In vivo intra-airway vector-free epigenetic editing

Abstract

Disclosed are methods and compositions for gene-specific epigenetic editing involving contacting a gene or gene promoter with a gene-specific fusion complex containing a DNA demethylase enzyme. The methods include demethylating one or more nucleotides within the gene, promoter, or anticipated regulatory region, resulting in epigenetic editing of the gene, nucleotides, regulatory motif, or promoter. This demethylation process serves to test the mechanistic significance of gene demethylation or enhance the transcriptional responsiveness of the gene.

Description

IN VIVO INTRA-AIRWAY VECTOR-FREE EPIGENETIC EDITING CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to United States Provisional Patent No.63 / 579,451, filed on 29-August-2023, the entirety of which is incorporated by reference as if fully reproduced and set forth herein in its entirety. This application also claims the benefit of priority to United States Provisional Patent No.63 / 646,659, filed on 13-May-2024, the entirety of which is incorporated by reference as if fully reproduced and set forth herein in its entirety. FIELD OF THE INVENTION

[0002] This invention generally relates to methods for epigenetic editing and compositions directed to epigenetic editing. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0003] Not applicable. BACKGROUND OF THE INVENTION

[0004] The Global Human Genome Project (HGP) was adopted globally in 1990 by many governments and countries. The HGP was an international effort to map every gene in the human genome. In 1984 (and earlier), it was widely hoped that having a complete map of every human gene would elucidate many (if not most) of human diseases and even human character traits. It was hoped that persons who have certain diseases / conditions would be found to have slight differences in their DNA (and genes) and that would explain their many differences or diseases / conditions.

[0005] In 1984 and earlier, it was also suspected that each person’s genes determine their destiny to a very large degree. Before the HGP was underway, it was thought that having elucidated every gene in the human genome, then, treating a disease in a patient would mainly be a matter of “fixing” the “slightly different” genes in that patient using “gene editing”. There are many different types of gene editing (e.g., CRISPR / Cas9, mRNA vaccines, delivery vectors, delivery formulations, and zinc finger nucleases), but most of the DNA gene editing techniques in common will: 1) “cut out” or remove the undesirable portion of DNA (remove the problem genes) from the actual DNA chain, then 2) copy in the desirable genes into the patient’s DNA (chain). As is usually the case, learning more about DNA sequences led to more questions.

[0006] As time progressed, the HGP became more complex as different countries worked to provide published DNA base-pair sequences for differing populations.

[0007] Adding to the complexity, the HGP eventually, over time, proved that the promise of the HGP is true to varying degrees for some conditions (e.g., such as being relatively tall or relatively short in height). As the HGP neared completion, the HGP eventually proved that environmental conditions (i.e., nurture or environment vs. nature or DNA) have much greater impact on diseases, conditions, and gene expression in humans than was previously imagined. Interestingly, it was found that some (or many) environmentally gained traits can be passed on, expanding the field of epigenetics and leading to revolutionary ideas in adaptation of organisms. As the technology has advanced, the ability to act with targeted purpose has not. What is urgently needed now are new methods and compositions for epigenetic editing. BRIEF SUMMARY OF THE INVENTION

[0008] The following presents a simplified summary of the innovation in order to provide a basic understanding of some aspects of the invention. This summary is not an extensive overview of the invention. It is intended to neither identify key or critical elements of the invention nor delineate the scope of the invention. Its sole purpose is to present some concepts of the invention in a simplified form as a prelude to the more detailed description that is presented later.

[0009] Previous approaches to gene-specific epigenetic editing have primarily focused on utilizing non-specific demethylating agents, such as DNA methyltransferase inhibitors, to induce global changes in DNA methylation patterns. These non-specific agents lack the precision required for targeted epigenetic modifications and may lead to unintended off-target effects throughout the genome. Additionally, these approaches do not provide a means to specifically target individual genes or regulatory regions for demethylation, limiting their utility in studying the functional consequences of gene-specific epigenetic modifications. In addition, previous approaches have utilized mRNA (or other) expression in target cells, which can have unpredictable results.

[0010] Other approaches have involved the use of gene-specific fusion proteins to target specific genes for epigenetic modifications. However, these fusion proteins have typically been designed to recruit histone-modifying enzymes rather than DNA demethylases, thereby focusing on histone modifications rather than direct DNA demethylation. While histone modifications play a crucial role in gene regulation, the direct demethylation of DNA at specific gene loci offers a more precise and targeted approach to modulating gene expression.

[0011] Furthermore, some methods have attempted to achieve gene-specific epigenetic editing by utilizing CRISPR-based technologies to recruit DNA demethylases to specific genomic loci. While CRISPR-based approaches have shown promise in enablingtargeted epigenetic modifications, they often rely on the recruitment of non-specific demethylases, limiting their specificity and precision in gene-specific epigenetic editing. Additionally, the efficiency and specificity of CRISPR-based epigenetic editing methods may vary depending on the target gene and the genomic context. However, none of these approaches have provided a comprehensive solution that combines the features described in this disclosure. The presently disclosed technology, in summary, solves the problems briefly mentioned above.

[0012] Epigenetics, in some discussions, is the study of heritable changes in gene expression that do not involve changes to the underlying DNA sequence. This field has gained significant attention due to its implications in various biological processes and diseases, including cancer, neurological disorders, and developmental abnormalities. One of the key mechanisms of epigenetic regulation is DNA methylation, which typically acts to repress gene expression. DNA methylation involves the addition of a methyl group to the DNA molecule, usually at cytosine bases in the context of CpG dinucleotides. This modification can alter the accessibility of the DNA to transcriptional machinery, thereby influencing gene expression.

[0013] Traditional methods of studying DNA methylation and its effects on gene expression have involved global demethylation or the use of non-specific demethylating agents. However, these approaches lack the precision needed to target specific genes or regulatory regions, leading to off-target effects and unintended consequences. The ability to perform gene-specific epigenetic editing has the potential to revolutionize the field by allowing researchers to investigate the functional significance of DNA methylation at specific loci. This targeted approach can provide insights into the role of epigenetic modifications in gene regulation and disease and may also offer new avenues for therapeutic interventions.

[0014] To further describe a brief summary of some embodiments herein, which can be combined with any other aspect, feature, embodiment, or description herein, the technology disclosed herein can be discussed by a discussion including the following list of features:

[0015] Feature 1. A method for a gene-specific epigenetic editing of a gene, the method comprising the steps of: (1) contacting the gene and / or a gene promoter with a gene-specific fusion complex comprising at least one enzyme functioning as a DNA demethylase; and (2) performing a demethylation of one or more nucleotides within a gene, a promoter, and / or an anticipated regulatory region; whereby the demethylation provides an epigenetic editing of the gene, one or more nucleotides, regulatory motif, and / or the promoter and provides a test of the mechanistic significance of the demethylation of the gene or provides enhancement of transcriptional responsiveness of the gene.

[0016] Feature 2. The method of feature 1, wherein the method includes one or more demethylases, and / or one or more DNA binding domains (DBDs) assembled in a construct, fusion protein, or RNA-protein complex.

[0017] Feature 3. The method of feature 1, wherein at least one enzymatic domain is derived from TDG (thymine DNA glycosylase), Tet1, Tet2 or Tet3 (ten-eleven translocation, TET, methylcytosine dioxygenase 1, 2 or 3), the entire coding sequence of mouse or human TDG, a derivate sequence of mouse or human TDG, the enzymatic domain of mouse or human Tet1, Tet2 or Tet3, or a derivative sequence of the enzymatic domain of mouse or human Tet1, Tet2 or Tet3; and / or wherein the gene-specific fusion complex is a gene- specific demethylase fusion complex.

[0018] Feature 4. The method of feature 1, wherein the method is utilized to change methylation of a promoter and / or a transcription of an associated gene.

[0019] Feature 5. The method of feature 1, wherein the fusion complex is including a demethylase enzyme with a sequence-specific DNA binding domain (DBD).

[0020] Feature 6. The method of feature 1, wherein the fusion complex includes a demethylase enzyme connected to a DBD; the DBD being a dCAS9-gRNA system, or a zinc finger protein array (ZFA).

[0021] Feature 7. The method of feature 6, wherein the dCAS9-based system comprises dCas9-Tet1 cat-NLS / Myc-SCP-HA / NLS / 6xHis-Stop Codon, SEQ ID NO: 9, dCas9-Tet1 mut-NLS / Myc-SCP-HA / NLS / 6xHis-Stop Codon, SEQ ID NO: 10; dCas9-TDG wt-NLS / Myc-SCP-HA / NLS / 6xHis-Stop Codon, SEQ ID NO: 11; and / or dCas9-TDG mut (N151A)-NLS / Myc-SCP-HA / NLS / 6xHis-Stop Codon, SEQ ID NO: 12.

[0022] Feature 8. The method of feature 7, wherein a supercharged peptide (SCP) is utilized to enhance a dCAS9-construct internalization into target or recipient cells.

[0023] Feature 9. The method of feature 8, wherein the SCP comprises SEQ ID NO: 13 (GSKPAKKGSKTPGTSKSAKPKKGPGSKPATKGSKKPGKSKSATPKKGPGSKPAKSGKKT PGTSKKPSKGKAPGTSKKPSKGKAPGSKPAKKGSKTPGTSKSAKPKKGPGKSKKATPKSG P).

[0024] Feature 10. The method of feature 1, including the method is utilized to edit a gene encoding CXCL11 (I-TAC).

[0025] Feature 11. The method of feature 1, wherein the fusion complex is including a demethylase enzyme with a DBD including the dCAS9-derived system tethered to the target DNA sequence via the guide RNAs (gRNA).

[0026] Feature 12. The method of feature 10 or 11, wherein guide RNAs designed for CXCL11 gene comprises: SEQ ID NO: 14 (AAAGCTACGTGAGTCAAGGA); SEQ ID NO: 15 (TGAGTGCTTTCACCTTCCAG); SEQ ID NO: 16 (AAAGATAGATGAAGGTCACC); SEQID NO: 17 (GTCCCACCAACTCAGGAAGA); SEQ ID NO: 18 (CCACAAGCCAGGAGATGCAG); or SEQ ID NO: 19 (CTGGGACCATCTTCCTGAGT).

[0027] Feature 13. The method of feature 1, wherein the method is executed as a gene-specific DNA demethylation.

[0028] Feature 14. The method of feature 1, wherein the method provides an epigenetic re-activation of a silenced or partially silenced gene, an epigenetic restoration of an adaptive, situation-appropriate responsiveness of intrinsic transcription and / or an epigenetic enhancement of a gene's expression.

[0029] Feature 15. The method of feature 1, with the proviso that the method does not require a vector and / or a transduction-based approach.

[0030] Feature 16. The method of feature 1, wherein the fusion complex is produced by an expression in a bacterial, insect or mammalian cell, then the fusion complex protein is purified and administered to a target cell or subject.

[0031] Feature 17. The method of feature 1, wherein the fusion complex includes a ZFA construct produced by recombinant expression in an E.coli cell, or by an expression in a human embryonic kidney (HEK) or Chinese Hamster Ovary (CHO) cell; wherein the expression in the E. coli cell includes a use of one or more molecular chaperones.

[0032] Feature 18. The method of feature 17, wherein one or more molecular chaperones includes pTf16 for ZFA-TDG and pKJE7 for ZFA-Tet.

[0033] Feature 19. The method of feature 1, wherein the method utilizes a combination of fusion proteins in each administration comprising ZFA-TDGs contacting 3 or more sites and / or ZFA-TET1 contacting 3 or more sites, all within the target promoter, or wherein the method is reduced to just a single fusion construct.

[0034] Feature 20. The method of feature 1, wherein the method provides one or more data including an epigenetic influence on disease risk and / or gene silencing.

[0035] Feature 21. The method of feature 10, wherein at least one enzyme includes TDG (G / T mismatch-specific thymine DNA glycosylase) and / or Tets 1, 2 or 3 (ten-eleven translocation, TET, methylcytosine dioxygenases 1, 2 or 3); wherein the gene-specific fusion complex is a gene-specific demethylation fusion complex; and where an AA sequence of one or more enzyme(s) comprises any ZFA-Tet1CD, any ZFA-TDG construct, ZF6-Tet cat (SEQ ID NO: 3), ZF8-Tet1 cat (SEQ ID NO: 4), ZF10-Tet cat (SEQ ID NO: 5), ZF6-Tet mut (SEQ ID NO: 6), ZF5-TDG wt (SEQ ID NO: 20), ZF7-TDG wt (SEQ ID NO: 21), ZF9-TDG wt (SEQ ID NO: 22), or ZF9-TDGmut (N151A) (SEQ ID NO: 23).

[0036] Feature 22. The method of feature 21 or feature 6, wherein the ZFA portion can be replaceable / variable depending on the target(s), can be altered to provide targeting of a different target, and / or to target a different gene; and / or wherein the ZFA part isreplaceable; and / or wherein the method provides a fusion of the ZFA portion with an enzyme via a polyaminoacid linker.

[0037] Feature 23. A method for treating and / or preventing any disease or disorder where a benefit can be obtained by epigenetic editing or transcriptional enhancement of a select gene, the method comprising the steps of: (1) obtaining a subject and; (A) contacting a gene and / or a gene promoter in the subject with a gene-specific fusion complex comprising at least one enzyme functioning as a DNA demethylase; and (B) performing a demethylation of one or more nucleotides within a gene, a promoter, and / or an anticipated regulatory region; whereby the demethylation provides an epigenetic editing of the gene, one or more nucleotides, regulatory motif, and / or the promoter; leading to therapeutic benefit via enhanced transcriptional responsiveness of the targeted gene; (2) wherein the contacting is executed by the step of administering the complex to the subject.

[0038] Feature 24. The method of feature 23, whereby epigenetic editing in contrast to other methods makes the gene 'open' to stimuli and provides adaptive, situation appropriate, context-dependent gene upregulation.

[0039] Feature 25. The method of feature 23, further comprising the step of: administering the fusion complex or complexes to a subject via an intra-airway administration into the subject operative to contacting lung cells of the subject.

[0040] Feature 26. The method of feature 23, further comprising the step of: administering one or more such fusion complexes to a subject via an intranasal administration into the subject.

[0041] Feature 27. The method of feature 23, further comprising administering the gene-specific fusion complex or complexes via instillation of the complex into a subject; or administering the gene-specific fusion complex or complexes in the form of an aerosol and / or in the form of an aerosol using a mesh-type nebulizer that is operative to minimizing degradation of the administered complex or complexes.

[0042] Feature 28. The method of feature 23, wherein the method provides a treatment for Alzheimer's disease, a cancer, Parkinson's disease, schizophrenia, , epilepsy, depression, fragile X syndrome, systemic lupus erythematosus, rheumatoid arthritis, systemic sclerosis, Sjogren's syndrome, Graves' disease, type 1 diabetes, Crohn's disease, osteoporosis, type 2 diabetes, asthma, allergy, hyperlipidemia, cardiomyopathy, cardiovascular disease, IgA nephropathy, diabetic nephropathy, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, lung fibrosis, or hepatitis C.

[0043] Feature 29. The method of feature 23, wherein a DBD of the fusion complex contacts one or more loci in a CXCL11 promoter.

[0044] Feature 30. The method of feature 23, with the proviso that the method does not require an adenoviral vector (AdV), an adeno-associated virus vector (AAV), a lentiviralvector (LV), or other viral or non-viral vector or delivery packaging vehicle to administer into subject.

[0045] Feature 31. A method for providing a research tool, providing experiments, a screening assay, or a research assay including a gene-specific epigenetic editing of a gene, the method comprising the steps of: (A) administering a fusion complex or combination of fusion complexes comprising at least one enzyme and DNA-binding domains, wherein the administering comprises: (1) contacting the gene and / or a gene promoter with a gene- specific fusion complex comprising at least one enzyme functioning as a DNA demethylase; and (2) performing a demethylation of one or more nucleotides within a gene, a promoter, and / or an anticipated regulatory region; whereby the demethylation provides an epigenetic editing of the gene or provides a test of the mechanistic significance of the demethylation of the gene, one or more nucleotides, regulatory motif, and / or the promoter; and (B) monitoring one or more parameter, signs and / or symptoms in the recipient cell, tissue, organ, animal or any experimental subject.

[0046] Feature 32. The method of feature 31, wherein at least one enzymatic domain is derived from TDG (thymine DNA glycosylase) and / or Tet1 (ten-eleven translocation, TET, methylcytosine dioxygenase 1), the entire wild type CDS (consensus coding sequence) of mouse TDG, a derivate sequence of mouse TDG, the enzymatic domain of human Tet1, or a derivative sequence of the enzymatic domain of human Tet1; and wherein the gene-specific fusion complex is a gene-specific demethylation fusion complex.

[0047] Feature 33. The method of feature 31, wherein the method is utilized to change a promoter methylation and / or a transcription of an associated gene.

[0048] Feature 34. The method of feature 31, wherein the fusion complex is including a demethylase enzyme fused with a sequence-specific DNA binding domain (DBD).

[0049] Feature 35. The method of feature 31, wherein the fusion complex includes a demethylase enzyme fused with a DBD comprised of, a zinc finger protein array (ZFA), or a dCAS9-gRNA system.

[0050] Feature 36. The method of feature 35, wherein the dCAS9-based system comprises dCas9-Tet1 cat-NLS / Myc-SCP-HA / NLS / 6xHis-Stop Codon, SEQ ID NO: 9, dCas9-Tet1 mut-NLS / Myc-SCP-HA / NLS / 6xHis-Stop Codon, SEQ ID NO: 10; dCas9-TDG wt-NLS / Myc-SCP-HA / NLS / 6xHis-Stop Codon, SEQ ID NO: 11; and / or dCas9-TDG mut (N151A)-NLS / Myc-SCP-HA / NLS / 6xHis-Stop Codon, SEQ ID NO: 12.

[0051] Feature 37. The method of feature 31, wherein a supercharged peptide (SCP) is utilized to enhance a dCAS9-construct internalization into cells.

[0052] Feature 38. The method of feature 37, wherein the SCP comprises SEQ ID NO: 13(GSKPAKKGSKTPGTSKSAKPKKGPGSKPATKGSKKPGKSKSATPKKGPGSKPAKSGKKT PGTSKKPSKGKAPGTSKKPSKGKAPGSKPAKKGSKTPGTSKSAKPKKGPGKSKKATPKSG P).

[0053] Feature 39. The method of feature 31, including the method is utilized to edit a gene encoding CXCL11 (I-TAC).

[0054] Feature 40. The method of feature 31, wherein the fusion complex is including a demethylase enzyme fused with a DBD wherein guide RNAs (gRNA) are tethered to target DNA sequence via dCAS9.

[0055] Feature 41. The method of feature 39 or 40, wherein guide RNA designed for CXCL11 gene comprises: SEQ ID NO: 14 (AAAGCTACGTGAGTCAAGGA); SEQ ID NO: 15 (TGAGTGCTTTCACCTTCCAG); SEQ ID NO: 16 (AAAGATAGATGAAGGTCACC); SEQ ID NO: 17 (GTCCCACCAACTCAGGAAGA); SEQ ID NO: 18 (CCACAAGCCAGGAGATGCAG); or SEQ ID NO: 19 (CTGGGACCATCTTCCTGAGT).

[0056] Feature 42. The method of feature 31, wherein the method is executed as a gene-specific DNA demethylation.

[0057] Feature 43. The method of feature 31, wherein the method provides an epigenetic re-activation of a silenced or partially silenced gene, an epigenetic restoration of an adaptive, situation-appropriate responsiveness of intrinsic transcription and / or an epigenetic enhancement of a gene's expression.

[0058] Feature 44. The method of feature 31, with the proviso that the method does not require a vector and / or a transduction-based approach.

[0059] Feature 45. The method of feature 31, wherein the fusion complex is produced by a recombinant expression in a bacterial, insect, yeast or mammalian cell.

[0060] Feature 46. The method of feature 31, wherein the fusion complex includes a ZFA construct produced by an expression in a target cell, a recombinant expression in an E.coli cell, or by an expression in a human embryonic kidney (HEK) or Chinese Hamster Ovary (CHO) cell; wherein the expression in the E. coli cell includes a use of one or more molecular chaperones.

[0061] Feature 47. The method of feature 45, wherein one or more molecular chaperones includes pTf16 for ZFA-TDG and pKJE7 for ZFA-Tet1.

[0062] Feature 48. The method of feature 31, further comprising the step of obtaining a subject and: administering the fusion complex or complexes to a subject via an intra- airway administering into the subject operative to contacting lung cells of the subject.

[0063] Feature 49. The method of feature 48, further comprising the step of: administering one or more such fusion complexes to the subject via an intranasal administration into the subject, and / or via aerosol administration into the subject..

[0064] Feature 50. The method of feature 49, wherein the method provides a treatment for any disease or disorder where a benefit can be obtained by epigenetic editing or transcriptional enhancement of a select gene.

[0065] Feature 51. The method of feature 50, wherein the method provides research directed to a treatment for Alzheimer's disease, a cancer, Parkinson's disease, schizophrenia, epilepsy, depression, fragile X syndrome, systemic lupus erythematosus, rheumatoid arthritis, systemic sclerosis, Sjogren's syndrome, Graves' disease, type 1 diabetes, Crohn's disease, osteoporosis, type 2 diabetes, asthma, allergy, hyperlipidemia, cardiomyopathy, cardiovascular disease, IgA nephropathy, diabetic nephropathy, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, lung fibrosis, or hepatitis C.

[0066] Feature 52. The method of feature 31, wherein the method is performed with a screening therapeutic agent, with a screening drug, with a screening compound, and / or with a screening condition.

[0067] Feature 53. The method of feature 31, with the proviso that the method does not require an adenoviral vector (AdV), an adeno-associated virus vector (AAV), a lentiviral vector (LV), or a non-viral vector to administer into subject.

[0068] Feature 54. The method of feature 39, wherein one or more loci in CXCL11 promoter for ZFA and gRNA are contacted.

[0069] Feature 55. The method of feature 54, wherein at least one enzyme includes TDG (G / T mismatch-specific thymine DNA glycosylase) and / or Tet1 (ten-eleven translocation, TET, methylcytosine dioxygenase 1); wherein the gene-specific fusion complex is a gene-specific demethylation fusion complex; and where an AA sequence of one or more enzyme(s) comprises any ZFA-Tet1CD, any ZFA-TDG construct, ZF6-Tet cat (SEQ ID NO: 3), ZF8-Tet1 cat (SEQ ID NO: 4), ZF10-Tet cat (SEQ ID NO: 5), ZF6-Tet mut (SEQ ID NO: 6), ZF5-TDG wt (SEQ ID NO: 20), ZF7-TDG wt (SEQ ID NO: 21), ZF9-TDG wt (SEQ ID NO: 22), or ZF9-TDGmut (N151A) (SEQ ID NO: 23).

[0070] Feature 56. A composition for a gene-specific epigenetic editing of a gene within a subject, the composition comprising: (1) a sequence-specific DBD; (2) at least one enzyme functioning as a DNA demethylase to perform a demethylation of at least a portion of the gene, one or more nucleotides in the gene or a promoter, and / or relevant CpG island(s), or other regulatory motif.

[0071] Feature 57. The composition of feature 56, wherein at least one enzymatic domain is derived from TDG (thymine DNA glycosylase) and / or Tet's 1, 2 or 3 (ten-eleven translocation, TET, methylcytosine dioxygenases 1, 2, 3), the entire coding sequence of mouse TDG, a derivate sequence of mouse TDG, the enzymatic domain of human Tet1, Tet2 or Tet3, or a derivative sequence of the enzymatic domain of human Tet1; or wherein the gene-specific fusion complex is a gene-specific demethylation fusion complex.

[0072] Feature 58. The composition of feature 56, wherein the composition is operative to change a promoter methylation and / or a transcription of an associated gene.

[0073] Feature 59. The composition of feature 56, wherein the fusion complex is including a demethylase enzyme with a sequence-specific DNA binding domain (DBD).

[0074] Feature 60. The composition of feature 56, wherein the fusion complex is including a demethylase enzyme with a DBD, or a DBD including a zinc finger protein array (ZFA), or a dCAS9 system.

[0075] Feature 61. The composition of feature 60, wherein the dCAS9-based system comprises dCas9-Tet1 cat-NLS / Myc-SCP-HA / NLS / 6xHis-Stop Codon, SEQ ID NO: 9, dCas9-Tet1 mut-NLS / Myc-SCP-HA / NLS / 6xHis-Stop Codon, SEQ ID NO: 10; dCas9-TDG wt-NLS / Myc-SCP-HA / NLS / 6xHis-Stop Codon, SEQ ID NO: 11; and / or dCas9-TDG mut (N151A)-NLS / Myc-SCP-HA / NLS / 6xHis-Stop Codon, SEQ ID NO: 12.

[0076] Feature 62. The composition of feature 56, further comprising wherein a supercharged peptide (SCP) is included to enhance a dCAS9-construct internalization into cells.

[0077] Feature 63. The composition of feature 62, wherein the SCP comprises SEQ ID NO: 13 (GSKPAKKGSKTPGTSKSAKPKKGPGSKPATKGSKKPGKSKSATPKKGPGSKPAKSGKKT PGTSKKPSKGKAPGTSKKPSKGKAPGSKPAKKGSKTPGTSKSAKPKKGPGKSKKATPKSG P).

[0078] Feature 64. The composition of feature 56, including the composition is utilized to edit a gene encoding CXCL11 (I-TAC).

[0079] Feature 65. The composition of feature 56, wherein the fusion complex is including a demethylase enzyme fused with a DBD including a CRISPR-derived system wherein guide RNAs (gRNA) are tethered to target DNA sequence via dCAS9.

[0080] Feature 66. The composition of feature 64 or 65, wherein guide RNA comprises: SEQ ID NO: 14 (AAAGCTACGTGAGTCAAGGA); SEQ ID NO: 15 (TGAGTGCTTTCACCTTCCAG); SEQ ID NO: 16 (AAAGATAGATGAAGGTCACC); SEQ ID NO: 17 (GTCCCACCAACTCAGGAAGA); SEQ ID NO: 18 (CCACAAGCCAGGAGATGCAG); or SEQ ID NO: 19 (CTGGGACCATCTTCCTGAGT).

[0081] Feature 67. The composition of feature 56, wherein the composition is operative to provide a gene-specific DNA demethylation.

[0082] Feature 68. The composition of feature 56, wherein the composition is operative to provide an epigenetic re-activation of a silenced or partially silenced gene, an epigenetic restoration of an adaptive, situation-appropriate responsiveness of intrinsic transcription and / or an epigenetic enhancement of a gene's expression.

[0083] Feature 69. The composition of feature 56, with the proviso that the composition does not require a vector and / or a transduction-based approach.

[0084] Feature 70. The composition of feature 56, wherein the fusion complex is produced by a recombinant expression in a cell.

[0085] Feature 71. The composition of feature 56, wherein the fusion complex includes a ZFA construct produced by an expression in a recombinant expression in a cell, a recombinant expression in an E.coli cell, or by an expression in a human embryonic kidney (HEK) or Chinese Hamster Ovary (CHO) cell; wherein the expression in the E. coli cell includes a use of one or more molecular chaperones.

[0086] Feature 72. The composition of feature 71, wherein one or more molecular chaperones include pTf16 for ZFA-TDG and pKJE7 for ZFA-Tet1.

[0087] Feature 73. The composition of feature 56, further comprising the composition is operative to provide the step of: administering the fusion complex or complexes to a subject via an intra-airway administering into the subject operative to contacting lung cells of the subject.

[0088] Feature 74. The composition of feature 73, further comprising the composition is operative to provide an execution of the step of: administering one or more such fusion complexes to a subject via an intranasal administration into the subject.

[0089] Feature 75. The composition of feature 56, wherein the composition provides a treatment for any disease or disorder where a benefit can be obtained by epigenetic editing or transcriptional enhancement of a select gene.

[0090] Feature 76. The composition of feature 56, wherein the composition provides a treatment for Alzheimer's disease, a cancer, Parkinson's disease, schizophrenia, epilepsy, depression, fragile X syndrome, systemic lupus erythematosus, rheumatoid arthritis, systemic sclerosis, Sjogren's syndrome, Graves' disease, type 1 diabetes, Crohn's disease, osteoporosis, type 2 diabetes, asthma, allergy, hyperlipidemia, cardiomyopathy, , cardiovascular disease, IgA nephropathy, diabetic nephropathy, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, lung fibrosis, or hepatitis C.

[0091] Feature 77. The composition of feature 56, further comprising the composition is operative to provide the step of: administering the gene-specific fusion complex or complexes into an airway of a subject; administering the gene-specific fusion complex or complexes via instillation of the complex into a subject; or administering the gene-specific fusion complex or complexes in the form of an aerosol and / or in the form of an aerosol using a mesh-type nebulizer that is operative to minimizing degradation of the administered complex or complexes.

[0092] Feature 78. The composition of feature 56, with the proviso that the composition does not require an adenoviral vector (AdV), an adeno-associated virus vector (AAV), a lentiviral vector (LV), or other viral or non-viral vector to administer into subject.

[0093] Feature 79. The composition of feature 56, wherein one or more loci in CXCL11 promoter for ZFA and gRNA are contacted utilizing the composition.

[0094] Feature 80. The composition of feature 79, wherein at least one enzyme includes TDG (G / T mismatch-specific thymine DNA glycosylase) and / or Tet1, Tet2 or Tet3 (ten-eleven translocation, TET, methylcytosine dioxygenase ); wherein the gene-specific fusion complex is a gene-specific demethylation fusion complex; and where an AA sequence of one or more enzyme(s) comprises any ZFA-Tet1CD, any ZFA-TDG construct, ZF6-Tet cat (SEQ ID NO: 3), ZF8-Tet1 cat (SEQ ID NO: 4), ZF10-Tet cat (SEQ ID NO: 5), ZF6-Tet mut (SEQ ID NO: 6), ZF5-TDG wt (SEQ ID NO: 20), ZF7-TDG wt (SEQ ID NO: 21), ZF9-TDG wt (SEQ ID NO: 22), or ZF9-TDGmut (N151A) (SEQ ID NO: 23).

[0095] In yet other embodiments, a method of making a composition is provided comprising a composition discussed in any of the features above and optionally adding one or more pharmaceutically acceptable excipients. Many of the compositions are pharmaceutically effective as a composition or “drug in capsule”, without needing excipients. As will be discussed in more detail below, the technology disclosed herein is surprisingly effective and solves many of the larger problems introduced above.

[0096] Other implementations are also described and recited herein. These and other features and advantages will be apparent from a reading of the following detailed description and a review of the associated drawings. It is to be understood that both the foregoing general description and the following detailed description are explanatory only and are not restrictive of aspects as claimed. BRIEF DESCRIPTION OF THE DRAWINGS

[0097] Solely for the purpose of illustration, certain embodiments of the present invention are explained using examples in the drawings described below. It should be understood, however, that the invention is not limited to the precise arrangements, dimensions, and configurations shown. In the figures:

[0098] FIG.1 shows reports on disease associated promoter DNA hypermethylation in noncancer diseases.

[0099] FIG.2 shows comparisons of three DNA-binding domains for vector-free demethylation.

[0100] FIG.3 shows an example summary of the characteristics of three vectors.

[0101] FIG.4 shows selected publications for targeted DNA demethylation / gene reactivation with research materials and year.

[0102] FIG.5A shows data showing vector-free epigenetic editing of CXCL11 in vitro. FIG.5B shows the 3T3 cells' transcriptional response, measured by real-time PCR. FIG.5C shows the MLE12 cells' transcriptional response, measured by real-time PCR. FIG. 5D shows CXCL11 protein secreted from cells following vector-free epigenetic editing, versus other conditions, measured by ELISA. FIG.5E shows cumulative target gene expression secreted versus conditions. FIG.5F and FIG.5G each show each show data from vector-free intra-airway in vivo epigenetic editing.

[0103] FIG.6A shows the site-specific DNA demethylation using ZFA-TETs fusion protein.

[0104] FIG.6B shows dCas9-TET based site-specific demethylation.

[0105] FIG.7A shows a diagram of ZFA-Tet1CD (left) and a diagram of dCAS9- Tet1CD (right). FIG.7B shows sequence-accurate to-scale 3D rendering of ZFA-TDGCD and dCAS9-TDGCD bound to a target gene promoter.

[0106] FIG.8A shows an example diagram of how six ZFA's were fused with Tet1CD or TDG (three each) or mutant controls, purified as recombinant proteins and administered to cells in the in vitro experiments or to mice in the in vivo experiments.

[0107] FIG.8B shows an example mCxcl11 promoter map and targeting loci.

[0108] FIG.8C shows an example for pre-screening of the targeting loci, 3T3 cells were transduced with lentiviral constructs encoding the 3 ZFA-Tet1CD and 3 ZFA-TDG fusions (or mutant controls) and tested for DNA methylation and IFNγ-stimulated expression.

[0109] FIG.8D shows comparative maps of the ZFA- and dCAS9- based fusion chimeras (e.g., structure and size comparison of ZFA-TDG and ZFA-TET1 constructs).

[0110] FIG.9A shows effects of ZFA-fused demethylases (in vitro).

[0111] FIG.9B shows effects (in vitro) of dCAS9 -fused demethylas

[0112] FIG.9C shows effects (in vitro) in control regions.

[0113] FIG.9D shows a synergistic action comparison (ZFA and dCAS9, in vitro).

[0114] FIG.9E shows a duration study (in vitro).

[0115] FIG.10A (in vitro) and FIG.10B (in vivo) show primary mouse lung epithelial cells after treatment with dCAS9-based demethylase proteins, vs catalytically inactive control; demonstrating intranuclear staining for the constructs 24 hours after the last administration in vitro (FIG.10A) or in vivo (FIG.10B, FIG.10C). In FIG.10C (in vivo), ZFA- 6xHis-demethylases are left panels; untreated is third from left panel, and ZFA-6xHis- demethylases with anti-histone-alexa467 are right panel.

[0116] FIG.11A, FIG.11B, FIG.11C, and FIG.11D show in vivo effects of vector- free epigenetic editors administered via the airways; results of in vivo administration of the ZFA- and dCAS9 / gRNA- demethylase proteins.

[0117] FIG.12 shows an example map of dCas9-TDG wt / SCP (11435 bp).

[0118] FIG.13 shows an example map of dCas9-Tet1 cat / SCP (12329 bp).

[0119] FIG.14 shows an example map of ZF5 TDG wt (6903 bp).

[0120] FIG.15 shows an example map of ZF6 Tet1 Cat (7782 bp).

[0121] FIG.16 shows an example map of ZF6 Tet1 mut (7782 bp).

[0122] FIG.17 shows an example map of ZF7 TDG wt (6891 bp).

[0123] FIG.18 shows an example map of ZF8 Tet1 cat (7782 bp).

[0124] FIG.19 shows an example map of ZF9 TDG mut (N151A) (6891 bp).

[0125] FIG.20 shows an example map of ZF9 TDG wt (6891 bp).

[0126] FIG.21 shows an example map of ZF10 Tet1 cat (7781 bp).

[0127] FIG.22A shows gel staining for SDS-PAGE of lysates.

[0128] FIG.22B shows a summary of the chaperone plasmids constructed with the bacterial expression vector pAR3.

[0129] FIG.23 shows features of Protein A and Protein B; a more noteworthy difference in features was that Protein A had a helix-based secondary structure, while Protein B had a higher proportion of strands; the expected secondary structures of the Protein A (544 amino acids) and Protein B (841 amino acids) are depicted.

[0130] FIG.24 shows SDS-PAGE analysis of Protein A constructed pET30a expression in E.coli BL21(DE3). Lane 1: Supernatant of cell lysate without induction, Lane 2: Supernatant of cell lysate with induction for 16 hrs at 15°C, Lane 3: Supernatant of cell lysate with induction for 3 hrs at 37°C, Lane 4: Pellet of cell lysate without induction, Lane 5: Pellet of cell lysate with induction for 16 hrs at 15°C, Lane 6: Pellet of cell lysate with induction for 3 hrs at 37°C. An arrow indicates an expected molecular weight of the recombinant protein.

[0131] For FIG.25A, FIG.25B, and FIG.25C, the data were analyzed using one- way analysis of variance (ANOVA) followed by LSD, Bonferroni and Holm multiple comparison tests using Js-STAR (KISNET; (kisnet.or.jp / nappa / software / star / ). Western blotting of the supernatants of protein lysates from chaperone vectors co-transformed E. coli BL21(DE3) with induction at 37°C for 3 hrs, using proteins (FIG.25A: Protein A and FIG. 25B: Protein B) and His-tag specific first antibodies. A lysate from naïve (non-transformed) BL(DE31) served as control. Gel staining of SDS-PAGE using the same sample at the same time (lower panels) shows the consistency of sample loading. Arrows indicate expected molecular weights of the recombinant proteins. In FIG.25C bar graphs represent ratio of band intensity to control in Protein A (left) and Protein B (right) western blotting (n = 4). *P < 0.05. **P < 0.01. Bar graphs are Mean±SEM..

[0132] As shown in FIG.26A and FIG.26B, proteins with >25% helix in their secondary structure (Protein C and D) showed better compatibility to chaperones pGro7 and pTf16. Western blotting of the supernatants of protein lysates from E. coli co-transformedwith chaperones and additional five proteins. FIG.26A: lysates from five proteins (Protein C- G) and chaperone vectors co-transformed BL21(DE3) E. coli were run on SDS-PAGE, and then blotted with anti-His-tag first antibody. FIG.26B: for each chaperone vector, the relationship between the helix content of the produced protein and the production efficiency is summarized in a graph. In the line graphs, the ratio of the western blot band intensity to control (non-transformed BL21) for each chaperone is plotted in the Y-axis and the helix contents of protein A-G is the X-axis. (n=3); Mean±SEM. FIG.26C: a summary plot integrating the efficiency of chaperones and helix content of all substrates. Purification of proteins in FIG.26A and FIG.25A, FIG.25B, and FIG.25C was subjectively scored blindly by a separate investigator on a scale of 1 to 5, 5 being the highest gel band intensity in the group and 1 being the lowest; ‘0’ was used when no band was visible. These scores were assembled into a data matrix and plotted as line charts with color coding, or hatching / shapes for each chaperon and with area under the curve indication. These results summarized in FIG.26C indicate that the helix content of the protein may be a more critical factor in predicting chaperone success.

[0133] A comparison of protein expression efficiencies between two conditions (3 hrs 37°C and 20 hrs 22.5°C) for supernatants from chaperone vector co-transformed E.coli BL21(DE3) is provided. FIG.27A shows pTf16 / Protein A co-transformed. FIG.27B shows pKJE7 / Protein B co-transformed. Arrows indicate expected molecular weights of the recombinant proteins. Bar graphs represent ratio of band intensity to 37°C 3hrs culture from densitometric analysis (n = 3). **P < 0.01. In the bar graphs, means are shown; error bars represent SEM. An unpaired, two-tailed Student's t-test was applied for FIG.27A and FIG.27B data using Excel in Office 365 (Microsoft).

[0134] Any / all trademarks mentioned or shown herein are used in fair use. It should be understood that while different depictions and / or numbers / numbering are / is sometimes used in some of the figures above to describe different embodiments and different aspects of the technology, any depiction and / or number from any figure can be inter-combined with a depiction or numbered aspect from any other figures. All trademarks, images, likenesses, words, and depictions in the drawings and the disclosure are plainly in fair use and are provided solely for the purposes of illustration of the invention in view of an urgent need to treat subjects as further discussed in detail below. DETAILED DESCRIPTION OF THE INVENTION

[0135] The subject innovation is now described in some instances, when necessary, with reference to the drawings. In the following description, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the present invention. It may be evident, however, that the present invention may be practicedwithout these specific details. In other instances, well-known structures, methods, and devices are shown in block diagram form or with illustrations in order to facilitate describing the present invention. It is to be appreciated that certain aspects, modes, embodiments, variations and features of the invention are described below in various levels of detail in order to provide a substantial understanding of the present invention. DEFINITIONS

[0136] For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims, are provided below. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Unless otherwise defined, 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. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided within the specification shall prevail. In general, chemical terminology is found in the International Union of Pure and Applied Chemistry GoldBook. This disclosure is purposefully in commonly understood words, known to a person of skill in the art, but Merriam-Webster’s Online Dictionary is used, when appropriate, for terms not specifically demonstrated herein or not known in the art.

[0137] As used in this specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the content clearly dictates otherwise. For example, reference to "a cell" includes a combination of two or more cells, and the like.

[0138] As used herein, the term "approximately" or "about" in reference to a value or parameter are generally taken to include numbers that fall within a range of 5%, 10%, 15%, or 20% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value). As used herein, reference to "approximately" or "about" a value or parameter includes (and describes) embodiments that are directed to that value or parameter. For example, description referring to "about X" includes description of "X".

[0139] As used herein, the term “or” means “and / or.” The term "and / or" as used in a phrase such as "A and / or B" herein is intended to include both A and B; A or B; A (alone); and B (alone). Likewise, the term "and / or" as used in a phrase such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0140] As used herein, the term "comprising" means that other elements can also be present in addition to the defined elements presented. The use of "comprising" indicatesinclusion rather than limitation. The term “including” can be interchanged with “comprising”.

[0141] The term "consisting of" refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

[0142] As used herein the term "consisting essentially of" refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention. For example, a pharmaceutical formulation can consist essentially of an active agent and another active ingredient, meaning that a variety of excipients or other additives can be present in the formulation, but no other active pharmaceutical ingredient (API) is present in the formulation, except in formulations wherein an intended synergistic effect is demonstrated by the claims or examples herein (e.g., a formulation consisting essentially of one, two, or three ingredients or pharmaceutically acceptable salts thereof). In another example, a pharmaceutical formulation can consist essentially of an active agent and another ingredient, meaning that the formulation is provided in the form of a nasal spray, an inhaled formulation, an orally administered formulation, or an injection formulation, each of which is tailored for a fast-acting agent, therapeutic agent, or antidote but not tailored for long-term administration (e.g., as a simultaneous treatment). In another example, in the case of a preventative therapy to purposefully prevent a cancer, the opposite, long-term combination therapy, can be referred to with “consisting essentially of”. This example could be applied to, for example, a cancer patient who is best treated by an evolving combination therapy. In another example, the term “consisting essentially of” can also be exemplified by plain language provided in the claims.

[0143] The term "statistically significant" or "significantly" refers to statistical significance and generally means a two-standard deviation (2SD) or greater difference.

[0144] As used herein, the term "subject" refers to a mammal, including but not limited to a dog, cat, horse, cow, pig, sheep, goat, rodent, or primate. Subjects can be house pets (e.g., dogs, cats), agricultural stock animals (e.g., cows, horses, pigs, chickens, etc.), laboratory animals (e.g., mice, rats, rabbits, etc.), but are not so limited. Subjects particularly include human subjects in urgent treatment as described herein. The human subject may be a pediatric, adult, or a geriatric subject. The human subject may be of any sex.

[0145] As used herein, the terms "effective amount", “therapeutically effective amount”, and “pharmaceutically effective amount” include an amount sufficient to prevent or ameliorate a manifestation of or a suspected manifestation of a medical condition, such as a cancer or a disease. The manifestation can be a sign or symptom or otherwise. It will be appreciated that there will be many ways known in the art to determine the effective amount for a given application. For example, the pharmacological methods for dosage determinationmay be used in the therapeutic context. In the context of therapeutic or prophylactic applications, the amount of a composition administered to the subject will depend on the type and severity of the medical condition and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. It will also depend on the degree, severity, and type of medical condition. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. Examples of other factors can be route of administration and length of administration(s). The compositions can also be administered in combination with one or more additional therapeutic compounds.

[0146] The term “protecting group” refers to a group of atoms that, when attached to a reactive functional group in a molecule, mask, reduce or prevent the reactivity of the functional group. Typically, a protecting group may be selectively removed as desired during the course of a synthesis. Examples of protecting groups can be found in Greene and Wuts, Protective Groups in Organic Chemistry, 3rdEd. and in Harrison, et al., Compendium of Synthetic Organic Methods, Vols.1-8. Examples of representative nitrogen protecting groups include, but are not limited to, formyl, acetyl, trifluoroacetyl, benzyl, benzyloxycarbonyl (“CBZ”), tert-butoxycarbonyl (“Boc”), trimethylsilyl (“TMS”), 2- trimethylsilyl-ethanesulfonyl (“TES”), trityl and substituted trityl groups, allyloxycarbonyl, 9- fluorenylmethyloxycarbonyl (“FMOC”), nitro-veratryloxycarbonyl (“NVOC”) and the like. Examples of representative hydroxylprotecting groups include, but are not limited to, those where the hydroxyl group is either acylated (esterified) or alkylated such as benzyl and trityl ethers, as well as alkyl ethers, tetrahydropyranyl ethers, trialkylsilyl ethers (e.g., TMS or TIPS groups), glycol ethers, such as ethylene glycol and propylene glycol derivatives and allyl ethers.

[0147] As used herein, a therapeutic that “prevents” a disorder or condition refers to a compound (or combination) that, in a statistical sample, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset or reduces the severity of one or more symptoms of the disorder or condition relative to the untreated control sample. For example, a compound (or combination) that prevents epilepsy may reduce the frequency of seizures and / or reduce the severity of seizures.

[0148] The term “treating” includes prophylactic and / or therapeutic treatments. The term “prophylactic or therapeutic” treatment is art-recognized and includes administration to the host of one or more of the subject compositions. If it is administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the host animal) then the treatment is prophylactic (i.e., it protects the host against developing the unwanted condition), whereas if it is administered after manifestation of the unwanted condition, the treatment is therapeutic, (i.e., it is intended to diminish, ameliorate, or stabilizethe existing unwanted condition or side effects thereof).

[0149] The phrases “conjoint administration” and “administered conjointly” refer to any form of administration of two or more different therapeutic compounds such that the second compound is administered while the previously administered therapeutic compound is still effective in the body (e.g., the two compounds are simultaneously effective in the patient, which may include synergistic effects of the two compounds). For example, the different therapeutic compounds can be administered either in the same formulation or in a separate formulation, either concomitantly or sequentially. In certain embodiments, the different therapeutic compounds can be administered at the same time, within one minute, 2 minutes, 4 minutes, 6 minutes, 10 minutes, 30 minutes, or an hour or 90 minutes of one another. In some embodiments, the different therapeutic compounds can be administered within 1 year of one another. Thus, an individual who receives such treatment can benefit from a combined effect of different therapeutic compounds.

[0150] As used herein, the terms “treat,” “treatment,” “treating,” or “amelioration” when used in reference to a disease, disorder, or medical condition, refer to therapeutic treatments for a condition, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a symptom or condition. The term “treating” includes reducing or alleviating at least one adverse effect or symptom of a condition. Treatment is generally “effective” if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective” if the progression of a condition is reduced or halted. That is, “treatment” includes not just the improvement of symptoms or markers, but also a cessation or at least slowing of progress or worsening of symptoms that would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), sign(s), diminishment of extent of the deficit, stabilized (i.e., not worsening) state of a symptom or condition, delay or slowing of onset of symptoms or indications, and an increased lifespan as compared to that expected in the absence of treatment.

[0151] As used herein, the term "long-term" administration means that the therapeutic agent or drug is administered for a period of at least 12 weeks. This includes that the therapeutic agent, combination, or drug is administered such that it is effective over, or for, a period of at least 12 weeks and does not necessarily imply that the administration itself takes place for 12 weeks, e.g., if sustained release compositions or long-acting therapeutic agent or drug is used. Thus, the subject is treated for a period of at least 12 weeks. In many cases, long-term administration is for at least 4, 5, 6, 7, 8, 9 months or more, or for at least 1, 2, 3, 5, 7 or 10 years, or more.

[0152] The administration of the compositions contemplated herein may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion,transfusion, implantation, application (e.g., topical, otic, or ocular), or transplantation. Administration can be accomplished by an implant. In some embodiments, compositions are administered parenterally. The phrases “parenteral administration” and “administered parenterally” as used herein refers to modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravascular, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intratumoral, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion. In one embodiment, the compositions contemplated herein are administered to a subject by direct injection into an artery, vein, lymph node, or organ (e.g., heart, muscle, organ).

[0153] The terms: “decrease”, “reduced”, “reduction”, or “inhibit” are all used herein to mean a decrease by a statistically significant amount. In some embodiments, “reduce,” “reduction" or “decrease" or “inhibit” typically means a decrease by at least 10% as compared to a reference level (e.g., the absence of a given treatment or agent) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% , or more. As used herein, “reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level. “Complete inhibition” is a 100% inhibition as compared to a reference level. A decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder.

[0154] The term “prodrug” is intended to encompass compounds which, under physiologic conditions, are converted into the therapeutically active agents of the present invention (e.g., any compound selected from this disclosure). A common method for making a prodrug is to include one or more selected moieties which are hydrolyzed under physiologic conditions to reveal the desired molecule. In other embodiments, the prodrug is converted by an enzymatic activity of the host animal. For example, esters or carbonates (e.g., esters or carbonates of alcohols or carboxylic acids) are preferred prodrugs of the present invention. In certain embodiments, some or all of the compounds selected from this disclosure in a formulation can be replaced with the corresponding suitable prodrug, e.g., wherein a hydroxyl in the parent compound is presented as an ester or a carbonate or carboxylic acid present in the parent compound is presented as an ester. In some embodiments, a “prodrug” is made by using an absorbing particle that subsequently releases an active form after administration.

[0155] In some embodiments, the decrease in the one or more signs or symptoms isevaluated according to the DSM-5. In some embodiments, signs are observed or measured by a health care provider. Symptoms can be reported by the subject. In some embodiments, the decrease of signs or symptoms occurs in less than about 120 minutes, 90 minutes, less than about 60 minutes, less than about 30 minutes, less than about 15 minutes, less than about 10 minutes, or less than about 5 minutes, or less than about 3 minutes, or less than about 1 minute. In some embodiments, the decrease of signs or symptoms occurs in less than 1 day, less than 1 week, less than 1 month, or in less than 1 year.

[0156] The terms “increased”, “increase”, “enhance”, or “activate” are all used herein to mean an increase by a statically significant amount. In some embodiments, the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level. In the context of a marker or symptom, a “increase” is a statistically significant increase in such level.

[0157] As used herein, an agent or a therapeutic agent provided to a subject and suspected to be or involved in a treatment can be a small molecule less than 1000 MW or a large molecule not less than 1000 MW including biologics, oligonucleotides, peptides, oligosaccharides, and larger molecules. Any of the therapeutic agents disclosed herein can be used as or in combination with small molecules and / or large molecules as discussed herein.

[0158] As used herein, a subject may or may not be aware of suffering from a cancer or a disease condition. A health care provider may suspect a disease or cancer or may have confirmed cancer or disease.

[0159] A subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment (e.g., a cancer or related disorder) or one or more complications related to such a condition, and optionally, but need not have already undergone treatment for a condition or the one or more complications related to the condition. Alternatively, a subject can also be one who has not been previously diagnosed as having a condition in need of treatment or one or more complications related to such a condition. For example, a subject can be one who exhibits one or more risk factors for a condition, or one or more complications related to a condition or a subject who does not exhibit risk factors. A “subject in need” of treatment for a particular condition can be a subject having that condition, diagnosed as having that condition, suspected as having, or at risk ofdeveloping that condition. In another example, the subject has been brought into a treatment situation entirely without the subject’s knowledge and / or intent. For example, a subject can obviously be in need of treatment but not be responsive to a treatment, and as described herein the present methods and formulations may save the subject’s life. Pharmaceutical Compositions

[0160] The compositions and methods of the present invention may be utilized to prevent a need for other treatment, to provide benefit when other treatment(s) fail, or to treat an individual in need thereof. In some embodiments, the individual is suspected of needing treatment. In certain embodiments, the individual is a mammal such as a human, or a non- human mammal. When administered to an animal, such as a human, the composition or the compound is preferably administered as a pharmaceutical composition comprising, for example, a compound of the invention and a pharmaceutically acceptable carrier. A compound can represent a combination therapy herein. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil, or injectable organic esters. In some embodiments, when such pharmaceutical compositions are for human administration, particularly for invasive routes of administration (i.e., routes, such as injection or implantation, that circumvent transport or diffusion through an epithelial barrier), the aqueous solution is pyrogen-free, or substantially pyrogen-free. The excipients can be chosen, for example, to effect delayed release of an agent or to selectively target one or more cells, tissues, or organs. The pharmaceutical composition can be in dosage unit form such as tablet, capsule (including sprinkle capsule and gelatin capsule), granule, lyophile for reconstitution, powder, solution, syrup, suppository, injection or the like. The composition can also be present in a transdermal delivery system, e.g., a skin patch. The composition can also be present in a solution suitable for topical administration, such as a lotion, cream, or ointment.

[0161] A pharmaceutically acceptable carrier can contain physiologically acceptable agents that act, for example, to stabilize, increase solubility or to increase the absorption of a compound such as a compound of the invention. Such physiologically acceptable agents include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients. The choice of a pharmaceutically acceptable carrier, including a physiologically acceptable agent, depends, for example, on the route of administration of the composition. The preparation or pharmaceutical composition can be a self-emulsifying drug delivery system or a self-micro emulsifying drug delivery system. The pharmaceutical composition (preparation) also can be a liposome or other polymer matrix, which can haveincorporated therein, for example, a compound of the invention. Liposomes, for example, which comprise phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer.

[0162] The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0163] The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible compositions employed in pharmaceutical formulations.

[0164] A pharmaceutical composition (preparation) can be administered to a subject by any of a number of routes of administration including, for example, orally, for example, drenches as in aqueous or non-aqueous solutions or suspensions, tablets, capsules including sprinkle capsules and gelatin capsules, boluses, powders, granules, pastes for application to the tongue; absorption through the oral mucosa (e.g., sublingually); subcutaneously; transdermal administration (for example as a patch applied to the skin); and topically (for example, as a cream, ointment or spray applied to the skin). The compound may also be formulated for inhalation. Inhalation can include inhalation of a liquid (droplets or aerosol). Inhalation can include a micronized powder adhered to carrier particles or can be without carrier particles. In certain embodiments, a compound may be simply dissolved or suspended in sterile water. Details of appropriate routes of administration and compositions suitable for same can be found in, for example, U.S. Patent Nos.6,110,973, 5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970 and 4,172,896, as well as in patents citedtherein.

[0165] The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 1 percent to about ninety-nine percent of active ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 10 percent to about 30 percent.

[0166] Methods of preparing these formulations or compositions include the step of bringing into association an active compound, such as a compound (or combination therapy) of the invention, with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0167] Formulations of the invention suitable for oral administration may be in the form of capsules (including sprinkle capsules and gelatin capsules), cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), lyophile, powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and / or as mouth washes and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient. Compositions or compounds may also be administered as a bolus, electuary or paste.

[0168] To prepare solid dosage forms for oral administration (capsules, including sprinkle capsules and gelatin capsules), tablets, pills, dragées, powders, granules and the like, the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and / or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and / or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate,solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; (10) complexing agents, such as, modified and unmodified cyclodextrins; and (11) coloring agents. In the case of capsules (including sprinkle capsules and gelatin capsules), tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

[0169] A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropyl methyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

[0170] The tablets, and other solid dosage forms of the pharmaceutical compositions, such as dragées, capsules (including sprinkle capsules and gelatin capsules), pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropyl methyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and / or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymers and waxes. The active ingredient can also be in micro- encapsulated form, if appropriate, with one or more of the above-described excipients.

[0171] Liquid dosage forms useful for oral administration include pharmaceutically acceptable emulsions, lyophiles for reconstitution, micro-emulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, cyclodextrins and derivatives thereof, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

[0172] Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

[0173] Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

[0174] Dosage forms for the topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that may be required.

[0175] The ointments, pastes, creams and gels may contain, in addition to an active compound, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

[0176] Powders and sprays can contain, in addition to an active compound, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

[0177] Transdermal patches have the added advantage of providing controlled delivery of a compound of the present invention to the body. Such dosage forms can be made by dissolving or dispersing the active compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the compound in a polymer matrix or gel.

[0178] The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intraocular (such as intravitreal), intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion. Pharmaceutical compositions suitable for parenteral administration comprise one or more active compounds in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render theformulation isotonic with the blood of the intended recipient or suspending or thickening agents.

[0179] Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0180] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.

[0181] In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.

[0182] Injectable depot forms are made by forming microencapsulated matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissue.

[0183] For use in the methods of this invention, active compounds can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.

[0184] Methods of introduction may also be provided by rechargeable or biodegradable devices. Various slow-release polymeric devices have been developed and tested in vivo in recent years for the controlled delivery of drugs, including proteinaceous biopharmaceuticals. A variety of biocompatible polymers (including hydrogels), including both biodegradable and non-degradable polymers, can be used to form an implant for the sustained release of a compound at a particular target site.

[0185] Actual dosage levels of the active ingredients in the pharmaceutical compositions may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

[0186] The selected dosage level will depend upon a variety of factors including the activity of the particular compound or combination of compounds employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound(s) being employed, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular compound(s) employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0187] A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the therapeutically effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the pharmaceutical composition or compound at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. By “therapeutically effective amount” is meant the concentration of a compound that is sufficient to elicit the desired therapeutic effect. It is generally understood that the effective amount of the compound will vary according to the weight, sex, age, and medical history of the subject. Other factors which influence the effective amount may include, but are not limited to, the severity of the patient's condition, the disorder being treated, the stability of the compound, and, if desired, another type of therapeutic agent being administered with the compound of the invention. A larger total dose can be delivered by multiple administrations of the agent. Methods to determine efficacy and dosage are known to those skilled in the art. See, e.g., Isselbacher, et al., (1996).

[0188] In general, a suitable daily dose of an active compound used in the compositions and methods of the invention will be that amount of the compound that is thelowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.

[0189] If desired, the effective daily dose of the active compound may be administered as one, two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. In certain embodiments of the present invention, the active compound may be administered two or three times daily. In other embodiments, the active compound will be administered once daily.

[0190] The subject or patient receiving this treatment is any animal in need, including primates, in particular humans; and other mammals such as equines bovine, porcine, sheep, feline, and canine; bird such as poultry; and pets in general.

[0191] In certain embodiments, compounds of the invention may be used alone or conjointly administered with another type of therapeutic agent.

[0192] The present disclosure includes the use of pharmaceutically acceptable salts of compounds of the invention in the compositions and methods of the present invention. In certain embodiments, contemplated salts of the invention include, but are not limited to, alkyl, dialkyl, trialkyl or tetra-alkyl ammonium salts. In certain embodiments, contemplated salts of the invention include, but are not limited to, L-arginine, benenthamine, benzathine, betaine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2-(diethylamino) ethanol, ethanolamine, ethylenediamine, N-methylglucamine, hydrabamine, 1H-imidazole, lithium, L-lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine, potassium, 1-(2- hydroxyethyl)pyrrolidine, sodium, triethanolamine, tromethamine, and zinc salts. In certain embodiments, contemplated salts of the invention include, but are not limited to, Na, Ca, K, Mg, Zn or other metal salts. In certain embodiments, contemplated salts of the invention include, but are not limited to, 1-hydroxy-2-naphthoic acid, 2,2-dichloroacetic acid, 2- hydroxyethanesulfonic acid, 2-oxoglutaric acid, 4-acetamidobenzoic acid, 4-aminosalicylic acid, acetic acid, adipic acid, l-ascorbic acid, l-aspartic acid, benzenesulfonic acid, benzoic acid, (+)-camphoric acid, (+)-camphor-10-sulfonic acid, capric acid (decanoic acid), caproic acid (hexanoic acid), caprylic acid (octanoic acid), carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, d-glucoheptonic acid, d-gluconic acid, d-glucuronic acid, glutamic acid, glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, l-malic acid, malonic acid, mandelic acid, methanesulfonic acid , naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, nicotinic acid, nitric acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, proprionic acid, l-pyroglutamic acid, salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, l-tartaric acid,thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, and undecylenic acid salts. In some embodiments, an N-H in elraglusib can become charged and a corresponding salt formed at or near the charge.

[0193] The pharmaceutically acceptable acid addition salts can also exist as various solvates, such as with water, methanol, ethanol, dimethylformamide, and the like. Mixtures of such solvates can also be prepared. The source of such solvate can be from the solvent of crystallization, inherent in the solvent of preparation or crystallization, or adventitious to such solvent. The invention contemplates polymorphs, cocrystals, and amorphous forms of all substances discussed herein. As discussed above, solvates and / or hydrates can be formed by, for example, a slow evaporation whereby water and / or solvent remain hydrogen bonded with OH groups in the molecule. The formation of a solvate / hydrate can be quickly confirmed after the evaporation by using attenuated total reflectance Fourier transform infra- red spectroscopy wherein the solid solvate / hydrate is directly placed on the instrument and the subsequent IR spectrum is compared to the IR spectrum of the solid non-solvate, non- hydrate.

[0194] Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

[0195] Examples of pharmaceutically acceptable antioxidants include: (1) water- soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal-chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[0196] As discussed above, unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art to which this disclosure belongs. It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims. Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy; The Encyclopedia of Molecular Cell Biology and Molecular Medicine; Molecular Biology and Biotechnology: a Comprehensive Desk Reference; Immunology; Janeway's Immunobiology; Lewin's Genes XI; Molecular Cloning: A Laboratory Manual.; Basic Methods in Molecular Biology; Laboratory Methods in Enzymology; CurrentProtocols in Molecular Biology (CPMB); Current Protocols in Protein Science (CPPS); and Current Protocols in Immunology (CPI).

[0197] In the embodiments discussed and in any of the aspects, the disclosure described herein does not directly concern a process for cloning human beings, processes for modifying the germ line genetic identity of human beings, uses of human embryos for industrial or commercial purposes or processes for modifying the genetic identity of animals which are likely to cause them suffering without any substantial medical benefit to man or animal, and also animals resulting from such processes.

[0198] Other terms are defined herein within the description of the various aspects of the invention. Unless otherwise defined, scientific and technical terms used with this application shall have the meanings commonly understood by persons having ordinary skill in the biomedical art. This invention is not limited to the method, protocols, reagents, etc., described herein and can vary. IN VIVO INTRA-AIRWAY VECTOR-FREE EPIGENETIC EDITING

[0199] The exciting paradigm of epigenetics that 'genes are not your destiny' has taken a novel turn with the development of epigenetic editing to selectively modulate the epigenetic status (promoter methylation) and thus transcription of genes. This methodology can resolve two major challenges in health research: A) the inability to test causality for exposure-related methylation changes that merely associate exposures with disease, and B) the need for novel therapeutic strategies to reverse the epigenetic alterations.

[0200] Aberrant DNA hypermethylation in key genes is well documented in a number of diseases; however, the mechanistic role and therapeutic applicability remain unclear due to a lack of tools. Here we apply epigenetic editing (in the direction of de-methylation) to address this issue in a transformative approach that overcomes the following key challenge. The need for viral vectors or similar genome integration steps for transduction of epigenetically acting constructs that has been suiting the in vitro epigenetic editing studies has hindered the translation to humans and is limiting experimentation with immunoregulatory genes, especially in vivo, because of related side-effects.

[0201] Our research group was among the first (simultaneously with two other unaffiliated groups) to demonstrate gene-specific DNA demethylation[1-4], which, unlike overexpression or treatment with exogenous product, epigenetically restores (or enhances for partially silenced genes) adaptive, situation-appropriate responsiveness of intrinsic transcription, heritable in a cell population for a prolonged effect. These prior publications used NOS2 gene as a target, which we no longer use, and employed a vectored, transduction-based approach which we also no longer use.

[0202] Here we describe the use of ‘custom-designed’ fusion complexes of demethylase enzymes (vs. controls catalytically inactivated by point mutation) with sequence-specific DNA binding domains (DBD) either a zinc finger protein array (ZFA) or a CRISPR-derived system where guide RNAs (gRNA) are tethered via dCAS9. Two enzymes, putatively functioning as DNA demethylases, are employed in combination: TDG and Tet1, which is one of the aspects of innovation (this combination has not been published before). These fusion constructs are then obtained as affinity- purified protein 'biologics' following recombinant expression in an E. coli or mammalian (HEK) systems, thus abrogating the need for viral or other vectors, which is the key aspect of the innovation. The E. coli system requires the use of molecular chaperons to assure proper folding of the protein, which we described in [5] without disclosing the purpose of the protein we were expressing.

[0203] This approach can broadly serve a dual purpose: A) makes a translational breakthrough advance for 'epigenetic editing', and B) serves to understand and test the mechanistic importance of methylation changes in a specific gene. FIG.8B shows an example of targeting loci in the CXCL11 promoter. The closer to TSS the more effective the construct will be. Constructs should not compete with TF’s. CXCL11 is well targetable by ZFA, especially in vicinity of TSS, and reasonably targetable with gRNAs.

[0204] A specific prototypical target employed here (one of the aspects of innovation) is the gene encoding CXCL11 (I-TAC) a chemokine postulated in prior literature to be protective against lung fibrosis. This is a secreted messenger molecule produced by certain cells in the lung and elsewhere. It is not known on what cells it acts and how it affect the course of the fibrosis besides it's documented action as a T-cell chemoattractant, but its abundance was linked to protective effect in humans [6] and in mice [7] leading us to hypothesize that creating an abundance of this protein by epigenetic editing can be therapeutic (no data yet). If true, this can potentially be used for various forms of lung fibrosis, from IPF to post-COVID fibrosis and could represent a potentially patentable aspect of the innovation. While the ZFA-based constructs have a natural ability to get into the nuclei of recipient cells, delivering fused enzymatic payload [8, 9], dCAS9 as a protein has not been shown to easily permeate into cell nuclei. This led to the development by others of various assistive sequences. We are employing the Supercharged Peptide (SCP) which has been shown by others to help dCAS9- constructs internalize into cells [10, 11].

[0205] An example aspect of our innovation is that we use it for epigenetic editing, not gene (nuclease) editing as reported by an original author. While the concept of epigenetic editing has advanced through the use of CRISPR-like binding domains (dCAS9) and multiple similar publications have appeared, the published works include in vitro, not in vivo work, and employ various viral or microvesicle / nanovesicle vectors rather than pure proteins as we detail here. Moreover, we believe we are the first to administer epigeneticallyacting proteins into live mice via a combination of intranasal instillations and a nebulized aerosol using a mesh-type nebulizer that minimizes the degradation of the administered protein; this intra-airway administration is another aspect of our innovation. As an overview, FIG.7A shows a diagram of ZFA-Tet1CD and a diagram of dCAS9-Tet1CD.

[0206] Example novelty aspect of this invention are: 1) the use of gene-specific demethylating fusion constructs as purified proteins, rather than encompassed in viral or other transfection / transduction vectors, which essentially gives a novel biologic class of drugs, and 2) employment of intra-airway administration (combination of intranasal instillation and nebulized aerosol) to target lung cells in vivo (have data in mice); the specific target used in the exemplary data is CXCL11 which is postulated to protect against lung fibrosis. Thus, our invention represents a novel treatment modality for fibrosis and, by extension, other diseases of the lung or other sites that are accessible to recombinant protein. Details of the invention which are novel include the combination of two different demethylase enzymes (Tet1 and TDG), targeting loci in CXCL11 promoter for ZFA and gRNA (sequences), fusion protein construct sequences, protocols including doses and time- kinetics for in vitro and in vivo use, as well as outcomes data (resultant methylation and expression effect via pyrosequencing and PCR). FIG.5A shows data showing vector-free epigenetic editing of CXCL11 in vitro. Six ZFA-TDG and ZFA-Tet1CD proteins were purified from E. coli and treated to naïve 3T3 or MLE12 cells, which were then stimulated with IFNγ vs. control (“CTR”): “wt”= working enzymes, “mut” = catalytically inactive controls, “buffer” – PBS only. FIG.5A shows pyrosequencing of CpG sites distal of TSS, proximal to it, and on the TSS of the target gene (see corresponding map). Note the control region “Upstream” which is >1000 bp away from the targeting area remained unaffected. FIG.5B shows the 3T3 cells’ transcriptional response, real-time PCR. FIG.5C shows the MLE12 cells’ transcriptional response, real-time PCR. FIG.5D shows ELISA target protein OD versus conditions. FIG.5E shows cumulative target gene expression secreted versus conditions.

[0207] The technology has been implemented and enabled. FIG.5F and FIG.5G show vector-free intra-airway in vivo epigenetic editing. Mice were treated to 6 affinity- purified ZFA-based constructs targeting CXCL11 administered via a combination of intranasal instillation + aerosol, followed by a transcriptional stimulus with IFNγ. FIG.5F shows ECs cells and FIG.5G shows EC-depleted cells were isolated using paramagnetic EpCAM beads, followed by pyrosequencing and real-time PCR. ***p<0.001, *p<0.05. FIG.9B shows illustrative In vitro vector-free epigenetic editing (effects) using dCAS9. dCAS9 fused with SCP and either TDG or Tet1CD were affinity purified as proteins, co- incubated with 6 gRNAs (vs. mock-transfected controls) and administered to cells in culture, followed next day by a transcriptional stimulus with IFNγ. *p<0.05. It is important to note that the custom fusion proteins that modify DNA methylation can be broadly used experimentallyand therapeutically to manipulate epigenetic status and thus transcriptional responsiveness of select genes. The scope of use can vary. TARGETED DNA DEMETHYLATION

[0208] Vectors, Effectors and Perspectives: Aberrant DNA hypermethylation at regulatory cis-elements of particular genes is seen in a plethora of pathological conditions including cardiovascular, neurological, immunological, gastrointestinal and renal diseases, as well as in cancer, diabetes and others. Thus, approaches for experimental and therapeutic DNA demethylation have a great potential to demonstrate mechanistic importance, and even causality of epigenetic alterations, and may open novel avenues to epigenetic cures. However, existing methods based on DNA methyltransferase inhibitors that elicit genome-wide demethylation are not suitable for treatment of diseases with specific epimutations and provide a limited experimental value. Therefore, gene-specific epigenetic editing is a critical approach for epigenetic re-activation of silenced genes. Site-specific demethylation can be achieved by utilizing sequence-dependent DNA-binding molecules such as zinc finger protein array (ZFA), transcription activator-like effector (TALE) and clustered regularly interspaced short palindromic repeat-associated dead Cas9 (CRISPR / dCas9). Synthetic proteins, where these DNA-binding domains are fused with the DNA demethylases such as ten-eleven translocation (Tet) and thymine DNA glycosylase (TDG) enzymes, successfully induced or enhanced transcriptional responsiveness at targeted loci. However, a number of challenges, including the dependence on transgenesis for delivery of the fusion constructs, remain issues to be solved. In this review, we detail current and potential approaches to gene-specific DNA demethylation as a novel epigenetic editing-based therapeutic strategy.

[0209] Introduction: the exciting paradigm of epigenetics, that ‘genes are not your destiny’, has taken a novel turn with the development of targeted molecular tools to selectively modulate the epigenetic status (e.g., promoter methylation) and thus transcription of genes. The ability to take control over the epigenome and ‘edit’ it for therapeutic and experimental benefit represents an enticing but complicated endeavor. This review focuses on the rapid progress of site-specific DNA demethylation, made possible by technological innovation since the 2010s, but also traces the footsteps of DNA demethylation research since its dawn in the 1980s. The article summarizes the current knowledge on the mechanisms of DNA demethylation and details how they are or can be exploited to re- activate epigenetically silenced genes. Going beyond an overview of ‘vectored’ delivery of epigenetic modifiers, the paper introduces the concept of vector-free epigenetically acting agents, with a view to future application in epigenetic medicine.

[0210] DNA Methylation as a Key Epigenetic Mechanism of Gene Function Control: DNA methylation that alters gene expression without a change in the DNA sequence is themost widely studied epigenetic mechanism involved in the pathogenesis of various diseases [12, 13]. DNA methylation is a covalent modification that occurs to cytosines in the context of cytosine-phosphate-guanosine (CpG) residues; when it occurs in key regulatory regions of the gene (e.g., promoter), methylation suppresses the expression of the gene

[0014] . Virtually all cells in an organism contain all the genes of that organism’s genome; epigenetic mechanisms are broadly involved in the silencing of ‘unused’ genes which underlies the difference in structure and function of cells [15, 16]. This silencing is a powerful and inheritable mechanism of gene function control

[0015] . DNA methylation is a key player in epigenetic silencing of transcription [14, 17]; conversely, lowered methylation of promoters or regulatory intragenic regions has been linked to enhanced transcription [16, 18, 19]. DNA methylation of CpG dinucleotides is catalyzed by DNMT3a and b, and DNMT1 maintains the methylation during semiconservative DNA replication

[0020] . In case this maintenance methylation fails for some reason, e.g., inhibition, depletion or nuclear exclusion of DNMT1, so-called “passive demethylation” occurs in dividing cells

[0021] . Passive demethylation is a relatively slow process and theoretically does not occur in terminally differentiated non- dividing cells

[0022] . Given these points, it is unlikely that passive demethylation plays a leading role in demethylation in most adult somatic cells that have completed mitosis

[0023] , although it plays an important role when genome wide DNMT inhibitors are employed therapeutically.

[0211] The first report on biological active DNA demethylation processes independent of cell replication in vertebrates was published in 1984

[0024] , in which HpaII site 611 base pairs upstream from the 5’ end of the of the chicken vitellogenin II gene were seen to exhibit estrogen-dependent demethylation independent of DNA synthesis, predicting the existence of a site-specific demethylase. However, no further characterization of this enzyme has been forthcoming. Numerous evidences of active demethylation have been reported since then [25-29]. Another common example of active demethylation is the demethylation of pluripotency genes in nuclear reprogramming. During normal development, genes related to pluripotency are constitutively silenced by histone and DNA methylation. For example, the proximal m5CpG of the oct4 gene regulatory site in thymocyte nuclei transplanted into Xenopus oocytes is immediately demethylated

[0030] . Demethylation usually occurs with changes in physiological cellular states. Examples include changes in cell differentiation, such as terminal differentiation, or when exposed to nuclear hormones, protein kinase C (PKC) activators, histone deacetylase inhibitors (HDACi) or changes in neural activity [31- 36]. These findings indicate that active demethylation is a widespread phenomenon in both dividing and differentiated cells.

[0212] Etiological Significance of DNA Methylation: In mammals, about 60–80% of the CpG sites in the genome are modified into 5mC

[0018] . CpGs outside promoter CpGislands are usually highly methylated, while those within promoter CpG islands are mostly unmethylated [37, 38]. However, in several diseases, the promoter CpG islands of genes ‘protective’ against the disease are found to be aberrantly methylated [39-41]. Epigenetic alterations in CpG islands and, more broadly, elsewhere across the epigenomic landscape have been increasingly reported, ranging from the most frequent examples, linking DNA hypermethylation to cellular carcinogenesis, to a plethora of cardiovascular, renal, hepatic, metabolic and infectious diseases and environmental exposures [42-48]. Thus, the scope of aberrant methylation in pathogenesis is becoming more and more elucidated. For example, aberrations of cell cycle checkpoints in cancer result in abnormal cell proliferation and chromosomal instability. Examples include abnormal methylation of the p53 gene [49-51], or hypermethylation of the checkpoint genes with Fork-head and Ring finger (CHFR) in cancer cells [52-60].

[0213] While methylation and transcription are usually inversely correlated, and this is typically true for promoter methylation, there are exceptional situations where an increase in methylation leads to an increase in transcription if this methylation change affects a key regulatory area of a special kind [61, 62], which can happen with enhancers, or where increased methylation inhibits binding of repressive factors or methylation-sensitive transcription factors. In general, however, selective demethylation of genes occurs during gene expression enhancement, but it is not certain to what extent demethylation is a primary factor versus a secondary consequence of enhanced transcription.

[0214] Moreover, DNA demethylation may not always be sufficient to increase transcription. For example, demethylation of the IL2 promoter is necessary but not sufficient for gene activation

[0063] . In this case, binding of the transcription factor Oct-1 to the promoter- enhancer region of the IL2 gene is a higher priority requirement for enhanced gene expression. When Oct-1 binds to the promoter-enhancer region as a result of additional stimulation, the IL2 gene becomes more sensitive to DNA demethylation and its gene expression is more highly upregulated. When the stimulus is removed, Oct-1 remains in the promoter-enhanced region, making the second and subsequent induction of gene expression faster and stronger

[0063] . Thus, priming of the locus occurs to prepare for subsequent external stimuli. The transcriptional result of gene demethylation is in this case dependent on the presence of a background stimulus: if present, the newly demethylated gene becomes intrinsically upregulated, otherwise it will ‘wait’ for an extrinsic trigger. This leads to an important potential benefit of epigenetic editing over traditional gene overexpression or a recombinant protein administration: demethylation opens the promoter to situation-appropriate, context-dependent intrinsic (or extrinsic, if desired) stimuli.

[0215] Aberrant hypermethylation occurs as a result of intrinsic deregulation or upon exposure to environmental toxicants; many environmental exposures have been linked toepigenomic effects [64, 65] leading to two major challenges in the field. First, while the epidemiologic data for association of exposures with epigenomic effects are strong, proof of causality for exposure-related methylation changes at specific loci has been impossible due to a lack of experimental tools to specifically revert these alterations. Second, there are no therapeutic agents that can gene-specifically reverse ‘harmful’ epigenetic changes and achieve epigenetic-based treatment. These challenges offer an attractive opportunity to employ DNA demethylation as a therapeutic strategy for diseases where DNA hypermethylation is involved. Until recently, only non-specific agents, such as cytidine analogs including 5-aza-2′-deoxycytidine, have been available for removing methyl groups from CpGs based on the inhibition of DNMTs, and they have been widely used to study the effects of demethylation on gene promoters [66-69]. However, these non-specific agents lead to global demethylation of CpGs, making it impossible to define the causal effects of specific CpG aberrations

[0070] . Following these experimental trials, active DNA demethylation by enzymatic activity has become the focus of attention as an experimental and potential future therapeutic strategy

[0071] , promoting interest in gene-specific (or site-specific) demethylation approaches

[0072] .

[0216] Biochemical Mechanisms of Active DNA Demethylation: It is increasingly recognized that active demethylation plays an important role in a wide variety of biological processes, and much attention and effort has been devoted to the elucidation of the mechanism of active demethylation, including the identification of the involved demethylase(s). DNA demethylase activity was noted for the first time in extracts of mouse erythroleukemia nuclei

[0073] . It was found that 5-methylcytosine (5mC) is finally replaced by C in a replication-independent manner, but this mechanism has not been elucidated in detail for a long time. Over the past years, several studies have proposed various possible mechanisms through which active DNA demethylation may take place. These include enzymatic removal of methyl groups at 5mC, a series of intermediary conversions, participation of the base excision repair (BER) and less well-known mechanisms.

[0217] Enzymatic Removal of the Methyl Group of 5mC: In the initial step of the envisioned DNA demethylation process, 5mC is converted to 5-hydroxymethyl cytosine (5hmC) by the addition of atomic oxygen; 5hmC is further oxidized to 5-formylcytosine (5fC) and finally 5-carboxylcytosine (5caC)

[0074] . This is an important step that allows for several alternative or duplicative methods to remake normal cytosine residues autonomously or in a DNA replication-dependent manner, ultimately removing the methyl mark [75-77]. The oxidized intermediates, 5fC and 5caC, are removed and can further regenerate unmethylated cytosine at the target site through the BER mechanism. The first half of the oxidation reactions are catalyzed by the ten-eleven translocation protein (TET) familyenzymes, while the second half, removal of 5fC and 5caC, is catalyzed by thymine DNA glycosylase (TDG)

[0078] .

[0218] The first member of TET family, TET1, was discovered in acute myeloid leukemia (AML) cells as a fusion partner for histone H3 Lys4 methyltransferase

[0079] . Subsequent in vitro studies have shown that the enzymatic activity of human TET1 includes the ability to hydrolyze 5mC

[0080] . TET protein can act on both fully methylated and hemimethylated DNA

[0081] . This ability of TETs to oxidize 5mC is particularly important in embryonic stem cells (ESCs), which must maintain the ability to self-renew and take on diverse 5mC patterns

[0082] . The TET protein is characterized by its distinctive core catalytic domain, and its isoforms are known to be cell type specific [83, 84]. Mammals encode three TET protein orthologues (TET1, TET2, and TET3)

[0085] . The three TET proteins have similar C-terminal catalytic domain-containing cysteine-rich (Cys) regions and a double-stranded b- helix (DSBH) fold which exhibits 2-oxoglutarate (2-OG)- and Fe(II)-dependent dioxygenase activity

[0086] .

[0219] BER Through Direct Excision of 5mC Converts: Following the sequential oxidation reactions described above, TDG initiates the removal of target bases to make abasic sites

[0087] . Subsequently, apyrimidinic acid (AP) lyase activity nicks the DNA backbone, producing 5′ phosphomonoesters and 3′ sugar phosphate residues. AP endonuclease then removes the 3′ sugar chain, leaving a single nucleotide gap that will eventually be filled by DNA repair polymerase or ligase

[0088] . Genetic and biochemical evidence suggested at first that this mechanism is used in plants

[0089] , whereas evidence supporting the existence of similar mechanisms in the vertebrates was less compelling. In 2000, a study using extracts from chick embryos suggested that this repair mechanism may contribute to DNA demethylation in vertebrates as well

[0090] . This study also demonstrated the existence of 5mC glycosylase activity against hemimethylated DNA in the extract and revealed that the enzyme responsible for this activity is a homologue of human TDG. Therefore, the 5mC glycosylase activity detected in this chicken embryo extract was assumed to be due to TDG. However, the excision activity of TDG against 5mC is only 1 / 30 to 1 / 40 of that against T / G mismatch, and TDG does not have the activity to cleave N- glycosidic bonds

[0091] . Thus, the details of the involvement of TDG in DNA demethylation needed to be further explored. Since TDG is known to act on hydroxymethylated cytosines as well as 5fC and 5caC via its specific recognition site, though not on 5mC [91-94], it suggests a reasonable concept of the demethylation process that entails starting with a conversion of 5mC to hmC / fC / caC by TETs, followed by TDG action. This is confirmed by the observation that, in TDG-deleted cells, an accumulation of 5fC / 5caC is observed

[0095] . Given that on average ~10% of mCs in mammals are hmCs (bisulfite-based methods, e.g., pyrosequencing, do not distinguish the two forms), this could explain the consistent but low-impact results of using TDG alone in epigenetic editing studies [1, 4]. Indeed, low levels of active, replication-independent DNA demethylation were seen to be mediated by TDG

[0095] .

[0220] The action of TDG as a demethylase, whether involving BER or not, is supported by epigenetic editing reports using solely this enzyme [1, 4]. Assessment of the relative input of TETs and TDG in demethylation can be complicated by the intrinsic presence of either enzyme in the nucleus. This has been addressed in a convincing demonstration that TET and TDG interact with each other in several ways in the demethylation process: TET1 and TDG interact physically, forming stable TET1–TDG complexes

[0096] . Moreover, TET1CD and TDG act in concert to release 5mC so that the excision of 5mC and 5hmC from DNA requires the catalytic activities of both TET1 and TDG; finally, TET stabilizes TDG activity, and their coordinated action engages BER in a way that avoids double strand breaks

[0097] . Hence, TDG is not simply a sequential follower of TET, as was previously considered.

[0221] This information about TDG is not inconsistent with its previously presumed role as a demethylase following the deamination of mC by the AID (activation-induced deaminase) / APOBEC (apolipoprotein B mRNA-editing enzyme complex) family of cytidine deaminases [80, 98]. Deamination-mediated demethylation could also involve the DNA damage response protein GADD45, or even methyl-binding domain protein MBD4 [30, 99, 100]. However, there is evidence that contradicts this premise [101, 102]. It therefore appears that deaminases are unlikely to be enzymatic effectors for epigenetic editing.

[0222] Potential for Enzymatic Demethylation without Excision and BER: Studies in embryonic cells allow the derivation of a mechanism in which initial conversion of mC to hmC by TET is followed by passive dilution where, as a result of replication, the unmodified C is regenerated [103, 104]. It remains unclear to what extent this occurs during epigenetic editing, but it is plausible in dividing cells. It has also been reported that DNMTs that normally methylate the cytosine can, in some conditions, have a reverse effect, directly transforming mC to C

[0105] .

[0223] AlkB homologs (ALKBH) are the other class (together with TET) of the Fe(II) / 2-Oxoglutarate (2OG)-dependent dioxygenase superfamily of enzymes that have demethylating activity, and unlike TET they are capable of directly demethylating some of their substrates

[0106] . Besides their action on 3mC and 4mC, ALKBH act on 5mC

[0107] , but while the outcome of the reaction for 3mC is unmethylated C, for 5mC it stops at 5caC, which then likely becomes the substrate for TDG.

[0224] Limited evidence suggests that 5caC may be converted into C directly and without excision: it was shown that both bacterial and mammalian C5-MTases can catalyze the direct decarboxylation of 5caC (but not of 5DC), yielding unmodified cytosine in the DNA

[0108] . It is possible that future studies will elaborate further on active enzymatic DNAdemethylation. The attractiveness of this or another direct enzymatic conversion mechanism that avoids base excision lies in the lower risk of strand breaks and potential mutagenesis, which may confound translational approaches in the future.

[0225] Biological Effects of De-Repression of Epigenetically Silenced Genes: De- repression of epigenetically silenced genes by active, targeted DNA demethylation can have direct, exciting potential applications in a number of experimental strategies and human diseases:

[0226] For Instructing Cells to Produce a Protective / Therapeutic Protein: One benefit of the re-activation of epigenetically silenced genes is to confer unusual (but desirable) property to cells, e.g., production of a protein that is not typically produced by a given cell type, for example, a cytokine or immunoregulatory molecule. As an example, targeted de- methylation of nitric oxide synthase silenced in fibroblasts [1, 4] confers to them an ability to produce nitric oxide (NO), which is a key component of innate immunity, including direct anti- microbial effects [109-112]. Enhancement of NO production by upregulation of NOS2 expression is a desirable therapeutic strategy [4, 113]; reactivation of the enzyme’s gene could lead to increased production at targeted tissue sites.

[0227] In Targeting Specific Monogenic Epigenetic Aberrations That Single- Handedly Cause Disease: For example, fragile X Syndrome is a form of autism linked to epigenetic silencing in the promoter of the FMR1 gene

[0114] ; demethylation of its promoter could restore transcription, and this presumption has recently been clearly demonstrated

[0115] .

[0228] To modify expression of cancer-related genes: silencing of tumor-suppressor promoters by DNA hypermethylation is an important mechanism of carcinogenesis

[0116] and could be selectively targeted.

[0229] In Experimental Strategies Such as Transdifferentiation and Regeneration; Transdifferentiation: Artificial overexpression of non-typical genes in fibroblasts transforms these cells into other types [117, 118]; epigenetic editing could provide heritable effect without the need for transgenesis. Proliferation: Specialized cells (cardiomyocytes or neurons) are non-dividing because genes responsible for cell division are switched off; epigenetic editing of cell cycle regulators could drive DNA synthesis and potentially lead to proliferation

[0119] .

[0230] However, until recently, most of the identified DNA methylation changes are used either correlatively, or as biomarkers for various diseases: 1. diagnostic markers

[0120] ; 2. prognostic markers [121, 122]; 3. markers for optimal treatment according to disease subclasses

[0123] ; 4. markers for monitoring treatment efficacy

[0124] ; 5. markers to identify genes to be examined for the development of epigenetically targeted therapies

[0116] . These DNA hypermethylated markers have been studied most extensively in cancer

[0125] .However, aberrant DNA hypermethylation is being implicated in other diseases. A selection of recently published reports on non-cancer disease-linked promoter DNA hypermethylation is shown in FIG.1. As shown, gene promoters are abnormally hypermethylated in many diseases, making them prime candidates for therapeutic targets. Therefore, developing strategies for site-specific CpG promoter demethylation of disease-associated genes is a critical and exciting challenge. FIG.1 shows details of reports on disease associated promoter DNA hypermethylation in noncancer diseases.

[0231] Gene-Specific DNA Demethylation: Site-specific gene targeting DNA demethylation is accomplished by fusing the demethylases to engineered DNA-binding domains (DBDs) that tether the enzyme to the desired DNA sequence

[0072] . Since it was first reported simultaneously by three independent groups [1-4], the development of this innovative technology has opened the possibility of epigenetic therapeutic applications for various diseases. Here, we review the latest findings on strategies for site-specific DNA demethylation and discuss the prospects for clinical translation in the future.

[0232] Selection of Demethylase for Targeted DNA Demethylation: The most frequently reported DNA demethylase for gene targeting are the TET family proteins. Among these, TET1 fusion protein is most commonly used. However, there are scattered reports using TET2 and TET3, which are considered to have enzymatic activity equivalent to that of TET1, at least in vitro

[0086] , although not in all experimental settings [2].

[0233] An increasing number of studies have reported the success of tethering TETs to DNA for targeted demethylation in recent years [150-154]. In the context of T cell differentiation, the dCas9-TET1catalytic domain (CD) demethylates the FOXP3-Treg specific demethylated region (TSDR) of Jurkat cells, leading to a Treg-like phenotype, suggested the feasibility of dCas9-TET1-mediated Treg programmation of primary T cells [155, 156].

[0234] In cancer cells, dCas9-TET1 enhances the cisplatin sensitivity of A549 cells (lung carcinoma epithelial cells) by promoting the expression of nicotinamide nucleotide transhydrogenase (NNT)

[0157] . dCas9-TET1CD was used to demethylate the EphA7 gene: the protein correlates with life expectancy in cervical cancer, indicating the potential of dCas9-TET1CD as a therapeutic strategy for cervical cancer

[0158] . Leucine-rich repeat and immunoglobulin-like domain (LRIG) 1 is a negative regulator of receptor tyrosine kinases and a tumor suppressor; decreased LRIG1 expression is consistently observed in various types of cancer and is linked to poor patient prognosis

[0159] . dCas9-TET1CD-mediated demethylation along with VP64-mediated transcriptional activation increased endogenous LRIG1 expression in breast cancer cells, and reduced cancer cell viability

[0160] . TET1CD reactivated X-linked endogenous FOXP3 in breast cancer

[0161] . The tripartite-motif (TRIM) family proteins contribute to cancer initiation, progress, or therapy resistance, exhibiting tumor-suppressive functions: they are frequently downregulated by promoter methylation incancerous tissues

[0162] . dCas9-TET1CD induced specific demethylation of TRIM58 in renal carcinoma cells (RCC)

[0163] . Promoter of the TMEM244 gene was demethylated to upregulate its expression in Sézary syndrome—an aggressive form of cutaneous T-cell lymphoma

[0164] . dCas9-TET1CD could demethylate hepatocyte nuclear factor (HNF)1A and Beta-1,4-mannosyl-glycoprotein 4-beta-N-acetylglucosaminyltransferase (MGAT3) genes in BG1 (human ovarian adenocarcinoma) cells

[0165] , and telomeric repeat-containing RNA (TERRA) in HeLa (human cervix carcinoma) and T98G (human glioblastoma) cell lines

[0166] .

[0235] The following reports have been published on the function of TET1 in non- tumor cells. TET1 ablation impairs cardiac differentiation of mouse embryonic stem cells and re-expression of the TET1CD rescued the differentiation defect in Tet-triple knockout mESCs

[0167] . Cyclin-dependent kinase-like (CDKL) 5 is associated with X-linked infantile spasm syndrome (ISSX). Halmai et al. performed artificial X chromosome inactivation (XCI) using dCas9-TET1CD targeting CDLK5

[0168] . Rett syndrome is an X-linked neurodevelopmental disorder caused by loss-of-function heterozygous mutations of methyl CpG-binding protein 2 (MECP2) on the X chromosome in young females

[0169] . Qian et al. reported reactivation of MECP2 in human embryonic stem cells (hESCs) derived from RTT by dCas9-TET1CD

[0170] . These two reports suggest the potential of dCas9-TET1CD for therapeutic application in X chromosome associated diseases. dCas9-TET1CD also could re-activate Oct4 in NIH- 3T3 cells

[0171] and beta-galactoside alpha-2,6-sialyltransferase 1 (ST6GAL1) in CHO cells

[0172] .

[0236] A few studies signal readiness to transition epigenetic editing experiments to in vivo. Fragile X syndrome (FXS), the most common genetic form of intellectual disability in males, is caused by silencing of the FMR1 gene associated with hypermethylation in the CGG expansion mutation in the 5′ UTR of FMR1. dCas9-TET1CD was used to demethylate the CGG repeats in the brain cells of mice so that FMR1 expression in edited neurons was maintained in vivo after engrafting into the mouse brain

[0115] . With dCas9-TET1CD system and hydrodynamic tail vein injection, Hanzawa et al. showed targeted DNA demethylation of the Fgf 21 promoter in the liver of PPARα-deficient mice

[0173] . Russell–Silver syndrome (RSS) is a rare disorder characterized by intrauterine growth restriction (IUGR) and poor growth after birth. The patients of RSS show H19 upregulation and insulin-like growth factor (Igf) 2 downregulation. Horii et al. developed RSS model mice via demethylated H19 promotor lesion with embryonic microinjection of dCas9-TET1CD

[0174] . Noack et al. delivered dCas9-TET1CD targeted Dchs1, a regulator of corticogenesis, to mouse brains by electroporation and verified the biological relevance of the aberrant methylation of Dchs1 in developmental malformations and cognitive impairment

[0175] .

[0237] On the other hand, TDG alone has been successful in demethylating and transcriptionally enhancing the NOS2 gene [1, 4]. Furthermore, targeted DNA demethylation with Release of Silencing 1 (ROS1) 5mC DNA glycosylase

[0176] fused to the DNA-binding domain of yeast GAL4 (GBD) has been reported

[0177] . These reports are supplemented by studies using targeted fusions with P300 acetyltransferase, which helps support the epigenetic editing effect on gene transcription [155, 178]. Thus, the selection of demethylases may be broadened in the future.

[0238] Given the multi-step nature of transformations from mC to C where TET proteins and TDG glycosylases interact in moving the atoms of the CH3 group, the strategies for application of multiple demethylases for epigenetic editing form a novel area of studies that may be called ‘atomic epigenetics’. When interpreting the reports of each single enzyme successfully employed for demethylation, it is important to consider that the other enzymes were intrinsically present as is natural for unmodified cells. At the same time, the paucity of studies on combinatorial use of different demethylases presents a space for future investigations.

[0239] Site-Specific DNA-Binding Domains: There are several types of DBDs that have been fused with the putative demethylases to tether them sequence-specifically to the promoters of interest. Aside from customizable DBDs aimed to allow single gene specificity, other naturally occurring DBDs have historically been the first used to affect a group of genes at once. For example, the Rel-homology domain (RHD) of NF-kB has been employed to target TDG to all NF-kB-dependent genes [1, 4]. This and similar systems (using for example transcription factor binding domains) may be employed in epigenetic editing for re- activation of intrinsic transcription using a ‘grouped’ approach.

[0240] Recently, more specific and customizable DBDs have become applied. One of these is the recombinant transcription activator-like effector (TALE) [3, 179]. TALE is a class of naturally occurring DNA-binding proteins found in the plant pathogen Xanthomonas sp. The DNA-binding domain of each TALE consists of 34-amino acid repeat modules arranged in tandem that can be rearranged according to a simple cipher to target new DNA sequences. Customized TALEs can be used for a wide variety of genome engineering applications, including transcriptional modulation and genome editing

[0180] . The molecular tool through which DNA demethylase is targeted to the disease-related gene promoters can be made by tethering human TET1 demethylase enzyme to a TALE repeat that has targeted DNA-binding specificities [3]. Modified TALE repeats provide an attractive platform for guiding TET1 activity. This is because assembly of individual repeat domains of known nucleotides can be used to produce large amounts of monomeric proteins that bind to virtually any target DNA sequence

[0181] . However, TALEs may be less suitable specifically for de-methylation, as they have difficulty binding richly methylated sequences

[0182] .

[0241] Concomitant to the TALEs, a well-known DNA-binding protein used for targeted editing is the zinc finger protein tandem array (ZFA). The DNA recognition domain of ZFA contains 3–6 or more Cys2-His2 zinc fingers. Each zinc finger in a ZFA recognizes a 3-bp DNA sequence via a single α-helix; tandem array assembly allows recognition of a longer sequence in the increments of 3, usually 9–18 bp in length

[0183] . ZFAs approach the DNA from the major groove

[0184] and bind with high specificity to DNA sequences [183, 185]. Different ZFA modules are used in combination, based on their respective affinities for a particular three-base sequence, to target specific genomic regions

[0186] . The site-specific DNA demethylation using ZFA-TETs fusion protein is shown in FIG.6A and FIG.6B. FIG. 6A shows ZFA-TETs fusion protein. FIG.6B shows CRISPR-dCas9-TET based site-specific demethylation.

[0242] Both ZFA and TALE were used as binding platforms for TET1 in site-specific DNA-demethylation studies, and both systems were equally effective in inducing transcription of targeted genes [182, 187, 188]. ZFAs and TALEs each have strengths and weaknesses regarding opportunities for targeting and specificity of binding; however, ZFAs provide a broader choice of specific arrays for a given target area. Novel algorithms simplify and improve the design process [189-191]. In a typical 3-finger array, 9 bp are recognized; however, it is possible to design 6-mers or longer molecules to target 20–30 bp sequences for better specificity. Longer ZFAs and TALEs, however, could still suffer from some non- specific binding, as off-target binding is known to occur both for ZFs

[0192] , and for TALE’s [193, 194]. To maximize single gene specificity, a ‘multiple hits’ approach may be used to target multiple fusion molecules to the same promoter in the near vicinity of each other, and in both strands [4], because the action of multiple demethylase molecules is cumulative, and it is unlikely that a combination of a dozen different constructs will bind in close proximity to an unrelated promoter.

[0243] In the last decade, the clustered regularly interspaced short palindromic repeat-associated ‘dead’ Cas9 (CRISPR / dCas9) module has received significant attention as a site-specific epigenome editing tool [173, 195]. The CRISPR module can be used as a site-specific demethylation tool by combining the specific DNA-binding ability of inactivated ‘dead’ cas9 (dCas9) with demethylase, such as TET1 or TDG. Quite a few targeted demethylation studies have been published using CRISPR-dCas9 systems [150-175, 196- 199]. Generally, CRISPR-dCas9-TET1CD fusion proteins are paired with sgRNAs that have programmable 20 nucleotide sequences homologous to the target loci (FIG.6B).

[0244] The first report on site-specific demethylation using the CRISPR-dCas9-TET1 system was published in 2016

[0199] . In that study, the demethylation and transcription of RANKL, MAGEB2 and MMP2 were promoted by the delivery of dCas9-TET1 constructs to cultured cells (HEK293, SH-SY5Y, HeLa) using plasmid vectors. They also modified thesgRNA by inserting bacteriophage MS2 RNA elements into the conventional sgRNA to improve the efficiency of delivery of the fused proteins to their targeted binding regions. Several other attempts have been made to amplify the response to associated effector modules in the CRSPR system. For example, Taghbalout et al. developed the Casilio-DNA Methylation Editing (ME) platform to amplify the efficacy of effector modules working in the targeted promotor loci [154, 200]. Nguyen et al. introduced the Sun Tag linker to the system to recruit multiple copies of VP64, a strong non-epigenetic transcriptional activation domain, at each locus of interest, which improved the efficiency of fused TET1 in inducing demethylation and transcription

[0199] . The characteristics of the three DNA-binding modalities are summarized in FIG.2.

[0245] Comparing ZFA with CRISPR, both have their own strengths and weaknesses. ZFA, being a protein, is more stable in vivo than a guide RNA. As shown in, for example, FIG.7A, ZFAs are much smaller than dCAS9, for which the size and structural complexity represent a significant problem. ZFAs can permeate into cells and further into their nuclei, spontaneously delivering a fused enzymatic payload

[0201] , whereas for dCAS9, additional measures for internalization and translocation are required. At the same time, gRNA DBD promises a better specificity of the binding; re-designing a ZFA-based construct to target a new gene requires re-expression and repurification of the entire fusion protein, whereas, with dCAS9-demethylases, only a replacement of gRNA is required to re-target; finally, the emerging wealth of tools for CRISPR editing creates a favorable landscape for the development of new applications. Nevertheless, the current enthusiasm for CRISPR technology in gene editing cannot be directly translated to vector-free epigenetic editing; the benefits of ZFA over dCAS9 (spontaneous internalization, small size, absence of the need to co-transfect gRNAs and protect them from the aggressive in vivo airway milieu) make it a valuable approach that remains a useful tool for epigenetic editing [202, 203].

[0246] FIG.7A shows anticipated 3D structural drawings of ZFA-TET1CD and dCAS9-TET1CD constructs. The images were generated with PHYRE2 Protein Fold Recognition Server. (sbg.bio.ic.ac.uk / phyre2 / html / page.cgi?id=index accessed on 20 April 2023). FIG.7B shows sequence-accurate to-scale 3D rendering of ZFA-TDGCD and dCAS9-TDGCD bound to a target gene promoter. The image was derived from ICM Browser using custom PDB files.

[0247] Finally, some older domains may become re-employed to support the transition of epigenetic editing from virally vectored to protein-only tools

[0204] .

[0248] In vivo Delivery: Most of the previous findings on site-targeted DNA demethylation have been based on in vitro experiments. As one of the few exceptions, Lei et al. introduced the CRISPR system into mice by microinjection of the dCas9-DNA methyltransferase MQ1 construct on a lentivirus vector into zygotes

[0205] . Hanzawa et al.also expressed plasmid-based CRISPR / dCas9 in mouse livers using the hydrodynamic tail vein injection

[0173] . These studies are highly relevant because they demonstrate that the approach of site-specific DNA demethylation can potentially work in vivo. However, laboratory techniques such as zygotes microinjection and hydrodynamic tail vein injection have problems in clinical application, such as high procedural difficulty and inability to ensure accurate organ-specific delivery. Methods such as microinjection and electroporation are more suitable in vitro [206, 207].

[0249] Since in vivo knowledge of site-specific DNA demethylation is so limited, an overview of in vivo site-specific demethylation will depend on what is known about targeted nucleases genome editing, which has similarities

[0205] . Here, we summarized current strategies of three main viral vectors and other non-viral technologies.

[0250] Viral Delivery: Viral transduction benefits from the ability of virions to incorporate their genome into the host for replication; thus, viruses are used as vectors to encode proteins and deliver them to target cells.

[0251] Adenoviral Vectors (AdVs): First-generation adenovirus vectors (AdVs) were designed to substitute a transgene for the E1 (3.15 kb) and / or E3 (3.1 kb) regions.

[0208] . The first-generation adenovirus vectors could transduce a wide variety of target cells, achieved high levels of gene expression and were sufficient for applications where transient high activity was required and an immune response to the vector or transgene was negligible. However, the first generation of adenoviral vectors had to overcome several safety concerns. At high ‘multiplicities of infection’ (MOI), certain host gene products with E1-like activity may be expressed, thereby unnecessarily inducing host cell proliferation

[0209] . Leakage of adenoviral DNA replication accumulating cytotoxic late gene products has a direct cytotoxic effect on transduced cells and triggers a host cellular immune response [210, 211]. To overcome these adverse effects, the second generation of AdVs were developed with extended genome packaging capacity by removing two or more initial genes, such as E2 and E4

[0212] . Several clinical trials of genome editing nucleases have been conducted using such AdVs. For systemic delivery of ZFAs, the expression unit coding the ZFAs is inserted into a serotype 5 AdV pseudotyped with serotype 35 fiber (AdV5 / 35) to repair autologous CD4+ helper T cells from HIV infected patients

[0213] . Despite improvements compared to the first generation, the problem of immune side effect in vivo has not been completely resolved in the second generation AdVs, and therefore they are not yet optimal vectors for therapeutic approaches

[0214] . A further evolution of AdVs is the high-capacity AdV (HCAdV), which removes all virus-specific genetic information, leaving only the inverse terminal repeat sequence (ITR) and packing signal. Compared to common AdVs, HCAdVs have the following advantages: they suppress both innate and adaptive immune responses,and their total packaging capacity has been expanded to a maximum of 36 kb, facilitating delivery of large TALE expression cassettes or the CRISPR / dCas system

[0215] .

[0252] One drawback of AdVs is that the level of coxsackie-adenovirus receptor (CAR), the receptor for Ad on cells, determines the delivery efficiency

[0216] . For example, AR expression in cancer cells is said to be negatively correlated with tumor grade, which means that high delivery efficiency may not be achieved when targeting cancer cells in the advanced clinical stage

[0217] . Therefore, the study of AdVs that could specifically act on cancer cells is taking on a new and important role in anti-tumor research.

[0253] Adeno-Associated Viruses Vectors (AAVs): Adeno-associated viral vectors (AAVs) are delivery vectors derived from icosahedral, non-enveloped viruses of the family Parvoviridae, genus Dependovirus, and are commonly used in genome editing technology because of their ability to be incorporated site-specifically and because of their low immunogenicity. The pathogenicity of AAV has not been confirmed so far. AAVs can deliver their genomes to mitotic and non-mitotic cells and can survive outside the chromosome without delivering their genome to the host cell

[0218] . A major drawback of the AAV vector system is its relatively small genome size (approximately 4.7 kb), which limits the size of the foreign genes that it can carry. However, with careful design, the sequences for expressing ZFA and TALE expression cassettes can be successfully encapsulated in the AAV vector

[0214] . Multiple studies on AAV / ZFA-mediated genome editing have shown positive results in experiments with animal models in vivo [219-221]. For example, in a study on hemophilia B using a humanized mouse model, the concentration of human factor IX in plasma recovered to 23% of normal on average by administering ZFA-carried AAV to the model mice

[0222] . Since the size of a typical spCas9 is about 4.2 kb, packaging it using an AAV with a cargo size of about 4.5 kb poses a challenge

[0222] . To solve this problem, a smaller saCas9 (~3.2 kb) was developed from Staphylococcus aureus species

[0223] . To date, AAV is established as a delivery vehicle for the CRISPR / Cas system and has been used to target brain cells, as well as skeletal and cardiac muscle cells [224, 225].

[0254] On another topic, AAV has been reported to be a promising vaccine vector. Taking advantage of this property, attempts are being made to develop a COVID-19 vaccine based on the AAV vector [226, 227]. The AAV vaccines reportedly have advantages over first-generation vaccines targeting the same spike protein, such as thermostability, high efficiency, safety and single-dose vaccination. This research demonstrates the potential and benefits of an AAV as a gene transfer vector.

[0255] Lentiviral Vectors (LVs): Lentiviruses are viruses belonging to the retroviridae family and are characterized by the stable insertion of viral genomic information into the hostgenome using viral RT (reverse transcriptase) and IN (integrase)

[0228] . Lentivirus-based vectors have attractive advantages as gene transfer vectors, including the following

[0229] :

[0256] (i) sustained gene transfer through stable integration of the vector into the host genome.

[0257] (ii) ability to infect both dividing and non-dividing cells.

[0258] (iii) broad tissue and cell orientation.

[0259] (iv) no expression of viral proteins in the host cells after vector transduction.

[0260] (v) ability to deliver complex gene elements.

[0261] (vi) a more secure integrated site profile.

[0262] (vii) relatively easy vector manipulation and production.

[0263] However, multiple patient deaths in early clinical trials have prevented lentiviral use as delivery vectors in humans [230, 231]. Therefore, safer integrase-deficient lentiviral vectors (IDLV) have been developed in recent years. For example, Lombardo et al. used IDLV as a delivery vector for ZFA-based genome editing in human stem cells

[0232] . However, the efficiency of IDLV may depend on the target tissue

[0231] . For example, IDLV is less efficient at transferring genes into mouse hepatocytes than a conventional lentiviral vector, but it is highly efficient at transferring genes into murine muscle [231, 233]. The characteristics of the three vectors mentioned above are summarized in FIG.3.

[0264] Although viral vectors are by far the most efficient tools for gene transfer, many issues preclude their in vivo and especially clinical use. Therefore, while the transduction / transfection-based approach to embedding epigenetically acting fusion proteins into recipient cells has served a useful purpose in the early stages of epigenetic editing, with experimentation almost exclusively being performed in vitro, it is linked to numerous problems when it comes to translating it into in vivo, and especially into human applications. The viral vector is a foreign invader; thus, the body may mount an immune response [234, 235]; a repeat administration or, in case of a pre-existing immunity to the viral vector, even the first administration will be ineffective for an immune subject

[0234] .

[0265] Especially with retroviral vectors, genes incorporated into chromosomes pose a risk of oncogenesis due to random gene transfer [236-238]. Moreover, insertional mutagenesis, viral persistence, accumulation of proto-oncogenic lesions, immunogenicity, cellular toxicity, risks related to immune surveillance and other problems common to gene therapy will similarly affect epigenetic therapy [239-242]. Finally, viral transfection can affect transcriptional activity of host genes, which creates the potential for misinterpretation of gene expression data generated from transfected cells

[0243] and impairs usefulness for experimentation on epigenetic control of transcription, even in vitro but especially in vivo.

[0266] Non-Viral Vectors: Non-viral vectors are classified into several subtypes based on their raw materials, sizes or production methods. For example, by raw materialthey can be classified into lipid-based vectors, polymer-based vectors, etc. Lipid-based vectors are further classified into lipid nanoparticles (LNPs) and liposomes, etc., depending on their manufacturing method and size. Liposomes, polymersomes, lipoplexes, polyplexes and dendrimers, are commonly used as gene delivery vectors and can enclose the mRNA encoding the fusion chimeras [244-247]. However, the low transfection efficiency and low gene expression rate compared to viral vectors have been obstacles to the clinical application of non-viral vectors. Considering this, various improvements of non-viral vectors have been attempted

[0248] . One example of these improvements is cationic liposomes that have been validated for their high transfection efficiency and biocompatibility

[0249] . Positively charged gene particles packaged in cationic lipids or cationic polymers are expected to be attracted to the negative charges of the cell membrane, thus increasing the efficiency of cellular uptake

[0250] . Studies on non-viral nanoparticle-mediated in vivo delivery are increasing in number, especially for the delivery of relatively small molecules such as RNAi [251-253]. However, there are few reports on non-viral vector-mediated in vivo delivery of megamolecules for genome editing. As an example, Han et al. recently reported using nanoparticles to deliver antithrombin-targeted CRISPR / Cas9 to the liver of a hemophiliac model mouse

[0254] . Several groups have now reported LNP formulations for successful expression in lungs following inhalation [255-259], indicating that the approach may be ready for therapeutic efforts in pulmonary disease. Specifically, in

[0260] , more efficient delivery to fibrotic than healthy lungs was reported, reached over 12% of epithelial cells and 10% endothelial cells in fibrotic lungs following intratracheal delivery.

[0267] Applications of programmable nuclease complexes are often hampered by the inability of the complex to reach the target tissue or, if it does reach the target, to pass through the cell membrane and then into the nucleus, and to exert therapeutic activity in vivo. Similar challenges can be expected to occur with site-specific demethylases.

[0268] Direct Delivery of Epigenetically Acting Fusion Proteins: Transition from viral vectors to the use of non-viral engineered packaging systems for epigenetically acting constructs could be a step forward in the field. It is important to consider, however, that the very need for genomic integration / transgenesis is linked to some of the side effects and challenges listed above for viral vectors. Lack of control on the site of insertion, problematic control of expression (both the duration of expression and its extent) including inability to stop the action if needed, immunogenicity of the mRNA, design challenges and finally susceptibility to degradation by nucleases are commonly seen with mRNA therapies and vaccines

[0261] . Thus, development of novel, safe and effective approaches, e.g., based on protein-only ‘biologics’ that act on DNA methylation, is enticing.

[0269] Key challenges that may be anticipated here will include ascertaining internalization of such fusion protein constructs into recipient cells and nuclei, andmaintaining their intactness in an in vivo environment. Proteins are usually less lipophilic, causing problems in the cell membrane permeation process, but a ZFA protein complex can, surprisingly, penetrate cell membranes due to the positive charge of Cys2-His2 zinc finger domains, and Gaj et al. showed that the direct delivery of ZFA-tethered nuclease protein can disrupt the CCR5 gene in both HEK-293 HDF cells, and human CD4+ T cells [8]. Unlike the naturally cell-penetrating ZFAs, for TALEs and CRISPR / Cas9, conjugation of cell- penetrating peptides (CPPs) is needed to facilitate cell entry

[0262] . In recent years, various CPPs, such as TAT peptide, SEQ ID NO: 46: (YGRKKRRQRRR), have been developed

[0263] .

[0270] Another problem with direct delivery, especially for in vivo applications, is protein stability. In the in vivo environment, unprotected proteins will come into direct contact with degrading enzymes. Therefore, it is important to not only expedite delivery, but also to increase the stability of the protein or otherwise protect it, e.g., by encapsulation into vesicles. One method utilized plasma membrane-derived extracellular microvesicles (ARMMs; arrestin domain-containing protein 1-mediated microvesicles) [264, 265] for delivery of CRISPR-Cas9 / guide RNA complex, which can be employed for epigenetic editing. For ZFAs, strategies to keep them stable have also been proposed, such as modifying the lysine residues necessary for degradation

[0266] . Finally, the LNP packaging discussed above can also be employed to package not only mRNA, but also the purified epigenetically acting constructs, provided their size allows.

[0271] At the moment, non-vectored (ribo)protein-only epigenetic editing is the cutting edge of the field.

[0272] Conclusions: The number of publications on targeted DNA demethylation has increased at an accelerating rate in recent years, reflecting the high level of interest in this field. Most of them are in vitro (FIG.4) or ex vivo

[0115] studies using plasmid or viral vectors as a carrier, but, recently, in vivo experiments have also been reported [153, 173]. Although neither of the gene delivery strategies are immediately applicable to clinical research, further expansion of in vivo applications of the targeted DNA demethylation is expected to follow in the future. FIG.4 shows selected publications for targeted DNA demethylation / gene reactivation.

[0273] Targeted DNA demethylation in key regulatory areas of genes allows re- activation of epigenetically silenced transcripts for experimental and therapeutic purposes. This technology can be broadly useful in biomedical research, and its current advances aim to overcome the key challenges of delivery, efficiency and gene-specificity of epigenetic effects.

[0274] Summary of benefits: Opens the gene to context-dependent, situation- appropriate stimuli; Potentially heritable (at least mitotically) effects in tissues; Mechanisticstudies in epigenetics (environmental epigenetics, immunotoxicology); Vector-free methods will allow studying causality of immunoregulatory genes; Forms a new class of ‘biologic’ drugs; Summary of challenges: Not every gene can likely be targeted; Gene-specificity / off- target effects; Non-viral delivery challenges; Cell-specific delivery

[0275] In some embodiments, the techniques described herein relate to a method for a gene-specific epigenetic editing of a gene, the method including the steps of: (1) contacting the gene and / or a gene promoter with a gene-specific fusion complex including at least one enzyme functioning as a DNA demethylase; and (2) performing a demethylation of one or more nucleotides within a gene, a promoter, and / or an anticipated regulatory region; whereby the demethylation provides an epigenetic editing of the gene, one or more nucleotides, regulatory motif, and / or the promoter and provides a test of the mechanistic significance of the demethylation of the gene or provides enhancement of transcriptional responsiveness of the gene.

[0276] According to some aspects, the techniques described herein relate to a method, wherein the method includes one or more demethylases, and / or one or more DNA binding domains (DBDs) assembled in a construct, fusion protein, or RNA-protein complex.

[0277] In some embodiments, the techniques described herein relate to a method, wherein at least one enzymatic domain is derived from TDG (thymine DNA glycosylase), Tet1, Tet2 or Tet3 (ten-eleven translocation, TET, methylcytosine dioxygenase 1, 2 or 3), the entire coding sequence of mouse or human TDG, a derivate sequence of mouse or human TDG, the enzymatic domain of mouse or human Tet1, Tet2 or Tet3, or a derivative sequence of the enzymatic domain of mouse or human Tet1, Tet2 or Tet3; and / or wherein the gene- specific fusion complex is a gene-specific demethylase fusion complex.

[0278] According to some aspects, the techniques described herein relate to a method, wherein the method is utilized to change methylation of a promoter and / or a transcription of an associated gene.

[0279] In some embodiments, the techniques described herein relate to a method, wherein the fusion complex is including a demethylase enzyme with a sequence-specific DNA binding domain (DBD).

[0280] According to some aspects, the techniques described herein relate to a method, wherein the fusion complex includes a demethylase enzyme connected to a DBD; the DBD being a dCAS9-gRNA system, or a zinc finger protein array (ZFA).

[0281] In some embodiments, the techniques described herein relate to a method, wherein the dCAS9-based system includes dCas9-Tet1 cat-NLS / Myc-SCP-HA / NLS / 6xHis- Stop Codon, SEQ ID NO: 9, dCas9-Tet1 mut-NLS / Myc-SCP-HA / NLS / 6xHis-Stop Codon, SEQ ID NO: 10; dCas9-TDG wt-NLS / Myc-SCP-HA / NLS / 6xHis-Stop Codon, SEQ ID NO: 11;and / or dCas9-TDG mut (N151A)-NLS / Myc-SCP-HA / NLS / 6xHis-Stop Codon, SEQ ID NO: 12.

[0282] According to some aspects, the techniques described herein relate to a method, wherein a supercharged peptide (SCP) is utilized to enhance a dCAS9-construct internalization into target or recipient cells.

[0283] In some embodiments, the techniques described herein relate to a method, wherein the SCP includes SEQ ID NO: 13 (GSKPAKKGSKTPGTSKSAKPKKGPGSKPATKGSKKPGKSKSATPKKGPGSKPAKSGKKT PGTSKKPSKGKAPGTSKKPSKGKAPGSKPAKKGSKTPGTSKSAKPKKGPGKSKKATPKSG P).

[0284] According to some aspects, the techniques described herein relate to a method, including the method is utilized to edit a gene encoding CXCL11 (I-TAC).

[0285] In some embodiments, the techniques described herein relate to a method, wherein the fusion complex is including a demethylase enzyme with a DBD including the dCAS9-derived system tethered to the target DNA sequence via the guide RNAs (gRNA).

[0286] According to some aspects, the techniques described herein relate to a method, wherein guide RNAs designed for CXCL11 gene includes: SEQ ID NO: 14 (AAAGCTACGTGAGTCAAGGA); SEQ ID NO: 15 (TGAGTGCTTTCACCTTCCAG); SEQ ID NO: 16 (AAAGATAGATGAAGGTCACC); SEQ ID NO: 17 (GTCCCACCAACTCAGGAAGA); SEQ ID NO: 18 (CCACAAGCCAGGAGATGCAG); or SEQ ID NO: 19 (CTGGGACCATCTTCCTGAGT).

[0287] In some embodiments, the techniques described herein relate to a method, wherein the method is executed as a gene-specific DNA demethylation.

[0288] According to some aspects, the techniques described herein relate to a method, wherein the method provides an epigenetic re-activation of a silenced or partially silenced gene, an epigenetic restoration of an adaptive, situation-appropriate responsiveness of intrinsic transcription and / or an epigenetic enhancement of a gene's expression.

[0289] In some embodiments, the techniques described herein relate to a method, with the proviso that the method does not require a vector and / or a transduction-based approach.

[0290] According to some aspects, the techniques described herein relate to a method, wherein the fusion complex is produced by an expression in a bacterial, insect or mammalian cell, then the fusion complex protein is purified and administered to a target cell or subject.

[0291] In some embodiments, the techniques described herein relate to a method, wherein the fusion complex includes a ZFA construct produced by recombinant expressionin an E.coli cell, or by an expression in a human embryonic kidney (HEK) or Chinese Hamster Ovary (CHO) cell; wherein the expression in the E. coli cell includes a use of one or more molecular chaperones.

[0292] According to some aspects, the techniques described herein relate to a method, wherein one or more molecular chaperones includes pTf16 for ZFA-TDG and pKJE7 for ZFA-Tet.

[0293] In some embodiments, the techniques described herein relate to a method, wherein the method utilizes a combination of fusion proteins in each administration including ZFA-TDGs contacting 3 or more sites and / or ZFA-TET1 contacting 3 or more sites, all within the target promoter, or wherein the method is reduced to just a single fusion construct.

[0294] According to some aspects, the techniques described herein relate to a method, wherein the method provides one or more data including an epigenetic influence on disease risk and / or gene silencing.

[0295] In some embodiments, the techniques described herein relate to a method, wherein at least one enzyme includes TDG (G / T mismatch-specific thymine DNA glycosylase) and / or Tets 1, 2 or 3 (ten-eleven translocation, TET, methylcytosine dioxygenases 1, 2 or 3); wherein the gene-specific fusion complex is a gene-specific demethylation fusion complex; and where an AA sequence of one or more enzyme(s) includes any ZFA-Tet1CD, any ZFA-TDG construct, ZF6-Tet cat (SEQ ID NO: 3), ZF8-Tet1 cat (SEQ ID NO: 4), ZF10-Tet cat (SEQ ID NO: 5), ZF6-Tet mut (SEQ ID NO: 6), ZF5-TDG wt (SEQ ID NO: 20), ZF7-TDG wt (SEQ ID NO: 21), ZF9-TDG wt (SEQ ID NO: 22), or ZF9- TDGmut (N151A) (SEQ ID NO: 23).

[0296] According to some aspects, the techniques described herein relate to a method or claim 6, wherein the ZFA portion can be replaceable / variable depending on the target(s), can be altered to provide targeting of a different target, and / or to target a different gene; and / or wherein the ZFA part is replaceable; and / or wherein the method provides a fusion of the ZFA portion with an enzyme via a polyaminoacid linker.

[0297] In some embodiments, the techniques described herein relate to a method for treating and / or preventing any disease or disorder where a benefit can be obtained by epigenetic editing or transcriptional enhancement of a select gene, the method including the steps of: (1) obtaining a subject and; (A) contacting a gene and / or a gene promoter in the subject with a gene-specific fusion complex including at least one enzyme functioning as a DNA demethylase; and (B) performing a demethylation of one or more nucleotides within a gene, a promoter, and / or an anticipated regulatory region; whereby the demethylation provides an epigenetic editing of the gene, one or more nucleotides, regulatory motif, and / or the promoter; leading to therapeutic benefit via enhanced transcriptional responsiveness ofthe targeted gene; (2) wherein the contacting is executed by the step of administering the complex to the subject.

[0298] According to some aspects, the techniques described herein relate to a method, whereby epigenetic editing in contrast to other methods makes the gene 'open' to stimuli and provides adaptive, situation appropriate, context-dependent gene upregulation.

[0299] In some embodiments, the techniques described herein relate to a method, further including the step of: administering the fusion complex or complexes to a subject via an intra-airway administration into the subject operative to contacting lung cells of the subject.

[0300] According to some aspects, the techniques described herein relate to a method, further including the step of: administering one or more such fusion complexes to a subject via an intranasal administration into the subject.

[0301] In some embodiments, the techniques described herein relate to a method, further including administering the gene-specific fusion complex or complexes via instillation of the complex into a subject; or administering the gene-specific fusion complex or complexes in the form of an aerosol and / or in the form of an aerosol using a mesh-type nebulizer that is operative to minimizing degradation of the administered complex or complexes.

[0302] According to some aspects, the techniques described herein relate to a method, wherein the method provides a treatment for Alzheimer's disease, a cancer, Parkinson's disease, schizophrenia, , epilepsy, depression, fragile X syndrome, systemic lupus erythematosus, rheumatoid arthritis, systemic sclerosis, Sjogren's syndrome, Graves' disease, type 1 diabetes, Crohn's disease, osteoporosis, type 2 diabetes, asthma, allergy, hyperlipidemia, cardiomyopathy, cardiovascular disease, IgA nephropathy, diabetic nephropathy, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, lung fibrosis, or hepatitis C.

[0303] In some embodiments, the techniques described herein relate to a method, wherein a DBD of the fusion complex contacts one or more loci in a CXCL11 promoter.

[0304] According to some aspects, the techniques described herein relate to a method, with the proviso that the method does not require an adenoviral vector (AdV), an adeno-associated virus vector (AAV), a lentiviral vector (LV), or other viral or non-viral vector or delivery packaging vehicle to administer into subject.

[0305] In some embodiments, the techniques described herein relate to a method for providing a research tool, providing experiments, a screening assay, or a research assay including a gene-specific epigenetic editing of a gene, the method including the steps of: (A) administering a fusion complex or combination of fusion complexes including at least one enzyme and DNA-binding domains, wherein the administering includes: (1) contacting thegene and / or a gene promoter with a gene-specific fusion complex including at least one enzyme functioning as a DNA demethylase; and (2) performing a demethylation of one or more nucleotides within a gene, a promoter, and / or an anticipated regulatory region; whereby the demethylation provides an epigenetic editing of the gene or provides a test of the mechanistic significance of the demethylation of the gene, one or more nucleotides, regulatory motif, and / or the promoter; and (B) monitoring one or more parameter, signs and / or symptoms in the recipient cell, tissue, organ, animal or any experimental subject.

[0306] According to some aspects, the techniques described herein relate to a method, wherein at least one enzymatic domain is derived from TDG (thymine DNA glycosylase) and / or Tet1 (ten-eleven translocation, TET, methylcytosine dioxygenase 1), the entire wild type CDS (consensus coding sequence) of mouse TDG, a derivate sequence of mouse TDG, the enzymatic domain of human Tet1, or a derivative sequence of the enzymatic domain of human Tet1; and wherein the gene-specific fusion complex is a gene- specific demethylation fusion complex.

[0307] In some embodiments, the techniques described herein relate to a method, wherein the method is utilized to change a promoter methylation and / or a transcription of an associated gene.

[0308] According to some aspects, the techniques described herein relate to a method, wherein the fusion complex is including a demethylase enzyme fused with a sequence-specific DNA binding domain (DBD).

[0309] In some embodiments, the techniques described herein relate to a method, wherein the fusion complex includes a demethylase enzyme fused with a DBD included of, a zinc finger protein array (ZFA), or a dCAS9-gRNA system.

[0310] According to some aspects, the techniques described herein relate to a method, wherein the dCAS9-based system includes dCas9-Tet1 cat-NLS / Myc-SCP- HA / NLS / 6xHis-Stop Codon, SEQ ID NO: 9, dCas9-Tet1 mut-NLS / Myc-SCP-HA / NLS / 6xHis- Stop Codon, SEQ ID NO: 10; dCas9-TDG wt-NLS / Myc-SCP-HA / NLS / 6xHis-Stop Codon, SEQ ID NO: 11; and / or dCas9-TDG mut (N151A)-NLS / Myc-SCP-HA / NLS / 6xHis-Stop Codon, SEQ ID NO: 12.

[0311] In some embodiments, the techniques described herein relate to a method, wherein a supercharged peptide (SCP) is utilized to enhance a dCAS9-construct internalization into cells.

[0312] According to some aspects, the techniques described herein relate to a method, wherein the SCP includes SEQ ID NO: 13 (GSKPAKKGSKTPGTSKSAKPKKGPGSKPATKGSKKPGKSKSATPKKGPGSKPAKSGKKT PGTSKKPSKGKAPGTSKKPSKGKAPGSKPAKKGSKTPGTSKSAKPKKGPGKSKKATPKSG P).

[0313] In some embodiments, the techniques described herein relate to a method, including the method is utilized to edit a gene encoding CXCL11 (I-TAC).

[0314] According to some aspects, the techniques described herein relate to a method, wherein the fusion complex is including a demethylase enzyme fused with a DBD wherein guide RNAs (gRNA) are tethered to target DNA sequence via dCAS9.

[0315] In some embodiments, the techniques described herein relate to a method, wherein guide RNA designed for CXCL11 gene includes: SEQ ID NO: 14 (AAAGCTACGTGAGTCAAGGA); SEQ ID NO: 15 (TGAGTGCTTTCACCTTCCAG); SEQ ID NO: 16 (AAAGATAGATGAAGGTCACC); SEQ ID NO: 17 (GTCCCACCAACTCAGGAAGA); SEQ ID NO: 18 (CCACAAGCCAGGAGATGCAG); or SEQ ID NO: 19 (CTGGGACCATCTTCCTGAGT).

[0316] According to some aspects, the techniques described herein relate to a method, wherein the method is executed as a gene-specific DNA demethylation.

[0317] In some embodiments, the techniques described herein relate to a method, wherein the method provides an epigenetic re-activation of a silenced or partially silenced gene, an epigenetic restoration of an adaptive, situation-appropriate responsiveness of intrinsic transcription and / or an epigenetic enhancement of a gene's expression.

[0318] According to some aspects, the techniques described herein relate to a method, with the proviso that the method does not require a vector and / or a transduction- based approach.

[0319] In some embodiments, the techniques described herein relate to a method, wherein the fusion complex is produced by a recombinant expression in a bacterial, insect, yeast or mammalian cell.

[0320] According to some aspects, the techniques described herein relate to a method, wherein the fusion complex includes a ZFA construct produced by an expression in a target cell, a recombinant expression in an E.coli cell, or by an expression in a human embryonic kidney (HEK) ) or Chinese Hamster Ovary (CHO) cell; wherein the expression in the E. coli cell includes a use of one or more molecular chaperones.

[0321] In some embodiments, the techniques described herein relate to a method, wherein one or more molecular chaperones includes pTf16 for ZFA-TDG and pKJE7 for ZFA-Tet1.

[0322] According to some aspects, the techniques described herein relate to a method, further including the step of obtaining a subject and: administering the fusion complex or complexes to a subject via an intra-airway administering into the subject operative to contacting lung cells of the subject.

[0323] In some embodiments, the techniques described herein relate to a method, further including the step of: administering one or more such fusion complexes to the subjectvia an intranasal administration into the subject, and / or via aerosol administration into the subject..

[0324] According to some aspects, the techniques described herein relate to a method, wherein the method provides a treatment for any disease or disorder where a benefit can be obtained by epigenetic editing or transcriptional enhancement of a select gene.

[0325] In some embodiments, the techniques described herein relate to a method, wherein the method provides research directed to a treatment for Alzheimer's disease, a cancer, Parkinson's disease, schizophrenia, epilepsy, depression, fragile X syndrome, systemic lupus erythematosus, rheumatoid arthritis, systemic sclerosis, Sjogren's syndrome, Graves' disease, type 1 diabetes, Crohn's disease, osteoporosis, type 2 diabetes, asthma, allergy, hyperlipidemia, cardiomyopathy, cardiovascular disease, IgA nephropathy, diabetic nephropathy, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, lung fibrosis, or hepatitis C.

[0326] According to some aspects, the techniques described herein relate to a method, wherein the method is performed with a screening therapeutic agent, with a screening drug, with a screening compound, and / or with a screening condition.

[0327] In some embodiments, the techniques described herein relate to a method, with the proviso that the method does not require an adenoviral vector (AdV), an adeno- associated virus vector (AAV), a lentiviral vector (LV), or a non-viral vector to administer into subject.

[0328] According to some aspects, the techniques described herein relate to a method, wherein one or more loci in CXCL11 promoter for ZFA and gRNA are contacted.

[0329] In some embodiments, the techniques described herein relate to a method, wherein at least one enzyme includes TDG (G / T mismatch-specific thymine DNA glycosylase) and / or Tet1 (ten-eleven translocation, TET, methylcytosine dioxygenase 1); wherein the gene-specific fusion complex is a gene-specific demethylation fusion complex; and where an AA sequence of one or more enzyme(s) includes any ZFA-Tet1CD, any ZFA- TDG construct, ZF6-Tet cat (SEQ ID NO: 3), ZF8-Tet1 cat (SEQ ID NO: 4), ZF10-Tet cat (SEQ ID NO: 5), ZF6-Tet mut (SEQ ID NO: 6), ZF5-TDG wt (SEQ ID NO: 20), ZF7-TDG wt (SEQ ID NO: 21), ZF9-TDG wt (SEQ ID NO: 22), or ZF9-TDGmut (N151A) (SEQ ID NO: 23).

[0330] According to some aspects, the techniques described herein relate to a composition for a gene-specific epigenetic editing of a gene within a subject, the composition including: (1) a sequence-specific DBD; (2) at least one enzyme functioning as a DNA demethylase to perform a demethylation of at least a portion of the gene, one or more nucleotides in the gene or a promoter, and / or relevant CpG island(s), or other regulatory motif.

[0331] In some embodiments, the techniques described herein relate to a composition, wherein at least one enzymatic domain is derived from TDG (thymine DNA glycosylase) and / or Tet's 1, 2 or 3 (ten-eleven translocation, TET, methylcytosine dioxygenases 1, 2, 3), the entire coding sequence of mouse TDG, a derivate sequence of mouse TDG, the enzymatic domain of human Tet1, Tet2 or Tet3, or a derivative sequence of the enzymatic domain of human Tet1; or wherein the gene-specific fusion complex is a gene-specific demethylation fusion complex.

[0332] According to some aspects, the techniques described herein relate to a composition, wherein the composition is operative to change a promoter methylation and / or a transcription of an associated gene.

[0333] In some embodiments, the techniques described herein relate to a composition, wherein the fusion complex is including a demethylase enzyme with a sequence-specific DNA binding domain (DBD).

[0334] According to some aspects, the techniques described herein relate to a composition, wherein the fusion complex is including a demethylase enzyme with a DBD, or a DBD including a zinc finger protein array (ZFA), or a dCAS9 system.

[0335] In some embodiments, the techniques described herein relate to a composition, wherein the dCAS9-based system includes dCas9-Tet1 cat-NLS / Myc-SCP- HA / NLS / 6xHis-Stop Codon, SEQ ID NO: 9, dCas9-Tet1 mut-NLS / Myc-SCP-HA / NLS / 6xHis- Stop Codon, SEQ ID NO: 10; dCas9-TDG wt-NLS / Myc-SCP-HA / NLS / 6xHis-Stop Codon, SEQ ID NO: 11; and / or dCas9-TDG mut (N151A)-NLS / Myc-SCP-HA / NLS / 6xHis-Stop Codon, SEQ ID NO: 12.

[0336] According to some aspects, the techniques described herein relate to a composition, further including wherein a supercharged peptide (SCP) is included to enhance a dCAS9-construct internalization into cells.

[0337] In some embodiments, the techniques described herein relate to a composition, wherein the SCP includes SEQ ID NO: 13 (GSKPAKKGSKTPGTSKSAKPKKGPGSKPATKGSKKPGKSKSATPKKGPGSKPAKSGKKT PGTSKKPSKGKAPGTSKKPSKGKAPGSKPAKKGSKTPGTSKSAKPKKGPGKSKKATPKSG P).

[0338] According to some aspects, the techniques described herein relate to a composition, including the composition is utilized to edit a gene encoding CXCL11 (I-TAC).

[0339] In some embodiments, the techniques described herein relate to a composition, wherein the fusion complex is including a demethylase enzyme fused with a DBD including a CRISPR-derived system wherein guide RNAs (gRNA) are tethered to target DNA sequence via dCAS9.

[0340] According to some aspects, the techniques described herein relate to a composition, wherein guide RNA includes: SEQ ID NO: 14 (AAAGCTACGTGAGTCAAGGA); SEQ ID NO: 15 (TGAGTGCTTTCACCTTCCAG); SEQ ID NO: 16 (AAAGATAGATGAAGGTCACC); SEQ ID NO: 17 (GTCCCACCAACTCAGGAAGA); SEQ ID NO: 18 (CCACAAGCCAGGAGATGCAG); or SEQ ID NO: 19 (CTGGGACCATCTTCCTGAGT).

[0341] In some embodiments, the techniques described herein relate to a composition, wherein the composition is operative to provide a gene-specific DNA demethylation.

[0342] According to some aspects, the techniques described herein relate to a composition, wherein the composition is operative to provide an epigenetic re-activation of a silenced or partially silenced gene, an epigenetic restoration of an adaptive, situation- appropriate responsiveness of intrinsic transcription and / or an epigenetic enhancement of a gene's expression.

[0343] In some embodiments, the techniques described herein relate to a composition, with the proviso that the composition does not require a vector and / or a transduction-based approach.

[0344] According to some aspects, the techniques described herein relate to a composition, wherein the fusion complex is produced by a recombinant expression in a cell.

[0345] In some embodiments, the techniques described herein relate to a composition, wherein the fusion complex includes a ZFA construct produced by an expression in a recombinant expression in a cell, a recombinant expression in an E.coli cell, or by an expression in a human embryonic kidney (HEK) ) or Chinese Hamster Ovary (CHO) cell; wherein the expression in the E. coli cell includes a use of one or more molecular chaperones.

[0346] According to some aspects, the techniques described herein relate to a composition, wherein one or more molecular chaperones include pTf16 for ZFA-TDG and pKJE7 for ZFA-Tet1.

[0347] In some embodiments, the techniques described herein relate to a composition, further including the composition is operative to provide the step of: administering the fusion complex or complexes to a subject via an intra-airway administering into the subject operative to contacting lung cells of the subject.

[0348] According to some aspects, the techniques described herein relate to a composition, further including the composition is operative to provide an execution of the step of: administering one or more such fusion complexes to a subject via an intranasal administration into the subject.

[0349] In some embodiments, the techniques described herein relate to a composition, wherein the composition provides a treatment for any disease or disorder where a benefit can be obtained by epigenetic editing or transcriptional enhancement of a select gene.

[0350] According to some aspects, the techniques described herein relate to a composition, wherein the composition provides a treatment for Alzheimer's disease, a cancer, Parkinson's disease, schizophrenia, epilepsy, depression, fragile X syndrome, systemic lupus erythematosus, rheumatoid arthritis, systemic sclerosis, Sjogren's syndrome, Graves' disease, type 1 diabetes, Crohn's disease, osteoporosis, type 2 diabetes, asthma, allergy, hyperlipidemia, cardiomyopathy, , cardiovascular disease, IgA nephropathy, diabetic nephropathy, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, lung fibrosis, or hepatitis C.

[0351] In some embodiments, the techniques described herein relate to a composition, further including the composition is operative to provide the step of: administering the gene-specific fusion complex or complexes into an airway of a subject; administering the gene-specific fusion complex or complexes via instillation of the complex into a subject; or administering the gene-specific fusion complex or complexes in the form of an aerosol and / or in the form of an aerosol using a mesh-type nebulizer that is operative to minimizing degradation of the administered complex or complexes.

[0352] According to some aspects, the techniques described herein relate to a composition, with the proviso that the composition does not require an adenoviral vector (AdV), an adeno-associated virus vector (AAV), a lentiviral vector (LV), or other viral or non- viral vector to administer into subject.

[0353] In some embodiments, the techniques described herein relate to a composition, wherein one or more loci in CXCL11 promoter for ZFA and gRNA are contacted utilizing the composition.

[0354] According to some aspects, the techniques described herein relate to a composition, wherein at least one enzyme includes TDG (G / T mismatch-specific thymine DNA glycosylase) and / or Tet1, Tet2 or Tet3 (ten-eleven translocation, TET, methylcytosine dioxygenase ); wherein the gene-specific fusion complex is a gene-specific demethylation fusion complex; and where an AA sequence of one or more enzyme(s) includes any ZFA- Tet1CD, any ZFA-TDG construct, ZF6-Tet cat (SEQ ID NO: 3), ZF8-Tet1 cat (SEQ ID NO: 4), ZF10-Tet cat (SEQ ID NO: 5), ZF6-Tet mut (SEQ ID NO: 6), ZF5-TDG wt (SEQ ID NO: 20), ZF7-TDG wt (SEQ ID NO: 21), ZF9-TDG wt (SEQ ID NO: 22), or ZF9-TDGmut (N151A) (SEQ ID NO: 23).According to some aspects, the method disclosed above can be further comprising the step of determining a therapy based upon one or more biomarkers and / orone or move proteins. A system can be implemented using these example methods and embodiments.

[0355] The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. Moreover, due to biological functional equivalency considerations, some changes can be made in protein structure without affecting the biological or chemical action in kind or amount. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims. In any of the embodiments disclosed herein the technology points (e.g., any feature herein) can be inter-combined and can be discussed along with the following example discussion points:

[0356] Discussion point 1. A method for gene-specific epigenetic editing of a gene, the method comprising: contacting the gene and / or a gene promoter with a gene-specific fusion complex comprising at least one enzyme functioning as a DNA demethylase; and performing a demethylation of one or more nucleotides within the gene, the promoter, and / or an anticipated regulatory region; wherein the demethylation provides an epigenetic editing of the gene, the one or more nucleotides, a regulatory motif, and / or the promoter and provides a test of the mechanistic significance of the demethylation of the gene or provides enhancement of transcriptional responsiveness of the gene.

[0357] Discussion point 2. The method of discussion point 1, wherein the method includes one or more demethylases, such as TET enzymes, which catalyze the oxidation of 5-methylcytosine to 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine; APOBEC enzymes, which can deaminate 5-methylcytosine to thymine, leading to base excision repair and replacement with unmodified cytosine; or AID enzymes, which can also deaminate 5-methylcytosine to thymine, initiating a base excision repair process that ultimately replaces the methylated cytosine with an unmodified cytosine, and / or one or more DNA binding domains (DBDs), such as zinc finger domains, which utilize a combination of zinc finger proteins to recognize specific DNA sequences; TALE domains, which can, forexample, consist of tandem repeats of a 33-35 amino acid motif that can be engineered to bind specific DNA sequences; or CRISPR / Cas systems, which employ a guide RNA to direct the Cas endonuclease to a specific genomic location, assembled in a construct, fusion protein, or RNA-protein complex to target specific genomic regions for demethylation.

[0358] Discussion point 3. The method of discussion point 1, wherein at least one enzymatic domain is derived from TDG (thymine DNA glycosylase), which catalyzes the removal of thymine and uracil from G:T and G:U mismatches, resulting from deamination of 5-methylcytosine and cytosine, respectively; Tet1, Tet2 or Tet3 (ten-eleven translocation, TET, methylcytosine dioxygenase 1, 2 or 3), which oxidize 5-methylcytosine to 5- hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine, leading to base excision repair and replacement with unmodified cytosine; the entire coding sequence of mouse or human TDG, a derivate sequence of mouse or human TDG, the enzymatic domain of mouse or human Tet1, Tet2 or Tet3, or a derivative sequence of the enzymatic domain of mouse or human Tet1, Tet2 or Tet3; and / or wherein the gene-specific fusion complex is a gene- specific demethylase fusion complex designed to target and demethylate specific genes or genomic regions, allowing for precise epigenetic editing and modulation of gene expression.

[0359] Discussion point 4. The method of discussion point 1, wherein the fusion complex is including a demethylase enzyme, such as a TET enzyme, which catalyzes the oxidation of 5-methylcytosine to 5-hydroxymethylcytosine, 5-formylcytosine, and 5- carboxylcytosine; an APOBEC enzyme, which can deaminate 5-methylcytosine to thymine, leading to base excision repair and replacement with unmodified cytosine; or an AID enzyme, which can also deaminate 5-methylcytosine to thymine, initiating a base excision repair process that ultimately replaces the methylated cytosine with an unmodified cytosine, with a sequence-specific DNA binding domain (DBD), such as a zinc finger domain, which utilizes a combination of zinc finger proteins to recognize specific DNA sequences; a TALE domain, which can, for example, consist of tandem repeats of a 33-35 amino acid motif that can be engineered to bind specific DNA sequences; or a CRISPR / Cas system, which employs a guide RNA to direct the Cas endonuclease to a specific genomic location, to direct the demethylase activity to specific genomic targets, enabling targeted epigenetic editing and modulation of gene expression.

[0360] Discussion point 5. The method of discussion point 1, wherein the fusion complex includes a demethylase enzyme, such as a TET enzyme, which catalyzes the oxidation of 5-methylcytosine to 5-hydroxymethylcytosine, 5-formylcytosine, and 5- carboxylcytosine; an APOBEC enzyme, which can deaminate 5-methylcytosine to thymine, leading to base excision repair and replacement with unmodified cytosine; or an AID enzyme, which can also deaminate 5-methylcytosine to thymine, initiating a base excision repair process that ultimately replaces the methylated cytosine with an unmodified cytosine,connected to a DBD (DNA binding domain); the DBD being a dCAS9-gRNA system, which utilizes a catalytically inactive Cas9 protein and a guide RNA to target specific DNA sequences, with the guide RNA containing a 20-nucleotide sequence complementary to the target DNA; or a zinc finger protein array (ZFA), which can, for example, consist of multiple zinc finger proteins designed to recognize specific DNA sequences, with each zinc finger recognizing a 3-4 base pair DNA sequence, providing a means to target the demethylase activity to specific genomic regions for precise epigenetic editing and modulation of gene expression.

[0361] Discussion point 6. The method of discussion point 1, wherein the fusion complex is including a demethylase enzyme, such as a TET enzyme, which catalyzes the oxidation of 5-methylcytosine to 5-hydroxymethylcytosine, 5-formylcytosine, and 5- carboxylcytosine; an APOBEC enzyme, which can deaminate 5-methylcytosine to thymine, leading to base excision repair and replacement with unmodified cytosine; or an AID enzyme, which can also deaminate 5-methylcytosine to thymine, initiating a base excision repair process that ultimately replaces the methylated cytosine with an unmodified cytosine, with a DBD including the dCAS9-derived system tethered to the target DNA sequence via the guide RNAs (gRNA), allowing for precise targeting of the demethylase activity to specific genomic loci, with the guide RNA containing a 20-nucleotide sequence complementary to the target DNA, enabling targeted epigenetic editing and modulation of gene expression at specific genes or regulatory elements.

[0362] Discussion point 7. The method of discussion point 1, wherein the demethylation enhances gene expression by reducing the repressive effects of DNA methylation on transcription, leading to increased accessibility of the gene promoter and regulatory elements to transcriptional machinery, as the removal of methyl groups from cytosine residues can prevent the binding of repressive proteins, such as methyl-CpG binding domain (MBD) proteins, and allow for the recruitment of transcriptional activators, potentially activating previously silenced genes or increasing the expression of genes with important cellular functions, such as tumor suppressor genes, cell cycle regulators, or genes involved in cell differentiation and development, providing a means to modulate gene expression for therapeutic purposes or to study the role of specific genes in cellular processes.

[0363] Discussion point 8. A gene-specific fusion complex for epigenetic editing of a gene, the gene-specific fusion complex comprising: at least one enzyme functioning as a DNA demethylase, wherein the gene-specific fusion complex is configured to contact the gene and / or a gene promoter and perform a demethylation of one or more nucleotides within the gene, the promoter, and / or an anticipated regulatory region; wherein the demethylation provides an epigenetic editing of the gene, the one or more nucleotides, a regulatory motif,and / or the promoter and provides a test of the mechanistic significance of the demethylation of the gene or provides enhancement of transcriptional responsiveness of the gene.

[0364] Discussion point 9. The gene-specific fusion complex of discussion point 8, wherein the enzyme catalyzes removal of methyl groups from DNA by oxidizing 5- methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5- carboxylcytosine (5caC), which can be subsequently replaced by unmodified cytosine through base excision repair mechanisms, with thymine DNA glycosylase (TDG) being a key enzyme in this process, or by deaminating 5mC to thymine, which is then replaced by unmodified cytosine through base excision repair, with activation-induced cytidine deaminase (AID) and apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) enzymes being involved in this process, ultimately resulting in the targeted demethylation of specific genomic regions and potentially leading to changes in gene expression.

[0365] Discussion point 10. The gene-specific fusion complex of discussion point 8, wherein the demethylation is of nucleotides within genetic elements such as promoters, which are located upstream of the transcription start site and contain binding sites for transcription factors that initiate transcription; enhancers, which are distal regulatory elements that can enhance transcription by looping into proximity with the promoter and recruiting transcriptional activators; silencers, which are regulatory elements that can repress transcription by recruiting repressive proteins or preventing the binding of activators; insulators, which are boundary elements that can prevent the spread of heterochromatin and maintain distinct chromatin domains; and other regulatory regions that control gene expression, allowing for targeted epigenetic modification of these elements to study their role in gene regulation and potentially modulate gene expression for therapeutic purposes or to investigate the mechanisms underlying cellular processes and disease states.

[0366] Discussion point 11. The gene-specific fusion complex of discussion point 8, wherein the demethylation provides epigenetic modification of genetic elements by altering the methylation status of cytosine residues, which can impact the binding of transcription factors, chromatin accessibility, and overall gene expression, as methylated cytosines are generally associated with transcriptional repression, while unmethylated cytosines are associated with active transcription, with methyl-CpG binding domain (MBD) proteins recognizing and binding to methylated cytosines, recruiting repressive chromatin remodeling complexes and histone deacetylases (HDACs) that promote a closed chromatin structure and inhibit transcription, while demethylation can lead to the dissociation of these repressive complexes and the recruitment of transcriptional activators, enabling the study of how changes in DNA methylation patterns influence gene expression and cellular processes, and potentially providing a means to modulate gene expression for therapeutic purposes.

[0367] Discussion point 12. The gene-specific fusion complex of discussion point 8, wherein the demethylation enables studying the functional effects of demethylation on gene regulation, cell fate determination, and disease pathogenesis by allowing targeted removal of methyl groups from specific genomic loci, which can help elucidate the role of DNA methylation in various biological processes, such as embryonic development, where DNA methylation patterns are dynamically regulated and play a crucial role in cell differentiation and organogenesis; X-chromosome inactivation, where one of the two X chromosomes in female mammals is silenced through extensive DNA methylation to achieve dosage compensation; genomic imprinting, where certain genes are expressed in a parent-of-origin- specific manner due to differential methylation patterns established during gametogenesis; and cancer, where aberrant DNA methylation patterns, such as hypermethylation of tumor suppressor gene promoters and hypomethylation of oncogene promoters, contribute to tumorigenesis, potentially identifying new therapeutic targets and advancing our understanding of the epigenetic mechanisms underlying these processes.

[0368] Discussion point 13. The gene-specific fusion complex of discussion point 8, wherein the demethylation enhances gene expression by reducing the repressive effects of DNA methylation on transcription, leading to increased accessibility of the gene promoter and regulatory elements to transcriptional machinery, as the removal of methyl groups from cytosine residues can prevent the binding of repressive proteins, such as methyl-CpG binding domain (MBD) proteins, which recognize and bind to methylated cytosines and recruit repressive chromatin remodeling complexes and histone deacetylases (HDACs) that promote a closed chromatin structure and inhibit transcription, and allow for the recruitment of transcriptional activators, such as histone acetyltransferases (HATs) and chromatin remodeling complexes that promote an open chromatin structure and facilitate transcription, potentially activating previously silenced genes or increasing the expression of genes with important cellular functions, such as tumor suppressor genes, cell cycle regulators, or genes involved in cell differentiation and development, providing a means to modulate gene expression for therapeutic purposes or to study the role of specific genes in cellular processes and disease states.

[0369] Discussion point 14. The method of discussion point 1, wherein the gene- specific fusion complex further comprises a DNA-binding domain that recognizes and binds to a specific DNA sequence, directing the demethylase enzyme to the target gene or genomic region, allowing for precise and targeted demethylation of the desired loci, which can be used to study the effects of demethylation on gene expression and cellular processes, such as cell differentiation, where targeted demethylation of lineage-specific genes can promote the differentiation of stem cells into specific cell types; cellular reprogramming, where targeted demethylation of pluripotency genes can facilitate thereprogramming of somatic cells into induced pluripotent stem cells (iPSCs); and disease modeling, where targeted demethylation of disease-associated genes in patient-derived iPSCs can help elucidate the epigenetic mechanisms underlying the disease phenotype, as well as potentially modulate gene expression for therapeutic purposes, such as reactivating silenced tumor suppressor genes in cancer or downregulating overexpressed genes in autoimmune disorders.

[0370] Discussion point 15. The method of discussion point 14, wherein the DNA- binding domain is selected from the group consisting of a zinc finger domain, which utilizes a combination of zinc finger proteins to recognize specific DNA sequences, with each zinc finger consisting of an α-helix and a β-sheet stabilized by a zinc ion and recognizing a 3-4 base pair DNA sequence, allowing for the modular assembly of multiple zinc fingers to target longer and more specific DNA sequences; a TALE domain, which can, for example, consist of tandem repeats of a 33-35 amino acid motif derived from transcription activator-like effectors (TALEs) of Xanthomonas bacteria, with each repeat containing a repeat variable diresidue (RVD) at positions 12 and 13 that determines the nucleotide binding specificity, allowing for the engineering of TALE arrays with desired DNA binding specificities; and a CRISPR / Cas system, which employs a guide RNA (gRNA) to direct the Cas endonuclease to a specific genomic location, with the gRNA containing a 20-nucleotide spacer sequence complementary to the target DNA and a scaffold sequence that binds to the Cas protein, allowing for the reprogramming of DNA targeting specificity by simply changing the spacer sequence of the gRNA, providing a versatile and programmable means to target specific genomic regions for demethylation and epigenetic editing.

[0371] Discussion point 16. The method of discussion point 1, wherein the enzyme is selected from the group consisting of a TET enzyme, which catalyzes the oxidation of 5- methylcytosine to 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine, with TET1, TET2, and TET3 being the three mammalian TET enzymes that belong to the Fe(II) / α-ketoglutarate-dependent dioxygenase superfamily and utilize molecular oxygen and α-ketoglutarate as co-substrates to catalyze the oxidation reaction, with subsequent rounds of oxidation converting 5hmC to 5fC and 5caC, which can be recognized and excised by thymine DNA glycosylase (TDG) and replaced with unmodified cytosine through base excision repair; an APOBEC enzyme, which can deaminate 5-methylcytosine to thymine, leading to base excision repair and replacement with unmodified cytosine, with APOBEC3A and APOBEC3B being the most active in this process and belonging to the larger APOBEC family of cytidine deaminases that play important roles in innate immunity and mRNA editing; and an AID enzyme, which can also deaminate 5-methylcytosine to thymine, initiating a base excision repair process that ultimately replaces the methylated cytosine with an unmodified cytosine, with AID being primarily active in B cells during antibody diversification throughsomatic hypermutation and class switch recombination, but also exhibiting off-target activity on non-immunoglobulin genes, providing a range of enzymatic options for targeted demethylation of specific genomic regions and epigenetic editing.

[0372] Discussion point 17. The method of discussion point 1, wherein the gene is associated with a disease or disorder, and the epigenetic editing of the gene through targeted demethylation can potentially modulate its expression and alleviate the associated disease phenotype, as aberrant DNA methylation patterns have been implicated in various diseases, such as cancer, where hypermethylation of tumor suppressor gene promoters and hypomethylation of oncogene promoters contribute to tumorigenesis; imprinting disorders, where defects in the establishment or maintenance of parental-specific methylation patterns lead to disorders such as Prader-Willi syndrome and Angelman syndrome; and neurological disorders, where altered DNA methylation patterns have been associated with conditions such as schizophrenia, bipolar disorder, and Alzheimer's disease, providing a potential therapeutic approach for these conditions by correcting the abnormal methylation patterns and restoring normal gene expression, as well as a means to study the epigenetic mechanisms underlying these disorders and identify novel therapeutic targets.

[0373] Discussion point 18. The method of discussion point 17, wherein the disease or disorder is selected from the group consisting of cancer, where aberrant DNA methylation patterns contribute to tumor suppressor gene silencing and oncogene activation, with examples including hypermethylation of the BRCA1 gene in breast and ovarian cancer, leading to impaired DNA repair and increased cancer risk; hypermethylation of the MLH1 gene in colorectal cancer, leading to microsatellite instability and increased mutation rates; and hypomethylation of the MAGE gene family in various cancers, leading to their aberrant expression and potential use as cancer biomarkers and immunotherapy targets; an autoimmune disorder, where altered DNA methylation may lead to dysregulated immune responses, with examples including hypomethylation of the IL-6 gene in rheumatoid arthritis, leading to increased IL-6 production and joint inflammation; hypomethylation of the CD40LG gene in systemic lupus erythematosus, leading to increased CD40 ligand expression and B cell hyperactivity; and hypermethylation of the FOXP3 gene in multiple sclerosis, leading to reduced regulatory T cell function and impaired immune suppression; and a neurodegenerative disorder, where changes in DNA methylation are implicated in the pathogenesis of conditions such as Alzheimer's disease, with examples including hypomethylation of the APP gene, leading to increased amyloid precursor protein production and amyloid plaque formation; hypermethylation of the BDNF gene, leading to reduced brain-derived neurotrophic factor expression and impaired neuronal survival; and hypomethylation of the SNCA gene, leading to increased α-synuclein expression and aggregation in Parkinson's disease, highlighting the potential of targeted demethylation as atherapeutic strategy for these diseases by correcting the aberrant methylation patterns and restoring normal gene expression.

[0374] Discussion point 19. The gene-specific fusion complex of discussion point 8, further comprising a DNA-binding domain that directs the demethylase enzyme to the target gene or genomic region by recognizing and binding to a specific DNA sequence, allowing for precise targeting of the demethylase activity to the desired loci, which can be used to study the effects of demethylation on gene expression and cellular processes, such as cell differentiation, where targeted demethylation of lineage-specific genes can promote the differentiation of stem cells into specific cell types; cellular reprogramming, where targeted demethylation of pluripotency genes can facilitate the reprogramming of somatic cells into induced pluripotent stem cells (iPSCs); and disease modeling, where targeted demethylation of disease-associated genes in patient-derived iPSCs can help elucidate the epigenetic mechanisms underlying the disease phenotype, as well as potentially modulate gene expression for therapeutic purposes, such as reactivating silenced tumor suppressor genes in cancer or downregulating overexpressed genes in autoimmune disorders.

[0375] Discussion point 20. The gene-specific fusion complex of discussion point 19, wherein the DNA-binding domain is selected from the group consisting of a zinc finger domain, which utilizes a combination of zinc finger proteins to recognize specific DNA sequences, with each zinc finger consisting of an α-helix and a β-sheet stabilized by a zinc ion and recognizing a 3-4 base pair DNA sequence, allowing for the modular assembly of multiple zinc fingers to target longer and more specific DNA sequences; a TALE domain, which can, for example, consist of tandem repeats of a 33-35 amino acid motif derived from transcription activator-like effectors (TALEs) of Xanthomonas bacteria, with each repeat containing a repeat variable di-residue (RVD) at positions 12 and 13 that determines the nucleotide binding specificity, allowing for the engineering of TALE arrays with desired DNA binding specificities; and a CRISPR / Cas system, which employs a guide RNA (gRNA) to direct the Cas endonuclease to a specific genomic location, with the gRNA containing a 20- nucleotide spacer sequence complementary to the target DNA and a scaffold sequence that binds to the Cas protein, allowing for the reprogramming of DNA targeting specificity by simply changing the spacer sequence of the gRNA, providing a versatile and programmable means to target specific genomic regions for demethylation and epigenetic editing.

[0376] Discussion point 21. The gene-specific fusion complex of discussion point 8, wherein the enzyme is selected from the group consisting of a TET enzyme, which catalyzes the oxidation of 5-methylcytosine to 5-hydroxymethylcytosine, 5-formylcytosine, and 5- carboxylcytosine, with TET1, TET2, and TET3 being the three mammalian TET enzymes that belong to the Fe(II) / α-ketoglutarate-dependent dioxygenase superfamily and utilize molecular oxygen and α-ketoglutarate as co-substrates to catalyze the oxidation reaction,with subsequent rounds of oxidation converting 5hmC to 5fC and 5caC, which can be recognized and excised by thymine DNA glycosylase (TDG) and replaced with unmodified cytosine through base excision repair; an APOBEC enzyme, which can deaminate 5- methylcytosine to thymine, leading to base excision repair and replacement with unmodified cytosine, with APOBEC3A and APOBEC3B being the most active in this process and belonging to the larger APOBEC family of cytidine deaminases that play important roles in innate immunity and mRNA editing; and an AID enzyme, which can also deaminate 5- methylcytosine to thymine, initiating a base excision repair process that ultimately replaces the methylated cytosine with an unmodified cytosine, with AID being primarily active in B cells during antibody diversification through somatic hypermutation and class switch recombination, but also exhibiting off-target activity on non-immunoglobulin genes, providing a range of enzymatic options for targeted demethylation of specific genomic regions and epigenetic editing.

[0377] In different embodiments, the method for gene-specific epigenetic editing involves contacting the gene and / or a gene promoter with a gene-specific fusion complex comprising at least one enzyme functioning as a DNA demethylase. In certain embodiments, the gene-specific fusion complex includes one or more demethylases and / or one or more DNA binding domains (DBDs) assembled in a construct, fusion protein, or RNA-protein complex. This step initiates the process of gene-specific epigenetic editing by performing demethylation. The gene-specific fusion complex may be derived from TDG (thymine DNA glycosylase), Tet1, Tet2, or Tet3, providing specific enzymatic activity for demethylation. The entire coding sequence of mouse or human TDG, a derivative sequence of mouse or human TDG, the enzymatic domain of mouse or human Tet1, Tet2, or Tet3, or a derivative sequence of the enzymatic domain of mouse or human Tet1, Tet2, or Tet3 may be used.

[0378] In certain embodiments, the fusion complex includes a demethylase enzyme with a sequence-specific DNA binding domain (DBD). The DBD may be a dCAS9-gRNA system or a zinc finger protein array (ZFA). The dCAS9-based system may comprise constructs such as dCas9-Tet1 cat-NLS / Myc-SCP-HA / NLS / 6xHis-Stop Codon, dCas9-Tet1 mut-NLS / Myc-SCP-HA / NLS / 6xHis-Stop Codon, dCas9-TDG wt-NLS / Myc-SCP- HA / NLS / 6xHis-Stop Codon, and / or dCas9-TDG mut (N151A)-NLS / Myc-SCP-HA / NLS / 6xHis- Stop Codon. These constructs provide specific targeting for demethylation.

[0379] Additionally, a supercharged peptide (SCP) may be utilized to enhance the internalization of the dCAS9 construct into target or recipient cells. The SCP comprises SEQ ID NO: 13 (GSKPAKKGSKTPGTSKSAKPKKGPGSKPATKGSKKPGKSKSATPKKGPGSKPAKSGKKT PGTSKKPSKGKAPGTSKKPSKGKAPGSKPAKKGSKTPGTSKSAKPKKGPGKSKKATPKSG P), providing a specific sequence for the supercharged peptide.

[0380] In certain embodiments, the fusion complex is produced by expression in a bacterial, insect, or mammalian cell. The fusion complex protein is then purified and administered to a target cell or subject. The fusion complex may include a ZFA construct produced by recombinant expression in an E. coli cell or by expression in a human embryonic kidney (HEK) ) or Chinese Hamster Ovary (CHO) cell. The expression in the E. coli cell may include the use of one or more molecular chaperones, such as pTf16 for ZFA-TDG and pKJE7 for ZFA-Tet, to assist in protein folding.

[0381] The method aims to achieve gene-specific epigenetic editing by contacting the gene and / or gene promoter with a gene-specific fusion complex and performing demethylation. This process provides an epigenetic editing of the gene, one or more nucleotides, regulatory motif, and / or the promoter. The demethylation provides a test of the mechanistic significance of the demethylation of the gene or enhances the transcriptional responsiveness of the gene.

[0382] In different embodiments, the method for gene-specific epigenetic editing includes the step of contacting the gene and / or a gene promoter with a gene-specific fusion complex comprising at least one enzyme functioning as a DNA demethylase. In certain embodiments, the gene-specific fusion complex includes one or more demethylases and / or one or more DNA binding domains (DBDs) assembled in a construct, fusion protein, or RNA- protein complex. This step facilitates the demethylation process. The gene-specific fusion complex is designed to perform targeted demethylation, which is necessary for achieving gene-specific epigenetic editing.

[0383] In certain embodiments, the gene-specific fusion complex includes a demethylase enzyme with a sequence-specific DNA binding domain (DBD). This configuration allows the complex to target specific DNA sequences for demethylation. The demethylase enzyme in the fusion complex is responsible for the demethylation process, which is a step in modifying the epigenetic state of the gene and its regulatory elements.

[0384] The gene-specific fusion complex may be derived from various sources to provide specific enzymatic activity for demethylation. For instance, the enzymatic domain can be derived from TDG (thymine DNA glycosylase), Tet1, Tet2, or Tet3. These enzymes are known for their role in DNA demethylation, and their inclusion in the fusion complex ensures the effective removal of methyl groups from the DNA.

[0385] Additionally, the gene-specific fusion complex may include a demethylase enzyme connected to a DBD, such as a dCAS9-gRNA system or a zinc finger protein array (ZFA). The dCAS9-gRNA system is particularly useful for providing sequence-specific targeting, as it can be tethered to the target DNA sequence via guide RNAs (gRNA). This system enhances the precision of the demethylation process, ensuring that only the intended DNA regions are modified.

[0386] In certain embodiments, the gene-specific fusion complex is produced by expression in a bacterial, insect, or mammalian cell. The fusion complex protein is then purified and administered to a target cell or subject. This production method ensures that the fusion complex is available in sufficient quantities for effective gene-specific epigenetic editing.

[0387] Furthermore, the fusion complex may include a ZFA construct produced by recombinant expression in an E. coli cell or by expression in a human embryonic kidney (HEK) ) or Chinese Hamster Ovary (CHO) cell. The expression in the E. coli cell may involve the use of one or more molecular chaperones, such as pTf16 for ZFA-TDG and pKJE7 for ZFA-Tet. These molecular chaperones assist in the proper folding and expression of the ZFA constructs, ensuring their functionality in the fusion complex.

[0388] Overall, the step involves the use of a gene-specific fusion complex that includes at least one enzyme functioning as a DNA demethylase to perform demethylation. This step is for achieving gene-specific epigenetic editing by contacting the gene and / or gene promoter with the fusion complex and performing demethylation. The inclusion of specific enzymatic domains, DNA binding domains, and production methods ensures the effectiveness and precision of the demethylation process.

[0389] In different embodiments, the gene-specific fusion complex may be derived from TDG (thymine DNA glycosylase), Tet1, Tet2, or Tet3. In certain embodiments, the gene-specific fusion complex comprises at least one enzyme functioning as a DNA demethylase. The enzymatic domain may be derived from TDG (thymine DNA glycosylase), Tet1, Tet2, or Tet3. The entire coding sequence of mouse or human TDG, a derivative sequence of mouse or human TDG, the enzymatic domain of mouse or human Tet1, Tet2, or Tet3, or a derivative sequence of the enzymatic domain of mouse or human Tet1, Tet2, or Tet3 may be utilized.

[0390] The gene-specific fusion complex is designed to perform targeted demethylation. The purpose is to use a gene-specific fusion complex that includes at least one enzyme functioning as a DNA demethylase to perform demethylation. This complex facilitates the demethylation process, which is for gene-specific epigenetic editing. The demethylation process involves contacting the gene and / or a gene promoter with the gene- specific fusion complex, which includes at least one enzyme functioning as a DNA demethylase. This process is aimed at achieving gene-specific epigenetic editing by demethylating one or more nucleotides within a gene, a promoter, and / or an anticipated regulatory region.

[0391] The demethylation provides an epigenetic editing of the gene, one or more nucleotides, regulatory motif, and / or the promoter. This editing can test the mechanistic significance of the demethylation of the gene or enhance the transcriptional responsivenessof the gene. The gene-specific fusion complex, therefore, plays a role in modifying the epigenetic state of the gene and its regulatory elements, understanding the functional impact of demethylation, or enhancing gene expression.

[0392] In summary, the gene-specific fusion complex derived from TDG, Tet1, Tet2, or Tet3, and comprising at least one enzyme functioning as a DNA demethylase, is utilized to perform targeted demethylation. This process is for gene-specific epigenetic editing, providing a means to test the mechanistic significance of demethylation or enhance transcriptional responsiveness of the gene.

[0393] In different embodiments, the method for gene-specific epigenetic editing includes a demethylase enzyme with a sequence-specific DNA binding domain (DBD). In certain embodiments, the gene-specific fusion complex comprises at least one enzyme functioning as a DNA demethylase. This complex performs the demethylation process. The demethylase enzyme is for the demethylation of one or more nucleotides within a gene, a promoter, and / or an anticipated regulatory region, thereby achieving epigenetic editing.

[0394] In certain embodiments, the demethylase enzyme is connected to a DBD, which provides sequence-specific targeting. The DBD can be a dCAS9-gRNA system or a zinc finger protein array (ZFA). The dCAS9-gRNA system is designed to tether the demethylase enzyme to the target DNA sequence via guide RNAs (gRNA). This system ensures that the demethylation process is specific to the desired DNA sequence, enhancing the precision of the epigenetic editing.

[0395] The gene-specific fusion complex, including the demethylase enzyme and the DBD, facilitates the demethylation process by targeting specific DNA sequences. This targeted approach allows for the modification of the epigenetic state of the gene and its regulatory elements, providing a test of the mechanistic significance of the demethylation of the gene or enhancing the transcriptional responsiveness of the gene.

[0396] In certain embodiments, the demethylase enzyme is derived from TDG (thymine DNA glycosylase), Tet1, Tet2, or Tet3. These enzymes provide specific enzymatic activity for demethylation, ensuring the effectiveness of the gene-specific fusion complex in performing the demethylation process. The entire coding sequence of mouse or human TDG, a derivative sequence of mouse or human TDG, the enzymatic domain of mouse or human Tet1, Tet2, or Tet3, or a derivative sequence of the enzymatic domain of mouse or human Tet1, Tet2, or Tet3 can be used in the gene-specific fusion complex.

[0397] The method for gene-specific epigenetic editing, therefore, includes a demethylase enzyme with a sequence-specific DNA binding domain (DBD) to target specific DNA sequences for demethylation. This approach ensures that the demethylation process is precise and effective, providing a means to test the mechanistic significance of the demethylation of the gene or to enhance the transcriptional responsiveness of the gene.

[0398] In different embodiments, the method for gene-specific epigenetic editing includes a demethylase enzyme with a DNA binding domain (DBD) including the dCAS9- derived system tethered to the target DNA sequence via the guide RNAs (gRNA). In certain embodiments, the demethylase enzyme is part of a gene-specific fusion complex that includes at least one enzyme functioning as a DNA demethylase. This complex performs demethylation, which is for gene-specific epigenetic editing. The demethylase enzyme, in this context, is designed to target specific DNA sequences, ensuring precise demethylation.

[0399] In certain embodiments, the dCAS9-derived system is utilized to provide sequence-specific targeting. This system comprises dCas9-Tet1 cat-NLS / Myc-SCP- HA / NLS / 6xHis-Stop Codon, dCas9-Tet1 mut-NLS / Myc-SCP-HA / NLS / 6xHis-Stop Codon, dCas9-TDG wt-NLS / Myc-SCP-HA / NLS / 6xHis-Stop Codon, and / or dCas9-TDG mut (N151A)- NLS / Myc-SCP-HA / NLS / 6xHis-Stop Codon. These constructs are designed to provide specific demethylation constructs, ensuring that the demethylation process is both targeted and efficient.

[0400] Additionally, the guide RNAs (gRNA) are designed to tether the dCAS9- derived system to the target DNA sequence. For instance, guide RNAs designed for the CXCL11 gene comprise SEQ ID NO: 14 (AAAGCTACGTGAGTCAAGGA), SEQ ID NO: 15 (TGAGTGCTTTCACCTTCCAG), SEQ ID NO: 16 (AAAGATAGATGAAGGTCACC), SEQ ID NO: 17 (GTCCCACCAACTCAGGAAGA), SEQ ID NO: 18 (CCACAAGCCAGGAGATGCAG), or SEQ ID NO: 19 (CTGGGACCATCTTCCTGAGT). These specific sequences ensure that the gRNA can accurately guide the dCAS9-derived system to the intended target, facilitating precise demethylation.

[0401] The method also includes the use of a supercharged peptide (SCP) to enhance the internalization of the dCAS9 construct into target or recipient cells. The SCP comprises SEQ ID NO: 13 (GSKPAKKGSKTPGTSKSAKPKKGPGSKPATKGSKKPGKSKSATPKKGPGSKPAKSGKKT PGTSKKPSKGKAPGTSKKPSKGKAPGSKPAKKGSKTPGTSKSAKPKKGPGKSKKATPKSG P), which is designed to improve the delivery efficiency of the dCAS9 construct, ensuring that it reaches the target cells effectively.

[0402] In summary, the method for gene-specific epigenetic editing involves a demethylase enzyme with a DBD, including the dCAS9-derived system tethered to the target DNA sequence via guide RNAs. This system is designed to provide targeted demethylation, with specific constructs and sequences ensuring precision and efficiency in the demethylation process. The use of a supercharged peptide further enhances the delivery of the dCAS9 construct into target cells, making the method effective for gene-specific epigenetic editing.

[0403] In different embodiments, the fusion complex is produced by an expression in a bacterial, insect, or mammalian cell, then the fusion complex protein is purified and administered to a target cell or subject. In certain embodiments, the bacterial cell, insect cell, and mammalian cell are utilized to express the gene-specific fusion complex. The gene- specific fusion complex comprises at least one enzyme functioning as a DNA demethylase. These cells produce the fusion complex efficiently and ensure its proper folding and functionality.

[0404] In certain embodiments, the gene-specific fusion complex includes a demethylase enzyme connected to a DNA binding domain (DBD). The DBD can be a dCAS9-gRNA system or a zinc finger protein array (ZFA). Including a DBD provides sequence-specific targeting for the demethylation process. The demethylase enzyme, such as those derived from TDG (thymine DNA glycosylase), Tet1, Tet2, or Tet3, provides the enzymatic activity necessary for demethylation.

[0405] In certain embodiments, the fusion complex is produced by recombinant expression in an E. coli cell or by expression in a human embryonic kidney (HEK) ) or Chinese Hamster Ovary (CHO) cell. The expression in the E. coli cell includes the use of one or more molecular chaperones, such as pTf16 for ZFA-TDG and pKJE7 for ZFA-Tet. Using molecular chaperones assists in the proper folding and expression of the ZFA constructs, ensuring their functionality in the demethylation process.

[0406] The fusion complex, once produced and purified, is administered to a target cell or subject. The administration of the fusion complex aims to perform gene-specific epigenetic editing by demethylating specific nucleotides within a gene, promoter, and / or anticipated regulatory region. This demethylation provides an epigenetic editing of the gene, one or more nucleotides, regulatory motif, and / or the promoter. It also provides a test of the mechanistic significance of the demethylation of the gene or enhances the transcriptional responsiveness of the gene.

[0407] In summary, the production and administration of the gene-specific fusion complex involve several steps, including expression in bacterial, insect, or mammalian cells, purification of the fusion complex protein, and its subsequent administration to target cells or subjects. The fusion complex includes a demethylase enzyme connected to a DBD, which provides sequence-specific targeting for the demethylation process. The use of molecular chaperones in the expression process ensures the proper folding and functionality of the fusion complex, enabling effective gene-specific epigenetic editing.

[0408] In different embodiments, the fusion complex includes a ZFA construct produced by recombinant expression in an E. coli cell, or by an expression in a human embryonic kidney (HEK) or Chinese Hamster Ovary (CHO) cell. In certain embodiments, the expression in the E. coli cell includes a use of one or more molecular chaperones. The ZFAconstruct is a zinc finger protein array (ZFA) that is utilized to provide sequence-specific targeting for demethylation. The ZFA construct is produced by recombinant expression, which involves the use of specific cells such as E. coli cells or human embryonic kidney (HEK) or Chinese Hamster Ovary (CHO) cells. The expression in the E. coli cell includes the use of molecular chaperones, which assist in the proper folding and functionality of the ZFA constructs.

[0409] In certain embodiments, the molecular chaperones include pTf16 for ZFA- TDG and pKJE7 for ZFA-Tet. The pTf16 chaperone is used specifically for the expression of ZFA-TDG constructs, while the pKJE7 chaperone is used for the expression of ZFA-Tet constructs. These molecular chaperones play a role in ensuring that the ZFA constructs are correctly folded and functional, thereby enhancing the efficiency of the demethylation process.

[0410] The ZFA constructs, once produced and properly folded, are included in the fusion complex, which also comprises at least one enzyme functioning as a DNA demethylase. The fusion complex performs targeted demethylation of specific DNA sequences within a gene, promoter, and / or anticipated regulatory region. The demethylation process provides an epigenetic editing of the gene, one or more nucleotides, regulatory motif, and / or the promoter. This epigenetic editing can be utilized to test the mechanistic significance of the demethylation of the gene or to enhance the transcriptional responsiveness of the gene.

[0411] In summary, the fusion complex includes a ZFA construct produced by recombinant expression in an E. coli cell or a human embryonic kidney (HEK) or Chinese Hamster Ovary (CHO) cell, with the expression in the E. coli cell involving the use of molecular chaperones such as pTf16 for ZFA-TDG and pKJE7 for ZFA-Tet. The ZFA constructs are included in the fusion complex to provide sequence-specific targeting for demethylation, thereby facilitating the epigenetic editing of the gene and its regulatory elements.

[0412] In different embodiments, the method for gene-specific epigenetic editing may include the use of molecular chaperones to assist in the expression of ZFA constructs. Specifically, in step 100-g, one or more molecular chaperones, such as pTf16 for ZFA-TDG and pKJE7 for ZFA-Tet, are utilized. These molecular chaperones play a role in ensuring the proper folding and functionality of the ZFA constructs during their production.

[0413] In certain embodiments,the molecular chaperone pTf16 is employed for the expression of ZFA-TDG constructs. The ZFA-TDG constructs are designed to target specific DNA sequences for demethylation, thereby facilitating the epigenetic editing process. The pTf16 chaperone assists in the correct folding of the ZFA-TDG proteins, ensuring their stability and activity.

[0414] Similarly, in other embodiments, the molecular chaperone pKJE7 is used for the expression of ZFA-Tet constructs. The ZFA-Tet constructs, like the ZFA-TDG constructs, are engineered to target specific DNA sequences for demethylation. The pKJE7 chaperone aids in the proper folding and functionality of the ZFA-Tet proteins, enhancing their effectiveness in the demethylation process.

[0415] The use of these molecular chaperones is for the successful production and application of the ZFA constructs in gene-specific epigenetic editing. By ensuring the correct folding and stability of the ZFA proteins, the chaperones contribute to the overall efficiency and precision of the demethylation process, ultimately leading to effective epigenetic modifications of the target genes.

[0416] In different embodiments, the method for gene-specific epigenetic editing involves performing a demethylation of one or more nucleotides within a gene, a promoter, and / or an anticipated regulatory region. In certain embodiments, the demethylation process is utilized to change the methylation of a promoter and / or the transcription of an associated gene. This process is executed as a gene-specific DNA demethylation. In certain embodiments, the method is utilized to edit a gene encoding CXCL11 (I-TAC). Additionally, the method may utilize a combination of fusion proteins in each administration, comprising ZFA-TDGs contacting three or more sites and / or ZFA-TET1 contacting three or more sites, all within the target promoter, or the method may be reduced to just a single fusion construct.

[0417] The demethylation process is performed to achieve epigenetic editing by demethylation, which involves the removal of methyl groups from the DNA, thereby altering the gene expression. The components involved in this step include the promoter, gene, nucleotides, and anticipated regulatory region. Performing demethylation on these components achieves gene-specific epigenetic editing by contacting the gene and / or gene promoter with a gene-specific fusion complex and performing demethylation.

[0418] The gene-specific fusion complex comprises at least one enzyme functioning as a DNA demethylase, which facilitates the demethylation process. The demethylation provides an epigenetic editing of the gene, one or more nucleotides, regulatory motif, and / or the promoter. This process provides a test of the mechanistic significance of the demethylation of the gene or provides enhancement of transcriptional responsiveness of the gene.

[0419] In certain embodiments, the method includes the use of guide RNAs designed for the CXCL11 gene, which comprises specific sequences such as SEQ ID NO: 14 (AAAGCTACGTGAGTCAAGGA), SEQ ID NO: 15 (TGAGTGCTTTCACCTTCCAG), SEQ ID NO: 16 (AAAGATAGATGAAGGTCACC), SEQ ID NO: 17 (GTCCCACCAACTCAGGAAGA), SEQ ID NO: 18 (CCACAAGCCAGGAGATGCAG), orSEQ ID NO: 19 (CTGGGACCATCTTCCTGAGT). These guide RNAs are used to target specific sequences within the gene for demethylation.

[0420] The method also includes the use of a dCAS9-derived system tethered to the target DNA sequence via the guide RNAs (gRNA). This system provides targeted demethylation by directing the DNA demethylase enzyme to specific sites within the gene, promoter, or anticipated regulatory region.

[0421] Overall, the method for gene-specific epigenetic editing involves a series of steps and sub-steps that utilize various components to achieve targeted demethylation and epigenetic editing of specific genes. The process is designed to alter gene expression, test the mechanistic significance of demethylation, and enhance transcriptional responsiveness, providing a comprehensive approach to gene-specific epigenetic editing.

[0422] In different embodiments, the method for gene-specific epigenetic editing may be utilized to change the methylation of a promoter and / or the transcription of an associated gene. In certain embodiments, the gene-specific fusion complex, which includes at least one enzyme functioning as a DNA demethylase, is employed to perform the demethylation process. The gene-specific fusion complex may include a demethylase enzyme connected to a DNA binding domain (DBD), such as a dCAS9-gRNA system or a zinc finger protein array (ZFA). This complex facilitates the demethylation process by targeting specific DNA sequences.

[0423] In certain embodiments, the demethylation process involves performing a demethylation of one or more nucleotides within a gene, a promoter, and / or an anticipated regulatory region. This process aims to achieve epigenetic editing by altering the methylation status of these regions. The demethylation provides an epigenetic editing of the gene, one or more nucleotides, regulatory motif, and / or the promoter. This editing can be used to test the mechanistic significance of the demethylation of the gene or to enhance the transcriptional responsiveness of the gene.

[0424] The gene-specific fusion complex may be derived from TDG (thymine DNA glycosylase), Tet1, Tet2, or Tet3, which provide specific enzymatic activity for demethylation. The entire coding sequence of mouse or human TDG, a derivative sequence of mouse or human TDG, the enzymatic domain of mouse or human Tet1, Tet2, or Tet3, or a derivative sequence of the enzymatic domain of mouse or human Tet1, Tet2, or Tet3 may be used in the fusion complex.

[0425] In certain embodiments, the method may be utilized to edit a gene encoding CXCL11 (I-TAC). The guide RNAs designed for the CXCL11 gene may comprise specific sequences such as SEQ ID NO: 14 (AAAGCTACGTGAGTCAAGGA), SEQ ID NO: 15 (TGAGTGCTTTCACCTTCCAG), SEQ ID NO: 16 (AAAGATAGATGAAGGTCACC), SEQ IDNO: 17 (GTCCCACCAACTCAGGAAGA), SEQ ID NO: 18 (CCACAAGCCAGGAGATGCAG), or SEQ ID NO: 19 (CTGGGACCATCTTCCTGAGT).

[0426] The method may also be executed as a gene-specific DNA demethylation, which involves the targeted demethylation of specific genes. This process can provide an epigenetic re-activation of a silenced or partially silenced gene, an epigenetic restoration of an adaptive, situation-appropriate responsiveness of intrinsic transcription, and / or an epigenetic enhancement of a gene’s expression.

[0427] In summary, the method for gene-specific epigenetic editing involves utilizing a gene-specific fusion complex comprising at least one enzyme functioning as a DNA demethylase to perform demethylation on one or more nucleotides within a gene, promoter, and / or anticipated regulatory region. This process aims to achieve epigenetic editing, test the mechanistic significance of demethylation, and enhance transcriptional responsiveness of the gene.

[0428] In different embodiments, the method is utilized to edit a gene encoding CXCL11 (I-TAC). In certain embodiments, the method includes performing a demethylation of one or more nucleotides within a gene, a promoter, and / or an anticipated regulatory region. The demethylation process is executed as a gene-specific DNA demethylation. The gene-specific fusion complex, which includes at least one enzyme functioning as a DNA demethylase, is employed to achieve this demethylation. The demethylation provides an epigenetic editing of the gene, one or more nucleotides, regulatory motif, and / or the promoter. This process provides a test of the mechanistic significance of the demethylation of the gene or provides enhancement of transcriptional responsiveness of the gene.

[0429] In certain embodiments, the method utilizes guide RNAs designed for the CXCL11 gene. These guide RNAs comprise specific sequences such as SEQ ID NO: 14 (AAAGCTACGTGAGTCAAGGA), SEQ ID NO: 15 (TGAGTGCTTTCACCTTCCAG), SEQ ID NO: 16 (AAAGATAGATGAAGGTCACC), SEQ ID NO: 17 (GTCCCACCAACTCAGGAAGA), SEQ ID NO: 18 (CCACAAGCCAGGAGATGCAG), or SEQ ID NO: 19 (CTGGGACCATCTTCCTGAGT). These sequences are designed to target the CXCL11 gene specifically, facilitating precise demethylation.

[0430] The method may also include the use of a dCAS9-derived system tethered to the target DNA sequence via the guide RNAs (gRNA). This system ensures that the demethylase enzyme is directed to the specific DNA sequences that require demethylation. The dCAS9-based system may comprise constructs such as dCas9-Tet1 cat-NLS / Myc-SCP- HA / NLS / 6xHis-Stop Codon, dCas9-Tet1 mut-NLS / Myc-SCP-HA / NLS / 6xHis-Stop Codon, dCas9-TDG wt-NLS / Myc-SCP-HA / NLS / 6xHis-Stop Codon, and / or dCas9-TDG mut (N151A)- NLS / Myc-SCP-HA / NLS / 6xHis-Stop Codon. These constructs are designed to provide specific demethylation activity.

[0431] Additionally, a supercharged peptide (SCP) may be utilized to enhance the internalization of the dCAS9-construct into target or recipient cells. The SCP comprises a specific sequence, SEQ ID NO: 13 (GSKPAKKGSKTPGTSKSAKPKKGPGSKPATKGSKKPGKSKSATPKKGPGSKPAKSGKKT PGTSKKPSKGKAPGTSKKPSKGKAPGSKPAKKGSKTPGTSKSAKPKKGPGKSKKATPKSG P), which facilitates the efficient delivery of the construct into the cells.

[0432] In summary, the method for gene-specific epigenetic editing of the CXCL11 gene involves the use of a gene-specific fusion complex comprising a DNA demethylase enzyme, guide RNAs for precise targeting, and potentially a dCAS9-derived system and supercharged peptide for enhanced delivery. This method aims to achieve targeted demethylation, providing epigenetic editing and potentially enhancing the transcriptional responsiveness of the gene.

[0433] In different embodiments, the method is executed as a gene-specific DNA demethylation. In certain embodiments, the gene-specific DNA demethylation involves performing a demethylation of one or more nucleotides within a gene, a promoter, and / or an anticipated regulatory region. The demethylation process is facilitated by contacting the gene and / or a gene promoter with a gene-specific fusion complex comprising at least one enzyme functioning as a DNA demethylase. The gene-specific fusion complex may include one or more demethylases and / or one or more DNA binding domains (DBDs) assembled in a construct, fusion protein, or RNA-protein complex. The demethylase enzyme may be derived from TDG (thymine DNA glycosylase), Tet1, Tet2, or Tet3, providing specific enzymatic activity for demethylation.

[0434] In certain embodiments, the gene-specific fusion complex is a gene-specific demethylase fusion complex, which includes a demethylase enzyme with a sequence- specific DNA binding domain (DBD). The DBD may be a dCAS9-gRNA system or a zinc finger protein array (ZFA). The dCAS9-based system may comprise constructs such as dCas9-Tet1 cat-NLS / Myc-SCP-HA / NLS / 6xHis-Stop Codon, dCas9-Tet1 mut-NLS / Myc-SCP- HA / NLS / 6xHis-Stop Codon, dCas9-TDG wt-NLS / Myc-SCP-HA / NLS / 6xHis-Stop Codon, and / or dCas9-TDG mut (N151A)-NLS / Myc-SCP-HA / NLS / 6xHis-Stop Codon. Additionally, a supercharged peptide (SCP) may be utilized to enhance the internalization of the dCAS9- construct into target or recipient cells. The SCP may comprise SEQ ID NO: 13 (GSKPAKKGSKTPGTSKSAKPKKGPGSKPATKGSKKPGKSKSATPKKGPGSKPAKSGKKT PGTSKKPSKGKAPGTSKKPSKGKAPGSKPAKKGSKTPGTSKSAKPKKGPGKSKKATPKSG P).

[0435] The method may also include guide RNAs designed for specific genes, such as the CXCL11 gene, comprising sequences like SEQ ID NO: 14 (AAAGCTACGTGAGTCAAGGA), SEQ ID NO: 15 (TGAGTGCTTTCACCTTCCAG), SEQ IDNO: 16 (AAAGATAGATGAAGGTCACC), SEQ ID NO: 17 (GTCCCACCAACTCAGGAAGA), SEQ ID NO: 18 (CCACAAGCCAGGAGATGCAG), or SEQ ID NO: 19 (CTGGGACCATCTTCCTGAGT).

[0436] In certain embodiments, the method utilizes a combination of fusion proteins in each administration, comprising ZFA-TDGs contacting three or more sites and / or ZFA- TET1 contacting three or more sites, all within the target promoter. Alternatively, the method may be reduced to just a single fusion construct. The demethylation provides an epigenetic editing of the gene, one or more nucleotides, regulatory motif, and / or the promoter, and may provide a test of the mechanistic significance of the demethylation of the gene or enhance the transcriptional responsiveness of the gene.

[0437] In different embodiments, the method utilizes a combination of fusion proteins in each administration comprising ZFA-TDGs contacting three or more sites and / or ZFA- TET1 contacting three or more sites, all within the target promoter, or wherein the method is reduced to just a single fusion construct. In certain embodiments, the ZFA-TDGs and ZFA- TET1 fusion proteins are designed to target multiple sites within the promoter region to achieve efficient and specific demethylation. The ZFA-TDGs and ZFA-TET1 fusion proteins may be produced by recombinant expression in an E. coli cell or by expression in a human embryonic kidney (HEK) or Chinese Hamster Ovary (CHO) cell, with the expression in the E. coli cell including the use of one or more molecular chaperones such as pTf16 for ZFA- TDG and pKJE7 for ZFA-Tet.

[0438] In certain embodiments, the ZFA-TDGs and ZFA-TET1 fusion proteins are assembled in a construct, fusion protein, or RNA-protein complex to facilitate the demethylation process. The fusion complex includes a demethylase enzyme with a sequence-specific DNA binding domain (DBD) to target specific DNA sequences for demethylation. The DBD may be a dCAS9-gRNA system or a zinc finger protein array (ZFA), providing sequence-specific targeting for demethylation. The dCAS9-based system may comprise constructs such as dCas9-Tet1 cat-NLS / Myc-SCP-HA / NLS / 6xHis-Stop Codon, dCas9-Tet1 mut-NLS / Myc-SCP-HA / NLS / 6xHis-Stop Codon, dCas9-TDG wt-NLS / Myc-SCP- HA / NLS / 6xHis-Stop Codon, and / or dCas9-TDG mut (N151A)-NLS / Myc-SCP-HA / NLS / 6xHis- Stop Codon. These constructs are designed to provide specific enzymatic activity for demethylation.

[0439] In certain embodiments, a supercharged peptide (SCP) is utilized to enhance the dCAS9-construct internalization into target or recipient cells. The SCP may comprise SEQ ID NO: 13 (GSKPAKKGSKTPGTSKSAKPKKGPGSKPATKGSKKPGKSKSATPKKGPGSKPAKSGKKT PGTSKKPSKGKAPGTSKKPSKGKAPGSKPAKKGSKTPGTSKSAKPKKGPGKSKKATPKSGP), providing a specific sequence for the supercharged peptide to improve delivery efficiency.

[0440] The method may also include guide RNAs designed for specific genes, such as the CXCL11 gene, comprising sequences like SEQ ID NO: 14 (AAAGCTACGTGAGTCAAGGA), SEQ ID NO: 15 (TGAGTGCTTTCACCTTCCAG), SEQ ID NO: 16 (AAAGATAGATGAAGGTCACC), SEQ ID NO: 17 (GTCCCACCAACTCAGGAAGA), SEQ ID NO: 18 (CCACAAGCCAGGAGATGCAG), or SEQ ID NO: 19 (CTGGGACCATCTTCCTGAGT). These guide RNAs provide specific sequences for targeting the CXCL11 gene, facilitating precise demethylation.

[0441] In certain embodiments, the method is executed as a gene-specific DNA demethylation, providing targeted DNA demethylation to achieve epigenetic editing. The demethylation process provides an epigenetic re-activation of a silenced or partially silenced gene, an epigenetic restoration of an adaptive, situation-appropriate responsiveness of intrinsic transcription, and / or an epigenetic enhancement of a gene’s expression. This method does not require a vector and / or a transduction-based approach, simplifying the process and making it more efficient.

[0442] Overall, the method described involves the use of a combination of fusion proteins, specific constructs, and guide RNAs to achieve targeted demethylation and epigenetic editing of genes, providing a powerful tool for gene-specific epigenetic modifications.

[0443] In different embodiments, the method provides an epigenetic editing of the gene, one or more nucleotides, regulatory motif, and / or the promoter. In certain embodiments, the gene-specific fusion complex is utilized to perform targeted demethylation. The gene-specific fusion complex may include at least one enzyme functioning as a DNA demethylase. The demethylation process is performed on one or more nucleotides within a gene, a promoter, and / or an anticipated regulatory region. This process provides an epigenetic editing of the gene, one or more nucleotides, regulatory motif, and / or the promoter.

[0444] In certain embodiments, the demethylation provides a test of the mechanistic significance of the demethylation of the gene or provides enhancement of transcriptional responsiveness of the gene. The purpose is to test the mechanistic significance of the demethylation of the gene or to enhance the transcriptional responsiveness of the gene. The demethylation process is achieved by contacting the gene and / or gene promoter with a gene-specific fusion complex comprising at least one enzyme functioning as a DNA demethylase.

[0445] In certain embodiments, the method provides an epigenetic re-activation of a silenced or partially silenced gene, an epigenetic restoration of an adaptive, situation-appropriate responsiveness of intrinsic transcription, and / or an epigenetic enhancement of a gene’s expression. The purpose is to modify gene expression and transcriptional responsiveness. Themethod may include the use of a gene-specific fusion complex that includes at least one enzyme functioning as a DNA demethylase to perform demethylation. The gene-specific fusion complex may be produced by expression in a bacterial, insect, or mammalian cell, then the fusion complex protein is purified and administered to a target cell or subject.

[0446] In certain embodiments, the method provides one or more data including an epigenetic influence on disease risk and / or gene silencing. The method may utilize a combination of fusion proteins in each administration comprising ZFA-TDGs contacting three or more sites and / or ZFA-TET1 contacting three or more sites, all within the target promoter, or the method may be reduced to just a single fusion construct.

[0447] In different embodiments, the method provides an epigenetic re-activation of a silenced or partially silenced gene, an epigenetic restoration of an adaptive, situation- appropriate responsiveness of intrinsic transcription, and / or an epigenetic enhancement of a gene’s expression. In certain embodiments, the epigenetic re-activation involves the use of a gene-specific fusion complex that includes at least one enzyme functioning as a DNA demethylase. This complex is designed to target specific DNA sequences and perform demethylation, thereby reactivating genes that have been silenced or partially silenced.

[0448] In certain embodiments, the epigenetic restoration of an adaptive, situation- appropriate responsiveness of intrinsic transcription is achieved by performing a demethylation of one or more nucleotides within a gene, a promoter, and / or an anticipated regulatory region. This demethylation process provides an epigenetic editing of the gene, one or more nucleotides, regulatory motif, and / or the promoter, which in turn restores the gene's transcriptional responsiveness to its natural, adaptive state.

[0449] In certain embodiments, the epigenetic enhancement of a gene’s expression is facilitated by the same demethylation process. The gene-specific fusion complex, which may include one or more demethylases and / or one or more DNA binding domains (DBDs) assembled in a construct, fusion protein, or RNA-protein complex, targets specific DNA sequences to remove methyl groups. This removal of methyl groups enhances the transcriptional responsiveness of the gene, leading to increased gene expression.

[0450] The method may utilize various enzymatic domains derived from TDG (thymine DNA glycosylase), Tet1, Tet2, or Tet3 to provide the necessary enzymatic activity for demethylation. These domains can be part of the gene-specific fusion complex, which is designed to perform targeted demethylation. The fusion complex may also include a demethylase enzyme connected to a DBD, such as a dCAS9-gRNA system or a zinc finger protein array (ZFA), to ensure sequence-specific targeting.

[0451] In certain embodiments, the method does not require a vector and / or a transduction-based approach, simplifying the process and reducing potential complications associated with vector-based methods. The fusion complex can be produced by expression in a bacterial, insect, or mammalian cell, then purified and administered to the target cell or subject. This approach ensures that the fusion complex is efficiently delivered to the target site, where it can perform the necessary demethylation to achieve the desired epigenetic modifications.

[0452] Overall, the method provides a versatile and efficient means of modifying gene expression through targeted demethylation, offering potential applications in gene therapy, research, and the study of epigenetic influences on disease risk and gene silencing.

[0453] In different embodiments, the method provides one or more data including an epigenetic influence on disease risk and / or gene silencing. In certain embodiments, the method involves contacting the gene and / or a gene promoter with a gene-specific fusion complex comprising at least one enzyme functioning as a DNA demethylase. This gene- specific fusion complex may include one or more demethylases and / or one or more DNA binding domains (DBDs) assembled in a construct, fusion protein, or RNA-protein complex. The demethylase enzyme may be derived from TDG (thymine DNA glycosylase), Tet1, Tet2, or Tet3, providing specific enzymatic activity for demethylation. The entire coding sequence of mouse or human TDG, a derivative sequence of mouse or human TDG, the enzymatic domain of mouse or human Tet1, Tet2, or Tet3, or a derivative sequence of the enzymatic domain of mouse or human Tet1, Tet2, or Tet3 may be utilized.

[0454] In certain embodiments, the method includes performing a demethylation of one or more nucleotides within a gene, a promoter, and / or an anticipated regulatory region. This demethylation provides an epigenetic editing of the gene, one or more nucleotides, regulatory motif, and / or the promoter. The method may also provide an epigenetic re- activation of a silenced or partially silenced gene, an epigenetic restoration of an adaptive, situation-appropriate responsiveness of intrinsic transcription, and / or an epigenetic enhancement of a gene’s expression.

[0455] In certain embodiments, the method utilizes a combination of fusion proteins in each administration comprising ZFA-TDGs contacting three or more sites and / or ZFA- TET1 contacting three or more sites, all within the target promoter, or the method may be reduced to just a single fusion construct. The fusion complex may be produced by expression in a bacterial, insect, or mammalian cell, then the fusion complex protein is purified and administered to a target cell or subject. The fusion complex may include a ZFA construct produced by recombinant expression in an E. coli cell, or by expression in a human embryonic kidney (HEK) or Chinese Hamster Ovary (CHO) cell, wherein theexpression in the E. coli cell includes the use of one or more molecular chaperones, such as pTf16 for ZFA-TDG and pKJE7 for ZFA-Tet.

[0456] The method does not require a vector and / or a transduction-based approach, simplifying the process. The method provides one or more data including an epigenetic influence on disease risk and / or gene silencing, allowing for the study of the epigenetic influence on disease risk and gene silencing.

[0457] In different embodiments, the method provides a test of the mechanistic significance of the demethylation of the gene or provides enhancement of transcriptional responsiveness of the gene. In certain embodiments, the gene-specific fusion complex is utilized to perform the demethylation process. The gene-specific fusion complex may include at least one enzyme functioning as a DNA demethylase. The DNA demethylase may be derived from TDG (thymine DNA glycosylase), Tet1, Tet2, or Tet3. The entire coding sequence of mouse or human TDG, a derivative sequence of mouse or human TDG, the enzymatic domain of mouse or human Tet1, Tet2, or Tet3, or a derivative sequence of the enzymatic domain of mouse or human Tet1, Tet2, or Tet3 may be used. The gene-specific fusion complex may be assembled in a construct, fusion protein, or RNA-protein complex, and may include one or more demethylases and / or one or more DNA binding domains (DBDs).

[0458] In certain embodiments, the method does not require a vector and / or a transduction-based approach. This simplifies the method and avoids the complexities associated with vector-based delivery systems. The gene-specific fusion complex may be produced by expression in a bacterial, insect, or mammalian cell. After expression, the fusion complex protein is purified and administered to a target cell or subject. This approach ensures that the fusion complex is efficiently produced and delivered to the target site.

[0459] The demethylation process provides an epigenetic editing of the gene, one or more nucleotides, regulatory motif, and / or the promoter. This epigenetic editing can be used to test the mechanistic significance of the demethylation of the gene or to enhance the transcriptional responsiveness of the gene. The method may provide an epigenetic re- activation of a silenced or partially silenced gene, an epigenetic restoration of an adaptive, situation-appropriate responsiveness of intrinsic transcription, and / or an epigenetic enhancement of a gene’s expression. This allows for precise control over gene expression and can be used to study the functional impact of demethylation or to enhance gene expression for therapeutic purposes.

[0460] In different embodiments, the method for gene-specific epigenetic editing may be executed without the requirement of a vector and / or a transduction-based approach. This is significant as it simplifies the method and potentially reduces the complexity and cost associated with vector-based delivery systems. The method described, with the proviso thatthe method does not require a vector and / or a transduction-based approach, aligns with the broader goal of achieving efficient and targeted gene-specific epigenetic editing.

[0461] In certain embodiments, the gene-specific fusion complex, which includes at least one enzyme functioning as a DNA demethylase, is utilized to perform the demethylation process. The gene-specific fusion complex may be produced by expression in a bacterial, insect, or mammalian cell, followed by purification and administration to the target cell or subject. This approach ensures that the fusion complex is efficiently delivered to the target site without the need for vectors or transduction methods.

[0462] The gene-specific fusion complex may include a demethylase enzyme connected to a DNA binding domain (DBD), such as a dCAS9-gRNA system or a zinc finger protein array (ZFA). These systems provide sequence-specific targeting, allowing for precise demethylation of the target gene or promoter. For instance, the dCAS9-based system may comprise constructs such as dCas9-Tet1 cat-NLS / Myc-SCP-HA / NLS / 6xHis-Stop Codon, dCas9-Tet1 mut-NLS / Myc-SCP-HA / NLS / 6xHis-Stop Codon, dCas9-TDG wt-NLS / Myc-SCP- HA / NLS / 6xHis-Stop Codon, and / or dCas9-TDG mut (N151A)-NLS / Myc-SCP-HA / NLS / 6xHis- Stop Codon. These constructs are designed to facilitate targeted demethylation and enhance the efficiency of the editing process.

[0463] Additionally, the method may utilize supercharged peptides (SCP) to enhance the internalization of the dCAS9-construct into target or recipient cells. The SCP may comprise specific sequences, such as SEQ ID NO: 13, which are designed to improve the delivery and uptake of the fusion complex.

[0464] By avoiding the use of vectors and transduction-based approaches, the method offers a streamlined and potentially more accessible means of achieving gene- specific epigenetic editing. This approach not only simplifies the delivery process but also reduces the potential for off-target effects and other complications associated with vector- based systems.

[0465] In different embodiments, the method for gene-specific epigenetic editing includes a demethylase enzyme connected to a DNA binding domain (DBD). The DBD may be a dCAS9-gRNA system or a zinc finger protein array (ZFA). In certain embodiments, the dCAS9-based system comprises constructs such as dCas9-Tet1 cat-NLS / Myc-SCP- HA / NLS / 6xHis-Stop Codon, dCas9-Tet1 mut-NLS / Myc-SCP-HA / NLS / 6xHis-Stop Codon, dCas9-TDG wt-NLS / Myc-SCP-HA / NLS / 6xHis-Stop Codon, and / or dCas9-TDG mut (N151A)- NLS / Myc-SCP-HA / NLS / 6xHis-Stop Codon. These constructs are designed to provide specific demethylation activities.

[0466] In certain embodiments, a supercharged peptide (SCP) is utilized to enhance the internalization of the dCAS9-construct into target or recipient cells. The SCP comprises SEQ ID NO: 13(GSKPAKKGSKTPGTSKSAKPKKGPGSKPATKGSKKPGKSKSATPKKGPGSKPAKSGKKT PGTSKKPSKGKAPGTSKKPSKGKAPGSKPAKKGSKTPGTSKSAKPKKGPGKSKKATPKSG P), which facilitates the efficient delivery of the dCAS9-construct into the cells.

[0467] In other embodiments, guide RNAs designed for the CXCL11 gene are utilized. These guide RNAs comprise specific sequences such as SEQ ID NO: 14 (AAAGCTACGTGAGTCAAGGA), SEQ ID NO: 15 (TGAGTGCTTTCACCTTCCAG), SEQ ID NO: 16 (AAAGATAGATGAAGGTCACC), SEQ ID NO: 17 (GTCCCACCAACTCAGGAAGA), SEQ ID NO: 18 (CCACAAGCCAGGAGATGCAG), or SEQ ID NO: 19 (CTGGGACCATCTTCCTGAGT). These sequences are designed to target the CXCL11 gene specifically for demethylation.

[0468] Additionally, in certain embodiments, one or more molecular chaperones such as pTf16 for ZFA-TDG and pKJE7 for ZFA-Tet are included to assist in the proper folding and expression of the ZFA constructs. These molecular chaperones ensure that the ZFA constructs are correctly folded and functional for the demethylation process.

[0469] The method described in these embodiments provides a comprehensive approach to gene-specific epigenetic editing by utilizing a combination of advanced molecular tools and techniques to achieve precise and efficient demethylation of target genes.

[0470] In different embodiments, the gene-specific epigenetic editing method includes the use of specific constructs for targeted demethylation. In certain embodiments, the method comprises dCas9-Tet1 cat-NLS / Myc-SCP-HA / NLS / 6xHis-Stop Codon, dCas9- Tet1 mut-NLS / Myc-SCP-HA / NLS / 6xHis-Stop Codon, dCas9-TDG wt-NLS / Myc-SCP- HA / NLS / 6xHis-Stop Codon, and / or dCas9-TDG mut (N151A)-NLS / Myc-SCP-HA / NLS / 6xHis- Stop Codon. These constructs are designed to provide specific demethylation activity.

[0471] The dCas9-Tet1 cat-NLS / Myc-SCP-HA / NLS / 6xHis-Stop Codon construct includes a catalytically active Tet1 domain fused with a dCas9 protein, which is further tagged with nuclear localization signals (NLS), Myc, supercharged peptide (SCP), hemagglutinin (HA), and a 6xHis tag followed by a stop codon. This construct is designed to target specific DNA sequences for demethylation, leveraging the sequence-specific binding capability of dCas9 guided by guide RNAs (gRNA).

[0472] Similarly, the dCas9-Tet1 mut-NLS / Myc-SCP-HA / NLS / 6xHis-Stop Codon construct includes a catalytically inactive (mutant) Tet1 domain, which serves as a control to distinguish the effects of demethylation from other potential influences of the fusion protein.

[0473] The dCas9-TDG wt-NLS / Myc-SCP-HA / NLS / 6xHis-Stop Codon construct includes a wild-type thymine DNA glycosylase (TDG) domain, which is known for its role in base excision repair and demethylation. This construct is also tagged with NLS, Myc, SCP,HA, and a 6xHis tag, ensuring efficient targeting and internalization into the nucleus of the target cells.

[0474] Lastly, the dCas9-TDG mut (N151A)-NLS / Myc-SCP-HA / NLS / 6xHis-Stop Codon construct includes a mutant TDG domain (N151A), which is catalytically inactive, serving as a control to validate the specific effects of TDG-mediated demethylation.

[0475] These constructs are utilized to enhance the specificity and efficiency of the demethylation process, providing a robust tool for gene-specific epigenetic editing. The inclusion of SCP in these constructs facilitates the internalization of the dCas9-construct into target or recipient cells, thereby improving the delivery efficiency of the demethylation machinery.

[0476] In summary, the method involves the use of specific dCas9-Tet1 and dCas9- TDG constructs, each designed with precise tags and modifications to ensure targeted and efficient demethylation of specific DNA sequences, thereby enabling gene-specific epigenetic editing.

[0477] In different embodiments, the method for gene-specific epigenetic editing includes the utilization of a supercharged peptide (SCP) to enhance the internalization of a dCAS9-construct into target or recipient cells. The SCP is specifically designed to improve the delivery efficiency of the dCAS9-construct, ensuring that the gene-specific fusion complex reaches the intended cellular targets effectively. The SCP comprises SEQ ID NO: 13, which is a specific peptide sequence: GSKPAKKGSKTPGTSKSAKPKKGPGSKPATKGSKKPGKSKSATPKKGPGSKPAKSGKKTP GTSKKPSKGKAPGTSKKPSKGKAPGSKPAKKGSKTPGTSKSAKPKKGPGKSKKATPKSGP . This sequence is utilized to enhance the internalization process, thereby facilitating the gene-specific epigenetic editing by ensuring that the dCAS9-construct is efficiently delivered into the cells where it can perform the demethylation process.

[0478] The method involves contacting the gene and / or a gene promoter with a gene-specific fusion complex comprising at least one enzyme functioning as a DNA demethylase. This step is for initiating the process of gene-specific epigenetic editing. The gene-specific fusion complex includes components such as the DNA demethylase and the SCP, which work together to perform the demethylation of one or more nucleotides within a gene, a promoter, and / or an anticipated regulatory region. The demethylation process provides an epigenetic editing of the gene, one or more nucleotides, regulatory motif, and / or the promoter, and it can also provide a test of the mechanistic significance of the demethylation of the gene or enhance the transcriptional responsiveness of the gene.

[0479] In certain embodiments, the SCP is utilized to enhance the internalization of the dCAS9-construct, which is an aspect of the method. The SCP's specific sequence, SEQ ID NO: 13, is designed to improve the efficiency of the delivery process, ensuring that thegene-specific fusion complex reaches the target cells effectively. This enhancement is for the successful execution of the gene-specific epigenetic editing, as it ensures that the dCAS9-construct can perform the demethylation process within the target cells.

[0480] Overall, the method for gene-specific epigenetic editing leverages the SCP to enhance the internalization of the dCAS9-construct, thereby facilitating the efficient delivery and subsequent demethylation process. This approach ensures that the gene-specific fusion complex can effectively reach and modify the target cells, providing a robust and efficient method for gene-specific epigenetic editing.

[0481] In different embodiments, the method for gene-specific epigenetic editing includes a demethylase enzyme connected to a DNA binding domain (DBD). In certain embodiments, the DBD is a dCAS9-gRNA system or a zinc finger protein array (ZFA). The dCAS9-based system may comprise constructs such as dCas9-Tet1 cat-NLS / Myc-SCP- HA / NLS / 6xHis-Stop Codon, dCas9-Tet1 mut-NLS / Myc-SCP-HA / NLS / 6xHis-Stop Codon, dCas9-TDG wt-NLS / Myc-SCP-HA / NLS / 6xHis-Stop Codon, and / or dCas9-TDG mut (N151A)- NLS / Myc-SCP-HA / NLS / ..

[0482] There are at least 5 problems in epigenetic editing and these are, for example, the off-target effects, undesired genomic mutations caused by the treatment, nuclear structure, cell types, and method of administration; this invention uses specific gene targeting to avoid off-target effects, causes no undesired genomic mutations and is applicable to almost any nuclear structure and cell type while providing a universal method of administration that includes intra-airway administration or any known.

[0483] A point of novelty, for discussions, of the technology herein is, for example, the use of a gene-specific fusion complex comprising a DNA demethylase to achieve targeted demethylation, allowing precise epigenetic editing and enhanced transcriptional responsiveness, while overcoming the current problems, examples discussed above, in the technology. The technology herein provides the future.

[0484] Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure. The methods, kits, formulations, and devices disclosed herein can be combined in any way into systems to address the current public health emergency.

[0485] The technology described herein is further illustrated by the following examples which in no way should be construed as being further limiting. The Examples are provided to demonstrate examples of future planned work, which in some experiments is emergency work. Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. EXAMPLES

[0486] In addition to the scientific experiments discussed above, the following Examples are provided to illustrate the invention, can be inter-combined with any other point herein and should not be considered to limit its scope. EXAMPLE 1. VECTOR-FREE INTRA-AIRWAY IN VIVO EPIGENETIC EDITING

[0487] Targeted modification of epigenetic landscape of a specific gene presents an exciting opportunity to take control over the epigenome. ‘Epigenetic editing’ is an emerging experimental strategy [72, 272-274] with the potential for novel therapeutic avenues. Of particular interest is DNA demethylation, which allows highly specific re-activation of epigenetically silenced genes or genes even partially muted by CpG methylation. Such re- activation allows context-dependent, situation-appropriate adaptive enhancement of stimulated transcriptional responsiveness of intrinsic genes in cells. This is situ activation can be used not only as a mechanistic tool to test causality of DNA methylation changes linked to an exposure or disease, but more broadly in a range of biomedical applications where enhancement of gene function is desired. Epigenetic editing can be accomplished by molecules where a sequence-specific DNA-binding domain (DBD) is fused with an enzymatic effector (e.g. a DNA demethylase). Earlier studies have shown proof-of-principle using zinc finger arrays (ZFA), TALE domains, or CRISPR-like dCAS9- gRNA systems for tethering epigenetically acting enzymes to DNA and have demonstrated the function of thymine-DNA-glycosylase (TDG) and ten-eleven translocation proteins (Tet) as DNA demethylases [1-4, 177, 199, 270].

[0488] However, an unresolved problem has been impairing future developments of this technology, particularly precluding its translation to a therapeutic approach. Currently, reports on epigenetic editing involve some form of transgenesis, usually by viral transduction or transfection, so that the epigenetically-acting constructs are expressed within the recipient cell. While this approach has served its useful purpose with the experimentation almost exclusively being done in vitro, it is linked to numerous problems when it comes to translating it into in vivo, and especially into human applications [239-243].

[0489] Here we obtained the epigenetically-acting fusion constructs as affinity- purified proteins using a common recombinant approach and administered them to live animals via the airways. We report promoter demethylation and resultant transcriptional enhancement of a prototypical gene target Cxcl11 in lung epithelial cells of mice, and in two cell line mimics of murine lung cells. We tested two types of constructs: one based on ZFAs that benefit from the zinc finger’s natural ability to permeate into cells and nuclei delivering afused enzymatic payload, and another based on dCAS9 enabled with additional internalization-promoting moieties.

[0490] Results; Overview, construct design and pre-screening in vitro; Study overview is presented in FIG.8A.

[0491] FIG.8A shows six ZFA’s were fused with Tet1CD or TDG (three each) or mutant controls, purified as recombinant proteins and administered to cells in the in vitro experiments or to mice in the in vivo experiments. In parallel, fusions of dCAS9 with Tet1CD or TDG (or mutant controls) were obtained as recombinant proteins, preincubated with six gRNAs and administered to cells in vitro or to mice in vivo. The effects were determined by assessing DNA methylation and transcriptional responsiveness of CXCL11. FIG.8B shows an example mCxcl11 promoter map and targeting loci. Six loci were selected for ZFA and six for gRNA, guided by predicted affinity of binding, specificity score, vicinity to TSS and avoidance of competition with known transcription factor –binding sites. Three pyrosequencing assays were designed to test DNA methylation effects in adjacent loci and a control assay for a remote site upstream. Note the coordinates used are mouse genome assembly 38. FIG.8C shows for pre-screening of the targeting loci, 3T3 cells were transduced with lentiviral constructs encoding the 3 ZFA-Tet1CD and 3 ZFA-TDG fusions (or mutant controls) and tested for DNA methylation and IFNγ-stimulated expression. FIG.8D shows comparative maps of the ZFA- and dCAS9- based fusion chimeras.

[0492] We fused TDG or Tet1 catalytic domain (Tet1CD), or their catalytically inactive mutants (1 amino acid replacement for TDG, 2 for Tet1) to two different types of DNA-binding domains (DBD) and thus obtained two types of epigenetic editor proteins. The first type comprised ZFAs as DBDs, the second was based on the nuclease-null ‘dead’ dCAS9 and guide RNAs (gRNA). To design these DBDs we analyzed the promoter sequence and chose the loci with the best combinations of predicted affinity and specificity scores in near vicinity to transcription start site (TSS) that did not occupy known transcription factor –binding sites. This resulted in 6 ZFA domains and 6 gRNAs targeting specific loci in Cxcl11 promoter (FIG.8B). Construct maps are shown in FIG.8D. We prescreened the effect of targeting these loci using a lentiviral transduction system for ZFA-based constructs. FIG.8C details the effects: we observed a drop of CpG methylation in all loci we tested (assays ‘Distal’, Proximal’ and ‘TSS’, FIG.8B) by ~15-30 percentage points, but not in a control region located more remotely upstream (‘Upstream’). The differences were significant when comparing values from cells expressing the working (wildtype, denoted ‘Wt’) constructs versus catalytically inactive mutant controls (denoted ‘Mut’) or truncated molecules comprising only ZFA but not the enzymes(s), or versus intact cells. This resulted in substantially enhanced transcriptional responsiveness of CXCL11 to IFNγ stimulus without affecting background transcription of the gene.

[0493] Intranuclear delivery in vitro and in vivo: We next obtained the ZFA-based and dCAS9-based demethylase fusion chimeras as recombinant proteins. To confirm that these protein fusions can be delivered into the nuclei of cells in vitro and in vivo we performed intranuclear staining of MLE12 and primary mouse airway cells after protein administrations. In vitro the proteins were added to culture for 1 hour, then the medium was replaced; 3 repeats for made ZFA, and 2 for dCAS9 proteins. In vivo the mice were given the proteins by a combination of intranasal instillations and nebulized aerosol, also with 3 repeats for ZFA, and 2 for dCAS9. FIG.10A, FIG.10A, and FIG.10C demonstrate intranuclear staining for the constructs 24 hours after the last administration in vitro (FIG.10A) or in vivo (FIG.10A, FIG.10C). Positive staining for His-tag – part of the epigenetic fusion chimeras - is seen in cells treated in culture or isolated from treated animals, but not in untreated controls stained identically, in the projection of DAPIstained nuclei. This staining is comparable to that for histone H3 – a positive control protein.

[0494] FIG.10A shows mouse lung epithelial cell line MLE12 cells treated in vitro with ZFA-based or dCAS9-based demethylase proteins, vs. untreated control. After permeabilization the cells were stained blue for nuclei (DAPI), green for cytoplasm (β- tubulin) and red either for histone H3 as a positive control (left set of images) or for His-tag domain of the demethylase fusion chimeras (right set of images). FIG.10A illustrates how mice were treated in vivo with dCAS9-based demethylase proteins, vs. untreated control; their primary lung epithelial cells were isolated as EpCAM-positive, and stained as in FIG. 10A. Panel C uses cells from a separate in vivo experiment with 1-time administration only and different staining procedure and antibodies: the primary EpCAM+ cells were stained for nuclei (DAPI, blue) and for His-tag domain of the proteins (green) or for histone H3 positive control (red). Untreated stained cells served as a negative control. Magnification X40.In vitro effects of vector-free epigenetic editor fusion proteins: The results of in vitro experiments are summarized in FIG.9A, FIG.9B, FIG.9C, FIG.9D, and FIG.9E. Both ZFA-based and dCAS9 / gRNA-based constructs (‘Wt’) accomplished a decrease in methylation and an increase in transcriptional responsiveness of CXCL11 in either 3T3 or MLE12 cells (FIG.9A and FIG.9B). Catalytically inactivated mutant constructs (‘Mut’) had no effect. The effects of the vector-free treatments were less pronounced compared to vectored delivery, however sufficiently impactful to allow hundreds of times more transcript. Only the loci in the vicinity of ZFA or gRNA binding were modulated; the control upstream loci remained unaffected (FIG. 9C).

[0495] To determine whether TDG and Tet1 synergistically contribute to the effect, and whether each alone is necessary and sufficient to attain artificial demethylation, we tested TDG-based and Tet1CD-based constructs separately, or together. In FIG.9D, both ZFA-based (to a lesser extent) and dCAS9-based (to a greater extent) demethylases weresubstantially more effective when both TDG and Tet1CD fusions were used, however each separately was also effective. In the case of dCAS9 constructs the combination effect was especially multiplicative.

[0496] To test duration of the effects, we continued to culture the cells after administration of the protein demethylase constructs and evaluated IFNγ-stimulated transcriptional responsiveness of CXCL11 at 1-, 2- and 3-week timepoints (FIG.9E). The fold upregulation over unstimulated background has progressively declined but remained elevated compared to mutant controls even at the 3-week timepoint. Because of the slight changes in background (unstimulated) transcription during the long culture both ratio and expression are shown. Note that MLE12 cells double every 7-9 hours and were split every 3 days.

[0497] FIG.9A, FIG.9B, FIG.9C, FIG.9D, and FIG.9E shows vector-free epigenetic editing in vitro. DNA methylation (via pyrosequencing, see loci map in FIG.8B) and gene expression effects of the ZFA-based and dCAS9-based fusion recombinant protein demethylases. Expression values and fold expression (IFNγ stimulated over unstimulated) are shown in order to control for any potential fluctuations in background expression. FIG. 9A shows effects of ZFA-fused demethylases. Methylation effect (3T3) and transcription effect (3T3, MLE12) of ZFA-demethylases in vitro. *p<0.05; ****p<0.0001. FIG.9B shows effects of dCAS9 -fused demethylases. Methylation and transcription effect of dCAS9- demethylases in vitro (MLE12). **p<0.01, +++p<0.0005 vs. Ctrl, *p<0.05. FIG.9C shows effects in control regions. Lack of effect in upstream control regions by either ZFA- or dCAS9-based constructs (3T3). FIG.9D shows synergistic action. Combination effect of TDG and Tet1 (3T3). ‘Mut’ not shown. *p<0.05 - ****p<0.0001. FIG.9E shows a duration study. Duration of the effect after a single set of administrations in MLE12 cells at 1-, 2- and 3-week timepoints. Note that Ratios (IFNγ stimulated divided by unstimulated) are plotted as MEAN without SEM as an integrated average. + p<0.05 +++p<0.001 in pairwise comparisons. +p<0.05; ++p<0.005, +++p<0.0005 pairwise.

[0498] In vivo effects of vector-free epigenetic editors administered via the airways: The results of in vivo administration of the ZFA- and dCAS9 / gRNA- demethylase proteins are summarized in FIG.11A, FIG.11B, FIG.11C, and FIG.11D. The treatments (FIG.11A) resulted in decreased methylation (FIG.11B, FIG.11C) in EpCAM-purified lung epithelial cells. The demethylation effect of the ZFA-based (FIG.11B) and of the dCAS9-based (FIG. 11C) wildtype (active, ‘Wt’) constructs was seen variably across the targeted areas compared to catalytically inactive mutant (‘Mut’) controls or naïve controls (‘Ctrl’), but not in the control remote regions (FIG.11D). IFNγ-stimulated transcriptional responsiveness of CXCL11 was significantly increased after treatment with Wt constructs versus inactivated Mut controls which had no (or trace) effect for either ZFA- (4B) or dCAS9- (FIG.11C)fusions. ZnCl2- containing buffer required for the ZFAs to maintain their natural conformation had no effect alone. In some but not other experiments we observed minimally elevated transcription of CXCL11 after IFNγ in untreated mice (vs. naïve controls), which remained insignificant.

[0499] Discussion: Targeted epigenetic editing has become an emerging approach to modulate transcriptional activity of intrinsic genes [274, 275]. From the early studies, including our own findings that sparked this research, the technology has developed from the use of ZFA or TALEs [1-4] to employ CRISPR-like dCAS9-based molecular constructs comprising epigenetically and non-epigenetically acting domains for transcriptional activation [199, 270, 276-279]. The impactful results of these and other studies bring hope of novel epigenetically–based therapeutics and have even started a discussion on the ethical and dual-purpose issues of epigenetic augmentation

[0280] .

[0500] At the same time, the published reports focus on in vitro work and employ viral or other complex methods to deliver epigenetic molecular constructs for stable or temporary incorporation into the genome of recipient cells. This poses a barrier to in vivo studies, and especially to therapeutic applications in humans: insertional mutagenesis, viral persistence, accumulation of proto-oncogenic lesions, immunogenicity, cellular toxicity, risks related to immune surveillance and other problems common to gene therapy [239-242]. Moreover, viral delivery by transfection can affect transcriptional activity of host genes which creates the potential for misinterpretation of gene expression data generated from transfected cells

[0243] and impairs usefulness for experimentation on epigenetic control of transcription particularly in immunologic studies.

[0501] In contrast, recombinant protein biologics are an established therapeutic approach. Because our target gene Cxcl11 encodes a protein that has potential therapeutic value in lung disease [6, 7] we focused on pulmonary administration for our in vivo experiments. Despite the challenges of protein delivery systemically (into blood) via the lungs, the airway lining itself represents an accessible target more easily reachable by a therapeutic [281, 282]. Limited but encouraging data show delivery of CAS9-type proteins into lung epithelial cells via intranasal instillation for gene editing

[0283] , which supported the feasibility of our plan.

[0502] Protein demethylases in our study understandably produced less powerful effect compared to transduced demethylases (FIG.9A, FIG.9B, FIG.9C, FIG.9D, and FIG. 9E vs. FIG.8C) although we did not aim for direct comparisons as it would be difficult to correlate the amount (or number) of proteins per cell produced after transduction vs. the amount of recombinant proteins administered. It remains to be determined whether delivery, intracellular internalization, intranuclear translocation or DNA binding are the areas for potential improvement, which could be the aim of future studies. It is of note however, thatsmall methylation changes can lead to large transcriptional effects – a premise that could be deduced from prior studies linking changed methylation and expression [2, 177, 284, 285] including data that even a 1% decrease in methylation leads to two-fold increase in transcription and a 5% - to 32- fold increase

[0286] .

[0503] The success of the two alternative DBDs we tested emphasizes the versatility of our vector-free approach. We note that each DBD has potential strengths and weaknesses in target binding, off-target effects, stability and delivery which will become more important when transitioning from in vitro approaches of the past to the vector-free in vivo studies. dCAS9 employs gRNAs which recognize a longer DNA sequence and are postulated to provide better gene-specificity than ZFA, however there is no obstacle to designing longer ZFAs that recognize e.g.20 or more base pairs for specificity similar to that of gRNA. gRNAs, even pre-mixed with dCAS9, may be fragile especially in the in vivo environment, whereas ZFA protein is more stable in vivo. The smaller size of ZFAs makes expression, purification and delivery less challenging than dCAS9. ZFAs enter into cells and further into their nuclei spontaneously, delivering a fused enzymatic payload [8, 9], whereas additional measures are needed for internalization and translocation of dCAS9, e.g. SCP [10, 11]. At the same time, re-designing a ZFA based construct to target a new gene requires re-expression and repurification of the entire fusion protein, whereas with dCAS9- demethylases only a replacement of gRNA’s is required to retarget. Finally, different gene promoters may present a varying set of targeting / binding loci which is dictated by the specific sequence of interest. Thus, the current enthusiasm for CRISPR technology in gene editing cannot be directly translated to vector-free epigenetics; the aforementioned benefits of ZFA make them a valuable approach that remains a useful tool for epigenetic editing

[0203] . Moreover, while we have not tested this, a combined use of ZFA-based and dCAS9-based constructs targeting the same promoter can enhance the effect, improve specificity, and broaden applicability, because the binding loci opportunities for either DBD usually differ and can present complementary opportunities.

[0504] Of note, ZFA and dCAS9 tests were performed as separate experiments, hence magnitude of the effects should not be directly compared. However, a similar extent and span of these effects suggests that the DBD’s binding alone is not part of the action, and that nature of the DBD has no or minimal impact on the outcome; lack of effect of the truncated ZFA-only constructs also supports this notion.

[0505] Cell composition: in live mice an influx of cells into the lungs could theoretically differ across study groups, which would contribute to methylation or expression difference. It is extremely unlikely that a one or two aminoacids mutation in the active center (as in our Mut controls) could result in differential cell recruitment activity between Mut and Wt protein –treated mice; hence Mut is an important and appropriate control. Themeasurements are done on purified EpCAM+ cells; we monitored cell purity by EpCAM+ staining and found no difference or bias comparing Mut vs. Wt samples. Moreover, we controlled for any unlikely technical bias related to the time it takes to complete the exposure: in some experiments we exposed Mut before Wt, in other experiments Wt before Mut. Moreover, in order to test whether heterogeneity of cell population could at all be a factor, we performed PCR on EpCAM-depleted flow-through fraction of lung cells and found Wt also enhanced CXCL11 responsiveness in these cells (data not shown). Finally, in vitro experiments additionally control for the unlikely bias due to difference in influx of cells.

[0506] In the in vitro experiments we have used MLE12 cells – a cell line of airway epithelial cells, and 3T3 cells – a fibroblast cell line, as two mimics of either airway lining or lung stromal cells. We observed similar to nearly identical results across all experiments (FIGs.9A-9E). In vitro studies have also allowed us to investigate the synergy and duration effects because they required a much smaller amount of epigenetic editor proteins than would be necessary for in vivo work.

[0507] Our duration experiment suggests that the effect of demethylation is mitotically heritable in a cell culture because MLE12 cells double approximately every 7-9 hours. Proteins were removed from the medium after initial administration(s), hence the observed effects are from a single set of treatments. This result is consistent with the postulated heritable nature of epigenetic effects and findings by others using vectored demethylation and hypermethylation systems

[0276] .

[0508] The synergistic effect of a combination of TDG and Tet1CD versus either enzyme alone is shown in FIG.9D. The combined effect was more pronounced in the dCAS9 system than in the ZFA system. Besides the known places of Tet’s and TDG in the demethylation mechanism with a postulated involvement of base-excision repair [287-290] it has been discussed whether they can act independently and to what extent

[0095] and whether both are needed for the outcome

[0291] . Building on the evidence that TDG physically interacts with Tet1, TET stabilizes TDG activity and that in general active DNA demethylation requires coordinated catalytic activities of both TET1 and TDG [96, 97], and on prior studies that overexpression of targeted TDG or similar glycosylase alone can accomplish artificial demethylation [1, 4, 177] we have expressed a premise that artificial overabundance of both enzymes may provide benefit

[0292] . Our data here present direct evidence that co- abundance of a combination of TDG with Tet1 at methylated sites provides a cooperative effect in epigenetic editing. Of course, intrinsic TDG and Tet1 assist our artificially overabundant TDG and Tet1 in performing their function, but from the point of view of maximizing the targeted demethylation effect for practical use combining both enzymes appears beneficial.

[0509] The practical benefits of epigenetic editors go beyond their value as a mechanistic tool for epigenetics research to test previously untestable mechanistic hypotheses. Un-silencing of desired intrinsic genes has advantages over the introduction of extraneous gene copies (overexpression) or recombinant protein treatment. Overexpression requires transgenesis (usually viral) and results in a constant high-level production of the encoded protein, usually entails no off-switch or regulation, however transgene promoter can become silenced over time and the function is lost. External recombinant protein therapeutics require repeated administrations (often lifetime) because of protein degradation (which raises cost and compliance issues), result in ‘pulsed’ high-dose fluxes of the agent from outside the cells with difficulties when intracellular effects are desired, or a difficult tissue morphology needs to be reached. In contrast, DNA demethylation opens the promoter to context-dependent, situation appropriate response to stimuli in situ. Epigenetic effects are long-lasting and mitotically heritable, and therefore offer the prospect for a stable increase of endogenous protein production in e.g. the airways as a more desirable, less expensive, mechanistically relevant and more effective treatment.

[0510] We hope our study is a step towards such therapeutic applications. Here we demonstrate for the first time the targeted epigenetic editing of a select gene in vivo performed without genome integration vectors via intra-airway administration of purified epigenetically acting recombinant protein biologics. This was accomplished by two types of fusion demethylase constructs based on either custom-designed ZFAs or CRISPR-like dCAS9-gRNAs targeting specific areas in the promoter of a prototype target gene Cxcl11. Pulmonary administration of such constructs to live animals or direct administration to two cell lines in culture resulted in significant decreases in DNA methylation and associated increases in stimulated, but not background, transcription of the gene of interest compared to catalytically inactive mutant control constructs or naïve background. The in vitro effects lasted for several weeks in a rapidly dividing culture, suggesting mitotic heritability. TDG and Tet1CD each allowed a fraction of the effects with a cooperative action when both were used in combination. EXAMPLE 2. CONSTRUCT DESIGNS Table 1. Selected Sequences Brief Overview

[0511] Beginning with some example enzymes, the Tet1 catalytic domain (wild type) DNA sequence (differing parts of wild type and mutant are indicated by bold or red letters) is as follows SEQ ID NO: 24: ctgcccacctgcagctgtcttgatcgagttatacaaaaagacaaaggcccatattatacaca ccttggggcaggaccaagtgttgctgctgtcagggaaatcatggagaataggtatggtcaaa aaggaaacgcaataaggatagaaatagtagtgtacaccggtaaagaagggaaaagctctcat gggtgtccaattgctaagtgggttttaagaagaagcagtgatgaagaaaaagttctttgttt ggtccggcagcgtacaggccaccactgtccaactgctgtgatggtggtgctcatcatggtgt gggatggcatccctcttccaatggccgaccggctatacacagagctcacagagaatctaaag tcatacaatgggcaccctaccgacagaagatgcaccctcaatgaaaatcgtacctgtacatg tcaaggaattgatccagagacttgtggagcttcattctcttttggctgttcatggagtatgt actttaatggctgtaagtttggtagaagcccaagccccagaagatttagaattgatccaagc tctcccttacatgaaaaaaaccttgaagataacttacagagtttggctacacgattagctcc aatttataagcagtatgctccagtagcttaccaaaatcaggtggaatatgaaaatgttgccc gagaatgtcggcttggcagcaaggaaggtcgtcccttctctggggtcactgcttgcctggac ttctgtgctcatcccCacagggAcattcacaacatgaataatggaagcactgtggtttgtac cttaactcgagaagataaccgctctttgggtgttattcctcaagatgagcagctccatgtgc tacctctttataagctttcagacacagatgagtttggctccaaggaaggaatggaagccaag atcaaatctggggccatcgaggtcctggcaccccgccgcaaaaaaagaacgtgtttcactca gcctgttccccgttctggaaagaagagggctgcgatgatgacagaggttcttgcacataaga taagggcagtggaaaagaaacctattccccgaatcaagcggaagaataactcaacaacaaca aacaacagtaagccttcgtcactgccaaccttagggagtaacactgagaccgtgcaacctga agtaaaaagtgaaaccgaaccccattttatcttaaaaagttcagacaacactaaaacttatt cgctgatgccatccgctcctcacccagtgaaagaggcatctccaggcttctcctggtccccg aagactgcttcagccacaccagctccactgaagaatgacgcaacagcctcatgcgggttttc agaaagaagcagcactccccactgtacgatgccttcgggaagactcagtggtgccaatgcag ctgctgctgatggccctggcatttcacagcttggcgaagtggctcctctccccaccctgtct gctcctgtgatggagcccctcattaattctgagccttccactggtgtgactgagccgctaac gcctcatcagccaaaccaccagccctccttcctcacctctcctcaagaccttgcctcttctc caatggaagaagatgagcagcattctgaagcagatgagcctccatcagacgaacccctatct gatgaccccctgtcacctgctgaggagaaattgccccacattgatgagtattggtcagacag tgagcacatctttttggatgcaaatattggtggggtggccatcgcacctgctcacggctcgg ttttgattgagtgtgcccggcgagagctgcacgctaccactcctgttgagcaccccaaccgt aatcatccaacccgcctctcccttgtcttttaccagcacaaaaacctaaataagccccaaca tggttttgaactaaacaagattaagtttgaggctaaagaagctaagaataagaaaatgaagg cctcagagcaaaaagaccaggcagctaatgaaggtccagaacagtcctctgaagtaaatgaa ttgaaccaaattccttctcataaagcattaacattaacccatgacaatgttgtcaccgtgtc cccttatgctctcacacacgttgcggggccctataaccattgggtc

[0512] The Tet1 catalytic domain (wild type) AA sequence is as follows SEQ ID NO: 1: LPTCSCLDRVIQKDKGPYYTHLGAGPSVAAVREIMENRYGQKGNAIRIEIVVYTGKEGKSSH GCPIAKWVLRRSSDEEKVLCLVRQRTGHHCPTAVMVVLIMVWDGIPLPMADRLYTELTENLK SYNGHPTDRRCTLNENRTCTCQGIDPETCGASFSFGCSWSMYFNGCKFGRSPSPRRFRIDPSSPLHEKNLEDNLQSLATRLAPIYKQYAPVAYQNQVEYENVARECRLGSKEGRPFSGVTACLD FCAHPHRDIHNMNNGSTVVCTLTREDNRSLGVIPQDEQLHVLPLYKLSDTDEFGSKEGMEAK IKSGAIEVLAPRRKKRTCFTQPVPRSGKKRAAMMTEVLAHKIRAVEKKPIPRIKRKNNSTTT NNSKPSSLPTLGSNTETVQPEVKSETEPHFILKSSDNTKTYSLMPSAPHPVKEASPGFSWSP KTASATPAPLKNDATASCGFSERSSTPHCTMPSGRLSGANAAAADGPGISQLGEVAPLPTLS APVMEPLINSEPSTGVTEPLTPHQPNHQPSFLTSPQDLASSPMEEDEQHSEADEPPSDEPLS DDPLSPAEEKLPHIDEYWSDSEHIFLDANIGGVAIAPAHGSVLIECARRELHATTPVEHPNR NHPTRLSLVFYQHKNLNKPQHGFELNKIKFEAKEAKNKKMKASEQKDQAANEGPEQSSEVNE LNQIPSHKALTLTHDNVVTVSPYALTHVAGPYNHWV

[0513] The Tet1 mutant H255Y-D257A (differing parts of wild type and mutant are indicated by bold or red letters) DNA sequence, SEQ ID NO: 25: ctgcccacctgcagctgtcttgaTcgagttatacaaaaagacaaaggcccatattatacaca ccttggggcaggaccaagtgttgctgctgtcagggaaatcatggagaataggtatggtcaaa aaggaaacgcaataaggatagaaatagtagtgtacaccggtaaagaagggaaaagctctcat gggtgtccaattgctaagtgggttttaagaagaagcagtgatgaagaaaaagttctttgttt ggtccggcagcgtacaggccaccactgtccaactgctgtgatggtggtgctcatcatggtgt gggatggcatccctcttccaatggccgaccggctatacacagagctcacagagaatctaaag tcatacaatgggcaccctaccgacagaagatgcaccctcaatgaaaatcgtacctgtacatg tcaaggaattgatccagagacttgtggagcttcattctcttttggctgttcatggagtatgt actttaatggctgtaagtttggtagaagcccaagccccagaagatttagaattgatccaagc tctcccttacatgaaaaaaaccttgaagataacttacagagtttggctacacgattagctcc aatttataagcagtatgctccagtagcttaccaaaatcaggtggaatatgaaaatgttgccc gagaatgtcggcttggcagcaaggaaggtcgtcccttctctggggtcactgcttgcctggac ttctgtgctcatcccTacagggCcattcacaacatgaataatggaagcactgtggtttgtac cttaactcgagaagataaccgctctttgggtgttattcctcaagatgagcagctccatgtgc tacctctttataagctttcagacacagatgagtttggctccaaggaaggaatggaagccaag atcaaatctggggccatcgaggtcctggcaccccgccgcaaaaaaagaacgtgtttcactca gcctgttccccgttctggaaagaagagggctgcgatgatgacagaggttcttgcacataaga taagggcagtggaaaagaaacctattccccgaatcaagcggaagaataactcaacaacaaca aacaacagtaagccttcgtcactgccaaccttagggagtaacactgagaccgtgcaacctga agtaaaaagtgaaaccgaaccccattttatcttaaaaagttcagacaacactaaaacttatt cgctgatgccatccgctcctcacccagtgaaagaggcatctccaggcttctcctggtccccg aagactgcttcagccacaccagctccactgaagaatgacgcaacagcctcatgcgggttttc agaaagaagcagcactccccactgtacgatgccttcgggaagactcagtggtgccaatgcag ctgctgctgatggccctggcatttcacagcttggcgaagtggctcctctccccaccctgtct gctcctgtgatggagcccctcattaattctgagccttccactggtgtgactgagccgctaac gcctcatcagccaaaccaccagccctccttcctcacctctcctcaagaccttgcctcttctc caatggaagaagatgagcagcattctgaagcagatgagcctccatcagacgaacccctatct gatgaccccctgtcacctgctgaggagaaattgccccacattgatgagtattggtcagacag tgagcacatctttttggatgcaaatattggtggggtggccatcgcacctgctcacggctcgg ttttgattgagtgtgcccggcgagagctgcacgctaccactcctgttgagcaccccaaccgt aatcatccaacccgcctctcccttgtcttttaccagcacaaaaacctaaataagccccaaca tggttttgaactaaacaagattaagtttgaggctaaagaagctaagaataagaaaatgaaggcctcagagcaaaaagaccaggcagctaatgaaggtccagaacagtcctctgaagtaaatgaa ttgaaccaaattccttctcataaagcattaacattaacccatgacaatgttgtcaccgtgtc cccttatgctctcacacacgttgcggggccctataaccattgggtc

[0514] The Tet1 mutant H255Y-D257A (differing parts of wild type and mutant are indicated by red letters) AA sequence, SEQ ID NO: 2: LPTCSCLDRVIQKDKGPYYTHLGAGPSVAAVREIMENRYGQKGNAIRIEIVVYTGKEGKSSH GCPIAKWVLRRSSDEEKVLCLVRQRTGHHCPTAVMVVLIMVWDGIPLPMADRLYTELTENLK SYNGHPTDRRCTLNENRTCTCQGIDPETCGASFSFGCSWSMYFNGCKFGRSPSPRRFRIDPS SPLHEKNLEDNLQSLATRLAPIYKQYAPVAYQNQVEYENVARECRLGSKEGRPFSGVTACLD FCAHPYRAIHNMNNGSTVVCTLTREDNRSLGVIPQDEQLHVLPLYKLSDTDEFGSKEGMEAK IKSGAIEVLAPRRKKRTCFTQPVPRSGKKRAAMMTEVLAHKIRAVEKKPIPRIKRKNNSTTT NNSKPSSLPTLGSNTETVQPEVKSETEPHFILKSSDNTKTYSLMPSAPHPVKEASPGFSWSP KTASATPAPLKNDATASCGFSERSSTPHCTMPSGRLSGANAAAADGPGISQLGEVAPLPTLS APVMEPLINSEPSTGVTEPLTPHQPNHQPSFLTSPQDLASSPMEEDEQHSEADEPPSDEPLS DDPLSPAEEKLPHIDEYWSDSEHIFLDANIGGVAIAPAHGSVLIECARRELHATTPVEHPNR NHPTRLSLVFYQHKNLNKPQHGFELNKIKFEAKEAKNKKMKASEQKDQAANEGPEQSSEVNE LNQIPSHKALTLTHDNVVTVSPYALTHVAGPYNHWV

[0515] The TDG wild type (active, differing parts of wild type and mutant are indicated by red letters) DNA sequence, SEQ ID NO: 26: gacgcagaggccgcgcgcagctattctctggagcaagttcaagctttgtattcatttccatt tcaacaaatgatggcagaagttcctaacatggcagtcacgactggacagcaggtgccagcag tagctcctaacatggcaaccgtgacagaacagcaggtgcccgaagacgctcctgtccaggaa cctgcaccagaagctccaaagagaaggaaaaggaaacccagagcagcagagccccaggaacc agtggagcccaaaaaacctgctacgtcgaagaaatccggcaagtctacaaaatcaaaggaaa agcaggagaaaatcacagacgcgtttaaagtgaaaaggaaagtggaccgcttcaacggcgtc tctgaagctgagcttctgaccaagactcttcctgacattttgaccttcaatctggatattgt gatcattggcattAAcccgggattaatggctgcttacaaaggacatcactaccctgggcctg gaaatcacttctggaagtgtctgttcatgtcggggctgagtgaggtgcagctgaatcacatg gatgaccacaccttacccggcaagtacggcatcggattcaccaacatggtggaacggacgac gccgggcagcaaggatctgtctagtaaagagttccgggaaggagggcgcatcctggtgcaga aactgcagaaatatcagccacgaatagcggtgtttaatggaaaatgtatttatgaaattttc agtaaagaagtttttggagtaaaggttaagaacttggaatttgggcttcaaccccacaagat cccagacacagaaactctgtgctacgtcatgccgtcgtccagcgccagatgtgctcagtttc cccgggcccaggacaaagttcattactacattaagctgaaggacttgagagaccaactgaaa ggcattgaacgcaacgcggacgttcaggaagtgcagtatacatttgacctgcagcttgcgca agaggacgcaaagaagatggctgttaaggaagaaaagtatgatccaggctatgaggcagctt acggcggtgcctatggggaaaacccatgtaatggggaaccttgtggcattgcttcaaatggg ctaacagctcacagtgcggagccgagaggagaagcggcccccagcgatgttccgaatgggca gtggatggcacagtcgtttgcagagcagatcccttcttttaataattgtgggacccgagagc aggaagaagagagccacgct

[0516] The TDG wild type (active) AA sequence, SEQ ID NO: 7:DAEAARSYSLEQVQALYSFPFQQMMAEVPNMAVTTGQQVPAVAPNMATVTEQQVPEDAPVQE PAPEAPKRRKRKPRAAEPQEPVEPKKPATSKKSGKSTKSKEKQEKITDAFKVKRKVDRFNGV SEAELLTKTLPDILTFNLDIVIIGINPGLMAAYKGHHYPGPGNHFWKCLFMSGLSEVQLNHM DDHTLPGKYGIGFTNMVERTTPGSKDLSSKEFREGGRILVQKLQKYQPRIAVFNGKCIYEIF SKEVFGVKVKNLEFGLQPHKIPDTETLCYVMPSSSARCAQFPRAQDKVHYYIKLKDLRDQLK GIERNADVQEVQYTFDLQLAQEDAKKMAVKEEKYDPGYEAAYGGAYGENPCNGEPCGIASNG LTAHSAEPRGEAAPSDVPNGQWMAQSFAEQIPSFNNCGTREQEEESHA

[0517] The TDG mutant N151A (inactive, differing parts of wild type and mutant are indicated by bold or red letters) DNA sequence, SEQ ID NO: 27: gacgcagaggccgcgcgcagctattctctggagcaagttcaagctttgtattcatttccatt tcaacaaatgatggcagaagttcctaacatggcagtcacgactggacagcaggtgccagcag tagctcctaacatggcaaccgtgacagaacagcaggtgcccgaagacgctcctgtccaggaa cctgcaccagaagctccaaagagaaggaaaaggaaacccagagcagcagagccccaggaacc agtggagcccaaaaaacctgctacgtcgaagaaatccggcaagtctacaaaatcaaaggaaa agcaggagaaaatcacagacgcgtttaaagtgaaaaggaaagtggaccgcttcaacggcgtc tctgaagctgagcttctgaccaagactcttcctgacattttgaccttcaatctggatattgt gatcattggcattGCcccgggattaatggctgcttacaaaggacatcactaccctgggcctg gaaatcacttctggaagtgtctgttcatgtcggggctgagtgaggtgcagctgaatcacatg gatgaccacaccttacccggcaagtacggcatcggattcaccaacatggtggaacggacgac gccgggcagcaaggatctgtctagtaaagagttccgggaaggagggcgcatcctggtgcaga aactgcagaaatatcagccacgaatagcggtgtttaatggaaaatgtatttatgaaattttc agtaaagaagtttttggagtaaaggttaagaacttggaatttgggcttcaaccccacaagat cccagacacagaaactctgtgctacgtcatgccgtcgtccagcgccagatgtgctcagtttc cccgggcccaggacaaagttcattactacattaagctgaaggacttgagagaccaactgaaa ggcattgaacgcaacgcggacgttcaggaagtgcagtatacatttgacctgcagcttgcgca agaggacgcaaagaagatggctgttaaggaagaaaagtatgatccaggctatgaggcagctt acggcggtgcctatggggaaaacccatgtaatggggaaccttgtggcattgcttcaaatggg ctaacagctcacagtgcggagccgagaggagaagcggcccccagcgatgttccgaatgggca gtggatggcacagtcgtttgcagagcagatcccttcttttaataattgtgggacccgagagc aggaagaagagagccacgct

[0518] The TDG mutant N151A (inactive, differing parts of wild type and mutant are indicated by red letters) AA sequence, SEQ ID NO: 8: DAEAARSYSLEQVQALYSFPFQQMMAEVPNMAVTTGQQVPAVAPNMATVTEQQVPEDAPVQE PAPEAPKRRKRKPRAAEPQEPVEPKKPATSKKSGKSTKSKEKQEKITDAFKVKRKVDRFNGV SEAELLTKTLPDILTFNLDIVIIGIAPGLMAAYKGHHYPGPGNHFWKCLFMSGLSEVQLNHM DDHTLPGKYGIGFTNMVERTTPGSKDLSSKEFREGGRILVQKLQKYQPRIAVFNGKCIYEIF SKEVFGVKVKNLEFGLQPHKIPDTETLCYVMPSSSARCAQFPRAQDKVHYYIKLKDLRDQLK GIERNADVQEVQYTFDLQLAQEDAKKMAVKEEKYDPGYEAAYGGAYGENPCNGEPCGIASNG LTAHSAEPRGEAAPSDVPNGQWMAQSFAEQIPSFNNCGTREQEEESHA

[0519] Super charged protein (SCP) DNA sequence– published by Yin / Gao et al., SEQ ID NO: 28:ggtagtaaacccgctaagaaaggaagcaagactcctgggacaagcaaaagtgccaaaccgaa gaaagggccgggaagcaagccagcgaccaaaggtagcaaaaagcccggcaaaagtaagagcg ctacgcctaagaagggaccagggagtaaaccggcaaagagcggcaaaaaaacaccgggcact agtaaaaagcccagtaagggaaaagcccctggaaccagcaagaaacctagcaaaggtaaggc gcccggcagcaagccagccaaaaagggcagtaagacgccgggtacgagtaaaagcgctaaac cgaaaaaagggcctggaaagagtaagaaggcaacaccgaaaagtggtcca

[0520] Super charged protein (SCP) AA sequence– published by Yin / Gao et al., SEQ ID NO: 13:TCATTAAAGACAAGGACTTCCTGGACAATGAGGAGAACGAGGACATTCTTGAGGACATTGTC CTCACCCTTACGTTGTTTGAAGATAGGGAGATGATTGAAGAACGCTTGAAAACTTACGCTCACTTAAGTCCGATGGATTTGCCAACAGGAACTTCATGCAGTTGATCCATGATGACTCTCTCAC CTTTAAGGAGGACATCCAGAAAGCACAAGTTTCTGGCCAGGGGGACAGTCTTCACGAGCACA TCGCTAATCTTGCAGGTAGCCCAGCTATCAAAAAGGGAATACTGCAGACCGTTAAGGTCGTG GATGAACTCGTCAAAGTAATGGGAAGGCATAAGCCCGAGAATATCGTTATCGAGATGGCCCG AGAGAACCAAACTACCCAGAAGGGACAGAAGAACAGTAGGGAAAGGATGAAGAGGATTGAAG AGGGTATAAAAGAACTGGGGTCCCAAATCCTTAAGGAACACCCAGTTGAAAACACCCAGCTT CAGAATGAGAAGCTCTACCTGTACTACCTGCAGAACGGCAGGGACATGTACGTGGATCAGGA ACTGGACATCAATCGGCTCTCCGACTACGACGTGGATGCTATCGTGCCCCAGTCTTTTCTCA AAGATGATTCTATTGATAATAAAGTGTTGACAAGATCCGATAAAAATAGAGGGAAGAGTGAT AACGTCCCCTCAGAAGAAGTTGTCAAGAAAATGAAAAATTATTGGCGGCAGCTGCTGAACGC CAAACTGATCACACAACGGAAGTTCGATAATCTGACTAAGGCTGAACGAGGTGGCCTGTCTG AGTTGGATAAAGCCGGCTTCATCAAAAGGCAGCTTGTTGAGACACGCCAGATCACCAAGCAC GTGGCCCAAATTCTCGATTCACGCATGAACACCAAGTACGATGAAAATGACAAACTGATTCG AGAGGTGAAAGTTATTACTCTGAAGTCTAAGCTGGTCTCAGATTTCAGAAAGGACTTTCAGT TTTATAAGGTGAGAGAGATCAACAATTACCACCATGCGCATGATGCCTACCTGAATGCAGTG GTAGGCACTGCACTTATCAAAAAATATCCCAAGCTTGAATCTGAATTTGTTTACGGAGACTA TAAAGTGTACGATGTTAGGAAAATGATCGCAAAGTCTGAGCAGGAAATAGGCAAGGCCACCG CTAAGTACTTCTTTTACAGCAATATTATGAATTTTTTCAAGACCGAGATTACACTGGCCAAT GGAGAGATTCGGAAGCGACCACTTATCGAAACAAACGGAGAAACAGGAGAAATCGTGTGGGA CAAGGGTAGGGATTTCGCGACAGTCCGGAAGGTCCTGTCCATGCCGCAGGTGAACATCGTTA AAAAGACCGAAGTACAGACCGGAGAGAAATACGGCGGATTCGATTCTCCTACAGTCGCTTAC AGTGTACTGGTTGTGGCCAAAGTGGAGAAAGGGAAGTCTAAAAAACTCAAAAGCGTCAAGGA ACTGCTGGGCATCACAATCATGGAGCGATCAAGCTTCGAAAAAAACCCCATCGACTTTCTCG AGGCGAAAGGATATAAAGAGGTCAAAAAAGACCTCATCATTAAGCTTCCCAAGTACTCTCTC TTTGAGCTTGAAAACGGCCGGAAACGAATGCTCGCTAGTGCGGGCGAGCTGCAGAAAGGTAA CGAGCTGGCACTGCCCTCTAAATACGTTAATTTCTTGTATCTGGCCAGCCACTATGAAAAGC TCAAAGGGTCTCCCGAAGATAATGAGCAGAAGCAGCTGTTCGTGGAACAACACAAACACTAC CTTGATGAGATCATCGAGCAAATAAGCGAATTCTCCAAAAGAGTGATCCTCGCCGACGCTAA CCTCGATAAGGTGCTTTCTGCTTACAATAAGCACAGGGATAAGCCCATCAGGGAGCAGGCAG AAAACATTATCCACTTGTTTACTCTGACCAACTTGGGCGCGCCTGCAGCCTTCAAGTACTTC GACACCACCATAGACAGAAAGCGGTACACCTCTACAAAGGAGGTCCTGGACGCCACACTGAT TCATCAGTCAATTACGGGGCTCTATGAAACAAGAATCGACCTCTCTCAGCTCGGTGGAGACg gtggtagtggaggttcaggaggatccggggggagcggagggagcgatccagcacagtggcgg ccgcTCCTGCCCACCTGCAGCTGTCTTGATCGAGTTATACAAAAAGACAAAGGCCCATATTA TACACACCTTGGGGCAGGACCAAGTGTTGCTGCTGTCAGGGAAATCATGGAGAATAGGTATG GTCAAAAAGGAAACGCAATAAGGATAGAAATAGTAGTGTACACCGGTAAAGAAGGGAAAAGC TCTCATGGGTGTCCAATTGCTAAGTGGGTTTTAAGAAGAAGCAGTGATGAAGAAAAAGTTCTTGGTGTGGGATGGCATCCCTCTTCCAATGGCCGACCGGCTATACACAGAGCTCACAGAGAAT CTAAAGTCATACAATGGGCACCCTACCGACAGAAGATGCACCCTCAATGAAAATCGTACCTGTACATGTCAAGGAATTGATCCAGAGACTTGTGGAGCTTCATTCTCTTTTGGCTGTTCATGGA GTATGTACTTTAATGGCTGTAAGTTTGGTAGAAGCCCAAGCCCCAGAAGATTTAGAATTGAT CCAAGCTCTCCCTTACATGAAAAAAACCTTGAAGATAACTTACAGAGTTTGGCTACACGATT AGCTCCAATTTATAAGCAGTATGCTCCAGTAGCTTACCAAAATCAGGTGGAATATGAAAATG TTGCCCGAGAATGTCGGCTTGGCAGCAAGGAAGGTCGTCCCTTCTCTGGGGTCACTGCTTGC CTGGACTTCTGTGCTCATCCCCACAGGGACATTCACAACATGAATAATGGAAGCACTGTGGT TTGTACCTTAACTCGAGAAGATAACCGCTCTTTGGGTGTTATTCCTCAAGATGAGCAGCTCC ATGTGCTACCTCTTTATAAGCTTTCAGACACAGATGAGTTTGGCTCCAAGGAAGGAATGGAA GCCAAGATCAAATCTGGGGCCATCGAGGTCCTGGCACCCCGCCGCAAAAAAAGAACGTGTTTATAAGATAAGGGCAGTGGAAAAGAAACCTATTCCCCGAATCAAGCGGAAGAATAACTCAACA ACAACAAACAACAGTAAGCCTTCGTCACTGCCAACCTTAGGGAGTAACACTGAGACCGTGCA ACCTGAAGTAAAAAGTGAAACCGAACCCCATTTTATCTTAAAAAGTTCAGACAACACTAAAA CTTATTCGCTGATGCCATCCGCTCCTCACCCAGTGAAAGAGGCATCTCCAGGCTTCTCCTGG TCCCCGAAGACTGCTTCAGCCACACCAGCTCCACTGAAGAATGACGCAACAGCCTCATGCGG GTTTTCAGAAAGAAGCAGCACTCCCCACTGTACGATGCCTTCGGGAAGACTCAGTGGTGCCA ATGCAGCTGCTGCTGATGGCCCTGGCATTTCACAGCTTGGCGAAGTGGCTCCTCTCCCCACC CTGTCTGCTCCTGTGATGGAGCCCCTCATTAATTCTGAGCCTTCCACTGGTGTGACTGAGCC GCTAACGCCTCATCAGCCAAACCACCAGCCCTCCTTCCTCACCTCTCCTCAAGACCTTGCCT CTTCTCCAATGGAAGAAGATGAGCAGCATTCTGAAGCAGATGAGCCTCCATCAGACGAACCC CTATCTGATGACCCCCTGTCACCTGCTGAGGAGAAATTGCCCCACATTGATGAGTATTGGTC AGACAGTGAGCACATCTTTTTGGATGCAAATATTGGTGGGGTGGCCATCGCACCTGCTCACG GCTCGGTTTTGATTGAGTGTGCCCGGCGAGAGCTGCACGCTACCACTCCTGTTGAGCACCCC AACCGTAATCATCCAACCCGCCTCTCCCTTGTCTTTTACCAGCACAAAAACCTAAATAAGCC CCAACATGGTTTTGAACTAAACAAGATTAAGTTTGAGGCTAAAGAAGCTAAGAATAAGAAAA TGAAGGCCTCAGAGCAAAAAGACCAGGCAGCTAATGAAGGTCCAGAACAGTCCTCTGAAGTA AATGAATTGAACCAAATTCCTTCTCATAAAGCATTAACATTAACCCATGACAATGTTGTCAC CGTGTCCCCTTATGCTCTCACACACGTTGCGGGGCCCTATAACCATTGGGTCCCTAAGAAAA AGAGGAAGGTGgcgggcgctGAGCAGAAATTGATTAGTGAAGAGGATCTCGGTAGTAAACCC GCTAAGAAAGGAAGCAAGACTCCTGGGACAAGCAAAAGTGCCAAACCGAAGAAAGGGCCGGG AAGCAAGCCAGCGACCAAAGGTAGCAAAAAGCCCGGCAAAAGTAAGAGCGCTACGCCTAAGA AGGGACCAGGGAGTAAACCGGCAAAGAGCGGCAAAAAAACACCGGGCACTAGTAAAAAGCCC AGTAAGGGAAAAGCCCCTGGAACCAGCAAGAAACCTAGCAAAGGTAAGGCGCCCGGCAGCAA GCCAGCCAAAAAGGGCAGTAAGACGCCGGGTACGAGTAAAAGCGCTAAACCGAAAAAAGGGC CTGGAAAGAGTAAGAAGGCAACACCGAAAAGTGGTCCAggcggagggTACCCATACGATGTT CCAGATTACGCTtcgCCGAAGAAAAAGCGCAAGGTCggattcCATCATCATCATCATCATTA G

[0522] For the dCAS9-based CRISPR-like system, incorporate the following sequence into the mammalian expression vector pcDNA3.1-His; dCas9-Tet1 cat-NLS / Myc- SCP-HA / NLS / 6xHis-Stop Codon; AA sequence is SEQ ID NO: 9: MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEAT RLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVD EVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGL TPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNT EITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEF YKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLK DNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMT NFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRK VTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIV LTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDF LKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVV DELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQL QNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSD NVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAV VGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLAN GEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVN...

Claims

CLAIMS We claim:

1. A method for a gene-specific epigenetic editing of a gene, the method comprising the steps of: (1) contacting the gene and / or a gene promoter with a gene-specific fusion complex comprising at least one enzyme functioning as a DNA demethylase; and (2) performing a demethylation of one or more nucleotides within a gene, a promoter, and / or an anticipated regulatory region; whereby the demethylation provides an epigenetic editing of the gene, one or more nucleotides, regulatory motif, and / or the promoter and provides a test of the mechanistic significance of the demethylation of the gene or provides enhancement of transcriptional responsiveness of the gene.

2. The method of claim 1, wherein the method includes one or more demethylases, and / or one or more DNA binding domains (DBDs) assembled in a construct, fusion protein, or RNA- protein complex.

3. The method of claim 1, wherein at least one enzymatic domain is derived from TDG (thymine DNA glycosylase), Tet1, Tet2 or Tet3 (ten-eleven translocation, TET, methylcytosine dioxygenase 1, 2 or 3), the entire coding sequence of mouse or human TDG, a derivate sequence of mouse or human TDG, the enzymatic domain of mouse or human Tet1, Tet2 or Tet3, or a derivative sequence of the enzymatic domain of mouse or human Tet1, Tet2 or Tet3; and / or wherein the gene-specific fusion complex is a gene-specific demethylase fusion complex.

4. The method of claim 1, wherein the method is utilized to change methylation of a promoter and / or a transcription of an associated gene.

5. The method of claim 1, wherein the fusion complex is including a demethylase enzyme with a sequence-specific DNA binding domain (DBD).

6. The method of claim 1, wherein the fusion complex includes a demethylase enzyme connected to a DBD; the DBD being a dCAS9-gRNA system, or a zinc finger protein array (ZFA).

7. The method of claim 6, wherein the dCAS9-based system comprises dCas9-Tet1 cat-NLS / Myc-SCP-HA / NLS / 6xHis-Stop Codon, SEQ ID NO: 9, dCas9-Tet1 mut-NLS / Myc-SCP-HA / NLS / 6xHis-Stop Codon, SEQ ID NO: 10; dCas9-TDG wt-NLS / Myc-SCP-HA / NLS / 6xHis-Stop Codon, SEQ ID NO: 11; and / or dCas9-TDG mut (N151A)-NLS / Myc-SCP-HA / NLS / 6xHis-Stop Codon, SEQ ID NO:

12.

8. The method of claim 7, wherein a supercharged peptide (SCP) is utilized to enhance a dCAS9-construct internalization into target or recipient cells.

9. The method of claim 8, wherein the SCP comprises SEQ ID NO: 13 (GSKPAKKGSKTPGTSKSAKPKKGPGSKPATKGSKKPGKSKSATPKKGPGSKPAKSGKKT PGTSKKPSKGKAPGTSKKPSKGKAPGSKPAKKGSKTPGTSKSAKPKKGPGKSKKATPKSG P).

10. The method of claim 1, including the method is utilized to edit a gene encoding CXCL11 (I-TAC).

11. The method of claim 1, wherein the fusion complex is including a demethylase enzyme with a DBD including the dCAS9-derived system tethered to the target DNA sequence via the guide RNAs (gRNA).

12. The method of claim 10 or 11, wherein guide RNAs designed for CXCL11 gene comprises: SEQ ID NO: 14 (AAAGCTACGTGAGTCAAGGA); SEQ ID NO: 15 (TGAGTGCTTTCACCTTCCAG); SEQ ID NO: 16 (AAAGATAGATGAAGGTCACC); SEQ ID NO: 17 (GTCCCACCAACTCAGGAAGA); SEQ ID NO: 18 (CCACAAGCCAGGAGATGCAG); or SEQ ID NO: 19 (CTGGGACCATCTTCCTGAGT).

13. The method of claim 1, wherein the method is executed as a gene-specific DNA demethylation.

14. The method of claim 1, wherein the method provides an epigenetic re-activation of a silenced or partially silenced gene, an epigenetic restoration of an adaptive, situation- appropriate responsiveness of intrinsic transcription and / or an epigenetic enhancement of a gene’s expression.

15. The method of claim 1, with the proviso that the method does not require a vector and / or a transduction-based approach.

16. The method of claim 1, wherein the fusion complex is produced by an expression in a bacterial, insect or mammalian cell, then the fusion complex protein is purified and administered to a target cell or subject.

17. The method of claim 1, wherein the fusion complex includes a ZFA construct produced by recombinant expression in an E.coli cell, or by an expression in a human embryonic kidney (HEK) or Chinese Hamster Ovary (CHO) cell; wherein the expression in the E. coli cell includes a use of one or more molecular chaperones.

18. The method of claim 17, wherein one or more molecular chaperones includes pTf16 for ZFA-TDG and pKJE7 for ZFA-Tet.

19. The method of claim 1, wherein the method utilizes a combination of fusion proteins in each administration comprising ZFA-TDGs contacting 3 or more sites and / or ZFA-TET1 contacting 3 or more sites, all within the target promoter, or wherein the method is reduced to just a single fusion construct.

20. The method of claim 1, wherein the method provides one or more data including an epigenetic influence on disease risk and / or gene silencing.

21. The method of claim 10, wherein at least one enzyme includes TDG (G / T mismatch- specific thymine DNA glycosylase) and / or Tets 1, 2 or 3 (ten-eleven translocation, TET, methylcytosine dioxygenases 1, 2 or 3); wherein the gene-specific fusion complex is a gene- specific demethylation fusion complex; and where an AA sequence of one or more enzyme(s) comprises any ZFA-Tet1CD, any ZFA-TDG construct, ZF6-Tet cat (SEQ ID NO: 3), ZF8-Tet1 cat (SEQ ID NO: 4), ZF10-Tet cat (SEQ ID NO: 5), ZF6-Tet mut (SEQ ID NO: 6), ZF5-TDG wt (SEQ ID NO: 20), ZF7-TDG wt (SEQ ID NO: 21), ZF9-TDG wt (SEQ ID NO: 22), or ZF9-TDGmut (N151A) (SEQ ID NO: 23).

22. The method of claim 21 or claim 6, wherein the ZFA portion can be replaceable / variable depending on the target(s), can be altered to provide targeting of a different target, and / or to target a different gene; and / or wherein the ZFA part is replaceable; and / or wherein the method provides a fusion of the ZFA portion with an enzyme via apolyaminoacid linker.

23. A method for treating and / or preventing any disease or disorder where a benefit can be obtained by epigenetic editing or transcriptional enhancement of a select gene, the method comprising the steps of: (1) obtaining a subject and; (A) contacting a gene and / or a gene promoter in the subject with a gene-specific fusion complex comprising at least one enzyme functioning as a DNA demethylase; and (B) performing a demethylation of one or more nucleotides within a gene, a promoter, and / or an anticipated regulatory region; whereby the demethylation provides an epigenetic editing of the gene, one or more nucleotides, regulatory motif, and / or the promoter; leading to therapeutic benefit via enhanced transcriptional responsiveness of the targeted gene; (2) wherein the contacting is executed by the step of administering the complex to the subject.

24. The method of claim 23, whereby epigenetic editing in contrast to other methods makes the gene ‘open’ to stimuli and provides adaptive, situation appropriate, context-dependent gene upregulation.

25. The method of claim 23, further comprising the step of: administering the fusion complex or complexes to a subject via an intra-airway administration into the subject operative to contacting lung cells of the subject.

26. The method of claim 23, further comprising the step of: administering one or more such fusion complexes to a subject via an intranasal administration into the subject.

27. The method of claim 23, further comprising administering the gene-specific fusion complex or complexes via instillation of the complex into a subject; or administering the gene-specific fusion complex or complexes in the form of an aerosol and / or in the form of an aerosol using a mesh-type nebulizer that is operative to minimizing degradation of the administered complex or complexes.

28. The method of claim 23, wherein the method provides a treatment for Alzheimer’s disease, a cancer, Parkinson’s disease, schizophrenia, , epilepsy, depression, fragile X syndrome, systemic lupus erythematosus, rheumatoid arthritis, systemic sclerosis, Sjogren’ssyndrome, Graves’ disease, type 1 diabetes, Crohn’s disease, osteoporosis, type 2 diabetes, asthma, allergy, hyperlipidemia, cardiomyopathy, cardiovascular disease, IgA nephropathy, diabetic nephropathy, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, lung fibrosis, or hepatitis C.

29. The method of claim 23, wherein a DBD of the fusion complex contacts one or more loci in a CXCL11 promoter.

30. The method of claim 23, with the proviso that the method does not require an adenoviral vector (AdV), an adeno-associated virus vector (AAV), a lentiviral vector (LV), or other viral or non-viral vector or delivery packaging vehicle to administer into subject.

31. A method for providing a research tool, providing experiments, a screening assay, or a research assay including a gene-specific epigenetic editing of a gene, the method comprising the steps of: (A) administering a fusion complex or combination of fusion complexes comprising at least one enzyme and DNA-binding domains, wherein the administering comprises: (1) contacting the gene and / or a gene promoter with a gene-specific fusion complex comprising at least one enzyme functioning as a DNA demethylase; and (2) performing a demethylation of one or more nucleotides within a gene, a promoter, and / or an anticipated regulatory region; whereby the demethylation provides an epigenetic editing of the gene or provides a test of the mechanistic significance of the demethylation of the gene, one or more nucleotides, regulatory motif, and / or the promoter; and (B) monitoring one or more parameter, signs and / or symptoms in the recipient cell, tissue, organ, animal or any experimental subject.

32. The method of claim 31, wherein at least one enzymatic domain is derived from TDG (thymine DNA glycosylase) and / or Tet1 (ten-eleven translocation, TET, methylcytosine dioxygenase 1), the entire wild type CDS (consensus coding sequence) of mouse TDG, a derivate sequence of mouse TDG, the enzymatic domain of human Tet1, or a derivative sequence of the enzymatic domain of human Tet1; and wherein the gene-specific fusion complex is a gene-specific demethylation fusion complex.

33. The method of claim 31, wherein the method is utilized to change a promoter methylation and / or a transcription of an associated gene.

34. The method of claim 31, wherein the fusion complex is including a demethylase enzyme fused with a sequence-specific DNA binding domain (DBD).

35. The method of claim 31, wherein the fusion complex includes a demethylase enzyme fused with a DBD comprised of, a zinc finger protein array (ZFA), or a dCAS9-gRNA system.

36. The method of claim 35, wherein the dCAS9-based system comprises dCas9-Tet1 cat- NLS / Myc-SCP-HA / NLS / 6xHis-Stop Codon, SEQ ID NO: 9, dCas9-Tet1 mut-NLS / Myc-SCP-HA / NLS / 6xHis-Stop Codon, SEQ ID NO: 10; dCas9-TDG wt-NLS / Myc-SCP-HA / NLS / 6xHis-Stop Codon, SEQ ID NO: 11; and / or dCas9-TDG mut (N151A)-NLS / Myc-SCP-HA / NLS / 6xHis-Stop Codon, SEQ ID NO:

12.

37. The method of claim 31, wherein a supercharged peptide (SCP) is utilized to enhance a dCAS9-construct internalization into cells.

38. The method of claim 37, wherein the SCP comprises SEQ ID NO: 13 (GSKPAKKGSKTPGTSKSAKPKKGPGSKPATKGSKKPGKSKSATPKKGPGSKPAKSGKKT PGTSKKPSKGKAPGTSKKPSKGKAPGSKPAKKGSKTPGTSKSAKPKKGPGKSKKATPKSG P).

39. The method of claim 31, including the method is utilized to edit a gene encoding CXCL11 (I-TAC).

40. The method of claim 31, wherein the fusion complex is including a demethylase enzyme fused with a DBD wherein guide RNAs (gRNA) are tethered to target DNA sequence via dCAS9.

41. The method of claim 39 or 40, wherein guide RNA designed for CXCL11 gene comprises: SEQ ID NO: 14 (AAAGCTACGTGAGTCAAGGA); SEQ ID NO: 15 (TGAGTGCTTTCACCTTCCAG); SEQ ID NO: 16 (AAAGATAGATGAAGGTCACC); SEQ ID NO: 17 (GTCCCACCAACTCAGGAAGA); SEQ ID NO: 18 (CCACAAGCCAGGAGATGCAG); or SEQ ID NO: 19 (CTGGGACCATCTTCCTGAGT).

42. The method of claim 31, wherein the method is executed as a gene-specific DNAdemethylation.

43. The method of claim 31, wherein the method provides an epigenetic re-activation of a silenced or partially silenced gene, an epigenetic restoration of an adaptive, situation- appropriate responsiveness of intrinsic transcription and / or an epigenetic enhancement of a gene’s expression.

44. The method of claim 31, with the proviso that the method does not require a vector and / or a transduction-based approach.

45. The method of claim 31, wherein the fusion complex is produced by a recombinant expression in a bacterial, insect, yeast or mammalian cell.

46. The method of claim 31, wherein the fusion complex includes a ZFA construct produced by an expression in a target cell, a recombinant expression in an E.coli cell, or by an expression in a human embryonic kidney (HEK) or Chinese Hamster Ovary (CHO) cell; wherein the expression in the E. coli cell includes a use of one or more molecular chaperones.

47. The method of claim 45, wherein one or more molecular chaperones includes pTf16 for ZFA-TDG and pKJE7 for ZFA-Tet1.

48. The method of claim 31, further comprising the step of obtaining a subject and: administering the fusion complex or complexes to a subject via an intra-airway administering into the subject operative to contacting lung cells of the subject.

49. The method of claim 48, further comprising the step of: administering one or more such fusion complexes to the subject via an intranasal administration into the subject, and / or via aerosol administration into the subject..

50. The method of claim 49, wherein the method provides a treatment for any disease or disorder where a benefit can be obtained by epigenetic editing or transcriptional enhancement of a select gene.

51. The method of claim 50, wherein the method provides research directed to a treatment for Alzheimer’s disease, a cancer, Parkinson’s disease, schizophrenia, epilepsy, depression, fragile X syndrome, systemic lupus erythematosus, rheumatoid arthritis, systemic sclerosis,Sjogren’s syndrome, Graves’ disease, type 1 diabetes, Crohn’s disease, osteoporosis, type 2 diabetes, asthma, allergy, hyperlipidemia, cardiomyopathy, cardiovascular disease, IgA nephropathy, diabetic nephropathy, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, lung fibrosis, or hepatitis C.

52. The method of claim 31, wherein the method is performed with a screening therapeutic agent, with a screening drug, with a screening compound, and / or with a screening condition.

53. The method of claim 31, with the proviso that the method does not require an adenoviral vector (AdV), an adeno-associated virus vector (AAV), a lentiviral vector (LV), or a non-viral vector to administer into subject.

54. The method of claim 39, wherein one or more loci in CXCL11 promoter for ZFA and gRNA are contacted.

55. The method of claim 54, wherein at least one enzyme includes TDG (G / T mismatch- specific thymine DNA glycosylase) and / or Tet1 (ten-eleven translocation, TET, methylcytosine dioxygenase 1); wherein the gene-specific fusion complex is a gene-specific demethylation fusion complex; and where an AA sequence of one or more enzyme(s) comprises any ZFA-Tet1CD, any ZFA-TDG construct, ZF6-Tet cat (SEQ ID NO: 3), ZF8- Tet1 cat (SEQ ID NO: 4), ZF10-Tet cat (SEQ ID NO: 5), ZF6-Tet mut (SEQ ID NO: 6), ZF5- TDG wt (SEQ ID NO: 20), ZF7-TDG wt (SEQ ID NO: 21), ZF9-TDG wt (SEQ ID NO: 22), or ZF9-TDGmut (N151A) (SEQ ID NO: 23).

56. A composition for a gene-specific epigenetic editing of a gene within a subject, the composition comprising: (1) a sequence-specific DBD; (2) at least one enzyme functioning as a DNA demethylase to perform a demethylation of at least a portion of the gene, one or more nucleotides in the gene or a promoter, and / or relevant CpG island(s), or other regulatory motif.

57. The composition of claim 56, wherein at least one enzymatic domain is derived from TDG (thymine DNA glycosylase) and / or Tet’s 1, 2 or 3 (ten-eleven translocation, TET, methylcytosine dioxygenases 1, 2, 3), the entire coding sequence of mouse TDG, a derivate sequence of mouse TDG, the enzymatic domain of human Tet1, Tet2 or Tet3, or a derivative sequence of the enzymatic domain of human Tet1; or wherein the gene-specific fusion complex is a gene-specific demethylation fusion complex.

58. The composition of claim 56, wherein the composition is operative to change a promoter methylation and / or a transcription of an associated gene.

59. The composition of claim 56, wherein the fusion complex is including a demethylase enzyme with a sequence-specific DNA binding domain (DBD).

60. The composition of claim 56, wherein the fusion complex is including a demethylase enzyme with a DBD, or a DBD including a zinc finger protein array (ZFA), or a dCAS9 system.

61. The composition of claim 60, wherein the dCAS9-based system comprises dCas9-Tet1 cat-NLS / Myc-SCP-HA / NLS / 6xHis-Stop Codon, SEQ ID NO: 9, dCas9-Tet1 mut-NLS / Myc-SCP-HA / NLS / 6xHis-Stop Codon, SEQ ID NO: 10; dCas9-TDG wt-NLS / Myc-SCP-HA / NLS / 6xHis-Stop Codon, SEQ ID NO: 11; and / or dCas9-TDG mut (N151A)-NLS / Myc-SCP-HA / NLS / 6xHis-Stop Codon, SEQ ID NO:

12.

62. The composition of claim 56, further comprising wherein a supercharged peptide (SCP) is included to enhance a dCAS9-construct internalization into cells.

63. The composition of claim 62, wherein the SCP comprises SEQ ID NO: 13 (GSKPAKKGSKTPGTSKSAKPKKGPGSKPATKGSKKPGKSKSATPKKGPGSKPAKSGKKT PGTSKKPSKGKAPGTSKKPSKGKAPGSKPAKKGSKTPGTSKSAKPKKGPGKSKKATPKSG P).

64. The composition of claim 56, including the composition is utilized to edit a gene encoding CXCL11 (I-TAC).

65. The composition of claim 56, wherein the fusion complex is including a demethylase enzyme fused with a DBD including a CRISPR-derived system wherein guide RNAs (gRNA) are tethered to target DNA sequence via dCAS9.

66. The composition of claim 64 or 65, wherein guide RNA comprises: SEQ ID NO: 14 (AAAGCTACGTGAGTCAAGGA); SEQ ID NO: 15 (TGAGTGCTTTCACCTTCCAG); SEQ ID NO: 16 (AAAGATAGATGAAGGTCACC); SEQ ID NO: 17 (GTCCCACCAACTCAGGAAGA);SEQ ID NO: 18 (CCACAAGCCAGGAGATGCAG); or SEQ ID NO: 19 (CTGGGACCATCTTCCTGAGT).

67. The composition of claim 56, wherein the composition is operative to provide a gene- specific DNA demethylation.

68. The composition of claim 56, wherein the composition is operative to provide an epigenetic re-activation of a silenced or partially silenced gene, an epigenetic restoration of an adaptive, situation-appropriate responsiveness of intrinsic transcription and / or an epigenetic enhancement of a gene’s expression.

69. The composition of claim 56, with the proviso that the composition does not require a vector and / or a transduction-based approach.

70. The composition of claim 56, wherein the fusion complex is produced by a recombinant expression in a cell.

71. The composition of claim 56, wherein the fusion complex includes a ZFA construct produced by an expression in a recombinant expression in a cell, a recombinant expression in an E.coli cell, or by an expression in a human embryonic kidney (HEK) or Chinese Hamster Ovary (CHO) cell; wherein the expression in the E. coli cell includes a use of one or more molecular chaperones.

72. The composition of claim 71, wherein one or more molecular chaperones include pTf16 for ZFA-TDG and pKJE7 for ZFA-Tet1.

73. The composition of claim 56, further comprising the composition is operative to provide the step of: administering the fusion complex or complexes to a subject via an intra-airway administering into the subject operative to contacting lung cells of the subject.

74. The composition of claim 73, further comprising the composition is operative to provide an execution of the step of: administering one or more such fusion complexes to a subject via an intranasal administration into the subject.

75. The composition of claim 56, wherein the composition provides a treatment for anydisease or disorder where a benefit can be obtained by epigenetic editing or transcriptional enhancement of a select gene.

76. The composition of claim 56, wherein the composition provides a treatment for Alzheimer’s disease, a cancer, Parkinson’s disease, schizophrenia, epilepsy, depression, fragile X syndrome, systemic lupus erythematosus, rheumatoid arthritis, systemic sclerosis, Sjogren’s syndrome, Graves’ disease, type 1 diabetes, Crohn’s disease, osteoporosis, type 2 diabetes, asthma, allergy, hyperlipidemia, cardiomyopathy, , cardiovascular disease, IgA nephropathy, diabetic nephropathy, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, lung fibrosis, or hepatitis C.

77. The composition of claim 56, further comprising the composition is operative to provide the step of: administering the gene-specific fusion complex or complexes into an airway of a subject; administering the gene-specific fusion complex or complexes via instillation of the complex into a subject; or administering the gene-specific fusion complex or complexes in the form of an aerosol and / or in the form of an aerosol using a mesh-type nebulizer that is operative to minimizing degradation of the administered complex or complexes.

78. The composition of claim 56, with the proviso that the composition does not require an adenoviral vector (AdV), an adeno-associated virus vector (AAV), a lentiviral vector (LV), or other viral or non-viral vector to administer into subject.

79. The composition of claim 56, wherein one or more loci in CXCL11 promoter for ZFA and gRNA are contacted utilizing the composition.

80. The composition of claim 79, wherein at least one enzyme includes TDG (G / T mismatch- specific thymine DNA glycosylase) and / or Tet1, Tet2 or Tet3 (ten-eleven translocation, TET, methylcytosine dioxygenase ); wherein the gene-specific fusion complex is a gene-specific demethylation fusion complex; and where an AA sequence of one or more enzyme(s) comprises any ZFA-Tet1CD, any ZFA-TDG construct, ZF6-Tet cat (SEQ ID NO: 3), ZF8- Tet1 cat (SEQ ID NO: 4), ZF10-Tet cat (SEQ ID NO: 5), ZF6-Tet mut (SEQ ID NO: 6), ZF5- TDG wt (SEQ ID NO: 20), ZF7-TDG wt (SEQ ID NO: 21), ZF9-TDG wt (SEQ ID NO: 22), or ZF9-TDGmut (N151A) (SEQ ID NO: 23).