metabolic dysfunction in vitro. FIG. 4A shows pathway enrichment analysis of UC13 labeled glucose metabolomics with in vitro experiments showing the processes most affected by the metabolic dysfunction in Rett neurons. FIG. 4B shows that a lack of MECP2 in patient derived neurons leads to a decreased incorporation of UC13 labeled glucose into the citric acid cycle. FIG. 4C shows a decreased ATP metabolite pool size in Rett neurons from a metabolomics experiment. FIG. 4D shows oxygen consumption rate (OCR) and Complex IV-specific activity is decreased in Reh neurons as detected by the Seahorse assay. FIG. 4E shows quantification of Seahorse experiment parameters in Rett neurons. FIG. 4F shows immunostaining Rett NPCs for mitochondrial marker Tom20. FIG. 4G shows quantification of mitochondrial morphology parameters of Rett NPCs and neurons. FIG. 4H shows quantification of mitochondrial reactive oxygen species in Rett neurons. FIG. 41 shows analysis of mitochondrial permeability using JC-1 dye which increase in green to red ratio indicates increased membrane permeability. FIG. 4J shows that Rett neurons were assayed by western blot to assess changes in mitochondrial mass as indicated by mitochondrial protein VDAC. Statistical significance for comet and immunostaining experiments was calculated using student’s t test, (* p < 0.05, ** p < 0.01, *** p < 0.001). Metabolomics (Control is R16, Mutant is R17), Seahorse (Control is R16, Mutant is R15), Tom20 NPCs (Control is R18, Mutant is R15), Tom20 Neurons, MitoSOX, and JC-1 (Control is R18, Mutant is R17), VDAC (Control is R18).

[0018] FIGS. 5A-5G show that DNA damage is strongly associated with loss of MECP2. FIG. 5A shows immunostaining of WT neurons after treatment with siRNA targeting MECP2 for MECP2 and gamma-H2AX with quantification of gamma-H2AX punch per cell on the right. FIG. 5B shows senescence activity assay on WT neurons after treatment with siRNA targeting MECP2. Statistical significance was calculated using chi-square test. FIG. 5C shows analysis of oxygen consumption rate by Seahorse assay on WT neurons treated with siMECP2. FIG. 5D shows analysis of oxygen consumption rate by Seahorse assay on WT neurons treated with etoposide (10 uM) for 24 hours. FIG. 5E shows quantification of Seahorse experiment parameters in Rett neurons treated with etoposide. FIG. 5F shows quantification of immunostaining experiment of WT neurons treated with antimycin A (1 uM) or phenformin (20 uM) for 24 hours for gamma-142 AX punch per cell. FIG. 5G shows UC 13 -glucose tracing metabolomics experiment of WT neurons treated with etoposide (10 uM) for 24 hours. Statistical significance for seahorse and immunostaining experiments was calculated using student’s t-test, (* p < 0.05, ** p < 0.01, *** p < 0.001).

[0019] FIGS. 6A-6J show that loss of PARP1 function recapitulates key Rett syndrome phenotypes in neurons. FIG. 6A shows the MECP2 protein interactome from OpenCell proteome database. FIG. 6B shows gene ontology analysis of MECP2 interactors and their interactomes. FIG. 6C shows confocal microscopy of WT neurons to observe degree of colocalization between MeCP2 and PARP1, NUCKS1, H3K9me2, and nucleolin. FIG. 6D shows, Left: Scatter plot of pixel intensities across two channels (red: x axis and green: y axis) for each confocal image; Right: Quantification of colocalization between MeCP2 and PARP1, NUCKS1, H3K9me2, and nucleolin based on Pearson’s Correlation Coefficient (PCC). FIG. 6E shows measurement of PARP activity of Rett syndrome neurons through an ELISA-based plate assay. FIG. 6F shows measurement of PARP activity of WT neurons after treatment with PARP inhibitor Olaparib. FIG. 6G shows WT neurons treated with Olaparib (500 nM) had increased incidence of senescence as shown quantification of betagalactosidase staining. FIG. 6H shows quantification of immunostaining ofWT neurons treated with Olaparib (500 nM) for gamma-H2AX. FIG. 61 shows Seahorse experiment showing decreased mitochondrial oxygen consumption rate of WT neurons after treatment with Olaparib (500 nM). FIG. 6 J shows UC13 glucose tracing metabolomics experiment shows decrease in glucose contribution to the TCA cycle and glutaminolysis of WT neurons after treatment with Olaparib (500 nM). Statistical significance was calculated using student’s t test, (* p < 0.05, ** p < 0.01, *** p < 0.001).

[0020] FIGS. 7A-7F show that stimulation ofPARPl activity through NAD supplementation rescues Rett syndrome deficiencies in neurons. FIG. 7A shows induction of PARP activity, as measured by ELISA-based plate assay, in MECP2 mutant neurons through supplementation of nicotinamide adenine dinucleotide hydrate (NAD) for 24 hours. FIG. 7B shows, Left: immunostaining of Rett neurons following treatment with NAD and NAD with Olaparib for gamma-H2AX; Right: Quantification of gamma-H2AX foci per cell. FIG. 7C shows quantification of comet assay experiment of Rett neurons following stimulation of PARP activity with NAD treatment. FIG. 7D shows quantification of beta-galactosidase experiment of Rett neurons following treatment with NAD. FIG. 7E shows UC13 glucose tracing metabolomics experiment on mutant neurons shows increased TCA flux after treatment with NAD. FIG. 7F shows measurement of dendritic complexity on Rett neurons following treatment with NAD for 96 hours. Statistical significance was calculated using student’s t test, (* p < 0.05, ** p < 0.01, *** p < 0.001). Mutant=R17, B,D: Control=R18, C, F: Control =R16.

[0021] FIGS. 8A-8D show a quality control analysis of the single nuclei RNA seq data. FIG. 8A shows a violin plot showing the number of counts per cell in the Drop Seq and lOx data. FIG. 8B shows the number of genes per cell in each sample. FIG. 8C shows the number of cells detected in each sample. FIG. 8D shows the percent mitochondrial reads per cell in each sample.

[0022] FIGS. 9A-9C show a reanalysis of published sc-RNA-seq data. FIG. 9A shows, Left: UMAP showing 13 unique clusters identified during the reanalysis of the Greenberg group data; Right: UMAP overlayed broad cell type categories. FIG. 9B shows UMAP highlighting cell subtypes defined by expression of specific markers. FIG. 9C shows the number of genes changes in representative cell clusters. FIGS. 10A-10E show evidence of ribosomal dysfunction in MECP2 mutant neurons. FIG. 10A shows, Left: immunostaining of Rett neurons with nucleolin; Right: Quantification of nucleolar area. FIG. 10B shows a list of downregulated genes in the VIP cluster corresponding to the top GO categories. FIG. IOC shows, Right: immunostaining of Rett neurons with phospho-EIFSl or PRKCG and neuronal marker MAP2; Left: Quantification of mean fluorescence intensity of nuclear markers. FIG. 10D shows pathway analysis on pool metabolomics data of Rett neurons. FIG. 10E shows changes in metabolite pool size of several amino acids in Rett neurons. For FIG. 10A, FIG. 10D-E: Control isR16, Mutant is R17; for FIG. IOC: Control isR18, Mutant isR15.

[0023] FIGs. 11A-11D show that DNA damage is strongly associated with loss of MECP2 in isogenic neurons in vitro. FIG. 11A shows immunostaining of neurons for phospho- ATR (pATR). FIG. 11B shows a COMET assay of wildtype and isogenic CDKL5 mutant neurons showing elevated DNA damage in the mutant. FIG. 11C shows senescence assay for wildtype and isogenic CDKL5 neurons indicating elevated senescence in the mutant. FIG. 11D shows the result of a short interfering RNA experiment with siRNA against MECP2 or a scrambled control. On the left, immunostaining for MECP2 shows that the protein was diminished by the siRNA. On the right, the fluorescence intensity was quantified.

[0024] FIGs. 12A-12C show the levels of reactive oxygen species and mitochondrial damage in wildtype and Rett cells. FIG. 12A is a FACS plot with a marker of reactive oxygen species. There was no difference in staining intensity between wildtype and mutant (R25 vs R26) either with or without Antimycin A, a stimulator of ROS accumulation. FIG. 12B shows staining for mitochondria to highlight any morphological change. FIG. 12C is a FACS plot with a marker of reactive oxygen species. There was no difference in staining intensity between wildtype and mutant (R25 vs R26) either with or without CCCP a mitochondrial uncoupler.

[0025] FIGs. 13A-13G show that MECP2 interacts with PARP1 in human neurons. FIG. 13A shows the MECP2 protein interactome from OpenCell proteome database. FIG. 13B shows a gene ontology analysis of MECP2 interactors and their interactomes. FIG. 13C shows the results of confocal microscopy of WT neurons to observe degree of colocalization between MeCP2 and PARP1, NUCKS1, H3K9me2, and nucleolin. FIG. 13D shows, Left: provides a scatter plot of pixel intensities across two channels (red: x axis and green: y axis) for each confocal image; Right: shows the quantification of colocalization between MeCP2 and PARP1, NUCKS1, H3K9me2, and nucleolin based on Pearson’s Correlation Coefficient (PCC) (n=60 images per condition). FIG. 13E shows that co-immunoprecipitation of MECP2 and PARP1 was performed using nuclear lysate from human neurons. MECP2 was immunoprecipitated and then western blot was performed with antibody against PARP1. As a control, pulldown with IgG did not precipitate MECP2 or PARP1. FIG. 13E shows a correlation plot of localization of MECP2 within the nucleus with Parpl, Nucksl, phosphor- RNAPolII, and H3K3Me3, and on the right the Pearsons correlation is plotted. FIG. 13F show the ontological analysis of the proteins pulled down by MECP2 in the Jaenisch analysis as well as in two distinct wildtype neuronal lines in our group. FIG. 13G shows the change in pulldown by MECP2 when it was mutated to R133C, and on the right the data for Parpl is plotted, showing that the MECP2 mutation did not affect the binding between MECP2 and Parpl.

[0026] FIG. 14 shows that Parpl activity is depressed in the absence of MECP2 in neurons. Depicted is the measurement of PARP activity of Rett syndrome neurons, CDKL5 mutant neural progenitor cells (NPCs), and Rett rat model through an ELISA-based plate assay. (n=3 wells per condition, data shown are representative of five independent experiments.)

[0027] FIGs. 15A-15F show that stimulation of PARP 1 activity through NAD supplementation rescues Rett syndrome deficiencies in neurons. FIG. 15A shows, Left: immunostaining of Rett neurons following treatment NAD and NAD with Olaparib for gamma-H2AX; Right: quantification of gamma-H2AX foci per cell. (n=100 cells per condition, data shown are representative of three independent experiments.) FIG. 15B shows the quantification of COMET assay experiment of Rett neurons following stimulation of PARP activity with NAD treatment. (n=100 cells per condition, data shown are representative of three independent experiments.) FIG. 15C shows the quantification of beta galactosidase experiment of Rett neurons following treatment with NAD. (n=150 cells per condition, data shown are representative of three independent experiments.) FIG. 15D shows DEGs from WT vs MECP2- neurons bulk RNAseq, along with treatment by NAD. A statistically significant portion of the genes downregulated by loss of MECP2 were upregulated by NAD treatment. FIG. 15E shows an ontological analysis of the genes upregulated in MECP2- neurons, and those downregulated by NAD. FIG. 15F shows the measurement of dendritic complexity on Rett neurons following treatment with NAD for 96 hours. (n=50 cells per condition, data shown are representative of at least two independent experiments.) DETAILED DESCRIPTION

[0028] MECP2 has been described as both a transcriptional stimulator and inhibitor, a regulator of RNA splicing, and a regulator of DNA methylation or reader of methylation. Moreover, loss of MECP2 has been shown to lead to a variety of cell physiological defects such as mitochondrial permeability, diminished dendritic branching, altered electrophysiological activity, and changes in nuclear and nucleolar size. Despite all these interesting observations, it is still not clear which of these are relevant for human Rett Syndrome patients, which are direct consequences of loss of MECP2 function, and which should serve as proxies for experimentally targeting and developing therapeutic strategies.

[0029] Studies with an in vitro model of Rett Syndrome indicated that specifically postmitotic neurons lacking MECP2 show defects in dendritic branching coincident with induction of p53 and cellular senescence. Importantly, blocking senescence by P53 inhibition restored dendritic branching in MECP2 Syndrome patient-derived neurons. These results were consistent with those of the Galderisi group who also showed senescence phenotypes in various loss of function MECP2 models in human and murine cells. Furthermore, a more recent study demonstrated that overexpression of MECP2 rescued brain function and abrogated senescence in a mouse model of age-related cognitive decline. While several groups have connected loss of MECP2 function in neuronal senescence, the primary trigger of the stress that leads to senescence has not been established. Employing isogenic cell lines, the present disclosure presents evidence for DNA damage as a trigger of senescence and identifies a role for a PARP1 / MECP2 interaction in dysfunction in Rett Syndrome.

[0030] To explore the possibility that DNA damage is a trigger for dysfunction in MECP2- null neurons, an in vitro model of Rett syndrome that allows for sophisticated investigation was explored. An isogenic in vitro system was created to model how the loss of MECP2 impacts development of human neural cell types by exploiting reprogramming of patient fibroblasts to a pluripotent state to create human pluripotent stem cells (hiPSCs), and differentiation towards particular neural lineages (Neural Progenitor Cells (NPCs) and interneurons). Isogenic comparisons of neurons with and without MECP2 showed that loss of MECP2 is correlated with increased DNA damage, senescence and elevated P53 activity. In addition, the inhibition of P53 pathways reversed most of the Rett neuron phenotypes indicating the importance of P53 in dysfunction in Rett Syndrome. It was sought to define the primary trigger of induction of P53 activity that appears to cause downstream neuronal dysfunction in Rett neurons. Known P53 activators include DNA damage, dysfunction of mitochondria or ribosomes, ROS induction, telomere shortening etc. The inventors were unable to find consistent differences in ROS levels or telomere length between neurons with and without MECP2, so it was asked whether induction of DNA damage or mitochondrial dysfunction in human neurons can drive phenotypes akin to those found after deletion of MECP2.

[0031] A deeper analysis of DNA damage and repair was first performed on hiPSC derived neurons, and it was found that activated ATR was increased along with H2AX in neurons lacking MECP2, indicative of elevated DNA damage (FIGs. 3G and 3F). To determine the extent of physical damage to the genome, a COMET assay was performed in neurons, NPCs and hiPSCs from the isogenic Rett lines described in FIG. 11A (FIG. 3A). To determine if DNA damage is potentially a trigger of Rett Syndrome phenotypes irrespective of the nature of the mutation, isogenic neurons from hiPSCs derived from patients with Rett Syndrome caused by mutations in CDKL5 were also assessed. These hiPSCs were created by Muotri et al, and CDKL5 has previously been implicated in DNA damage and repair. Here it was found that Neurons lacking CDKL5 expression displayed elevated increased tail length in a COMET assay, a hallmark DNA damage assay (FIG. 11B, left). In addition, the CDKL5 neurons also showed elevated neuronal senescence (FIG. 11C). Therefore, it is possible that the phenotypes observed in Rett caused by mutations in either MECP2 or CDKL5 could be due primarily to elevated DNA damage.

[0032] The fact that elevated DNA damage correlated with loss of MECP2 in all models tested does not by itself prove that loss of MECP2 leads directly to increased DNA damage, nor does it discern whether loss of MECP2 function directly increases the number of breaks or inhibits DNA repair. In addition, this phenotype could be an indirect consequence of loss of MECP2 downstream of other defects. To determine whether DNA damage is a primary consequence of loss of MECP2 in human neurons, RNA interference was employed (FIG. HD) After suppressing MECP2 expression for 72 hours by RNAi, an elevation of P-H2AX foci (FIG. 5A) and senescence (FIG. 5B) was observed. These experiments identified DNA damage as an early consequence of loss of MECP2 function.

[0033] Senescence and P53 activation can also be due to mitochondrial dysfunction. This experiment began by measuring metabolism in neurons lacking MECP2 by metabolomics, glucose tracing and Seahorse analysis. Metabolic tracing showed that C-13-Glucose produced TCA metabolites at a much lower level in MECP2-null neurons (FIG. 4A). Plotting the fractional contribution of glucose carbons into TCA metabolites shows diminished TCA cycle activity (FIG. 4B). Metabolomics for total ATP levels also showed evidence of decreased TCA cycle output in MECP2 null neurons (FIG. 4C). Measuring oxygen consumption with a Seahorse assay showed that MECP2 mutant neurons from two distinct MECP2 Syndrome patients are defective at oxygen consumption, a readout of TCA cycle activity (FIG. 4D). Further analysis of Seahorse data from these experiments showed that these same MECP2 neurons from two patients had a reduced ATP consumption rate relative to isogenic neurons expressing MECP2 (FIG. 4E).

[0034] In some pathologies, mitochondrial dysfunction can be ascribed to changes in mitochondrial mass, complexity, or morphology. The volume and morphology of mitochondria were measured by high resolution microscopy (FIGs. 4F and 4G) and it was found that MECP2-null neurons showed increased volume and surface area of mitochondria, a phenotype previously associated with senescent cells. On the other hand, mitochondrial reactive oxygen species (FIG. 4H), permeability (FIG. 41), and expression of key proteins were measured (FIG. 4J), but did not reveal differences between neurons with or without MECP2 expression. Together, these data reinforce previous data pointing towards diminished mitochondrial activity, and lead to important questions about how mitochondrial function can be influenced by the loss of a nuclear-localized protein like MECP2.

[0035] Next, it was investigated whether metabolic dysfunction is also a primary response to loss of MECP2. To do this, an RNAi approach was employed to acutely suppress MeCP2 expression (FIG. 5C). It was found that mitochondrial activity was not changed after knockdown (FIG. 5D). Because previous experiments identified DNA damage as a first consequence of loss of MECP2 function, it was sought to assess whether directly targeting DNA damage in wildtype human neurons could phenocopy the loss of MECP2. Inducing DNA damage by Etoposide treatment diminished both the Oxygen Consumption Rate (OCR) and TCA metabolism (FIGs. 5G and 5D). With metabolomics, it was found that induction of DNA damage leads to diminished TCA cycle activity (FIG. 5G), again suggesting that the primary trigger of MECP2 Syndrome phenotypes is more likely to be DNA damage than mitochondrial dysfunction. Conversely, deliberately inducing metabolic dysfunction in wildtype neurons did not increase DNA damage (FIG. 5F), consistent with the notion that metabolic defects are a result of DNA damage and not a driver of MECP2 Syndrome like phenotypes. Therefore, a role for MECP2 in DNA damage and / or repair was probed for.

[0036] To identify potential interactions between MECP2 and the DNA repair machinery, immunoprecipitation (IP) coupled with Mass Spectrometry was performed. IP with an MECP2 antibody with nuclei from two wildtype neuronal lines was carried out (R16 / R18), and mass spectrometry was used to characterize interacting proteins (FIGs. 13A and 13B). Detection showed 250-350 potential interactors with MECP2 antibody versus samples performed without antibody. Between neurons from both cell lines, 89 proteins were shared (FIG. 13C) The same procedure was also carried out with nuclei from neurons lacking MECP2 to further identify specific interactors, and yielded a list of 45 interactors across all replicates and not pulled down from MECP2-mutant neurons (FIG. 13D). An ontological analysis of these interactors highlighted categories of genes such as Gene Expression, Nucleosome Organization and Transcription, mostly due to a large number of members of the BAG / BAF complex including at least one member from each of the SMARCA / B / C / DZE families (FIG. 13E). This is interesting as it was proposed previously, and subsequently became the subject of some controversy. Certain work reported that MECP2 interacts with the BAG / BAF complex, while other research has shown that there is no interaction. In addition, categories of genes such as Nucleotide Excision Repair and Double Strand Break Repair also came out of this analysis due to the combination of BAF complex proteins with PARP1. The BAF complex is known to open chromatin to facilitate DNA repair, and PARP1 is a well-established mediator of DNA repair through its ability to PARylate proteins required for DNA repair.

[0037] Recent data from the Jaensich group was also mined. The Jaensich group performed mass spectrometry on affinity purified MECP2 interactors from wildtype neurons that found roughly 1000 proteins (mining results of the Jaenisch data are shown in FIG. 13F). Overlapping these lists of interactors identified in these experiments yielded 40-50 proteins with high probability of MECP2 binding. Included in this list are members of the BAF complex, ribosomal proteins, and several DNA repair pathway proteins such as XRCC5, DDB1, TOPI and PARP1 (FIG. 13F). The Jaenisch study also used several tagged MECP2 mutants as a bait, and surprisingly only a few direct interactions appeared to be affected. For those described here, such as TOPI, XRCC5 and PARP1, the mutations did not appear to prevent binding to MECP2 (FIG. 13).

[0038] Finally, data in OpenCell was mined (FIG. 6A), a database summarizing results from a study that used Crispr / Cas9 to introduce peptide tags into individual genes of interest to facilitate both subcellular localization and physical interactions through tandem mass spectrometry pulldown (FIG. 6A). MECP2 was one of 1306 tagged genes in the database. While this study was carried out in HeLA cells, several of the proteins found to interact with MECP2 were related to DNA repair (FIG. 6A), such as PARP1 and NUCKS1.

[0039] From the pulldown of this disclosure, the Jaenisch pulldown, and the OpenCell pulldown, a particularly notable interaction for MECP2 was with PARP1, an enzyme that adds a poly-ADP- Ribose moiety to proteins and is known to have a key role in both DNA repair and transcription. This led to the reciprocal experiment. PARP1 antibody was used to pull down PARP1 as a bait, and mass spectrometry was performed to identify interacting proteins. Over 300 proteins were identified as compared to “minus antibody” condition, and MECP2 was amongst the top proteins identified (FIG. 6A). Gene ontological analysis of the proteins identified show that several proteins shared similarity with known MECP2 interactors, and the proteins sharing these similarities include proteins with functions related to Transcription, chromatin regulation, and DNA repair (FIG. 6B).

[0040] PARP1 has been shown to regulate MECP2 through PAR-ylation, which in turn modulates MECP2’s interactions with chromatin, but PARP1 has not been shown to be regulated by MECP2. Near-super-resolution microscopy was then used to determine whether MECP2 co-localizes in human neurons with PARP1, and other proteins previously proposed to interact with MECP2 (FIG. 13G). This approach showed that MECP2 also co-localized with the DNA repair proteins PARP1 and NUCKS1 to a very high degree, with over 0.5-0.6 Pearson’s Correlation Coefficient across multiple wildtype neurons. As a measure of the specificity of these interactions, it was also attempted to co-localize MECP2 with several other nuclear proteins related to heterochromatin or subnuclear domains (FIG. 13G), none of which overlapped as significantly as PARP1. Together, these 3 lines of evidence indicate a direct interaction between PARP1 and MECP2, along with other proteins related to DNA repair in the nucleus.

[0041] It was next sought to determine whether MECP2 and PARP1 functionally interact. PARP1 activity can be measured via ELISA assay to detect PAR-ylation of a protein substrate. PARP activity was assessed in neurons with and without MECP2, and it was consistently found that there are lower levels of activity in the absence of MECP2 across multiple genetic backgrounds representing distinct MECP2 mutations, including in neural cells from CDKL5 syndrome, and in MECP2-null Rat brain (FIG. 14). This suggests a functional connection between the two proteins in various contexts associated with Rett-like phenotypes. It is worth noting that both MECP2 and CDKL5 have been shown previously to be PARylated by PARP1, and further, CDKL5 has even been implicated in PARP1- dependent DNA repair.

[0042] PARP1 inhibitors are used to block DNA repair, leading to catastrophic DNA damage in cancer cells. Accordingly, they are known to be an effective treatment adjuvant. In addition, it is known that PARP1 inhibition with Olaparib can induce cellular senescence in various cell types as measured by P-galactosidase assay. Therefore, it was sought to determine whether PARP inhibition in normal neurons can phenocopy the outcome of loss of MECP2. First, an effective dose of Olaparib to decrease PARP1 activity in human neurons was identified (FIG. 6F). DNA damage and senescence was then measured in wildtype neurons treated with Olaparib, and it was found that PARP inhibition can indeed drive DNA damage and senescence (FIGs. 6G and 6H), in a similar fashion to loss of MECP2 by mutation or siRNA. In addition, PARP1 inhibition by Olaparib diminished oxygen consumption and ATP production as measured by Seahorse (FIG. 61), and metabolomics showed a similar phenotype on TCA cycle activity as shown for loss of MECP2 (FIG. 6 J).

[0043] To further probe for a functional interaction between PARP1 and MECP2, it was sought to test whether stimulation of PARP 1 activity in neurons lacking MECP2 can reverse the phenotypes caused by loss of MECP2. PARP1 is known to be stimulated in vivo and in vitro by elevated levels of NAD, a key co-factor for its enzymatic activity. An effective concentration for PARP1 stimulation by NAD was first assayed for, and it was found that addition of lOmM NAD can dramatically stimulate PARP activity in MECP2 mutant neurons (FIG. 7A). The effect of lOmM NAD on MECP2-null neurons was then assessed to determine whether PARP1 stimulation could diminish Rett Syndrome relevant defects. NAD treatment significantly decreased DNA damage as measured both by p-H2AX staining (FIG. 15A) and COMET assay (FIG. 15B). Because MECP2-null neurons treated with NAD showed less DNA damage, senescence was assessed in these cells under PARP1 stimulation. Indeed, by fluorescent Beta-gal activity assay, it was found that NAD treatment could diminish senescence in MECP2-null neurons (FIG. 15C). In addition, these effects of NAD on DNA damage and senescence both were blocked by the addition of Olaparib, demonstrating that the effect of NAD was indeed due to upregulation of PARP 1 (FIGs. 15A- 15C). Using RNA-seq from MECP2 wt vs mutant neurons, many genes were identified as up or downregulated in the mutant (FIG. 15D). Ontological analysis of the DEGs showed that those that were upregulated in the absence of MECP2 were mostly related to DNA damage and repair, and that NAD treatment diminished the expression of genes related to DNA damage and repair (FIG. 15E). In other words, many of the upregulated genes were related to DNA damage and repair such as RAD51, BRCA1, ERCC1, MRE11 that were induced by loss of MECP2 were suppressed by NAD treatment (FIGs. 15D and 15E), consistent with the diminished DNA damage shown by H2AX staining and COMET assay.

[0044] To determine if PARP1 stimulation in Rett neurons could reverse the physiological manifestations of loss of MECP2, MECP2-null neurons were treated with NAD and dendritic branching and metabolism was measured. Perhaps the defining characteristic of Rett Syndrome neurons is defects in dendritic branching, which has been described in essentially all models to date both in vivo and in vitro. Importantly, MECP2 null neurons treated with NAD showed a statistically significant increase in dendritic complexity (FIG. 15F). This is a key phenotype thought to mediate many of the electrophysiological manifestations of Rett Syndrome, as it is linked to neuronal network activity, and the present disclosure and others previously have defined this as a critical feature of the syndrome. Similarly, it was demonstrated that mitochondrial function is diminished in the absence of MECP2, but treatment with NAD reversed much of this defect (FIG. 7E). Together, the data in FIGs. 14 and 15 demonstrate that PARP1 activity is potentially vital to understand how loss of function mutations in MECP2 lead to patient phenotypes and point towards novel interventional approaches.

[0045] The present disclosure sheds new light on the etiology of Rett Syndrome by molecular interrogation of individual neurons in isogenic neurons in vitro. It was found that DNA damage is a primary trigger for dysfunction in MECP2 neurons, and that PARP1 is a key mediator of this effect. While previous efforts have described DNA damage as a phenotype in Rett Syndrome models, it was not previously implicated as a driver of phenotypes. The majority of previous studies argue that patient phenotypes occur due to misregulation of gene expression as a consequence of loss of MECP2 function resulting from altered chromatin structure or function, or because MECP2 is thought to serve as a transcriptional repressor. Instead, the data presented here suggest that patient phenotypes are instead the result of a stress response of neurons to dysfunctional DNA repair, because reversal of the stress response or alleviation of DNA damage by restoration of PARP activity (FIG. 15) appears to restore normal function to neurons lacking MECP2.

[0046] As disclosed herein, MECP2 closely associates and co-localizes with PARP1, a key regulator of DNA repair. Furthermore, MECP2 and PARP1 are demonstrated herein to functionally interact; stimulation of PARP 1 activity in neurons lacking MECP2 is shown to reverse the phenotypes caused by loss of MECP2. As PARP1 activity is known to be stimulated by elevated levels of nicotinamide adenine dinucleotide (NAD+), this disclosure demonstrates that treatment with NAD+ or NAD+ precursors is effective at reducing the physiological manifestations of loss of MECP2 and may therefore reverse the disease processes associated with Rett syndrome.

[0047] In some aspects, the present disclosure provides methods for treating a disease or condition characterized by a mutation of the MECP2 gene in a subject in need thereof, the method comprising administering an effective amount of nicotinamide adenine dinucleotide (NAD+) or a precursor or salt thereof to the subject.

[0048] In some embodiments, administration of NAD+ or a precursor or salt thereof improves at least one symptom of the disease or condition characterized by a mutation of the MECP2 gene in the subj ect.

[0049] In some embodiments, the disease or condition characterized by a mutation of the MECP2 gene is selected from classic Rett syndrome, variant Rett syndrome, PPM-X syndrome, MECP2 -related severe neonatal encephalopathy, nonsyndromic X-linked intellectual disability, and MECP2 duplication syndrome. In some embodiments, the disease or condition characterized by a mutation of the MECP2 gene is classic Rett syndrome.

[0050] In some embodiments, NAD+ is administered to the subject. In some embodiments, a precursor of NAD+ is administered to the subject. In some embodiments, the precursor of NAD+ is NMN (nicotinamide mononucleotide), NR (nicotinamide riboside), NA (nicotinic acid), Nam (nicotinamide), or Trp (tryptophan), or a salt thereof.

[0051] In some embodiments, the subject is a human.

[0052] In some embodiments, administration of NAD+ or a precursor or salt thereof reduces DNA damage in a cell of the subject. In some embodiments, administration of NAD+ or a precursor or salt thereof increases PARP1 expression or activity in a cell of the subject.

[0053] In some embodiments, the cell is a neuron.

[0054] In some embodiments, the effective amount is provided as a pharmaceutical composition comprising a pharmaceutically acceptable excipient, diluent, or carrier.

[0055] In some aspects, the present disclosure provides methods for treating a disease or condition characterized by a mutation of the MCEP2 gene in a subject in need thereof, the method comprising administering an effective amount of an agent that promotes DNA damage repair to the subj ect. In some embodiments, the disease or condition characterized by a mutation of the MECP2 gene is selected from classic Rett syndrome, variant Rett syndrome, PPM-X syndrome, MECP2 -related severe neonatal encephalopathy, nonsyndromic X-linked intellectual disability, and MECP2 duplication syndrome. In some embodiments, the disease or condition characterized by a mutation of the MECP2 gene is classic Rett syndrome.

[0056] In some embodiments, the agent increases expression or activity of PARP1 in a cell of the subject.

[0057] In some embodiments, the agent is NAD+. In some embodiments, the agent is a precursor of NAD+. In some embodiments, the precursor of NAD+ is NMN (nicotinamide mononucleotide), NR (nicotinamide riboside), NA (nicotinic acid), Nam (nicotinamide), or Trp (tryptophan), or a salt thereof.

[0058] In some embodiments, administration of the agent improves at least one symptom of the disease or condition characterized by a mutation of the MECP2 gene in the subject.

[0059] In some embodiments, the subject is a human.

[0060] In some embodiments, the effective amount is provided as a pharmaceutical composition comprising a pharmaceutically acceptable excipient, diluent, or carrier.

[0061] In some aspects, the present disclosure provides methods of treating CDKL5 disorder in a subject in need thereof, the method comprising administering an effective amount of nicotinamide adenine dinucleotide (NAD+) or a precursor or salt thereof to the subject.

[0062] In some embodiments, the agent is NAD+. In some embodiments, the agent is a precursor of NAD+. In some embodiments, the precursor of NAD+ is NMN (nicotinamide mononucleotide), NR (nicotinamide riboside), NA (nicotinic acid), Nam (nicotinamide), or Trp (tryptophan), or a salt thereof.

[0063] In some aspects, the present disclosure provides compositions comprising an agent that promotes DNA damage repair for treating a subject having a disease or condition characterized by a mutation of the MECP2 gene; optionally wherein the agent is NAD+ or a precursor or salt thereof.

[0064] In some embodiments, the disease or condition characterized by a mutation of the MECP2 gene is selected from classic Rett syndrome, variant Rett syndrome, PPM-X syndrome, MECP2 -related severe neonatal encephalopathy, nonsyndromic X-linked intellectual disability, and MECP2 duplication syndrome.

[0065] In some embodiments, the disease or condition characterized by a mutation of the MECP2 gene is classic Rett syndrome. In some aspects, the present disclosure provides compositions comprising an agent that promotes DNA damage repair for treating a subject having CDKL5 disorder; optionally wherein the agent is NAD+ or a precursor or salt thereof.

[0066] In some aspects, the present disclosure provides the use of an agent that promotes DNA damage repair for treating a subject having a disease or condition characterized by a mutation of the MECP2 gene; optionally wherein the agent is NAD+ or a precursor or salt thereof.

[0067] In some embodiments, the disease or condition characterized by a mutation of the MECP2 gene is selected from classic Rett syndrome, variant Rett syndrome, PPM-X syndrome, MECP2 -related severe neonatal encephalopathy, nonsyndromic X-linked intellectual disability, and ML sfJ2 duplication syndrome.

[0068] In some embodiments, the disease or condition characterized by a mutation of the MECP2 gene is classic Rett syndrome.

[0069] In some aspects, the present disclosure provides the use of an agent that promotes DNA damage repair for treating a subject having CDKL5 disorder; optionally wherein the agent is NAD+ or a precursor or salt thereof.

[0070] Dosing of Agents of this Disclosure

[0071] The agents of this disclosure, which may include NAD+, NMN (nicotinamide mononucleotide), NR (nicotinamide riboside), NA (nicotinic acid), Nam (nicotinamide), or Trp (tryptophan), may be administered to a subject in need thereof at a dosage determined to be effective by one of skill in the art by any means known in the art.

[0072] In some embodiments, an effective dose of NAD+ may range from 1-2000 mg / day, such as 1-1000 mg / day, 1-500 mg / day, or 250-300 mg / day.

[0073] In some embodiments, an effective dose of NMN may range from 1-2000 mg / day, such as 250-1500 mg / day, 500-1200 mg / day, or 500-750 mg / day.

[0074] In some embodiments, an effective dose of NR may range from 1-1000 mg / day, such as 1-500 mg / day, or 1-250 mg / day.

[0075] In some embodiments, an effective dose of NA may range from 1-2000 mg / day, such as 250-2000 mg / day, 500-1000 mg / day, or 250-500 mg / day.

[0076] In some embodiments, an effective dose of Nam may range from 1-2000 mg / day, such as 250-2000 mg / day, 500-1000 mg / day, or 250-500 mg / day. In some embodiments, an effective dose of Trp may range from 1-20 g / day, such as 1- 15 g / day, 5-15 g / day, or 8-12 g / day.

[0077] Pharmaceutical Compositions

[0078] The compositions and methods of the present disclosure may be utilized to treat an individual in need thereof. In some embodiments, the individual is a mammal such as a human, or a non-human mammal. When administered to an animal, such as a human, the composition may be administered as a pharmaceutical composition comprising, for example, nicotinamide adenine dinucleotide and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil, or injectable organic esters. In some embodiments, when such pharmaceutical compositions are for human administration, particularly for invasive routes of administration (i.e., routes, such as injection or implantation, that circumvent transport or diffusion through an epithelial barrier), the aqueous solution is pyrogen-free, or substantially pyrogen-free. The excipients can be chosen, for example, to effect delayed release of an agent or to selectively target one or more cells, tissues or organs. The pharmaceutical composition can be in dosage unit form such as tablet, capsule (including sprinkle capsule and gelatin capsule), granule, lyophile for reconstitution, powder, solution, syrup, suppository, injection or the like. The composition can also be present in a transdermal delivery system, e.g., a skin patch. The composition can also be present in a solution suitable for topical administration, such as a lotion, cream, or ointment.

[0079] A pharmaceutically acceptable carrier can contain physiologically acceptable agents that act, for example, to stabilize, increase solubility or to increase the absorption of an agent such as an agent described herein. Such physiologically acceptable agents include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients. The choice of a pharmaceutically acceptable carrier, including a physiologically acceptable agent, depends, for example, on the route of administration of the composition. The preparation or pharmaceutical composition can be a self-emulsifying drug delivery system or a self-microemulsifying drug delivery system. The pharmaceutical composition (preparation) also can be a liposome or other polymer matrix, which can have incorporated therein, for example, an agent of the dislcosure. Liposomes, for example, which comprise phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer.

[0080] The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0081] The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.

[0082] A pharmaceutical composition (preparation) can be administered to a subject by any of a number of routes of administration including, for example, orally (for example, drenches as in aqueous or non-aqueous solutions or suspensions, tablets, capsules (including sprinkle capsules and gelatin capsules), boluses, powders, granules, pastes for application to the tongue); absorption through the oral mucosa (e.g., sublingually); subcutaneously; transdermally (for example as a patch applied to the skin); and topically (for example, as a cream, ointment or spray applied to the skin). The agent may also be formulated for inhalation. In some embodiments, an agent may be simply dissolved or suspended in sterile water. Details of appropriate routes of administration and compositions suitable for same can be found in, for example, U.S. Pat. Nos. 6,110,973, 5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970 and 4,172,896, as well as in patents cited therein.

[0083] The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the agent which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 1 percent to about ninety-nine percent of active ingredient, e.g., from about 5 percent to about 70 percent, or from about 10 percent to about 30 percent.

[0084] Methods of preparing these formulations or compositions include the step of bringing into association an active agent, such as an agent described herein, with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association an agent described herein with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0085] Formulations suitable for oral administration may be in the form of capsules (including sprinkle capsules and gelatin capsules), cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), lyophile, powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water- in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and / or as mouth washes and the like, each containing a predetermined amount of an agent described herein as an active ingredient. Compositions may also be administered as a bolus, electuary or paste.

[0086] To prepare solid dosage forms for oral administration (capsules (including sprinkle capsules and gelatin capsules), tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and / or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and / or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; (10) complexing agents, such as, modified and unmodified cyclodextrins; and (11) coloring agents. In the case of capsules (including sprinkle capsules and gelatin capsules), tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

[0087] A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

[0088] The tablets, and other solid dosage forms of the pharmaceutical compositions, such as dragees, capsules (including sprinkle capsules and gelatin capsules), pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and / or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient can also be in micro- encapsulated form, if appropriate, with one or more of the above-described excipients.

[0089] Liquid dosage forms useful for oral administration include pharmaceutically acceptable emulsions, lyophiles for reconstitution, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, cyclodextrins and derivatives thereof, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, com, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

[0090] Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

[0091] Suspensions, in addition to the active agent, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

[0092] Dosage forms for the topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active agent may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that may be required.

[0093] The ointments, pastes, creams and gels may contain, in addition to an active agent, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

[0094] Powders and sprays can contain, in addition to an active agent, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

[0095] Transdermal patches have the added advantage of providing controlled delivery of an agent described hereinto the body. Such dosage forms can be made by dissolving or dispersing the active agent in the proper medium. Absorption enhancers can also be used to increase the flux of the agent across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the agent in a polymer matrix or gel.

[0096] The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrastemal injection and infusion. Pharmaceutical compositions suitable for parenteral administration comprise one or more active agent in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

[0097] Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions of the disclosure include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0098] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.

[0099] In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.

[0100] Injectable depot forms are made by forming microencapsulated matrices of the subject agents in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissue.

[0101] For use in the methods of the disclosure, active agents can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% or 0.5 to 90% of active ingredient in combination with a pharmaceutically acceptable carrier.

[0102] Methods of introduction may also be provided by rechargeable or biodegradable devices. Various slow release polymeric devices have been developed and tested in vivo in recent years for the controlled delivery of drugs, including proteinaceous biopharmaceuticals. A variety of biocompatible polymers (including hydrogels), including both biodegradable and non-degradable polymers, can be used to form an implant for the sustained release of an active ingredient at a particular target site.

[0103] Actual dosage levels of the active ingredients in the pharmaceutical compositions may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

[0104] The selected dosage level will depend upon a variety of factors including the activity of the particular agent or combination of agents employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound(s) being employed, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular agent(s) employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0105] A physician or veterinarian having ordinary skill in the art can readily determine and prescribe effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the pharmaceutical composition or agent at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. By “effective amount” it is meant the concentration of an agent that is sufficient to elicit the desired therapeutic effect, such as improving at least one symptom of the disease or condition to be treated. It is generally understood that the effective amount of the agent will vary according to the weight, sex, age, and medical history of the subject. Other factors which influence the effective amount may include, but are not limited to, the severity of the patient's condition, the disorder being treated, the stability of the compound, and, if desired, another type of therapeutic agent being administered with an agent described herein. A larger total dose can be delivered by multiple administrations of the agent. Methods to determine efficacy and dosage are known to those skilled in the art (Isselbacher et al. (1996) Harrison’s Principles of Internal Medicine 13 ed., 1814-1882, herein incorporated by reference).

[0106] In general, a suitable daily dose of an active agent used in the compositions and methods of the disclosure will be that amount of the agent that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.

[0107] If desired, the effective daily dose of the active agent may be administered as one, two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. In some embodiments of the present disclosure, the active agent may be administered two or three times daily. In some embodiments, the active agent will be administered once daily.

[0108] The patient receiving this treatment is any animal in need, including primates, in particular humans; and other mammals such as equines, cattle, swine, sheep, cats, and dogs; poultry; and pets in general.

[0109] In some embodiments, an agent described herein may be used alone or conjointly administered with another type of therapeutic agent.

[0110] Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

[0111] Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal-chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like. Definitions

[0112] Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings that are commonly understood by those of ordinary skill in the art. Generally, nomenclature used in connection with, and techniques of, chemistry, cell and tissue culture, molecular biology, cell and cancer biology, neurobiology, neurochemistry, virology, immunology, microbiology, pharmacology, genetics and protein and nucleic acid chemistry, described herein, are those well-known and commonly used in the art.

[0113] The methods and techniques of the present disclosure are generally performed, unless otherwise indicated, according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout this specification. See, e.g. “Principles of Neural Science”, McGraw-Hill Medical, New York, N.Y. (2000); Motulsky, “Intuitive Biostatistics”, Oxford University Press, Inc. (1995); Lodish et al., “Molecular Cell Biology, 4th ed ”, W. H. Freeman & Co., New York (2000); Griffiths et al., “Introduction to Genetic Analysis, 7th ed ”, W. H. Freeman & Co., N.Y. (1999); and Gilbert et al., “Developmental Biology, 6th ed ”, Sinauer Associates, Inc., Sunderland, MA (2000).

[0114] As used herein, the term "ameliorating" refers to any therapeutically beneficial result in the treatment of a disease state, e.g., cancer, including prophylaxis, lessening in the severity or progression, remission, or cure thereof.

[0115] “Treating” a condition or patient refers to taking steps to obtain beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (z.e., not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0116] The term “preventing” is art-recognized, and when used in relation to a condition, such as a local recurrence (e.g., pain), a disease such as cancer, a syndrome complex such as heart failure or any other medical condition, is well understood in the art, and includes administration of a composition which reduces the frequency of, or delays the onset of, symptoms of a medical condition in a subject relative to a subject which does not receive the composition. Thus, prevention of cancer includes, for example, reducing the number of detectable cancerous growths in a population of patients receiving a prophylactic treatment relative to an untreated control population, and / or delaying the appearance of detectable cancerous growths in a treated population versus an untreated control population, e.g., by a statistically and / or clinically significant amount.

[0117] “Administering” or “administration of’ a substance, or an agent to a subject can be carried out using one of a variety of methods known to those skilled in the art. For example, an agent can be administered, intravenously, arterially, intradermally, intramuscularly, intraperitoneally, subcutaneously, ocularly, sublingually, orally (by ingestion), intranasally (by inhalation), intraspinally, intracerebrally, and transdermally (by absorption, e.g., through a skin duct). An agent can also appropriately be introduced by rechargeable or biodegradable polymeric devices or other devices, e.g., patches and pumps, or formulations, which provide for the extended, slow or controlled release of the agent. Administering can also be performed, for example, once, a plurality of times, and / or over one or more extended periods.

[0118] Appropriate methods of administering a substance, or an agent to a subject will also depend, for example, on the age and / or the physical condition of the subject and the chemical and biological properties of the agent (e.g., solubility, digestibility, bioavailability, stability and toxicity). In some embodiments, an agent is administered orally, e.g., to a subject by ingestion. In some embodiments, the orally administered agent is in an extended release or slow release formulation, or administered using a device for such slow or extended release.

[0119] As used herein, the phrase “conjoint administration” refers to any form of administration of two or more different therapeutic agents such that the second agent is administered while the previously administered therapeutic agent is still effective in the body (e.g., the two agents are simultaneously effective in the patient, which may include synergistic effects of the two agents). For example, the different therapeutic compounds can be administered either in the same formulation or in separate formulations, either concomitantly or sequentially. Thus, an individual who receives such treatment can benefit from a combined effect of different therapeutic agents.

[0120] The term “modulate” as used herein includes the inhibition or suppression of a function or activity (such as cell proliferation) as well as the enhancement of a function or activity.

[0121] The phrase “pharmaceutically acceptable” is art-recognized. In some embodiments, the term includes compositions, excipients, adjuvants, polymers and other materials and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0122] “Pharmaceutically acceptable salt” or “salt” is used herein to refer to an acid addition salt or a basic addition salt which is suitable for or compatible with the treatment of patients.

[0123] The phrase “pharmaceutically acceptable carrier” as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filter, diluent, excipient, solvent or encapsulating material useful for formulating a drug for medicinal or therapeutic use.

[0124] As used herein, the term "in vivo" refers to processes that occur in a living organism.

[0125] As used herein, the terms "mammal" or "subject" or "patient" includes both humans and non-humans and include, but are not limited to, humans, non-human primates, canines, felines, mouse, bovine, equines, and porcines.

[0126] As used herein, the terms “Rett Syndrome” or “classic Rett syndrome” refer to the condition characterized especially by a loss-of-function genetic mutation in the MECP2 gene. Numerous genetic mutations are known to cause classic Rett syndrome, including loss-of- function genetic mutations that cause a loss of the entire gene copy. It will be understood to those of skill in the art how to appropriately determine whether a subject has and / or diagnose a subject with such a condition. Non-limiting examples of symptoms of classic Rett syndrome include delayed growth, loss of movement and coordination abilities, loss of communication abilities, unusual hand movements, unusual eye movements, breathing difficulties, irritability and crying, intellectual disabilities, seizures, scoliosis, irregular heartbeat, sleep disturbances, decreased response to pain, difficulties with chewing and / or swallowing, difficulties with bowel function, and teeth grinding.

[0127] As used herein, the term “variant Rett syndrome” refers to a cluster of Rett-like conditions that do not meet all diagnostic criteria for classic Rett syndrome. Non-limiting examples of variant Rett syndrome include late-onset Rett syndrome, preserved-speech Rett syndrome, late childhood regression type Rett syndrome, early-onset seizure type Rett syndrome, and congenital type Rett syndrome. It will be understood to those of skill in the art how to appropriately determine whether a subject has and / or diagnose a subject with such a condition.

[0128] As used herein, the term “MECP2 -related severe neonatal encephalopathy” refers to a neurological disorder that primarily affects males and causes brain dysfunction (encephalopathy). Affected males have a small head size (microcephaly), poor muscle tone (hypotonia) in infancy, movement disorders, rigidity, and seizures. It will be understood to those of skill in the art how to appropriately determine whether a subject has and / or diagnose a subject with such a condition. Non-limiting examples of symptoms of MECP2 -related severe neonatal encephalopathy include seizures, unusual movements, diminished or no responsiveness to touch and sound, apnea, irregular breathing, low heart rate, hypotension, low muscle tone, blue or pale skin, stained meconium, unusual eye motion or dilation, absence of reflexes, and difficulties with feeding.

[0129] As used herein, the term “nonsyndromic X-linked intellectual disability” refers to a condition which may be characterized by a missense mutation in the MECP2 gene resulting in severe intellectual disability in males. It will be understood to those of skill in the art how to appropriately determine whether a subject has and / or diagnose a subject with such a condition. Non-limiting examples of symptoms of nonsyndromic X-linked intellectual disability include delayed development of motor skills, delayed development of speech, low muscle tone, short stature, microcephaly, skeletal abnormalities, genital abnormalities, cleft lip, cleft palate, and distinctive facial features (e.g., long face, sloping forehead, broad nasal bridge, and / or upslanting palpebral fissures).

[0130] As used herein, the term “PPM-X syndrome” refers to the condition associated with a mutation in the MECP2 gene and characterized especially by psychotic disorders (most commonly bipolar disorder), a pattern of movement abnormalities known as parkinsonism, and mild to severe intellectual disability with impaired language development. It will be understood by those of skill in the art how to appropriately determine whether a subject has and / or diagnose a subject with such a condition. Non-limiting examples of symptoms of PPM-X syndrome include psychiatric disorders (e.g., bipolar disorder), parkinsonism, delayed intellectual development, delayed development of speech, muscle stiffness, exaggerated reflexes, microcephaly, tremors, and mood and / or behavioral disorders.

[0131] As used herein, the term "MECP2 duplication syndrome” refers to the condition characterized especially by a gain-of-function duplication of the MECP2 gene. It will be understood to those of skill in the art how to appropriately determine whether a subject has and / or diagnose a subject with such a condition. Non-limiting examples of symptoms of MECP2 duplication syndrome include low muscle tone, muscle rigidity, recurrent respiratory infection, delayed growth, delayed intellectual development, abnormal gait, seizures, gastrointestinal dysmotility, stereotypic hand movements, decreased sensitivity to pain, and scoliosis.

[0132] As used herein, the terms “CDKL5 disorder”, “CDKL5 syndrome” and “CDLK5 deficiency disorder” refer to the condition characterized especially by a deficiency in the cyclin-dependent kinase-like 5 protein. It will be understood to those of skill in the art how to appropriately determine whether a subject has and / or diagnose a subject with such a condition. Non-limiting examples of symptoms of CDKL5 disorder include early onset epilepsy (for example, up to 90% of children with CDKL5 disorder will develop seizures before 3 months of age), impaired gross motor skills, impaired fine motor skills, global developmental and intellectual delays, impaired language and communication skills, impaired social skills, low muscle tone, poor eye contact and visual fixation, sleep abnormalities, swallowing and feeding difficulties, growth restriction, gastrointestinal reflux, constipation, scoliosis, hip dysplasia, microcephaly, stereotypic movements, cold hands and feet, unexplained laughing or screaming spells, irregular breathing, and teeth grinding.

[0133] EXAMPLES

[0134] The present disclosure now being generally described, it will be more readily understood by reference to the following examples which are included merely for purposes of illustration of certain aspects and embodiments of the present disclosure and are not intended to limit the disclosure.

[0135] Example 1: Experimental Model and Subject Details

[0136] Human tissue transcriptomic profiling was performed from 5 individual Rett patient cortical tissue samples. Individual tissue samples were collected from the cortical brain repository at the NIH BioBrainBank.

[0137] Example 2: Animals

[0138] Generation of Rett syndrome rat model consisted of crosses of Mecp2ZFN / + females (SD- Mecp2tmlsage) to wildtype (WT) SI 00b eGFP to produce Mecp2ZFN / y and Mecp2ZFN / + rats. Details about this model are found in Patterson et al.

[0139] Example 3: Generation of In Vitro Neurons

[0140] First, isogenic Rett Syndrome and wild type human induced pluripotent stem cells (hiPSCs) were derived from Rett patient fibroblasts as described previously [Ohashi], hiPSCs were maintained on plates coated with matrigel (Coming) in mTeSRl (StemCell Technologies) until 80% confluency. Neural progenitor cell (NPC) fate was induced using StemDiff manufacturer’s protocol. Briefly, 2 million cells were passaged per well using Accutase (StemCell Technologies) into the STEMdiff SMADi neural induction medium (StemCell Technologies). Cells were passaged this way once a week, two more times to induce NPC fate. Next, NPCs were differentiated to a Cajal-Retzius neuronal cell type using growth factor withdrawal method. EGF and FGF were removed from the media, and the cells were cultured in DMEMF12 supplemented with N2 and B27 (Thermo Fisher).

[0141] Example 4: Nuclei Isolation from Frozen Brain Samples

[0142] Nuclei were isolated as described previously (Krishnaswami et al. 2016). Briefly, brain tissue was cut on ice using a scalpel and homogenized in a glass dounce. The homogenate was then filtered through a 40 um cell strainer and centrifuged at 1000g for 8 min at 4°C. The pellet was resuspended in a homogenization buffer and mixed with equal amounts of iodixanol. This mixture was then gently placed in a new tube over 29% iodixanol. The nuclei were centrifuged at 13500g for 20 min at 4°C. The pellet was resuspended in the immunostaining buffer and incubated for 15 min at 4°C. Next, primary antibody was added to the nuclei pellet and incubated on a rotator at 4°C for 40 min. The following primary antibodies were used: rabbit MECP2 (Diagenode Cl 5410052, 1 :250), chicken Neun (Millipore Sigma MAB377 1 :250). Nuclei were centrifuged at 400 g for 5 min at 4°C and washed with PBS / BSA twice. Secondary antibodies conjugated with Alexa 488 and 594 (1 :500, Life Technologies A-21203, A21202) were added, accompanied by DAPI (Invitrogen 1 : 500), the nuclei were incubated on a rotator at 4°C for 30 min. Immunostained nuclei were subjected to FACS.

[0143] Example 5: Library Preparation and Sequencing

[0144] Sorted nuclei were delivered to the UCLA Technology Center for Genomics & Bioinformatics where libraries for RNA sequencing were prepared. The samples were sequenced using NovaSeq 6000 S2 PE 2x50 with 50,000 reads per cell.

[0145] Example 6: Western Blot

[0146] Cell lysate was prepared using RIPA buffer (Pierce) supplemented with Halt Protease Inhibitor Cocktail (ThermoFisher Scientific) and Halt Phosphatase Inhibitor Cocktail (ThermoFisher Scientific). Total protein concentration was determined using BCA Protein Assay Kit (ThermoFisher Scientific) following the manufacturer’s protocol. Equal protein concentrations were loaded onto the NuPAGE 4-12% Bis-Tris gel (ThermoFisher Scientific) and run at 150 Volts for 90 minutes in running buffer, containing 25 mL of 20x NuPAGE MOPS SDS Running Buffer (ThermoFisher Scientific) and 475 mL of mili-Q water. Next, the protein was transferred onto the nitrocellulose membrane at 30 Volts for 60 minutes in transfer buffer, containing 25 mL 20x NuPAGE Transfer Buffer (ThermoFisher Scientific), 100 mL Methanol (ThermoFisher Scientific), 375 mL mili-Q water. The membrane was blocked overnight at 4°C in OneBlock Westem-FL Blocking Buffer (Genesee Scientific), then incubated in the primary antibody at 4°C overnight. The following primary antibodies were used: rabbit MECP2 (Diagenode C15410052, 1 : 1000), rabbit anti-histone H3 (Abeam abl791, 1 : 1000), rabbit VDAC (Cell Signaling Technology #4661, 1 : 1000), mouse beta-actin (SCBT sc-47778, 1 :500). The membrane was washed twice with 0.1% PBST and incubated in anti-rabbit or anti-mouse secondary HRP -labeled secondary antibody (ThermoFisher Scientific 31460, 31430 1 : 100000) for 1 hour at room temperature. The membrane was washed twice with 0.1% PBST and SuperSignal West Femto Maximum Sensitivity Substrate (ThermoFisher Scientific) was added to the membrane and subjected to film exposure.

[0147] Example 7: Immunofluorescence and Image Quantification

[0148] Cells grown on coverslips were washed with PBS and fixed with 4% paraformaldehyde (Electron Microscopy Sciences) for 15 minutes at room temperature. Next, the cells were washed with 0.1 % PBST three times and blocked in MAXblock Blocking Medium (Active Motif) for 1 hour at room temperature, then incubated overnight at 4°C in the primary antibody. The following primary antibodies were used: rabbit MECP2 (Diagenode C15410052, 1 : 1000), mouse phospho-H2AX (Millipore 05-636, 1 :2000), rabbit pATR (Abeam ab227851, 1 :500), mouse PARP1 (LS Bio LS-C41045, 1 :500), mouse NUCKS1 (US Biological 249527, 1 : 100), mouse H3K9me2 (Abeam abl220, 1 :300), mouse nucleolin (Abeam abl36649, 1 : 1000), mouse coilin (Abeam abl l822, 1 :2000), chicken MAP2 (Novus Bio NB300-213, 1 :2000), mouse pATM (Abeam ab81292, 1 :500), mouse SC35 (Abeam abl 1826, 1 : 100), rat phospho-RNA Polymerase II (Millipore MABE953, 1 :500), mouse Tom20 (Santa Cruz Biotechnology scl7764, 1 :50). Next, the slides were washed three times with 0.1% PBST and secondary antibody conjugated with Alexa 488, 568, 594 or 647 (1 :500, Life Technologies A-21203, A21202, A31571, A-21207) was used, accompanied by DAPI (Invitrogen 1 : 500). Slides were then washed three times with 0.1% PBST and mounted using Prolong Gold (Invitrogen). Mean fluorescence intensity and / or puncta number per cell were quantified using Imaged in blind analysis. Example 8: Disruption of Mitochondrial Function

[0149] Cells were washed with PBS and treated with either DMSO, 1 uM Phenformin, 20 uM Phenformin, 0.1 uM Rotenone or 5 uM Rotenone for three days. Media was changed every day.

[0150] Example 9:13C-Labeled Glucose Incorporation

[0151] Cells were fed with DMEM (ThermoFisher Scientific) supplemented with 4 mM glutamine (ThermoFisher Scientific), 1 mM pyruvate (ThermoFisher Scientific), and 10 mM13C labeled glucose (Cambridge Isotope Laboratories). 24 hours later the cells were washed twice with ammonium acetate (ThermoFisher Scientific) on ice and 80% methanol (ThermoFisher Scientific) was added to the cells. The plates were placed in -80°C for 15 minutes. Next, the cells were scraped off the plate into eppendorf tubes, vortexed and centrifuged at 17000 g for 10 minutes at 4°C. The methanol was then evaporated using the EZ-Lite evaporator. Dried samples were analyzed using mass spectrometry with cell count normalization between samples.

[0152] Example 10: Seahorse Assay

[0153] Cells were plated at a density 50,000-90,000 cells per well in a XF96 microplate (Agilent) and placed in the 37°C 5% CO2 incubator overnight. The next day, the cells were washed twice with the assay medium (Dulbecco’s Modified Eagle’s Medium supplemented with 10 mM glucose, 2 mM L-glutamine, 1 mM pyruvate and 5 mM HEPES, pH 7.4), and the microplate was placed in a 37°C incubator without CO2. 30 minutes later, the plate was loaded into the Seahorse XF96 Extracellular Flux Analyzer (Agilent Technologies). The following compounds were Injected during the assay: 2 uM oligomycin, 0.75 and 1.35 uM FCCP; 2 uM rotenone and antimycin A. When the measurements were done, cells were fixed with 4% paraformaldehyde, stained with Hoechst, and cell number per well was determined using an Operetta High-Content Imaging System (PerkinElmer). Oxygen consumption rates (OCR) were normalized to cell number per well.

[0154] Example 11: JC-1 Mitochondrial Permeability Assay

[0155] Mitochondrial permeability was measured using ThermoFisher’s MitoProbe JC-1 Assay Kit (M34152) according to the manufacturer’s protocol. A measure of permeability was calculated by a ratio of green and red flow cytometry events. Example 12: Reactive Oxygen Species Measurement

[0156] Detection of ROS in live cells was conducted using ThermoFisher’s MitoSOX Mitochondrial Superoxide Indicators for live-cell imaging (M36007) according to manufacturer’s directions.

[0157] Example 13: siRNA Gene Silencing

[0158] MeCP2 protein knockdown was performed using Lipofectamine RNAiMAX Transfection Reagent (Invitrogen) kit. A reverse transfection method was used with a ratio of 1 pl lipofectamine:20 nM siRNA for each well of a 24-well plate. Briefly, Lipofectamine and oligos were premixed in 100 pl of OptiMEM (Gibco) for 20 min in the precoated receiving plate. Cells were then passaged with TrypLE (Gibco), resuspended in 500 pl of cell media without antibiotics, and plated on top of transfection media. Transfections were incubated overnight at 37°C after which time media was replaced with standard cell culturing media with antibiotics for the indicated lengths of time.

[0159] Example 14: B-Galactosidase senescence assay

[0160] P-Galactosidase senescence assay was performed using either the Senescence P- Galactosidase Staining Kit from Cell Signaling or CellEvent Senescence Green Detection Kit from Invitrogen following respective manufacturers’ protocol. The number of blue / green cells and number of total cells were quantified using the Cell Counter plugin in ImageJ. Example 15: Alkaline Comet Assay

[0161] The alkaline comet assay was performed as previously described (See the World Wide Web at doi.org / 10.1093 / hmg / ddw395), with some minor changes. From a 24-well tissue culture plates (Greiner), cells were then harvested with TrypLE, spun down, and resuspended in 0.5% low-melting-point agarose at 37°C in a 1 to 10 ratio. Cell suspension was spread onto agarose-coated slides and allowed to polymerize for 20 minutes in the dark. After agarose solidification, samples were incubated in lysis buffer (10 mM Tris-HCl, pH 10, 2.5 M NaCl, 0.1 M EDTA, 1% Triton X-100) for 2 hours. Sorted nuclei from the brain were incubated in modified lysis buffer (1 mM Tris-HCl, pH 10, 2.5 M NaCl, 0.1 M EDTA, 1% Triton X-100) for 1 hour. Following removal of lysis buffer, samples were incubated in alkaline running buffer (0.3 M NaOH, 1 mM EDTA) for 30 min and finally electrophoresed at 300 mA for 30 min at 4°C. Slides are washed three times with distilled water (dH2O) and fixed with cold 70% ethanol. Cells were stained with Vista Green DNA staining solution (Abeam) for 15 minutes at room temperature, washed with dH2O and allowed for agarose to dry overnight. Images were acquired on the Zeiss Axio Imager A2. Images were analyzed using the

[0162] CometScore 2.0 software.

[0163] Example 16: Cell Culture Treatment

[0164] To reduce PARP activity, cells were treated with Olaparib (Selleck) at a concentration of 500 nM at indicated time points. To induce PARP activity, P-Nicotinamide adenine dinucleotide hydrate (Sigma) was used at 10 mM at indicated time points. To induce DNA damage, cells were treated with etoposide (MP Biomedicals) at a concentration of 10 pM for 24 hours.

[0165] Example 17: PARP Activity Measurements

[0166] PARP activity was analyzed from lysed cultured cells using PARP1 Chemiluminescent kit (RnD System) according to manufacturer instructions. Samples were normalized based on protein concentration according to the Pierce BCA Protein Assay (ThermoFisher). Rat brain samples Rett rat brains were provided by Dr. Michelle Olsen, Virginia Tech University. Brains were prepared in cell lysis buffer for Parpl activity assays. Example 18: Co-Immunoprecipitation

[0167] Nuclear lysate was prepared from human wildtype neurons using the same approach as described above for the brain samples. The lysate was probed with either IgG or an antibody against MECP2. These were incubated overnight and then precipitated with Protein- A beads. The beads were then run on a western blot and probed with an antibody against PARP1.

[0168] Example 19: Quantification and Statistical Analysis

[0169] Single-cell RNA-sequencing analysis

[0170] Aligned counts were clustered using Seurat v3 and the standard clustering pipeline and parameters. Cluster markers were calculated using the Wilcoxon rank sum test to compare distributions across individual clusters and the rest of the population. All differential expression analyses were also calculated using the Wilcoxon rank sum test. Venn diagrams were generated using bioVenn, a web application for the comparison and visualization of biological lists using proportional Venn diagrams (T. Hulsen, J. de Vlieg and W. Alkema, BMC Genomics 2008). Gene ontology visualizations were generated using ggplot2, with geometric points and a scaled color gradient.

[0171] Gene Ontology and Transcription Factor Analysis

[0172] Analysis was performed using Enrichr, a gene list enrichment analysis tool. Metabolic Pathway Analysis

[0173] Analysis was performed using pathway analysis module of the MetaboAnalyst platform.

[0174] Senescence Signatures

[0175] Senescence gene programs were obtained from published datasets such as The TP53 Database. Gene programs were evaluated in each condition with a module eigengene calculation implemented from the WGCNA package. Significance was evaluated using two- way ANOVA analysis. Violin plots for GLB1 expression were generated based upon singlecell analysis, using Seurat v3.

[0176] Mitochondrial Morphology Analysis

[0177] Mitochondrial morphology parameters were quantified using the MitochondrialAnalyzer plugin in Image!

[0178] Confocal Colocalization Analysis

[0179] The cells were analyzed by confocal microscopy using inverted confocal laser microscope Zeiss LSM880 with Airyscan at 100X. Pearson’s Correlation Coefficient and was quantified using Zeiss’s ZEN Blue’s colocalization module.

[0180] Dendritic Branching Analysis

[0181] The stained cells were then imaged at 20*, and dendritic arbors of individual cells were traced using Image! The number of dendritic ends per cell were counted using the Cell Counter plugin for Imaged. The number of dendritic ends per cell are presented as mean ends per cell ± SEM.

[0182] INCORPORATION BY REFERENCE

[0183] All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

[0184] EQUIVALENTS

[0185] While specific embodiments of the subject disclosure have been discussed, the above specification is illustrative and not restrictive. Many variations of the disclosure will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the disclosure should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

Claims

CLAIMSWhat is claimed is:

1. A method for treating a disease or condition characterized by a mutation of the MECP2 gene in a subject in need thereof, the method comprising administering an effective amount of nicotinamide adenine dinucleotide (NAD+) or a precursor or salt thereof to the subject.

2. The method of claim 1, wherein administration of NAD+ or a precursor or salt thereof improves at least one symptom of the disease or condition characterized by a mutation of the MECP2 gene in the subj ect.

3. The method of claim 1 or 2, wherein the disease or condition characterized by a mutation of the MECP2 gene is selected from classic Rett syndrome, variant Rett syndrome, PPM-X syndrome, MECP2 -related severe neonatal encephalopathy, nonsyndromic X-linked intellectual disability, and MECP2 duplication syndrome.

4. The method of claim 3, wherein the disease or condition characterized by a mutation of the MECP2 gene is classic Rett syndrome.

5. The method of any one of claims 1-4, wherein NAD+ is administered to the subject.

6. The method of any one of claims 1-4, wherein a precursor of NAD+ is administered to the subject.

7. The method of claim 6, wherein the precursor of NAD+ is NMN (nicotinamide mononucleotide), NR (nicotinamide riboside), NA (nicotinic acid), Nam (nicotinamide), or Trp (tryptophan), or a salt thereof.

8. The method of any one of claims 1-7, wherein the subject is a human.

9. The method of any one of claims 1-8, wherein administration of NAD+ or a precursor or salt thereof reduces DNA damage in a cell of the subject.

10. The method of any one of claims 1-9, wherein administration of NAD+ or a precursor or salt thereof increases PARP1 expression or activity in a cell of the subject.

11. The method of claim 9 or 10, wherein the cell is a neuron.

12. The method of any one of claims 1-11 wherein the effective amount is provided as a pharmaceutical composition comprising a pharmaceutically acceptable excipient, diluent, or carrier.

13. A method for treating a disease or condition characterized by a mutation of the MCEP2 gene in a subject in need thereof, the method comprising administering an effective amount of an agent that promotes DNA damage repair to the subject.

14. The method of claim 13, wherein the disease or condition characterized by a mutation of the MECP2 gene is selected from classic Rett syndrome, variant Rett syndrome, PPM-X syndrome, MECP2 -related severe neonatal encephalopathy, nonsyndromic X-linked intellectual disability, and MECP2 duplication syndrome.

15. The method of claim 14, wherein the disease or condition characterized by a mutation of the MECP2 gene is classic Rett syndrome.

16. The method of any one of claims 13-15, wherein the agent increases expression or activity of PARP1 in a cell of the subject.

17. The method of any one of claims 13-16, wherein the agent is NAD+.

18. The method of any one of claims 13-16, wherein the agent is a precursor of NAD+.

19. The method of claim 18, wherein the precursor of NAD+ is NMN (nicotinamide mononucleotide), NR (nicotinamide riboside), NA (nicotinic acid), Nam (nicotinamide), or Trp (tryptophan), or a salt thereof.

20. The method of any one of claims 13-19, wherein administration of the agent improves at least one symptom of the disease or condition characterized by a mutation of the MECP2 gene in the subject.

21. The method of any one of claims 13-20, wherein the subject is a human.

22. The method of any one of claims 13-21, wherein the effective amount is provided as a pharmaceutical composition comprising a pharmaceutically acceptable excipient, diluent, or carrier.

23. A method of treating CDKL5 disorder in a subject in need thereof, the method comprising administering an effective amount of nicotinamide adenine dinucleotide (NAD+) or a precursor or salt thereof to the subject.

24. The method of claim 23, wherein the agent is NAD+.

25. The method of claim 23, wherein the agent is a precursor of NAD+.

26. The method of claim 25, wherein the precursor of NAD+ is NMN (nicotinamide mononucleotide), NR (nicotinamide riboside), NA (nicotinic acid), Nam (nicotinamide), or Trp (tryptophan), or a salt thereof.

27. A composition comprising an agent that promotes DNA damage repair for treating a subject having a disease or condition characterized by a mutation of the MECP2 gene; optionally wherein the agent is NAD+ or a precursor or salt thereof.

28. The composition of claim 27, wherein the disease or condition characterized by a mutation of the MECP2 gene is selected from classic Rett syndrome, variant Rett syndrome, PPM-X syndrome, MECP2 -related severe neonatal encephalopathy, nonsyndromic X-linked intellectual disability, and MECP2 duplication syndrome.

29. The composition of claim 28, wherein the disease or condition characterized by a mutation of the MECP2 gene is classic Rett syndrome.

30. A composition comprising an agent that promotes DNA damage repair for treating a subject having CDKL5 disorder; optionally wherein the agent is NAD+ or a precursor or salt thereof.

31. Use of an agent that promotes DNA damage repair for treating a subject having a disease or condition characterized by a mutation of the MECP2 gene; optionally wherein the agent is NAD+ or a precursor or salt thereof.

32. The use of claim 31, wherein the disease or condition characterized by a mutation of the MECP2 gene is selected from classic Rett syndrome, variant Rett syndrome, PPM-X syndrome, MECP2 -related severe neonatal encephalopathy, nonsyndromic X-linked intellectual disability, and MECP2 duplication syndrome.

33. The use of claim 32, wherein the disease or condition characterized by a mutation of the MECP2 gene is classic Rett syndrome.

34. Use of an agent that promotes DNA damage repair for treating a subject having CDKL5 disorder; optionally wherein the agent is NAD+ or a precursor or salt thereof.

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