DNA probes for targeting methylation in mammalian cells

Abstract

The invention provides that the induction of DNA methylation changes at a target gene that undergoes loss of DNA methylation may reverse this methylation loss to normalize gene function. The present disclosure provides methods of treating a disease or disorder using a pharmaceutical composition comprising: a methylated DNA probe complementary to a target genomic locus comprising a target gene, and a delivery vehicle and methods of forming said pharmaceutical composition.

Description

DNA PROBES FOR TARGETING METHYLATION IN MAMMALIAN CELLSBACKGROUND OF THE INVENTIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of priority under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 737,176, filed December 20, 2024. The contents of the prior application are considered part of and are hereby incorporated by reference in their entirety.INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0002] The material in the accompanying sequence listing is hereby incorporated by reference into this application. The accompanying sequence listing xml file, name 20251218_JHU4820-lWO_SL.xml, was created on December 11, 2025 and is 32,003 bytes.BACKGROUNDFIELD OF THE INVENTION

[0003] The present invention relates generally to tools for epigenetic modulation, and more specifically to DNA probes for modulating targeted DNA methylation.BACKGROUND INFORMATION

[0004] Work in plants has demonstrated the ability of plants to silence genes by directing small RNAs to target-specifically induce DNA methylation. A simple tool to alter DNA methylation in mammalian cells has continued to remain elusive. Most of the work in gene silencing in mammalian systems has focused on microRNAs that do not induce DNA methylation changes but impede translation by binding to mRNA. However, this process is more transient compared to more persistent changes in gene function afforded by DNA methylation.

[0005] Recent advances in CRISPR have enabled locus-specific DNA methylation. However, this technology involves the use of viral vectors and may not be safe in humans. There is a need for targeted-specific induction of DNA methylation in mammalian cells.

[0006] Epigenetic modifications, such as DNA methylation (DNAm), can lead to alterations in nuclear architecture and landscape, which can in turn affect the accessibility of transcription and regulatory factors to genes. DNA methylation typically occurs at 5’ end cytosine residues within CpG dinucleotides and is generally associated with transcriptional repression, which is mediated by the binding of methyl-CpG binding proteins and recruitment of histone-modifyingPATENT ATTORNEY DOCKET NO. JHU4820-1WO enzymes. Aberrant DNA methylation has been linked to many diseases from cancer to neurodegenerative disorders, but also psychiatric diseases such as depression, anxiety, and PTSD.

[0007] Epigenetic mechanisms can also mediate the impact of adverse environmental conditions on gene function. For instance, it is well known that environmental stressors can cause epigenetic changes in the brain. In such instances, it has been shown that the glucocorticoid (GC) receptor that binds to cortisol can directly alter DNA methylation of genes that are targets of GC signaling, thus, raising the possibility of mitigating disease symptoms by potentially reversing these epigenetic marks.

[0008] Targeted manipulation of DNA methylation at specific gene loci can modulate gene function and provide new therapeutic strategies for disorders associated with aberrant epigenetic regulation. In plants, small RNAs have been shown to direct DNA methylation and gene silencing. Studies have shown the possibility of using pharmacological methods, such as DNA methyltransferase enzyme (DNMT) inhibitors, to impact DNA methylation in mammalian systems. However, these drugs target the epigenetic machinery and are not locusspecific. Although recent advances in CRISPR-based technologies have enabled locus-specific DNA methylation editing in mammalian systems, these methods may have limited clinical applicability due to their immunogenicity and off targeting. However. CRISPR-based therapies are now on the horizon for the treatment of debilitating diseases such as sickle cell-disease and cancer. Until these technologies are further refined to minimize their own disease burden and gain traction for the treatment of more common disorders, there is a need for safer methods.

[0009] The ability of the glucocorticoid receptor to induce loss-of-DNA methylation lends itself as a useful phenomenon in which to test alternative epigenetic tools. In particular, the FK506 binding protein 5 (FKBP5) gene encodes a co-chaperone of the glucocorticoid receptor and has been identified as a key regulator of the stress response. It is thought that GC-induced increase in FKBP5 levels leads to attenuated intracellular signaling and GC resistance. Chronic exposure to stress or excess glucocorticoids can induce the persistent demethylation of intronic glucocorticoid response elements (GREs) in the FKBP5 gene. This demethylation allows for increased binding of the GR to the GREs and a more robust transcription of FKBP5, w hich in turn leads to decreased sensitivity to GCs and GC resistance. Epigenetic alterations in FKBP5 have been linked to several stress-related psychiatric disorders such as PTSD, depression, anxiety, and alcohol abuse. The protein encoded by the FKBP5 gene acts as the primary11627337384.1331323.990775PATENT ATTORNEY DOCKET NO. JHU4820-1WO regulator of stress hormone (glucocorticoid) signaling and has been implicated in several stress-related disorders. Chronic exposure to stress leads to persistent loss of DNA methylation and transcriptional upregulation at FKBP5. There is a need for a simpler tool for site- specifically altering DNA methylation.SUMMARY

[0010] The present invention is based on the seminal discovery that methylated DNA probes may serve as a tool for targeted epigenetic manipulation that can reverse the persistent loss of DNA methylation caused by chronic exposure to GCs.

[0011] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising: a methylated DNA probe complementary to a target genomic locus comprising a target gene, and a delivery vehicle.

[0012] In some aspects, the target genomic locus comprises a promoter region, an enhancer region, a silencer region, an insulator region, an intron, or an exon. In one aspect, the target genomic locus comprises an intron. In another aspect, the target genomic locus comprises a promoter.

[0013] In some aspects, the methylated DNA probe comprises a methylated single-stranded DNA (ssDNA).

[0014] In some aspects, the delivery vehicle comprises an extracellular vesicle (EV). In some aspects, the EV comprises a tissue-specific EV. In some aspects, the EV comprises a brain-specific EV. In some aspects, the EV comprises an exosome. In some aspects, the brainspecific EV comprises a NlE-115 cell line-derived exosome.

[0015] In some embodiments, the present disclosure provides a method of producing a methylated DNA probe comprising contacting a nucleic acid sequence complementary to a target genomic locus comprising a target gene with a methyltransferase enzyme, wherein the methyltransferase enzyme methylates the nucleic acid sequence, thereby producing the methylated probe.

[0016] In some aspects, production of the methylated DNA probe further comprises denaturing the methylated DNA probe to generate a methylated single-stranded DNA (ssDNA).

[0017] In some aspects, the methyltransferase enzyme comprises a DNA methyltransferase enzyme (DNMT). In some aspects, the DNMT comprises a bacterial Sssl methylase enzyme.

[0018] In some aspects, the nucleic acid sequence is generated using a method comprising21627337384.1331323.990775PATENT ATTORNEY DOCKET NO. JHU4820-1WOPCR amplification, oligonucleotide synthesis, gene synthesis, hybridization capture, or a combination thereof.

[0019] In some embodiments, the present disclosure provides a method of altering DNA methylation in a cell comprising contacting the cell with any of the pharmaceutical compositions above, thereby altering DNA methylation.

[0020] In some aspects, altering DNA methylation comprises altering DNA methylation at a target genomic locus comprising a target gene.

[0021] In some aspects, altering DNA methylation comprise increasing DNA methylation at the target genomic locus.

[0022] In some aspects, altering DNA methylation comprises decreasing expression of the target gene.

[0023] In some aspects, the target genomic locus comprises a psychiatric disorder-related gene, infectious disorder-related gene, metabolic disorder-related gene, developmental disorder-related gene, or cancer-related gene.

[0024] In some aspects, the target gene is selected from the group consisting of glucocorticoid receptor (GR or NR3C1) gene, catechol-O-methyltransferase (COMT) gene, mineralocorticoid receptor (NR3C2), corticotropin-releasing hormone (CRH) gene, FKBP5, heat shock proteins (HSPs), and MAO A.

[0025] In some embodiments, the present disclosure provides a method of decreasing expression of a target gene in a cell comprising contacting the cell with any of the pharmaceutical compositions described above, thereby decreasing expression of the target gene in the cell.

[0026] In some aspects, decreasing expression of the target gene comprises altering DNA methylation at a target genomic locus comprising the target gene.

[0027] In some aspects, decreasing expression of the target gene comprises increasing DNA methylation at the target genomic locus.

[0028] In some aspects, the target gene comprises a psychiatric disorder-related gene, infectious disorder-related gene, metabolic disorder-related gene, or cancer-related gene.

[0029] In some aspects, the target gene is selected from the group consisting of glucocorticoid receptor (GR or NR3C1) gene, catechol-O-methyltransferase (COMT) gene, mineralocorticoid receptor (NR3C2), corticotropin-releasing hormone (CRH) gene, FKBP5. heat shock proteins (HSPs), MAO A, serotonin transporter gene (SLC6A4).31627337384.1331323.990775PATENT ATTORNEY DOCKET NO. JHU4820-1WO

[0030] In some embodiments, the present disclosure provides a method of regulating stress response in a subj ect comprising administering to the subj ect a therapeutically effective amount of any of the pharmaceutical composition described above, thereby regulating stress response in the subject.

[0031] In some aspects, regulating stress response comprises altering DNA methylation at a target genomic locus comprising a target gene.

[0032] In some aspects, altering DNA methylation comprises increasing DNA methylation at the target genomic locus.

[0033] In some aspects, altering DNA methylation comprises decreasing expression of the target gene.

[0034] In some aspects, the target gene comprises a stress response-related gene. In some aspects, the stress response-related gene is selected from the group consisting of glucocorticoid receptor (GR orNR3Cl) gene. catechol-O-methyltransferase (COMT) gene, mineralocorticoid receptor (NR3C2), corticotropin-releasing hormone (CRH) gene, FKBP5, and heat shock proteins (HSPs).

[0035] In some aspects, the subject is a mammal. In one aspect, the mammal is a human.

[0036] In some embodiments, the present disclosure provides a method of treating a disease or disorder in a subject, comprising administering to the subject a therapeutically effective amount of any the pharmaceutical composition described above, thereby treating the disease or disorder in the subject.

[0037] In some aspects, treating the disease or disorder comprises altering DNA methylation at a target genomic locus comprising a target gene.

[0038] In some aspects, altering DNA methylation comprise increasing DNA methylation at the genomic locus.

[0039] In some aspects, altering DNA methylation comprises decreasing expression of the target gene.

[0040] In some aspects, the disease or disorder comprises a psychiatric disease or disorder.

[0041] In some aspects, the disease or disorder comprises an infectious disorder.

[0042] In some aspects, the disease or disorder comprises a metabolic disorder.

[0043] In some aspects, the disease or disorder comprises cancer.

[0044] In some aspects, the target gene comprises a psychiatric disorder-related gene.

[0045] In some aspects, the target gene comprises an infectious disorder-related gene.41627337384.1331323.990775PATENT ATTORNEY DOCKET NO. JHU4820-1WO

[0046] In some aspects, the target gene comprises a metabolic disorder-related gene.

[0047] In some aspects, the target gene comprises a cancer-related gene.

[0048] In some aspects, the subject is a mammal. In some aspects, the mammal is a human.

[0049] In some embodiments, the present disclosure provides a kit comprising: a methylatedDN A probe, wherein the methylated DN A probe comprises a methylated nucleic acid sequence complementary to a target genomic locus comprising a target gene, and a delivery vehicle.

[0050] In some aspects, the kit further comprises a methyltransferase enzyme. In some aspects, the methyltransferase enzy me comprises a DNA methyltransferase enzyme (DNMT).

[0051] In some aspects, the methylated DNA probe comprises a methylated single-stranded DNA (ssDNA).

[0052] In some aspects, the kit further comprising primers selected from Table 1.

[0053] In some aspects, the delivery vehicle comprises an extracellular vesicle (EV). In some aspects, the EV comprises an exosome. In some aspects, the EV comprises a tissuespecific EV. In some aspects, the EV comprises a brain-specific EV. In some aspects, the brainspecific EV comprises a NlE-115 cell line-derived exosome.

[0054] In some aspects, the delivery7vehicle comprises lipofectamine.

[0055] Presented below are examples discussing use of methylated DNA probes to alter methylation of DNA at target genes contemplated for the discussed applications. The following examples are provided to further illustrate the embodiments of the present invention but are not intended to limit the scope of the invention. While they are typical of those that might be used, other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used.BRIEF DESCRIPTION OF THE DRAWINGS

[0056] FIGs. 1A-1E illustrate the methylated probe’s efficacy in increasing DNA methylation at two target genes (MAOA and FKBP5) in vitro. FIG. 1A. illustrates a graph showing that methylated single-stranded (ss) DNA probes induce methylation at several CpG dinucleotides at the FKBP5 GRE (glucocorticoid response element, first bar vs. third bar) and that this ability was specific to methylated probes. FIG. IB illustrates a graph that shows cells with increased DNA methylation levels were less likely to undergo DEX-induced transcription of FKBP5, making the epigenetic changes functionally relevant. FIG. 1C illustrates a graph to further demonstrate the utility7of this tool, a regulatory7, non-GRE region of the human51627337384.1331323.990775PATENT ATTORNEY DOCKET NO. JHU4820-1WO monoamine oxidase A gene (MA OA) was also targeted. Several CpGs underwent >10% increase in DNA methylation. FIG. ID illustrates a graph showing MAOA expression measured by qRT-PCR in the same groups of 293HEK cells. Expression levels of MAOA were normalized to those of P-actin and are shown relative to samples transfected with unmethylated probe (-Sssl). FIG IE illustrates a graph showing typical tracing obtained from pyrosequencing. Bar graphs represent mean ± SEM from samples processed in triplicate. *P<0.05 and **P<0.01 by Student's t-test.

[0057] FIGs. 2A-2C illustrate dose-dependent DNA methylation and gene expression changes following transfection of methylated DNA probes against FKBP5. FIG. 2A illustrates a graph that shows 293 Human Embryonic Kidney (HEK) cells transfected with singlestranded unmethylated probe (500 ng -Sssl), single-stranded methylated probe at two different concentrations (250 ng and 500 ng +SssI), or double-stranded methylated probe (500 ng +SssI +DS), and untransfected cells serving as controls (Control). DNA methylation levels of five CpG sites at the conserved glucocorticoid response element (GRE) of human FKBP5 intron 5 were analyzed by bisulfite pyrosequencing. Data for the first two CpGs are shown. FIG. 2B illustrates a ty pical pyrograms obtained from bisulfite pyrosequencing shown for each group at CpG-1. The percent DNA methylation determination occurs when R (or A / G) is dispensed, and it corresponds to the reverse complement of T / C (T for unmethylated and C for methylation CpG, respectively). FIG. 2C illustrates a graph that shows FKBP5 expression measured by qRT-PCR in the same groups of 293 HEK cells as in FIG. 2A treated with 1 pM dexamethasone (DEX) for four hours prior to collection. For each sample, mRNA was extracted, cDNA was synthesized, qRT-PCR was performed. Expression levels of FKBP5 were normalized to those of P-actin and are shown relative to untreated controls (Control). Bar graphs represent mean ± SEM from samples processed in triplicate. ***P<0.001, **P<0.01, and *P<0.05 by Student’s t-test.

[0058] FIGs. 3A-3B illustrate DNA methylation and gene expression analysis following transfection of methylated DNA probes against mouse Fkbp5. FIG. 3A illustrates a graph showing mouse AtT-20 pituitary cells transfected with single-stranded unmethylated probe (500 ng -Sssl), single-stranded methylated probe (500 ng +SssI), or double-stranded methylated probe (500 ng +SssI +DS). Untransfected cells served as controls (Control). DNA methylation levels of four CpG sites at the conserved glucocorticoid response element (GRE) of human FKBP5 intron 5 were analyzed by bisulfite pyrosequencing. Data for all four GRE61627337384.1331323.990775PATENT ATTORNEY DOCKET NO. JHU4820-1WOCpGs are shown. FIG. 3B illustrates a graph showing Fkbp5 expression measured by qRT- PCR in the same groups of AtT-20 cells as in FIG. 3A treated with 1 .M dexamethasone (DEX) for four hours prior to collection. For each sample, mRNA was extracted, cDNA was synthesized, qRT-PCR was performed. Expression levels of Fkbp5 were normalized to those of 0-actin and are shown relative to untreated controls (Control). Bar graphs represent mean ± SEM from samples processed in triplicate. ***P<0.001, **P<0.01, and *P<0.05 by Student’s t-test.

[0059] FIG. 4 illustrates DNA methylation analysis of human FKBP5 GRE at intron 7. Another intronic GRE downstream from the human FKBP5 intron 5 GRE was also analyzed in probe-transfected 293 HEK samples. Of the seven CpGs within the intron 7 GRE, only CpG- 6 could undergo non-specific DNA methylation increase.

[0060] FIG. 5 illustrates DNA methylation and gene expression analysis following transfection of methylated DNA probes against MAO A. 293HEK cells were transfected with single-stranded unmethylated probe (-Sssl) or single-stranded methylated probe (+SssI). DNA methylation levels of 14 CpG sites at a regulatory' region of human MAO A were analyzed by bisulfite pyrosequencing. Bar graphs represent mean ± SEM from samples processed in triplicate. *P<0.05 and **P<0.01 by Student's t-test.

[0061] FIGs. 6A-6C illustrate Tissue type-specific delivery of ExoVivo dye and DNA probes via exosomes. FIG. 6A illustrates images showing EVs labeled with ExoGlow-Vivo dye were retro-orbitally injected into 10-week-old C57 mice (1x109 EV particles per injection per mouse). IVIS Spectrum imaging data was collected at 24 hrs. and 48 hrs. FIG. 6B illustrates a graph showing IVIS imaging data collected at Day 11 and shows the decay of the ExoGlow dye in six major tissues (brain, heart, lung, liver, spleen, and kidney). As expected, most of the EVs ended up in the lung, liver, and kidney. However, substantial fraction of EVs remained in the brain. It should be noted that Exo-Glow EVs derived from 293HEK cells show no signal in the brain (System Biosciences). FIG. 6C illustrates a graph showing exosomes derived from N1E-115 cells electroporated (Neon Transfection System, ThermoFisher Scientific) with electroporation buffer containing lOpg of mouse Fkbp5 GRE probe (2x1010 EVs per electroporation). EVs electroporated with methylated or unmethylated and (both singlestranded) probe were retro-orbitally injected into 10-week-old C57B1 / 6 mice (1x109 EV particles per injection per mouse). Mouse tissues were harvested 48 hrs. following injection, and DNA from the hypothalamus was extracted. Increased DNA methylation was observed at71627337384.1331323.990775PATENT ATTORNEY DOCKET NO. JHU4820-1WO the Fkbp5 GRE. As previously done, assay design for the endogenous sequence precluded the amplification of the DNA probe. Methylation analysis of CpGs 200 bps upstream of the Fkbp5 GRE showed no change in DNAm (data not shown). Data are mean ± SEM, N=4 per treatment group, and *P<0.05, **P<0.01, and ***P<0.001.DETAILED DESCRIPTION

[0062] The present invention is based on the seminal discovery that methylated DNA probes may serve as a tool for targeted epigenetic manipulation that can reverse the persistent loss of DNA methylation caused by chronic exposure to GCs.

[0063] Before the present compositions and methods are described, it is to be understood that this invention is not limited to particular compositions, methods, and experimental conditions described, as such compositions, methods, and conditions may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only in the appended claims.

[0064] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, references to “the method” includes one or more methods, and / or steps of the type described herein which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

[0065] As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items.

[0066] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0067] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, it will be understood that modifications and variations are encompassed within the spirit and scope of the instant disclosure. The preferred methods and materials are now described.

[0068] Described herein is the induction of DNA methylation changes at a target gene that underwent loss of DNA methylation and reversing this methylation loss to normalize gene81627337384.1331323.990775PATENT ATTORNEY DOCKET NO. JHU4820-1WO function. The present disclosure provides methylated DNA probes that can induce DNA methylation of its complementary target. Described herein are methylated DNA probes that may serve as a promising tool for targeted epigenetic manipulation and have potential therapeutic applications for mitigating the impact of environmental stressors in many psychiatric and non-psychiatric disorders associated with aberrant DNA methylation patterns.

[0069] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising: a methylated DNA probe complementary' to a target genomic locus comprising a target gene, and a delivery vehicle.

[0070] As used herein the term "‘DNA probe” refers to a single-stranded DNA fragment that can bind to complementary nucleic acid sequences on a target DNA molecule in a sample of DNA.

[0071] As used herein the term “pharmaceutical composition” refers to the combination of substances that make up a medicinal product. This includes the active ingredients, which are the main components of the product, and excipients, which are additional materials that help control dosage, improve performance, and make the product easier to manufacture.

[0072] The pharmaceutical composition may also contain other therapeutic agents, and maybe formulated, for example, by employing conventional vehicles or diluents, as well as pharmaceutical additives of a type appropriate to the mode of desired administration (for example, excipients, preservatives, etc.) according to techniques known in the art of pharmaceutical formulation. In certain embodiments, the compositions disclosed herein are formulated with additional agents that promote entry into the desired cell or tissue. Such additional agents include micelles, liposomes, extracellular vesicles, and dendrimers.

[0073] As used herein the term “methylated DNA” refers to DNA that has had a methyl group added to it through a chemical reaction. This process is called DNA methylation and can affect how genes are expressed and proteins produced. DNA methylation is an epigenetic mechanism that plays a role in many biological functions, including but not limited to gene regulation, embryonic development, cellular identity, and disease risk. DNA methylation occurs when enzymes called DNA methyltransferases (DNMTs) add methyl groups to DNA nucleotides. The methyl group can attach to either adenine or cytosine, but adenine methylation is more common in prokaryotes, while cytosine methylation is more common in eukaryotes.

[0074] As used herein the term “delivery vehicle” refers to a structure or cell that transports molecules, like proteins, nucleic acids, drugs, genes, or other therapeutic agents into cells or91627337384.1331323.990775PATENT ATTORNEY DOCKET NO. JHU4820-1WO specific tissues within the body. Non limiting examples include vesicles within a cell, extracellular vesicles that travel between cells, or specific types of cells like red blood cells which can be used to deliver drugs to targeted tissues. Examples of delivery vesicles may include but are not limited to vesicles, liposomes, nanoparticles, exosomes, red blood cells, stem cells, bacteria, cell-penetrating peptides, microspheres, and microcapsules.

[0075] As used herein, the term “genomic locus” refers to a specific location on a chromosome, which may encode a gene or genetic marker. The location and DNA sequence of each locus are unique and distinctive from other loci. A locus can be associated with a specific phenotype or disease. The DNA sequence may encompass a gene, including regulatory elements such as a promoter region, an enhancer region, a silencer region, an insulator region; exons encoding a gene; and introns non-coding.

[0076] As used herein, the term “induce” refers to triggering or initiating a specific cellular process by the influence of an agent or molecule, thereby activating a particular biological pathway or gene expression.

[0077] In some aspects, the target genomic locus comprises a promoter region, an enhancer region, a silencer region, an insulator region, an intron, or an exon. In one aspect, the target genomic locus is an intron. In another aspect, the target genomic locus is a promoter. In one aspect, the target genomic locus is an exon.

[0078] In some aspects, the methylated DNA probe comprises a methylated single-stranded DNA (ssDNA).

[0079] As used herein the term “single-stranded DNA” or “ssDNA” refers to a linear chain of nucleotides, forming just one strand of DNA. ssDNA is essentially a single linear chain of nucleotides without a complementary strand, lacking the double helix structure found in double-stranded DNA (dsDNA).

[0080] In some aspects, the delivery vehicle comprises an extracellular vesicle (EV). In some aspects, the EV comprises a tissue-specific EV. In some aspects, the EV comprises a brain-specific EV. In some aspects, the EV comprises an exosome. In some aspects, the brainspecific EV comprises a NlE-115 cell line-derived exosome.

[0081] As used herein the term “extracellular vesicle” or “EV” refers to membrane-bound structures that are secreted by cells and contain a variety of molecules, including proteins, lipids, and nucleic acids. EVS are involved in many physiological and pathological processes and are being investigated as potential diagnostic and therapeutic tools. Non limiting examples101627337384.1331323.990775PATENT ATTORNEY DOCKET NO. JHU4820-1WO of EVs may include exosomes, arrestin domain-containing protein 1 -mediated microvesicles, small ectosomes, microvesicles, apoptotic bodies, apoptotic vesicles, migrasomes, exophers, large oncosomes, ectosomes, exomeres, supermeres, and membrane particles.

[0082] As used herein the term "‘tissue-specific EV” or “CTS-EV” refers to lipid-bilay er membrane structures that are secreted by most cell types and are isolated directly from tissues. CTS-EVs are also known as cell type-specific extracellular vesicles. Examples of tissue-specific EVs include but are not limited to brain-derived EVs, liver-derived EVs, kidney- derived EVs, lung-derived EVs, heart-derived EVs, muscle-derived EVs, adipose tissue- derived EVs. platelet-derived EVs, intestinal EVs, pancreatic EVs. prostate EVs, breast EVs, blood vessel EVs, and immune cell-derived EVs.

[0083] As used herein the term “exosome” refers to nanosized particles that are secreted from cells and carry proteins, lipids, nucleic acids, and metabolites. Exosomes range in size from 30 nm to 150 nm. Exosomes are a subtype of extracellular vehicles that can be released by several cell types.

[0084] In some embodiments, the present disclosure provides a method of producing a methylated DNA probe comprising contacting a nucleic acid sequence complementary to a target genomic locus comprising a target gene with a methyltransferase enzyme, wherein the methyltransferase enzyme methylates the nucleic acid sequence, thereby producing the methylated probe.

[0085] As used herein the term “methyltransferase enzyme" refers to enzymes that add methyl groups to various substrates, including proteins, phospholipids, and nucleotides. Methyl groups are chemical groups made up of one carbon and three hydrogen atoms. Examples of methyltransferase enzymes include but are not limited to DNA methyltransferase (DNMT), histamine N-methyltransferase, catechol-O-methyltransferase (COMT), thiopurine methyltransferase (TPMT), thiol methyltransferase (TMT), nicotinamide n-methyltransferase (NNMT), histamine n-methyltransferase (EINMT). METTL5, histone methyltransferase, 5- Methyltetrahydrofolate-homocysteine methyltransferase, O-methyltransferase, methionine synthase, and corrinoid-iron sulfur protein.

[0086] In some aspects, production of the methylated DNA probe further comprises denaturing the methylated DNA probe to generate a methylated single-stranded DNA (ssDNA).

[0087] As used herein the term “denaturing DNA” refers to the process of separating a1 11627337384.1331323.990775PATENT ATTORNEY DOCKET NO. JHU4820-1WO double-stranded DNA molecule into two single strands by breaking the hydrogen bonds that hold the base pairs together, typically achieved by applying heat. When heat is applied, the hydrogen bonds between complementary base pairs (A-T and C-G) weaken and eventually break, causing the DNA strands to separate.

[0088] In some aspects, the methyltransferase enzyme comprises a DNA methyltransferase enzyme (DNMT). In some aspects, the DNMT comprises a bacterial Sssl methylase enzyme.

[0089] In some aspects, the nucleic acid sequence is generated using a method comprising PCR amplification, oligonucleotide synthesis, gene synthesis, hybridization capture, or a combination thereof.

[0090] In some embodiments, the present disclosure provides a method of altering DNA methylation in a cell comprising contacting the cell with any of the pharmaceutical compositions above, thereby altering DNA methylation.

[0091] In some aspects, altering DNA methylation comprises altering DNA methylation at a target genomic locus comprising a target gene.

[0092] In some aspects, altering DNA methylation comprise increasing DNA methylation at the target genomic locus.

[0093] In some aspects, altering DNA methylation comprises decreasing expression of the target gene.

[0094] In some aspects, the target genomic locus comprises a psychiatric disorder-related gene, infectious disorder-related gene, metabolic disorder-related gene, developmental disorder-related gene, or cancer-related gene.

[0095] As used herein, the term “psychiatric disorder’ or “mental illness” refers to a mental health condition that significantly impacts a person’s thoughts, feelings, behavior, or mood, psychiatric disorders can cause distress or impairment in personal functioning, and increase the risk of disability, pain, death, or loss of freedom. Examples of psychiatric disorders include but are not limited to schizophrenia, bipolar disorder, depression, anxiety disorders, mood disorders, eating disorders, personality disorders, post-traumatic stress disorder (PTSD), psychotic disorders, substance use disorders, disruptive behavior disorders, neurodevelopmental disorders, sleep disorders, attention-deficit / hyperactivity disorder (ADEID). body dysmorphic disorder, caffeine addiction, cannabis addiction, catatonic disorder, Prader-Willi syndrome, and childhood disintegrative disorder.

[0096] As used herein the term “psychiatric disorder-related gene” refers to a specific121627337384.1331323.990775PATENT ATTORNEY DOCKET NO. JHU4820-1WO genetic variant that is associated with an increased risk of developing a mental health condition, while no single gene directly causes these disorders, various genes can contribute to vulnerability when combined with environmental factors. Examples of psychiatric disorder- related genes include but are not limited to CACNA1 C, CACNB2, 5-HTT (SLC6A4), MAO A, reelin, COMT. ANK3, TPH2. 1TPR2. SHANK2. TH, DISCI, FKBP5, NR3C1, and NCAN.

[0097] As used herein, the term “infectious disorder” refers to a disorder caused by harmful organisms, or pathogens, that enter the body or cell and multiply. These organisms can be bacteria, viruses, fungi, or parasites. Examples of infectious disorders include but are not limited to viral infections (e.g. the common cold, the flu. COVID- 19, hepatitis B and C, HIV and norovirus), bacterial infections (e g. strep throat, salmonella, tuberculosis, whooping cough, E. coli, and urinary tract infections (UTIs), fungal infections (e.g. ringworm, fungal nail infections, vaginal candidiasis, and thrush), parasitic infections (e g. giardiasis, toxoplasmosis, hookworms, and pinworms), sexually transmitted infections (STIs) (e.g. chlamydia, gonorrhea, and genital herpes), chickenpox, measles, mumps, rubella, monkeypox, mononucleosis, and rabies.

[0098] As used herein the term “infectious disorder-related gene” refers to a specific genetic variant that is associated with an increased risk of developing an infectious disorder. Non limiting examples of infectious disorder-related genes include HLA class I, HLA class II, IL4 / IL13, IFNG, STAT1, NEMO, UNC93B, TLR3, Duffy antigen receptor (DARC), PRiON protein (PRNP), Band 3 anion transport protein (SLC4A1), Chemokine receptor 5 (CCR5), and ITGB2

[0099] As used herein, the term “metabolic disorder” refers to a condition that occurs when the body's chemical reactions are abnormal, which disrupts the process of converting food into energy, metabolic disorders can lead to the body having too much or too little of certain substances, or to organ dysfunction. Examples of metabolic disorders include but are not limited to diabetes, metabolic syndrome, inherited metabolic disorders, fabry disease, niemann- Pick disease, mucolipidoses, mucopolysaccharidoses, mitochondrial disorders, memochromatosis, porphyria, and Wilson disease.

[0100] As used herein the term “metabolic disorder-related gene” refers to a specific genetic variant that is associated with an increased risk of developing a metabolic disorder. Examples of metabolic disorder-related genes include but are not limited to FTO. MC4R, IRS 1 , ADIPOQ. NR3C1, FOXC2, SREBP1, GNB3, LDLR, GBE1, IL1R1, TGFB1, IL6, COL5A2, SELE,131627337384.1331323.990775PATENT ATTORNEY DOCKET NO. JHU4820-1WOLIPC, LRPAP1, PRCP, AARS2, AASS, ABAT, ABCA12, acid sphingomyelinase (SMPD1), phenylalanine hydroxylase (PAEI), uroporphyrinogen III synthase (UROS), betahexosaminidase A (HEXA), fatty acid desaturase 1, 2 and 3, apolipoprotein A1-C3-A4-A5, zinc finger protein 259, BUD 13 homolog, lipase hepatic, dopamine receptor D2, leptin, leptin receptor, and GLP1R.

[0101] As used herein, the term “developmental disorder” refers to a condition that impacts a child's physical, cognitive, language, or behavioral development, often beginning during the developmental period and potentially causing significant limitations in daily functioning throughout a person’s life. Developmental disorders can affect various areas like motor skills, communication, learning, and social interaction, with causes ranging from genetic factors to environmental exposures, and symptoms varying in severity depending on the specific disorder. Non limiting examples of developmental disorders include autism spectrum disorder, cerebral palsy, fetal alcohol spectrum disorders (FASD). fragile X syndrome, intellectual disability, hearing loss, learning disability, spina bifida, Tourette syndrome, Rett syndrome, Prader-Willi Syndrome, and visions impairment.

[0102] As used herein the term “developmental disorder-related gene” refers to a specific genetic variant that is associated with an increased risk of developing a developmental disorder. Non limiting examples of developmental disorder-related genes include ARID! A, ARID IB, ASH1L, ASXL3, AARS1, AASS, ABAT, ABCB1 1, HRAS, EED, GPC3, U2AF2, PRPF19, RBFOX1, FMRI, SHANK3, CHD2, CHD7, CHD8, and PAX6.

[0103] The term “cancer” refers to a group of diseases characterized by abnormal and uncontrolled cell proliferation starting at one site (primary site) with the potential to invade and to spread to others sites (secondary sites, metastases) which differentiate cancer (malignant tumor) from benign tumor. Virtually all the organs can be affected, leading to more than 100 types of cancer that can affect humans. Cancers can result from many causes including genetic predisposition, viral infection, exposure to ionizing radiation, exposure environmental pollutant, tobacco and or alcohol use, obesity, poor diet, lack of physical activity or any combination thereof. Non limiting examples of cancer include Acute Lymphoblastic Leukemia, Adult; Acute Lymphoblastic Leukemia, Childhood; Acute Myeloid Leukemia, Adult; Adrenocortical Carcinoma; Adrenocortical Carcinoma. Childhood; AIDS-Related Lymphoma; AIDS-Related Malignancies; Anal Cancer; Astrocytoma, Childhood Cerebellar; Astrocytoma, Childhood Cerebral; Bile Duct Cancer, Extrahepatic; Bladder Cancer; Bladder141627337384.1331323.990775PATENTATTORNEY DOCKET NO. JHU4820-1WOCancer, Childhood; Bone Cancer, Osteosarcoma / Malignant Fibrous Histiocytoma; Brain Stem Glioma, Childhood; Brain Tumor, Adult; Brain Tumor, Brain Stem Glioma, Childhood; Brain Tumor, Cerebellar Astrocytoma, Childhood; Brain Tumor, Cerebral Astrocytoma / Malignant Glioma, Childhood; Brain Tumor, Ependymoma, Childhood; Brain Tumor, Medulloblastoma, Childhood; Brain Tumor, Supratentorial Primitive Neuroectodermal Tumors, Childhood; Brain Tumor, Visual Pathway and Hypothalamic Glioma, Childhood; Brain Tumor, Childhood (Other); Breast Cancer; Breast Cancer and Pregnancy; Breast Cancer, Childhood; Breast Cancer, Male; Bronchial Adenomas / Carcinoids, Childhood: Carcinoid Tumor, Childhood; Carcinoid Tumor. Gastrointestinal; Carcinoma, Adrenocortical; Carcinoma, Islet Cell; Carcinoma of Unknown Primary; Central Nervous System Lymphoma, Primary; Cerebellar Astrocytoma, Childhood; Cerebral Astrocytoma / Malignant Glioma, Childhood; Cervical Cancer; Childhood Cancers; Chronic Lymphocytic Leukemia; Chronic Myelogenous Leukemia; Chronic Myeloproliferative Disorders; Clear Cell Sarcoma of Tendon Sheaths; Colon Cancer; Colorectal Cancer, Childhood; Cutaneous T-Cell Lymphoma; Endometrial Cancer; Ependymoma, Childhood; Epithelial Cancer, Ovarian; Esophageal Cancer; Esophageal Cancer, Childhood; Ewing's Family of Tumors; Extracranial Germ Cell Tumor, Childhood; Extragonadal Germ Cell Tumor; Extrahepatic Bile Duct Cancer; Eye Cancer, Intraocular Melanoma; Eye Cancer, Retinoblastoma; Gallbladder Cancer; Gastric (Stomach) Cancer; Gastric (Stomach) Cancer, Childhood; Gastrointestinal Carcinoid Tumor; Germ Cell Tumor, Extracranial, Childhood; Germ Cell Tumor, Extragonadal; Germ Cell Tumor, Ovarian; Gestational Trophoblastic Tumor; Glioma. Childhood Brain Stem; Glioma. Childhood Visual Pathway and Hypothalamic; Hairy Cell Leukemia; Head and Neck Cancer; Hepatocellular (Liver) Cancer, Adult (Primary); Hepatocellular (Liver) Cancer, Childhood (Primary); Hodgkin's Lymphoma, Adult; Hodgkin's Lymphoma, Childhood; Hodgkin's Lymphoma During Pregnancy; Hypophar ngeal Cancer; Hypothalamic and Visual Pathway Glioma, Childhood; Intraocular Melanoma; Islet Cell Carcinoma (Endocrine Pancreas); Kaposi's Sarcoma; Kidney Cancer; Laryngeal Cancer; Laryngeal Cancer, Childhood; Leukemia, Acute Lymphoblastic, Adult; Leukemia, Acute Lymphoblastic, Childhood; Leukemia, Acute Myeloid, Adult; Leukemia, Acute Myeloid, Childhood; Leukemia, Chronic Lymphocytic; Leukemia, Chronic Myelogenous; Leukemia, Hairy Cell; Lip and Oral Cavity Cancer; Liver Cancer, Adult (Primary); Liver Cancer. Childhood (Primary); Lung Cancer. Non-Small Cell; Lung Cancer, Small Cell; Lymphoblastic Leukemia, Adult Acute; Lymphoblastic Leukemia,151627337384.1331323.990775PATENTATTORNEY DOCKET NO. JHU4820-1WOChildhood Acute; Lymphocytic Leukemia, Chronic; Lymphoma, AIDS — Related; Lymphoma, Central Nervous System (Primary); Lymphoma, Cutaneous T-Cell; Lymphoma, Hodgkin's, Adult; Lymphoma, Hodgkin's; Childhood; Lymphoma, Hodgkin's During Pregnancy; Lymphoma. Non-Hodgkin's. Adult; Lymphoma, Non-Hodgkin's, Childhood; Lymphoma, Non-Hodgkin's During Pregnancy; Lymphoma, Primary Central Nervous System; Macroglobulinemia, Waldenstrom's; Male Breast Cancer; Malignant Mesothelioma, Adult; Malignant Mesothelioma, Childhood; Malignant Thymoma; Medulloblastoma, Childhood; Melanoma; Melanoma, Intraocular; Merkel Cell Carcinoma; Mesothelioma, Malignant; Metastatic Squamous Neck Cancer with Occult Primary; Multiple Endocrine Neoplasia Syndrome, Childhood; Multiple Myeloma / Plasma Cell Neoplasm; Mycosis Fungoides; Myelodysplasia Syndromes; Myelogenous Leukemia, Chronic; Myeloid Leukemia, Childhood Acute; Myeloma, Multiple; Myeloproliferative Disorders, Chronic; Nasal Cavity and Paranasal Sinus Cancer; Nasopharyngeal Cancer; Nasopharyngeal Cancer. Childhood; Neuroblastoma; Non-Hodgkin's Lymphoma, Adult; Non-Hodgkin's Lymphoma, Childhood; Non-Hodgkin's Lymphoma During Pregnancy; Non-Small Cell Lung Cancer; Oral Cancer, Childhood; Oral Cavity7and Lip Cancer; Oropharyngeal Cancer; Osteosarcoma / Malignant Fibrous Histiocytoma of Bone; Ovarian Cancer, Childhood; Ovarian Epithelial Cancer; Ovarian Germ Cell Tumor; Ovarian Low Malignant Potential Tumor; Pancreatic Cancer; Pancreatic Cancer, Childhood', Pancreatic Cancer, Islet Cell; Paranasal Sinus and Nasal Cavity Cancer; Parathyroid Cancer; Penile Cancer; Pheochromocytoma; Pineal and Supratentorial Primitive Neuroectodermal Tumors, Childhood; Pituitary Tumor; Plasma Cell Neoplasm / Multiple Myeloma; Pleuropulmonary Blastoma; Pregnancy and Breast Cancer; Pregnancy and Hodgkin's Lymphoma; Pregnancy and Non-Hodgkin's Lymphoma; Primary Central Nervous System Lymphoma; Primary Liver Cancer, Adult; Primary Liver Cancer, Childhood; Prostate Cancer; Rectal Cancer; Renal Cell (Kidney) Cancer; Renal Cell Cancer, Childhood; Renal Pelvis and Ureter, Transitional Cell Cancer; Retinoblastoma; Rhabdomyosarcoma, Childhood; Salivary Gland Cancer; Salivary Gland’Cancer, Childhood; Sarcoma, Ewing's Family of Tumors; Sarcoma, Kaposi's; Sarcoma (Osteosarcoma) Malignant Fibrous Histiocytoma of Bone; Sarcoma, Rhabdomyosarcoma, Childhood; Sarcoma, Soft Tissue, Adult; Sarcoma, Soft Tissue, Childhood; Sezary Syndrome; Skin Cancer; Skin Cancer, Childhood; Skin Cancer (Melanoma); Skin Carcinoma. Merkel Cell; Small Cell Lung Cancer; Small Intestine Cancer; Soft Tissue Sarcoma, Adult; Soft Tissue Sarcoma, Childhood; Squamous Neck Cancer with161627337384.1331323.990775PATENT ATTORNEY DOCKET NO. JHU4820-1WOOccult Primary, Metastatic; Stomach (Gastric) Cancer; Stomach (Gastric) Cancer, Childhood; Supratentorial Primitive Neuroectodermal Tumors, Childhood; T-Cell Lymphoma, Cutaneous; Testicular Cancer; Thymoma, Childhood; Thymoma, Malignant; Thyroid Cancer; Thyroid Cancer, Childhood; Transitional Cell Cancer of the Renal Pelvis and Ureter; Trophoblastic Tumor, Gestational; Unknown Primary Site, Cancer of. Childhood; Unusual Cancers of Childhood; Ureter and Renal Pelvis, Transitional Cell Cancer; Urethral Cancer; Uterine Sarcoma; Vaginal Cancer; Visual Pathway and Hypothalamic Glioma, Childhood; Vulvar Cancer; Waldenstrom's Macro globulinemia; and Wilms' Tumor.

[0104] In some aspects, the target gene is selected from the group consisting of glucocorticoid receptor (GR or NR3C1) gene, catechol-O-methyltransferase (COMT) gene, mineralocorticoid receptor (NR3C2), corticotropin-releasing hormone (CRH) gene, FKBP5, heat shock proteins (HSPs), and MAO A.

[0105] As used herein the term “cancer-related gene” refers to a specific genetic variant that is associated with an increased risk of developing cancer. Examples of cancer-related genes include but are not related to BRCA1, BRCA2, TP53, APC, ATM, MLH1, MSH2, MSH6, PMS2, PTEN, CHEK2, PALB2, KRAS, EGFR, ERBB2, MYC, RBI, SMAD4, CDKN2A, STK11, MUTYH, POLE, EPCAM, BMPR1A, RAD51C, and RAD51D. In some aspects, the target genomic locus comprises repetitive elements found in the human genome. Repetitive elements may be hypomethylated in cancer. Examples of repetitive elements in the human genome include but are not limited to Long Interspersed Nuclear Element (LINE), Short Interspersed Nuclear Element (SINE), and SINE-VNTR-Alu (SVA).

[0106] In one aspect, altering DNA methylation comprises using more than one methylated DNA probe to target multiple genes.

[0107] In certain aspects, two to ten methylated DNA probes may be used to target multiple genes. In certain aspects, two methylated DNA probes may be used to target multiple genes. In certain aspects, three methylated DNA probes may be used to target multiple genes. In certain aspects, four methylated DNA probes may be used to target multiple genes. In certain aspects, five methylated DNA probes may be used to target multiple genes. In certain aspects, six methylated DNA probes may be used to target multiple genes. In certain aspects, seven methylated DNA probes may be used to target multiple genes. In certain aspects, eight methylated DNA probes may be used to target multiple genes. In certain aspects, nine methylated DNA probes may be used to target multiple genes. In certain aspects, ten171627337384.1331323.990775PATENT ATTORNEY DOCKET NO. JHU4820-1WO methylated DNA probes may be used to target multiple genes.

[0108] In some embodiments, the present disclosure provides a method of decreasing expression of a target gene in a cell comprising contacting the cell with any of the pharmaceutical compositions described above, thereby decreasing expression of the target gene in the cell.

[0109] Decreasing expression of a gene refers to the process by which turning a gene’s information into a functional protein is reduced, resulting in a lower amount of that protein being produced within a cell. Decreasing expression of a gene is often referred to as “downregulation” and can be caused by various mechanisms like regulatory proteins binding to the gene’s DNA to inhibit transcription.

[0110] In some aspects, decreasing expression of the target gene comprises altering DNA methylation at a target genomic locus comprising the target gene.[OHl] In some aspects, decreasing expression of the target gene comprises increasing DNA methylation at the target genomic locus.

[0112] In some aspects, the target gene comprises a psychiatric disorder-related gene, infectious disorder-related gene, metabolic disorder-related gene, developmental disorder- related gene, or cancer-related gene.

[0113] In some aspects, the target gene is selected from the group consisting of glucocorticoid receptor (GR or NR3C1) gene, catechol-O-methyltransferase (COMT) gene, mineralocorticoid receptor (NR3C2), corticotropin-releasing hormone (CRH) gene, FKBP5, heat shock proteins (HSPs), MAO A, serotonin transporter gene (SLC6A4).

[0114] In some embodiments, the present disclosure provides a method of regulating stress response in a subj ect comprising administering to the subj ect a therapeutically effective amount of any of the pharmaceutical composition described above, thereby regulating stress response in the subject.

[0115] As used herein, the term “subject” refers to any individual or patient to which the disclosed methods are performed, to whom the disclosed compositions are administered, or from whom a biological material (e.g., the sample from the subject in the disclosed methods) is obtained. Generally, the subject is human, although as will be appreciated by those in the art, the subject may be a non-human animal. Thus, other animals, including vertebrate such as rodents (including mice, rats, hamsters and guinea pigs), cats, dogs, rabbits, farm animals including cows, horses, goats, sheep, pigs, chickens, etc., and primates (including monkeys,181627337384.1331323.990775PATENTATTORNEY DOCKET NO. JHU4820-1WO chimpanzees, orangutans and gorillas) are included within the definition of subject.

[0116] The terms “administration of’ and or “administering” should be understood to mean providing a compound or pharmaceutical composition in a therapeutically effective amount to the subject in need of treatment. As non-limiting examples, administration routes can be enteral, topical, or parenteral. As such, administration routes include but are not limited to intracutaneous, subcutaneous, intravenous, intraperitoneal, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, transdermal, transtracheal, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal, oral, sublingual buccal, rectal, vaginal, nasal ocular administrations, as well infusion, inhalation, nebulization. otic, buccal, conjunctival, dental, endocervical, endosinusial, endotracheal, enteral, epidural, extraamniotic, extracorporeal, hemodialysis, infiltration, interstitial, intraabdominal, intraamniotic, intraarticular, intrabiliary . intrabronchial, intrabursal, intracartilaginous, intracaudal, intracavemous, intracavitary, intracerebroventricular, intracistemal, intracorneal, intracoronal, intracoronary, intracorpous cavemaosum, intradiscal, intraductal, intraduodenal, intradural, intraepidermal, intraesophageal, intragastric, intragingival, intrahippocampal, intraileal, intralesional, intraluminal, intralymphatic, intramedullary, intrameningeal, intramuscular, intraocular, intraovarian, intrapericardial, intrapleural, intraprostatic, intrapulmonary, intrasinal, intrasynovial, intratendinous. intratesticular, intrathoracic, intratubular, intratumor, intratympanic, intrauterine, intravascular, intravenous bolus, intravenous drip, intravesical, intravitreal, iontophoresis, irrigation, lary ngeal, nasogastric, ophthalmic, oropharyngeal, parenteral, percutaneous, periarticular, peridural, perineural, periodontal, retrobulbar, subconjunctival, sublingual, submucosal, topical, transmucosal, transplacental, transtympamc. ureteral, urethral, infraorbital, intraparenchymal, intraventricular, stereotactic administration subcuticular, or any combination thereof. A form of administration can be tailored for a particular disorder based on the properties and localization of the disorder and drug.

[0117] The terms “therapeutically effective amount”, “effective dose,” “therapeutically effective dose”, “effective amount,” or the like refer to that amount of the subject agent that will elicit the biological or medical response of a tissue, system, animal or human that is being sought by the researcher, veterinarian, medical doctor or other clinician. Generally, the response is either amelioration of symptoms in a patient or a desired biological outcome (e.g.. treatment of the disease). Such amount should be sufficient to eliminate aberrant response. The191627337384.1331323.990775PATENT ATTORNEY DOCKET NO. JHU4820-1WO effective amount can be determined as described herein.

[0118] In some aspects, regulating stress response comprises altering DNA methylation at a target genomic locus comprising a target gene.

[0119] In some aspects, altering DNA methylation comprises increasing DNA methylation at the target genomic locus.

[0120] In some aspects, altering DNA methylation comprises decreasing expression of the target gene.

[0121] In some aspects, the target gene comprises a stress response-related gene. In some aspects, the stress response-related gene is selected from the group consisting of glucocorticoid receptor (GR orNR3Cl) gene, catechol-O-methyltransferase (COMT) gene, mineralocorticoid receptor (NR3C2), corticotropin-releasing hormone (CRH) gene, FKBP5, and heat shock proteins (HSPs).

[0122] In some aspects, the subject is a mammal. In one aspect, the mammal is a human.

[0123] In some embodiments, the present disclosure provides a method of treating a disease or disorder in a subject, comprising administering to the subject a therapeutically effective amount of any the pharmaceutical composition described above, thereby treating the disease or disorder in the subject.

[0124] The term "treatment” is used interchangeably herein with the term "therapeutic method" or “therapy” and refers to 1) therapeutic treatments or measures that cure, slow down, lessen symptoms of, and / or halt progression of a diagnosed pathologic conditions or disorder (e.g., idiopathic pulmonary fibrosis), and / or 2) prophylactic / preventative measures. Those in need of treatment may include individuals already having a particular medical disorder as well as those who may ultimately acquire the disorder (i.e., those needing preventive measures).

[0125] In some aspects, treating the disease or disorder comprises altering DNA methylation at a target genomic locus comprising a target gene.

[0126] In some aspects, altering DNA methylation comprise increasing DNA methylation at the genomic locus.

[0127] In some aspects, altering DNA methylation comprises decreasing expression of the target gene.

[0128] In some aspects, the disease or disorder comprises a psychiatric disease or disorder.

[0129] In some aspects, the disease or disorder comprises an infectious disorder.

[0130] In some aspects, the disease or disorder comprises a metabolic disorder.201627337384.1331323.990775PATENT ATTORNEY DOCKET NO. JHU4820-1WO

[0131] In some aspects, the disease or disorder comprises a developmental disorder.

[0132] In some aspects, the disease or disorder comprises cancer.

[0133] In some aspects, the target gene comprises a psychiatric disorder-related gene.

[0134] In some aspects, the target gene comprises an infectious disorder-related gene.

[0135] In some aspects, the target gene comprises a metabolic disorder-related gene.

[0136] In some aspects, the target gene comprises a developmental disorder-related gene.

[0137] In some aspects, the target gene comprises a cancer-related gene.

[0138] In some aspects, the methylated DNA probe comprise a DNA fragment about 200 bp. In some aspects, the methylated DNA probe comprise a DNA fragment about less than 200 bp. Small sized DNA probes can easily be accommodated into delivery vesicles such as EVs.

[0139] In some aspects, the pharmaceutical composition induces less side effects than the available treatments for the disease or disorder.

[0140] In some aspects, the subject is a mammal. In some aspects, the mammal is a human.

[0141] In some embodiments, the present disclosure provides a kit comprising: a methylated DNA probe, wherein the methylated DNA probe comprises a methylated nucleic acid sequence complementary to a target genomic locus comprising a target gene, and a delivery7vehicle.

[0142] In some aspects, the kit further comprises a methyltransferase enzyme. In some aspects, the methyltransferase enzyme comprises a DNA methyltransferase enzyme (DNMT).

[0143] In some aspects, the methylated DNA probe comprises a methylated single-stranded DNA (ssDNA).

[0144] In some aspects, the kit further comprising primers used to produce the DNA methylated probe.

[0145] In some aspects, the delivery7vehicle comprises an extracellular vesicle (EV). In some aspects, the EV comprises an exosome. In some aspects, the EV comprises a tissuespecific EV. In some aspects, the EV comprises a brain-specific EV.

[0146] In some aspects, the ssDNA were introduced into a N1E-115 cell line-derived exosome. The ssDNA maybe introduced in the exosome by any methods known in the art including but not limited to electroporation, transfection (e.g., using lipid-based reagents), sonication, extrusion, freeze-thaw cycles, and chemical permeabilization.

[0147] In some aspects, the delivery vehicle comprises lipofectamine.

[0148] Presented below are examples discussing use of methylated DNA probes to alter methylation of DNA at target genes contemplated for the discussed applications. The following211627337384.1331323.990775PATENT ATTORNEY DOCKET NO. JHU4820-1WO examples are provided to further illustrate the embodiments of the present invention but are not intended to limit the scope of the invention. While they are typical of those that might be used, other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used.EXAMPLESEXAMPLE 1MethodsProbe Design and Amplification

[0149] Methylated DNA probes targeting the conserved glucocorticoid response element (GRE) in intron 5 of the human (chr6:35, 601,961 - 35,602,194; GRCh38 / hg38, 256 bp) and mouse (chrl7: 28,639,321 - 28,639,560; GRCm39 / mm39, 239 bp) of FKBP5 were designed. An intronic, regulatory region of the human MAOA gene (chrX:43,656,383 - 43,656,553; GRCh38 / hg38. 170 bp) was also designed tested as an alternate region29. Primers targeted smaller regions than those targeted by bisulfite sequencing to preclude the amplification of probe DNA during methylation analysis. Probes were amplified using Taq DNA Polymerase with ThermoPol Buffer under following conditions: initial denaturation at 95°C for 4 minutes, followed by 40 cycles of denaturation at 95°C for 1 minute, annealing at 60°C for 30 seconds, and extension at 72°C for 1 minute, with a final extension at 72°C for 8 minutes. PCR products were run on an agarose gel to verify correct amplicon size, and remaining PCR products were purified using the QIAquick PCR Purification Kit according to the manufacturer’s instructions (Qiagen, Germantown, MD). Concentrations of purified probes were determined using a Qubit 4 fluorometer. Primers used for generating the probes are shown in Table 1.In Vitro Methylation of DNA Probes

[0150] Purified FKBP5 probes were subjected to in vitro methylation using the bacterial CpG methyltransferase (M.SssI, New England Biolabs). One pg of the probe w as incubated with 4 units of M.SssI. 160 pM S-adenosylmethionine (SAM), and IX NEBuffer 2 in a total reaction volume of 100 pL at 37°C for two 1-hour cycles followed by 20 minutes at 65°C for enzyme inactivation. A negative control reaction (FKBP5 Sssl-) was performed in parallel, where the M.SssI enzyme was replaced with an equal volume of water. Following in vitro methylation, probes were purified again and eluted in 20 pL of EB buffer. The concentrations of the methylated (FK Sssl+) and unmethylated (FK Sssl-) probes were measured using Qubit 4.221627337384.1331323.990775PATENT ATTORNEY DOCKET NO. JHU4820-1WOCell Culture and Transfection

[0151] Human embryonic kidney 293 (293HEK) cells and mouse pituitary AtT-20 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Cells were maintained at 37°C in a humidified incubator with 5% CO2. To induce demethylation at the GRE of the endogenous FKBP5 GRE, cells were treated with 1 pM dexamethasone (DEX) for 5 days and cultured for an additional 5 days without any DEX24. Separate wells of cells were left untreated as negative controls. Prior to transfection, cells were seeded in 24-well plates at a density' of 2 * 105 cells per well and allowed to adhere overnight in DMEM free of antibiotics. The transfection of the methylated and unmethylated probes was performed in triplicate (500 ng of probe per well) using X-tremeGENE 360 according to the manufacturer’s instructions. Cells were fed fresh DMEM media one day after transfection and harvested on the second day for collecting gDNA. For the assessment of glucocorticoid-induced gene expression, a subset of the transfected 293HEK cells was treated with 1 pM DEX for 4 hours prior to harvesting. Cells were harvested for total RNA extraction using RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. AtT-20 mouse pituitary cell lines were subjected to similar procedures.DNA Extraction and Methylation Analysis by Bisulfite Pyrosequencing

[0152] Genomic DNA was extracted from 293HEK and AtT20 cells using the DNeasy Blood & Tissue Kit (Qiagen) according to the manufacturer's instructions. The extracted DNA was bisulfite-converted using the EZ DNA Methylation-Gold Kit according to the manufacturer’s instructions. Bisulfite-converted DNA was amplified using primers specific for human FKBP5 and MAOA and mouse Fkbp5 genes under following conditions: initial denaturation at 95°C for 4 minutes, followed by 40 cycles of denaturation at 95°C for 1 minute, annealing at 53°C for 30 seconds, and extension at 72°C for 1 minute, with a final extension at 72°C for 8 minutes. Two pL of the reaction was used to perform a second round of PCR with nested primers with one primer being biotinylated. Biotinylated nested PCR products were immobilized on Streptavidin Sepharose High Performance beads and purified using the PyroMark Q96 Vacuum Workstation. Pyrosequencing was performed on the PyroMark Q96 MD Pyrosequencer. The PyroMark QCpG software was used to generate pyrosequencing data and quantify the methylation levels at each CpG site. Bisulfite primers used for assessing DNA methylation levels of FKBP5 and MAOA genes are shown in Table 1.Table 1. Sequence of primers for Probe Design and Pyrosequencing231627337384.1331323.990775PATENTATTORNEY DOCKET NO. JHU4820-1WO241627337384.1331323.990775PATENTATTORNEY DOCKET NO. JHU4820-1WO251627337384.1331323.990775PATENTATTORNEY DOCKET NO. JHU4820-1WO

[0153] *These primers are biotinylated and HPLC-purified for subsequent pyrosequencingGene Expression Analysis

[0154] Reverse Transcription Quantitative Real-Time PCR (RT-qPCR) was performed to assess the impact of targeted DNA methylation on gene expression. Complementary DNA (cDNA) was synthesized from 293HEK RNA samples using QuantiTect Reverse Transcription Kit according to the manufacturer’s instructions. Negative reverse transcriptase samples were used to ensure the absence of contaminating genomic DNA. All reactions were carried out in triplicate using l x TaqMan Master Mix, Taqman probes for each transcript (FKBP5 and ACTB), and 30 ng of cDNA template in a total volume of 20 pl. Expression levels of the housekeeping gene ACTB (beta-actin) were used for normalization. RT-qPCR was performed on an Applied Biosystems QuantStudio 5 Real-time PCR System under standard qPCR conditions (50oC for 2 min; 95oC for 10 min; and 60oC for 1 min for 40 cycles). Each sample was tested in triplicate, and each replicate was checked to ensure that the threshold cycle (Ct) values were all within 0.25 Ct of the other two replicates. For the determination of relative expression values, the -AACt method was used31, where triplicate Ct values for each sample were averaged and subtracted from those derived from ACTB. The Ct difference for an arbitrary calibrator sample, usually an untreated control sample, was subtracted from those of the test samples, and the resulting -AACt values were raised to a power of 2 to determine normalized relative expression.In Vivo Testing:

[0155] Brain-specific extracellular vesicles (EVs) were extracted from a mouse neuronal cell line using polyethylene glycol precipitation. 400 nM of prepared probe was introduced into prepared EVs by electroporation. Mouse was injected retro-orbitally with methylated probe and control. Methylation and gene expression changes were tested through genomic DNA and26 1627337384.1 331323.990775PATENT ATTORNEY DOCKET NO. JHU4820-1WORNA extracted from mouse brain samples.EXAMPLE 2The methylated probe’s efficacy in increasing DNA methylation at two target genes (MAOA and FKBP5) in vitro

[0156] Introducing methylated DNA probes can alter DNA methylation levels of their targets. Methylated DNA altered the response of the FKBP5 gene to glucocorticoid treatment (FIGs. 1A-1E). Small DNA molecules can be encapsulated in lipid particles such as Lipofectamine or EVs to enable In vivo testing in mice.EXAMPLE 3The use of methylated, single-stranded probe to induce target-specific DNA methylation.

[0157] To test whether a simple DNA probe can target-specifically alter DNA methylation, a probe against specific CpG sites within the glucocorticoid response element (GRE) of the human FKBP5 gene was generated. In wfro-methylated, single- or double-stranded DNA probes were generated and transfected into human FIEK293 cells. Two days post-transfection, cells were analyzed for DNA methylation levels of the FKBP5 GRE by bisulfite pyrosequencing. Pyrosequencing analysis showed an increase of 20.5% increase in DNA methylation at CpG-1 (P=7.0xl0‘4) and an increase of 15.0% at CpG-2 (P= P=4.9xl0‘4) in 500 ng of methylated, single-stranded probe compared to untransfected samples (FIG. 2A). A doseresponse was also observed when transfections was performed with only 250 ng of probe, with CpG-1 showing a more modest increase of 8.6% (P=0.0066) and CpG-2 showing an increase of 7.6% (P=l. lxl0-4) compared to untransfected samples. Samples transfected with unmethylated, single-stranded, or methylated, double-stranded probes did not lead to an appreciable increase in DNA methylation (P>0.05).EXAMPLE 4The effect of probe-induced DNA methylation on gene expression

[0158] Whether the increase in DNA methylation was associated with differential gene expression was also tested. Previous studies have shown that while DNA methylation changes may not immediately result in changes in gene expression, they can modulate the level to which a gene can respond to a stimulus. In this case, the GREs of FKBP5 are responsive to glucocorticoids in a methylation-specific way. Treatment of 293HEK cells with dexamethasone (DEX) caused a significant increase in FKBP5 expression (2.5-fold, P=0.036).271627337384.1331323.990775PATENT ATTORNEY DOCKET NO. JHU4820-1WOWhile transfection with unmethylated, single-stranded, or methylated, double-stranded probes did not lead to an appreciable attenuation of DEX-induced increase in expression, methylated, single-stranded probes significantly reduced the DEX-induced increase in FKBP5. Specifically, cells treated with methylated, single-stranded 500 ng of probe DNA showed 76.2% reduction in expression compared to that of unmethylated, single-stranded 500 ng of probe DNA treated with DEX. Furthermore, a dose-response was observed with methylated, single-stranded 250 ng of probe DNA, where 29.2% reduction in expression was observed compared to unmethylated, single-stranded 500 ng of probe DNA treated with DEX (FIG. 2C).EXAMPLE 5Epigenetic and transcriptional effects of methylated, single-stranded probe in a mouse pituitary cell line

[0159] Mouse pituitary cells were also tested to determine whether the findings in 293HEK can be recapitulated in a different cell-type and species. Mouse AtT-20 cells were transfection with the mouse version of the probe against the conserved Fkbp5 intron 5 GRE. Results show that the use of methylated, single-stranded probes increased DNA methylation at three of the four CpGs when compared to unmethylated single-stranded probes: CpG-2 (9.5%, P=0.003), CpG-3 (6.1%, P=0.003), and CpG-4 (7.2%, P=0.03). There were no appreciable differences in DNAm between unmethylated, single-stranded probes and methylated, double-stranded probes (FIG. 3A). Then whether transfected DNA probes can modulate DEX-inducibility as the human probes had in the 293HEK cells was tested. Fkbp5 expression analysis shows that samples treated with no DNA probe, methylated, double-stranded probe, or unmethylated, single-stranded probe showed similar levels of gene induction by DEX treatment (7.8-. 6.3-, 6.7-fold induction, respectively, and P<1.3xl0'4). However, samples transfected with methylated, single-stranded probe showed a significant reduction in DEX-induced expression compared to samples transfected with unmethylated, single-stranded probe (42.5% reduction, P=6.4xl0’5, FIG. 3B).EXAMPLE 6Effect of DNA probes in non-targeted regions

[0160] The effect of the targeted probe on non-targeted regions was also tested. In particular, it has been theorized that GREs that are scattered across several intronic regions of a glucocorticoid-responsive gene interact in 3-D space of the nucleus to coordinate glucocorticoid-induced gene expression5. Therefore, the DNA methylation levels of the nearby281627337384.1331323.990775PATENT ATTORNEY DOCKET NO. JHU4820-1WOFKBP5 intron 7 GRE for any evidence of epigenetic changes due to the introduction of the intron 5 GRE probe was assayed. DNA methylation analysis shows that of the seven CpGs tested, six CpGs are too hypermethylated (>80% DNA methylation levels for all treatment groups) to undergo probe-induced methylation changes. CpG-6, which was strangely hypomethyated (<2% for all treatment groups) did not show any increase in DNA methylation (FIG. 4)EXAMPLE 7Additional genomic target of DNA methylation probe: MAOA

[0161] To demonstrate the broader applicability of the methylated probe approach beyond the FKBP5 gene, whether targeted methylation could be achieved at the regulatory intronic region of Monoamine Oxidase A (MAOA) was tested, a gene whose encoded protein metabolizes monoamine neurotransmitters. Following the transfection of methylated singlestranded probes targeting the CpG-dense MAOA intronic region into 293HEK cells, significant increases in DNA methylation across multiple CpG sites were observed compared to cells transfected with unmethylated probes: CpG-1 (10.1%, P=0.01), CpG-5 (12.7%, P=0.04), CpG- 6 (11.2%, P=0.02), CpG-8 (9.3%, P=0.006), CpG-10 (11.8%, P=0.006), and CpG-12 (14.3%, P=0.01) (FIG. 5). A one-way ANOVA revealed a significant difference between the two treatments (F(l, 26) = 6.25, P = 0.019), where methylation levels were higher in the methylated probe-treated group (M = 58.68, SD = 7.52) compared to the unmethylated probe-treated group (M = 52.31, SD = 10.25). Consistent with the increased methylation, a corresponding decrease in MAOA gene expression was observed. qRT-PCR analysis revealed that cells transfected with methylated probes showed a 27.3% reduction m MAOA expression compared to cells transfected with unmethylated probes (P=0.041) (FIG. ID). These results demonstrate that the herein described methylated probe approach can be effectively applied to different genomic regions beyond FKBP5 and can modulate gene expression through targeted methylation.EXAMPLE 8 Brain-specific exosomes

[0162] To find an appropriate mechanism capable of tissue-specifically delivering the DNA probe, exosomes were isolated, a subpopulation of EVs 30-150 nm in size, and tested whether exosomes derived from cells of a neuronal origin can properly deliver their cargo to the brain. It was first tested the ability of such EVs to deliver fluorescent molecules and then mouse Fkbp5 GRE probe into the brain of mice. Exosomes were cultured and isolated from the mouse291627337384.1331323.990775PATENT ATTORNEY DOCKET NO. JHU4820-1WO hypothalamic N1E-1 15 cell line by differential centrifugation, were labeled with ExoGlow- Vivo dye (System Biosciences), and injected into 10-week-old C57B1 / 6 mice. The ExoGlow dye only produces light when EVs are internalized by target tissue. IVIS near-infrared imaging results showed relatively low EV signal indicative of low uptake at 24 hrs, followed by an intense EV signal in the brain area at 48 hrs (FIG. 6A). EV accumulation at the site of injection (retro- orbital) was ruled out, since they are dispersed into the circulatory system and only shows a weak signal in the head after 24 hrs. Quantitation of ExoGlow dye decay after 11 days post-injection showed the highest light intensity in the liver but also substantial signal in the brain (FIG. 6B). These results demonstrate the successful targeting of N1E-115 cell line- derived EVs to the brain and their uptake by brain tissue. Methylated or unmethylated (+Sss / - Sss), single-stranded mouse Fkbp5 probe, were then introduced into N1E-115 cell line-derived exosomes by electroporation. Each mouse was injected once, and the hypothalamus was dissected for methylation analysis. Bisulfite pyrosequencing results show subtle but significant increase in DNAm at all four CpGs associated with the Fkbp5 GRE in mice injected with methylated probes (FIG. 6C). Results demonstrate that small nucleic acids can be packaged into exosomes and delivered in a tissue-specific way, presumably via receptor-ligand mediated endocytosis by cells that have the most similar cell surface protein profiles as exosomes.EXAMPLE 9Discussion

[0163] Described herein is a simple approach for inducing targeted DNA methylation using methylated DNA probes. This method offers several advantages over existing techniques, such as CRISPR-based tools or those for altering global DNA methylation. The recent development of dCas9-DNMT or dCas9-TET constructs, which have fused the deactivated CRISPR Cas9 nuclease with DNA methyltransferase or TET demethylase protein domains, has generated much optimism that “epigenetic therapies” are on the horizon. However, CRISPR-based methods, while powerful, are still prone to off-target effects due to the reduced targetspecificity afforded by short (~20 nt) guide RNA sequence. More importantly, CRISPR-based methods require the introduction into cells of relatively large DNA plasmids that encode both the dCas9 construct and the guide RNA. In contrast, global DNA methylation modifiers, such as DNA methyltransferase inhibitor 5-aza-2'-deoxy cytidine. lack target specificity and can lead to unintended changes in methylation patterns across the genome.

[0164] This approach consists of the PCR amplification against a region of interest, which301627337384.1331323.990775PATENT ATTORNEY DOCKET NO. JHU4820-1WO in this case was a methylation-sensitive, glucocorticoid response element in the intronic region of the FKBP5 gene. The PCR product was first in vzfro-methylated using the bacterial methyltransferase M.SssI and denatured to yield single-stranded DNA probes. Using the DNA probes against the FKBP5 GRE. appreciable increase in DNA methylation across at least two of the five CpGs across the GRE in the human 293HEK cells was observed. The observed increase in DNA methylation was specific to single-stranded DNA probes, as double-stranded, methylated, and single-stranded unmethylated proves did not provoke an increase in DNA methylation. Importantly, increase in DNA methylation at these CpGs was able to attenuate DEX-induced increase in FKBP5 levels. Further, the gene regulation by the DNA probe was dose-dependent, as the introduction of half of the amount of DNA probe resulted in a reduced addition of DNA methylation and reduced attenuation of FKBP5 induction by DEX compared to the full amount of DNA probe. Investigation of another nearby GRE in intron 7, which has been thought to physically interact in 3D-space with the intronic GRE (intron 5) targeted by the probe, did not show any significant changes in DNA methylation, presumably due to the dissimilar sequences.

[0165] It was then tested whether this phenomenon was unique to human cells by testing the mouse version of the Fkbp5 probe in the AtT-20 mouse pituitary cell line. Although more subtle than those observed in the human cell lines, a significant increase in DNA methylation was observed. Similarly, this increase in DNA methylation was able to thwart DEX-induction of Fkbp5.

[0166] An additional genomic region was tested to further generalize the probe-induced increase in DNA methylation. This time a probe against an important regulatory region of MAOA whose methylation levels correlated with enzy mes levels determined by PET brain imaging was designed.

[0167] The present disclosure highlights the potential of this method to modulate stress response pathways, as demonstrated by the targeted methylation of the FKBP5 gene. The FKBP5 gene is known to be influenced by glucocorticoid exposure and methylation status, with demethylation of the glucocorticoid response element leading to increased gene expression upon subsequent glucocorticoid exposure. By inducing targeted methylation of this region, the glucocorticoid-induced upregulation of FKBP5 expression was attenuated, suggesting that described approach could be used to fine-tune stress response pathways and potentially mitigate the detrimental effects of chronic stress or glucocorticoid exposure.311627337384.1331323.990775PATENT ATTORNEY DOCKET NO. JHU4820-1WO

[0168] The success of method described herein in both human and mouse cell lines indicates its potential for translation to in vivo models and eventual clinical applications. Small extracellular vesicles (EVs) such as exosomes hold immense therapeutic value as delivery devices, as their vesicle cargo space can easily accommodate a -200 bp DNA fragment and their surface proteins profiles can be modified to alter tissue targeting (REF).

[0169] The presently described study provides a proof-of-concept for the use of methylated DNA probes as a targeted epigenetic therapy. The ability to induce site-specific DNA methylation changes opens up new avenues for the treatment of a wide range of diseases characterized by aberrant methylation patterns.

[0170] In conclusion, described herein is a novel method for targeted induction of DNA methylation using methylated DNA probes. This approach offers a promising tool for modulating gene expression and function, with potential therapeutic applications in various diseases characterized by aberrant methylation patterns. Targeted epigenetic therapies, such as described herein, hold immense promise for revolutionizing the treatment of a broad spectrum of disorders.

[0171] References1. Binder, E.B., et al., Polymorphisms in FKBP5 are associated with increased recurrence of depressive episodes and rapid response to antidepressant treatment. Nat Genet, 2004. 36(12): p. 1319-25.2. Binder, E.B., The role of FKBP5, a co-chaperone of the glucocorticoid receptor in the pathogenesis and therapy of affective and anxiety disorders. Psychoneuroendocrinology, 2009. 34 Suppl 1 : p. S I 86-95.3. Binder, E.B., et al.. Association of FKBP5 polymorphisms and childhood abuse with risk of posttraumatic stress disorder symptoms in adults. JAMA, 2008. 299(11): p. 1291-305.4. Blair. L.J., et al., The Disease- Associated Chaperone FKBP51 Impairs Cognitive Function by Accelerating AMPA Receptor Recycling. eNeuro, 2019. 6(1).5. Bond, D.M. and D.C. Baulcombe, Epigenetic transitions leading to heritable, RNA- mediated de novo silencing in Arabidopsis thaliana. Proc Natl Acad Sci U S A, 2015. 112(3): p. 917-22.6. Cox, O.H., et al.. Characterization of glucocorticoid-induced loss of DNA methylation of the stress-response gene Fkbp5 in neuronal cells. Epigenetics, 2021 . 16(12): p. 1377-321627337384.1331323.990775PATENT ATTORNEY DOCKET NO. JHU4820-1WO1397.7. Cuerda-Gil, D. and R.K. Slotkin, Non-canonical RNA-directed DNA methylation. Nat Plants, 2016. 2(11): p. 16163.8. Elliott, E., et al., Resilience to social stress coincides with functional DNA methylation of the Crf gene in adult mice. Nat Neurosci, 2010. 13(11): p. 1351-3.9. Kawasaki, IT. and K. Taira, Induction of DNA methylation and gene silencing by short interfering RNAs in human cells. Nature, 2004. 431(7005): p. 211-7.10. Lee, R.S., et al., Chronic corticosterone exposure increases expression and decreases deoxyribonucleic acid methylation of Fkbp5 in mice. Endocrinology. 2010. 151(9): p. 4332-43.11. Lee, R.S., et al., A measure of glucocorticoid load provided by DNA methylation of Fkbp5 in mice. Psychopharmacology (Berl), 2011. 218(1): p. 303-12.12. Liu, X.S., et al., Editing DNA Methylation in the Mammalian Genome. Cell, 2016. 167(1): p. 233-247 el7.13. Niwa, M., et al., Adolescent stress-induced epigenetic control of dopaminergic neurons via glucocorticoids. Science, 2013. 339(6117): p. 335-9.14. Niwa, M., et al., A critical period of vulnerability to adolescent stress: epigenetic mediators in mesocortical dopaminergic neurons. Hum Mol Genet, 2016. 25(7): p. 1370-81.15. Sabbagh, J.J., et al., Age-associated epigenetic upregulation of the FKBP5 gene selectively impairs stress resiliency. PLoS One, 2014. 9(9): p. el07241.16. Sawamura, T., et al., Dexamethasone Treatment Leads to Enhanced Fear Extinction and Dynamic Fkbp5 Regulation in Amygdala. Neuropsychopharmacology, 2016. 41(3): p. 832-46.17. Scammell, J.G., et al., Overexpression of the FK506-binding immunophilin FKBP51 is the common cause of glucocorticoid resistance in three New World primates. Gen Comp Endocrinol, 2001. 124(2): p. 152-65.18. Taira, K., Induction of DNA methylation and gene silencing by short interfering RNAs in human cells. Nature, 2006. 441(7097): p. 1176.19. Ting, A.H., et al.. Short double-stranded RNA induces transcriptional gene silencing in human cancer cells in the absence of DNA methylation. Nat Genet, 2005. 37(8): p. 906- 10.331627337384.1331323.990775PATENT ATTORNEY DOCKET NO. JHU4820-1WO20. Wu, Y., et al.. Early-life stress reduces DNA methylation of the Pome gene in male mice. Endocrinology, 2014. 155(5): p. 1751-62.21. Yehuda, R., et al., Hydrocortisone responsiveness in Gulf War veterans with PTSD: effects on ACTH, declarative memory’ hippocampal [(18)F]FDG uptake on PET. Psychiatry Res, 2010. 184(2): p. 117-27.22. Jones PL, Veenstra GJ, Wade PA, Vermaak D, Kass SU, Landsberger N, Strouboulis J, Wolffe AP. Methylated DNA and MeCP2 recruit histone deacetylase to repress transcription. Nat Genet 1998; 19: 187-91.23. Baylin SB. Tying it all together: epigenetics, genetics, cell cycle, and cancer. Science 1997; 277: 1948-9.24. Jones PA, Baylin SB. The fundamental role of epigenetic events in cancer. Nat Rev Genet 2002; 3:415-28.25. Landgrave-Gomez J, Mercado-Gomez O, Guevara-Guzman R. Epigenetic mechanisms in neurological and neurodegenerative diseases. Front Cell Neurosci 2015; 9:58. PMC4343006.26. Klengel T, Mehta D, Anacker C, Rex-Haffner M, Pruessner JC, Pariante CM, Pace TW, Mercer KB, Mayberg HS, Bradley B, et al. Allele-specific FKBP5 DNA demethylation mediates gene-childhood trauma interactions. Nat Neurosci 2013; 16:33-41. PMC4136922.27. Uddin M, Aiello AE, Wildman DE, Koenen KC, Pawelec G, de Los Santos R, Goldmann E, Galea S. Epigenetic and immune function profiles associated with posttraumatic stress disorder. Proc Natl Acad Sci U S A 2010; 107:9470-5. PMC2889041.28. Murphy TM, O'Donovan A, Mullins N, O'Farrelly C, McCann A, Malone K. Anxiety’ is associated with higher levels of global DNA methylation and altered expression of epigenetic and interleukin-6 genes. Psychiatr Genet 2015; 25:71-8.29. Tsankova NM, Berton O, Renthal W, Kumar A, Neve RL, Nestler EJ. Sustained hippocampal chromatin regulation in a mouse model of depression and antidepressant action. Nat Neurosci 2006; 9:519-25.30. Weaver IC, Cervoni N, Champagne FA, D'Alessio AC, Sharma S, Seckl JR, Dymov S, Szyf M, Meaney MJ. Epigenetic programming by maternal behavior. Nat Neurosci 2004; 7:847-54.341627337384.1331323.990775PATENTATTORNEY DOCKET NO. JHU4820-1WO31. Erdmann RM, Picard CL. RNA-directed DNA Methylation. PLoS Genet 2020; 16:el009034. PMC7544125.32. Yang X, Lay F, Han H, Jones PA. Targeting DNA methylation for epigenetic therapy. Trends Pharmacol Sci 2010; 31 :536-46. PMC2967479.33. Liu XS, Wu H, Ji X, Stelzer Y, Wu X, Czaudema S, Shu J, Dadon D, Young RA, Jaenisch R. Editing DNA Methylation in the Mammalian Genome. Cell 2016; 167:233- 47 el7. PMC5062609.34. Vojta A, Dobrinic P, Tadic V, Bockor L, Korac P, Julg B, Klasic M, Zoldos V. Repurposing the CRISPR-Cas9 system for targeted DNA methylation. Nucleic Acids Res 2016; 44:5615-28. PMC4937303.35. Xu X, Tao Y, Gao X, Zhang L, Li X, Zou W, Ruan K, Wang F, Xu GL, Hu R. A CRISPR-based approach for targeted DNA demethylation. Cell Discov 2016; 2: 16009. PMC4853773.36. Kim S, Koo T, Jee HG, Cho HY, Lee G, Lim DG, Shin HS, Kim JS. CRISPR RNAs trigger innate immune responses in human cells. Genome Res 2018; 28:367-73. PMC5848615.37. Ewaisha R, Anderson KS. Immunogenicity of CRISPR therapeutics-Critical considerations for clinical translation. Front Bioeng Biotechnol 2023; 11: 1138596. PMC9978118.38. Hakim CH, Kumar SRP, Perez-Lopez DO, Wasala NB, Zhang D, Yue Y, Teixeira J, Pan X, Zhang K, Million ED, et al. Cas9-specific immune responses compromise local and systemic AAV CRISPR therapy in multiple dystrophic canine models. Nat Commun 2021 ; 12:6769. PMC8613397.39. Zhang XH, Tee LY, Wang XG, Huang QS, Yang SH. Off-target Effects in CRISPR / Cas9-mediated Genome Engineering. Mol Ther Nucleic Acids 2015; 4:e264. PMC4877446.40. Hoijer I, Emmanouilidou A, Ostlund R, van Schendel R, Bozorgpana S, Tijsterman M, Feuk L, Gyllensten U, den Hoed M, Ameur A. CRISPR-Cas9 induces large structural variants at on-target and off-target sites in vivo that segregate across generations. Nat Commun 2022; 13:627. PMC8810904.41. Frangoul H, Altshuler D, Cappellini MD, Chen YS. Domm J, Eustace BK. Foell J, de la Fuente J, Grupp S, Handgretinger R, et al. CRISPR-Cas9 Gene Editing for Sickle351627337384.1331323.990775PATENT ATTORNEY DOCKET NO. JHU4820-1WOCell Disease and beta-Thalassemia. N Engl J Med 2021 ; 384:252-60.42. Stadtmauer EA, Fraietta JA, Davis MM, Cohen AD, Weber KL, Lancaster E, Mangan PA, Kulikovskaya I, Gupta M, Chen F, et al. CRISPR-engineered T cells in patients with refractory cancer. Science 2020; 367. PMC11249135.43. Scammell JG, Denny WB, Valentine DL, Smith DF. Overexpression of the FK506- binding immunophilin FKBP51 is the common cause of glucocorticoid resistance in three New World primates. Gen Comp Endocrinol 2001; 124:152-65.44. Lee RS, Tamashiro KL, Y ang X, Purcell RH, Harvey A, Willour VL, Huo Y, Rongione M, Wand GS, Potash JB. Chronic corticosterone exposure increases expression and decreases deoxyribonucleic acid methylation of Fkbp5 in mice. Endocrinology 2010; 151 :4332-43. PMC2940504.45. Cox OH, Song HY, Garrison-Desany HM, GadiwallaN, Carey JL, Menzies J, Lee RS. Characterization of glucocorticoid-induced loss of DNA methylation of the stressresponse gene Fkbp5 in neuronal cells. Epigenetics 2021; 16: 1377-97. PMC8813076.46. Wochnik GM, Ruegg J, Abel GA, Schmidt U, Holsboer F, Rein T. FK506-binding proteins 51 and 52 differentially regulate dynein interaction and nuclear translocation of the glucocorticoid receptor in mammalian cells. J Biol Chem 2005; 280:4609-16.47. Hawn SE, Sheerin CM, Lind MJ, Hicks TA, Marraccini ME, Bountress K, Bacanu SA, Nugent NR, Amstadter AB. GxE effects of FKBP5 and traumatic life events on PTSD: A meta-analysis. J Affect Disord 2019; 243:455-62. PMC6487483.48. Qiu B, Luczak SE, Wall TL, Kirchhoff AM, Xu Y, Eng MY, Stewart RB, Shou W, Boehm SL. Chester JA, et al. The FKBP5 Gene Affects Alcohol Drinking in Knockout Mice and Is Implicated in Alcohol Drinking in Humans. Int J Mol Sci 2016; 17. PMC5000669.49. Tozzi L, Farrell C, Booij L, Doolin K, Nemoda Z, Szy f M, Pomares FB, Chiarella J, O'Keane V, Frodl T. Epigenetic Changes of FKBP5 as a Link Connecting Genetic and Environmental Risk Factors with Structural and Functional Brain Changes in Major Depression. Neuropsychopharmacology 2018; 43:1138-45. PMC5854813.50. Shumay E, Logan J, VolkowND, Fowler JS. Evidence that the methylation state of the monoamine oxidase A (MAOA) gene predicts brain activity’ of MAO A enzyme in healthy men. Epigenetics 2012; 7: 1151-60. PMC3469457.51. Renbaum P, Abrahamove D, Fainsod A, Wilson GG, Rottem S, Razin A. Cloning,361627337384.1331323.990775PATENTATTORNEY DOCKET NO. JHU4820-1WO characterization, and expression in Escherichia coli of the gene coding for the CpG DNA methylase from Spiroplasma sp. strain MQl(M.SssI). Nucleic Acids Res 1990; 18: 1145-52. PMC330428.52. Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 2008; 3: 1101-8.53. Cox OH, Song HY, Garrison-Desany HM, GadiwallaN, Carey JL, Menzies J, Lee RS. Characterization of glucocorticoid-induced loss of DNA methylation of the stressresponse gene Fkbp5 in neuronal cells. Epigenetics 2021: 1-21.54. Guo C. Ma X, Gao F, Guo Y. Off-target effects in CRISPR / Cas9 gene editing. Front Bioeng Biotechnol 2023; 1 1 : 1143157. PMC 10034092.55. Christman JK. 5-Azacytidine and 5-aza-2'-deoxycytidine as inhibitors of DNA methylation: mechanistic studies and their implications for cancer therapy. Oncogene 2002: 21:5483-95.

[0172] Although the invention has been described with reference to the above examples, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the following claims.371627337384.1331323.990775

Claims

What is claimed is:

1. A pharmaceutical composition comprising: a methylated DNA probe complementary to a target genomic locus comprising a target gene, and a delivery vehicle.

2. The pharmaceutical composition of claim 1. wherein the target genomic locus comprises a promoter region, an enhancer region, a silencer region, an insulator region, an intron or an exon.

3. The pharmaceutical composition of claim 2, wherein the target genomic locus comprises an intron.

4. The pharmaceutical composition of claim 2. wherein the target genomic locus comprises a promoter.

5. The pharmaceutical composition of claim 1, wherein the methylated DNA probe comprises a methylated single-stranded DNA (ssDNA).

6. The pharmaceutical composition of claim 1, wherein the delivery vehicle comprises an extracellular vesicle (EV).

7. The method of claim 6, wherein the EV comprises a tissue-specific EV.

8. The pharmaceutical composition of claim 7, wherein the EV comprises a brain-specific EV.

9. The pharmaceutical composition of claim 6. wherein the EV comprises an exosome.

10. The pharmaceutical composition of claim 8, wherein the brain-specific EV comprises aNlE-115 cell line-derived exosome.

11. A method of producing a methylated DNA probe comprising contacting a nucleic acid sequence complementary to a target genomic locus comprising a target gene with a methyltransferase enzy me, wherein the methyltransferase enzyme methylates the nucleic acid sequence, thereby producing the methy lated probe.

12. The method of claim 11. further comprising denaturing the methylated DNA probe to generate a methylated single-stranded DNA (ssDNA).

13. The method of claim 11, wherein the methyltransferase enzyme comprises a DNA methyltransferase enzy me (DNMT).

14. The method of claim 13, wherein the DNMT comprises a bacterial Sssl methylase enzyme.

15. The method of claim 11 , wherein the nucleic acid sequence is generated using a method38PATENTATTORNEY DOCKET NO. JHU4820-1WO comprising PCR amplification, oligonucleotide synthesis, gene synthesis, hybridization capture, or a combination thereof.

16. A method of altering DNA methylation in a cell comprising contacting the cell with the pharmaceutical compositions of any of claims 1-10, thereby altering DNA methylation.

17. The method of claim 16, wherein altering DNA methylation comprises altering DNA methylation at a target genomic locus comprising a target gene.

18. The method of claim 17, wherein altering DNA methylation comprise increasing DNA methylation at the target genomic locus.

19. The method of claim 17, wherein altering DNA methylation comprises decreasing expression of the target gene.

20. The method of claim 17, wherein the target genomic locus comprises a psychiatric disorder-related gene, or cancer-related gene.

21. The method of claim 17, wherein the target gene is selected from the group consisting of glucocorticoid receptor (GR or NR3C1) gene, catechol-O-methyltransferase (COMT) gene, mineralocorticoid receptor (NR3C2), corticotropin-releasing hormone (CRH) gene, FKBP5, heat shock proteins (HSPs), and MA OA.

22. A method of decreasing expression of a target gene in a cell comprising contacting the cell with the pharmaceutical compositions of any of claims 1-10, thereby decreasing expression of the target gene in the cell.

23. The method of claim 22. wherein decreasing expression of the target gene comprises altering DNA methylation at a target genomic locus comprising the target gene.

24. The method of claim 22, wherein decreasing expression of the target gene comprises increasing DNA methylation at the target genomic locus.

25. The method of claim 22, wherein the target gene comprises a psychiatric disorder- related gene or cancer-related gene.

26. The method of claim 22, wherein the target gene is selected from the group consisting of glucocorticoid receptor (GR or NR3C1) gene, catechol-O-methyltransferase (COMT) gene, mineralocorticoid receptor (NR3C2), corticotropin-releasing hormone (CRH) gene, FKBP5, heat shock proteins (HSPs), MAOA, serotonin transporter gene (SLC6A4).

27. A method of regulating stress response in a subject comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of any of claims391627337384.1331323.990775PATENTATTORNEY DOCKET NO. JHU4820-1WO1-10, thereby regulating stress response in the subject.

28. The method of claim 27, wherein regulating stress response comprises altering DNA methylation at a target genomic locus comprising a target gene.

29. The method of claim 28, wherein altering DNA methylation comprises increasing DNA methylation at the target genomic locus.

30. The method of claim 28, wherein altering DNA methylation comprises decreasing expression of the target gene.

31. The method of claim 28, wherein the target gene comprises a stress response-related gene.

32. The method of claim 28, wherein the stress response-related gene is selected from the group consisting of glucocorticoid receptor (GR or NR3CJ) gene, catechol-O- methyltransferase (COMT) gene, mineralocorticoid receptor (NR3C2), corticotropin-releasing hormone (CRH) gene, FKBP5, and heat shock proteins (HSPs).

33. The method of any of claims 27-32, wherein the subject is a mammal.

34. The method of claim 33, wherein the mammal is a human.

35. A method of treating a disease or disorder in a subject, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of any of claims 1-10. thereby treating the disease or disorder in the subject.

36. The method of claim 35, wherein treating the disease or disorder comprises altering DNA methylation at a target genomic locus comprising a target gene.

37. The method of claim 36, wherein altering DNA methylation comprise increasing DNA methylation at the genomic locus.

38. The method of claim 36, wherein altering DNA methylation comprises decreasing expression of the target gene.

39. The method of claim 35, wherein the disease or disorder comprises a psychiatric disease or disorder.

40. The method of claim 35, wherein the disease or disorder comprises cancer.

41. The method of claim 38, wherein the target gene comprises a psychiatric disorder- related gene.

42. The method of claim 38. wherein the target gene comprises a cancer-related gene.

43. The method of any of claims 35-42, wherein the subject is a mammal.401627337384.1331323.990775PATENTATTORNEY DOCKET NO. JHU4820-1WO44. The method of claim 43, wherein the mammal is a human.

45. A kit comprising: a. a methylated DNA probe, wherein the methylated DNA probe comprises a methylated nucleic acid sequence complementary to a target genomic locus comprising a target gene, and b. a deliver}7vehicle.

46. The kit of claim 45, further comprising a methyltransferase enzyme.

47. The kit of claim 46, wherein the methyltransferase enzy me comprises a DNA methyltransferase enzyme (DNMT).

48. The kit of claim 45, wherein the methylated DNA probe comprises a methylated singlestranded DNA (ssDNA).

49. The kit of claim 45, further comprising primers selected from Table 1.

50. The kit of claim 45, wherein the delivery7vehicle comprises an extracellular vesicle (EV).

51. The kit of claim 50, wherein the EV comprises an exosome.

52. The kit of claim 50, wherein the EV comprises a tissue-specific EV.

53. The kit of claim 40, wherein the EV comprises a brain-specific EV.

54. The kit of claim 53, wherein the brain-specific EV comprises a N1E-115 cell line- derived exosome.

55. The kit of claim 45, wherein the delivery7vehicle comprises lipofectamine.411627337384.1331323.990775

Images