Engineered DNA-binding proteins

Abstract

To provide engineered DNA binding proteins. [Solution] Provided herein are compositions comprising non-naturally occurring DNA-binding proteins that modulate the expression of endogenous genes, and methods of using the same. In one aspect, the application provides an expression cassette comprising a sequence encoding a non-naturally occurring transcription factor that increases the expression of the SCN1A gene in a cell. In certain embodiments, the transcription factor that increases the expression of SCN1A binds to a target site capable of increasing SCN1A expression by at least 20% in a transcription activation assay.

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 62 / 593,824, filed December 1, 2017; U.S. Provisional Patent Application No. 62 / 610,014, filed December 22, 2017; U.S. Provisional Patent Application No. 62 / 646,198, filed March 21, 2018; U.S. Provisional Patent Application No. 62 / 618,966, filed January 18, 2018; U.S. Provisional Patent Application No. 62 / 641,806, filed March 12, 2018; U.S. Provisional Patent Application No. 62 / 664,814, filed April 30, 2018; and U.S. Provisional Patent Application No. 62 / 664,817, filed April 30, 2018, each of which is incorporated herein by reference in its entirety. Sequence Listing

[0002] This application contains a Sequence Listing that has been submitted electronically in ASCII format, and is hereby incorporated by reference in its entirety. This ASCII copy was created on November 30, 2018, is named 46482-711_601_SL.txt, and is 864,453 bytes in size. [Background technology]

[0003] A wide range of human diseases are associated with abnormal gene expression. In some cases, genetic mutations in a gene cause the gene to become dysregulated, downregulated, or not expressed at all, resulting in haploinsufficiency. In some cases, genetic mutations in a gene cause the gene to become upregulated, resulting in overexpression of the gene. There are many challenges in treating hereditary disorders or diseases. One approach is gene therapy, which involves the therapeutic delivery of nucleic acids into a patient's cells. However, various challenges associated with gene therapy remain unresolved, such as undesirable immune responses elicited by gene therapy, off-target effects, limitations on the cloning capacity of gene therapy vehicles (e.g., viruses), and sustaining therapeutic effects over longer periods of time. The central nervous system (CNS) poses many unique challenges for the development of therapies that address underlying dysfunctions in gene and / or protein expression. Although there are drugs that help manage the symptoms of CNS diseases / disorders, many CNS diseases / disorders, such as Dravet syndrome or frontotemporal dementia, lack specific treatments or cures.Therefore, there is a need for novel compositions and methods that can modulate the expression of any endogenous gene to help reverse the effects of disease or disorder, particularly those that have reduced immunogenicity, reduced off-target effects, increased specificity for target genes, and / or increased therapeutic efficacy. Summary of the Invention [Means for solving the problem]

[0004] In one aspect, the present application provides an expression cassette comprising a sequence encoding a non-naturally occurring transcription factor that increases expression of the SCN1A gene in a cell. In certain embodiments, the transcription factor that increases expression of SCN1A binds to a target site capable of increasing SCN1A expression by at least 20% in a transcription activation assay. In certain embodiments, the transcription factor that increases expression of SCN1A is capable of reducing seizure frequency in a mouse model of Dravet syndrome by at least 20% in a hyperthermic seizure assay. In certain embodiments, the transcription factor that increases expression of SCN1A binds to a target site capable of increasing SCN1A expression by at least 20% in a transcription activation assay and is capable of reducing seizure frequency in a mouse model of Dravet syndrome by at least 20% in a hyperthermic seizure assay.

[0005] In certain embodiments, the transcription factor that increases the expression of SCN1A binds to a genomic location on chromosome 2. In certain embodiments, the transcription factor that increases the expression of SCN1A binds to a genomic location on chromosome 2 that is within 110 kb upstream or downstream of the transcription start site of the SCN1A gene. In certain embodiments, the transcription factor that increases the expression of SCN1A binds to a genomic location on chromosome 2 that is within the range of positions 166179652 to 165989571 (with reference to GRCh38.p12). In certain embodiments, the transcription factor that increases the expression of SCN1A binds to a genomic location on chromosome 2 that is within the range of positions 166128050 to 166127958 (with reference to GRCh38.p12). In certain embodiments, the transcription factor that increases the expression of SCN1A binds to a genomic location on chromosome 2 that is within the range of positions 166155414 to 166140590 (with reference to GRCh38.p12). In certain embodiments, a transcription factor that increases expression of SCN1A binds to a genomic location on chromosome 2 within the range of positions 166179652-1661777272 (with reference to GRCh38.p12). In certain embodiments, a transcription factor that increases expression of SCN1A binds to a genomic location on chromosome 2 within the range of positions 1659990246-165989592 (with reference to GRCh38.p12). In certain embodiments, a transcription factor that increases expression of SCN1A binds to a genomic region within 200 bp of a genomic location having any one of the sequences set forth in SEQ ID NOs: 35-37, 105-111, 136, 195-211, 224-238, and 240-267. In certain embodiments, a transcription factor that increases expression of SCN1A binds to a genomic region that at least partially overlaps with a genomic location having any one of the sequences set forth in SEQ ID NOs: 35-37, 105-111, 136, 195-211, 224-238, and 240-267. In certain embodiments, a transcription factor that increases expression of SCN1A binds to a genomic region having any one of the sequences set forth in SEQ ID NOs: 35-37, 105-111, 136, 195-211, 224-238, and 240-267.In certain embodiments, the transcription factor that increases expression of SCN1A binds to a genomic region having 18 to 27 nucleotides (e.g., at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides).

[0006] In certain embodiments, the transcription factor that increases SCN1A expression comprises a DNA-binding domain. In certain embodiments, the transcription factor that increases SCN1A expression comprises a DNA-binding domain and a transcription activation domain. In certain embodiments, the DNA-binding domain comprises at least 80% sequence identity to its closest human counterpart. In certain embodiments, the DNA-binding domain comprises at least 90% sequence identity to its closest human counterpart. In certain embodiments, the DNA-binding domain and the transcription activation domain both comprise at least 80% sequence identity to their closest human counterparts. In certain embodiments, the DNA-binding domain and the transcription activation domain both comprise at least 90% sequence identity to their closest human counterparts. In certain embodiments, the DNA-binding domain comprises a guide RNA and a nuclease-inactivated Cas protein. In certain embodiments, the nuclease-inactivated Cas protein is nuclease-inactivated Cas9. In certain embodiments, the DNA-binding domain comprises a zinc finger domain. In certain embodiments, the DNA-binding domain comprises between 6 and 9 zinc finger domains. In certain embodiments, the DNA-binding domain comprises six zinc fingers. In certain embodiments, a DNA-binding domain comprising six fingers binds to a genomic region having 18 nucleotides. In certain embodiments, a DNA-binding domain comprises nine zinc fingers. In certain embodiments, a DNA-binding domain comprising nine zinc fingers binds to a genomic region having 27 nucleotides. In certain embodiments, the DNA-binding domain comprises a sequence having at least 95% sequence identity to any of SEQ ID NOs: 135, 371, 372, or 376. In certain embodiments, the DNA-binding domain comprises a sequence having any one of SEQ ID NOs: 135, 371, 372, or 376. In certain embodiments, the DNA-binding domain is derived from human EGR1 or human EGR3.In certain embodiments, the transcription activation domain comprises a VPR, VP64, CITED2 or CITED4 sequence, or a functional fragment thereof. In certain embodiments, the transcription activation domain comprises a human CITED2 or CITED4 sequence, or a functional fragment thereof.

[0007] In certain embodiments, the expression cassette encoding a transcription factor that increases SCN1A expression further comprises a regulatory element that drives expression of the transcription factor at a higher level in PV neurons than in other cell types. In certain embodiments, the regulatory element comprises a sequence having any one of SEQ ID NOs: 183, 184, 185, or 417. In certain embodiments, the regulatory element comprises a sequence having SEQ ID NO: 183 or 185.

[0008] In certain embodiments, the expression cassette that encodes the transcription factor that increases the expression of SCN1A is part of a viral vector.In certain embodiments, the viral vector is AAV virus.In certain embodiments, the AAV virus is AAV9 virus or scAAV9 virus.

[0009] In another aspect, the application provides a method of increasing expression of SCN1A in a cell by administering any of the expression cassettes described herein that encode a transcription factor that increases expression of SCN1A. In certain embodiments, the cell is a PV neuron. In certain embodiments, the cell is within a subject. In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a human. In certain embodiments, increasing expression of SCN1A treats a disease, disorder, or condition. In certain embodiments, the disorder is a central nervous system disorder. In certain embodiments, the disorder is Dravet syndrome. In certain embodiments, the central nervous system disorder is neuronal hyperactivity. In certain embodiments, treating the central nervous system disorder comprises reducing neuronal hyperactivity. In certain embodiments, the symptom of the central nervous system disorder is seizures. In certain embodiments, treating the central nervous system disorder comprises reducing the frequency of seizures. In certain embodiments, treating the central nervous system disorder comprises reducing the severity of seizures. In certain embodiments, treating the central nervous system disorder comprises reducing the severity and frequency of seizures.

[0010] In another aspect, the present application provides an expression cassette comprising a sequence encoding a non-naturally occurring transcription factor that increases expression of a GRN gene in a cell.

[0011] In certain embodiments, the transcription factor that increases expression of the GRN binds to a genomic region having 18 to 27 nucleotides.

[0012] In certain embodiments, the transcription factor that increases the expression of GRN comprises a DNA-binding domain. In certain embodiments, the transcription factor that increases the expression of GRN comprises a DNA-binding domain and a transcription activation domain. In certain embodiments, the DNA-binding domain comprises a guide RNA and a nuclease-inactivating Cas protein. In certain embodiments, the nuclease-inactivating Cas protein is nuclease-inactivating Cas9. In certain embodiments, the DNA-binding domain comprises a zinc finger domain. In certain embodiments, the DNA-binding domain comprises 6 to 9 zinc finger domains. In certain embodiments, the DNA-binding domain comprises 6 zinc fingers. In certain embodiments, a DNA-binding domain comprising 6 zinc fingers binds to a genomic region having 18 nucleotides. In certain embodiments, the DNA-binding domain comprises 9 zinc fingers. In certain embodiments, a DNA-binding domain comprising 9 zinc fingers binds to a genomic region having 27 nucleotides. In certain embodiments, the DNA-binding domain comprises a sequence having at least 95% sequence identity to any of SEQ ID NOs: 171 or 412-416. In certain embodiments, the DNA-binding domain comprises a sequence having any one of SEQ ID NOs: 171, or 412-416. In certain embodiments, the DNA-binding domain is derived from human EGR1 or human EGR3. In certain embodiments, the transcription activation domain comprises a VPR, VP64, CITED2, or CITED4 sequence, or a functional fragment thereof.

[0013] In certain embodiments, the expression cassette encoding a transcription factor that increases expression of a GRN further comprises a regulatory element that drives expression of a non-naturally occurring transcriptional modulator. In certain embodiments, the regulatory element is a cell-type-selective regulatory element.

[0014] In certain embodiments, the expression cassette that encodes the transcription factor that increases the expression of GRN is part of a viral vector.In certain embodiments, the viral vector is AAV virus.In certain embodiments, the AAV virus is AAV9 virus or scAAV9 virus.

[0015] In another aspect, the present application provides a method for increasing the expression of GRN in a cell by administering any of the expression cassettes described herein that encode a transcription factor that increases the expression of GRN. In certain embodiments, increasing the expression of GRN treats a disease, disorder, or condition. In certain embodiments, the cell is within a subject. In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a human. In certain embodiments, the cell is selected from the group consisting of a central nervous system cell, a frontal cortex cell, a glial cell, a microglial cell, and a striatal cell. In certain embodiments, modulating the expression of GRN treats a disease or disorder. In certain embodiments, the disorder is a central nervous system disorder. In certain embodiments, the disorder is frontotemporal degeneration (FTD), Parkinson's disease, Alzheimer's disease, or atherosclerosis. In certain embodiments, the symptoms of central nervous system disorders are the presence of Lewy bodies, haploinsufficiency of progranulin (GRN), social deficit, lysosomal abnormalities, memory loss, loss of motor coordination, or muscle tremors. In certain embodiments, treating central nervous system disorders includes reducing the frequency of muscle tremors and / or reducing the severity of muscle tremors.

[0016] In another aspect, the present application provides an expression cassette encoding a non-naturally occurring DNA-binding protein comprising three or more DNA-binding domains, wherein the DNA-binding protein increases or represses expression of an endogenous gene by at least five-fold and has 90% or greater sequence identity to its closest human counterpart. In an exemplary embodiment, the DNA-binding domain comprises three or more zinc finger domains.

[0017] In certain embodiments, a DNA binding protein having 90% or more sequence identity to its closest human counterpart binds to a target site in the human genome to which the human counterpart protein does not naturally bind. In certain embodiments, the human counterpart is EGR1. In certain embodiments, the human counterpart is EGR3. In certain embodiments, a DNA binding protein having 90% or more sequence identity to its closest human counterpart comprises at least six zinc finger domains. In certain embodiments, a DNA binding protein having 90% or more sequence identity to its closest human counterpart comprises nine zinc finger domains. In certain embodiments, a DNA binding domain having 90% or more sequence identity to its closest human counterpart comprises at least one amino acid substitution at position -1, 2, 3, or 6 of the recognition helix of one or more zinc finger domains. In certain embodiments, the DNA binding protein having 90% or greater sequence identity to its closest human counterpart comprises one or more zinc finger domains derived from EGR1 or EGR3.

[0018] In certain embodiments, the DNA-binding protein having 90% or more sequence identity to its closest human counterpart further comprises a transcriptional effector domain. In certain embodiments, the transcriptional effector domain is derived from CITED2 or CITED4. In certain embodiments, the transcriptional effector domain is located at the C-terminus of the DNA-binding domain in the DNA-binding protein. In certain embodiments, the effector domain is located at the N-terminus of the DNA-binding domain in the DNA-binding protein. In certain embodiments, the DNA-binding protein having 90% or more sequence identity to its closest human counterpart comprises an effector domain located at the N-terminus of the DNA-binding domain in the DNA-binding protein and an effector domain located at the C-terminus of the DNA-binding domain in the DNA-binding protein.

[0019] In certain embodiments, a DNA binding protein with 90% or more sequence identity to its closest human counterpart recognizes a target binding site of 18 bp or longer, hi certain embodiments, a DNA binding protein with 90% or more sequence identity to its closest human counterpart recognizes a target binding site of 27 bp.

[0020] In certain embodiments, an expression cassette encoding a DNA-binding protein having 90% or more sequence identity to its closest human counterpart further comprises a cell-type-selective regulatory element. In certain embodiments, an expression cassette encoding a DNA-binding protein having 90% or more sequence identity to its closest human counterpart further comprises a regulatory element that drives expression of the DNA-binding protein at a higher level in PV neurons than in other cell types. In certain embodiments, the regulatory element comprises a sequence having any one of SEQ ID NOs: 183, 184, 185, or 417. In certain embodiments, the regulatory element comprises a sequence having SEQ ID NO: 183 or 185.

[0021] In certain embodiments, the expression cassette that encodes the DNA binding protein that has 90% or more sequence identity with its closest human counterpart is in a viral vector.In certain embodiments, the viral vector is AAV.In certain embodiments, the AAV is AAV9 or scAAV9.

[0022] In another aspect, the present application provides a method of treating a disease or condition, comprising administering a gene therapy comprising any of the expression cassettes provided herein encoding a DNA-binding protein having 90% or more sequence identity to its closest human counterpart. In certain embodiments, the DNA-binding protein having 90% or more sequence identity to its closest human counterpart elicits a reduced or minimal immune response when expressed in a cell or in vivo. In certain embodiments, the cell is within a subject. In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a human. In certain embodiments, the DNA-binding protein having 90% or more sequence identity to its closest human counterpart increases expression of endogenous SCN1A. In certain embodiments, the cell is a PV neuron. In certain embodiments, increasing expression of SCN1A treats a disease, disorder, or symptom. In certain embodiments, the disorder is a central nervous system disorder. In certain embodiments, the disorder is Dravet syndrome. In certain embodiments, the central nervous system disorder is neuronal hyperactivity. In certain embodiments, treating a central nervous system disorder comprises reducing neuronal hyperactivity. In certain embodiments, a symptom of a central nervous system disorder is seizures. In certain embodiments, treating a central nervous system disorder comprises reducing the frequency of seizures. In certain embodiments, treating a central nervous system disorder comprises reducing the severity of seizures. In certain embodiments, treating a central nervous system disorder comprises reducing the severity and frequency of seizures. In certain embodiments, a DNA-binding protein having 90% or more sequence identity to its closest human counterpart increases the expression of endogenous GRN. In certain embodiments, the cell is selected from the group consisting of a central nervous system cell, a frontal cortex cell, a glial cell, a microglial cell, and a striatal cell. In certain embodiments, modulating the expression of GRN treats a disease or disorder. In certain embodiments, the disorder is a central nervous system disorder.In certain embodiments, the disorder is frontotemporal degeneration (FTD), Parkinson's disease, Alzheimer's disease or atherosclerosis.In certain embodiments, the symptom of central nervous system disorder is the presence of Lewy body, haploinsufficiency of progranulin (GRN), lack of sociality, lysosomal abnormality, memory loss, loss of motor coordination or muscle tremor.In certain embodiments, treating central nervous system disorder comprises reducing the frequency of muscle tremor and / or reducing the severity of muscle tremor. Incorporation by Reference

[0023] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0024] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative instances in which the principles of the invention are utilized, and the accompanying drawings. The present invention provides, for example, the following items. (Item 1) 1. An expression cassette comprising a sequence encoding a non-naturally occurring transcription factor that increases expression of the SCN1A gene in a cell, wherein the transcription factor binds to a target site that is capable of increasing SCN1A expression by at least 20% in a transcription activation assay and is capable of reducing seizure frequency by at least 20% in a mouse model of Dravet syndrome in a hyperthermic seizure assay. (Item 2) 2. The expression cassette according to item 1, wherein the transcription factor binds to a genomic region having 18 to 27 nucleotides. (Item 3) 3. The expression cassette of any one of items 1 or 2, wherein the transcription factor comprises a DNA-binding domain. (Item 4) 4. The expression cassette of any one of items 1 to 3, wherein the transcription factor comprises a DNA-binding domain and a transcription activation domain. (Item 5) 5. The expression cassette of any one of items 3 or 4, wherein the DNA-binding domain comprises at least 80% sequence identity to its closest human counterpart. (Item 6) 6. The expression cassette of item 5, wherein the DNA-binding domain comprises at least 90% sequence identity to its closest human counterpart. (Item 7) 6. The expression cassette of any one of items 4 or 5, wherein the DNA binding domain and the transcription activation domain together comprise at least 80% sequence identity to their closest human counterparts. (Item 8) 8. The expression cassette of item 7, wherein the DNA binding domain and the transcription activation domain together comprise at least 90% sequence identity to their closest human counterparts. (Item 9) 5. The expression cassette of any one of items 3 or 4, wherein the DNA-binding domain comprises a guide RNA and a nuclease-inactivating Cas protein. (Item 10) 10. The expression cassette of item 9, wherein the nuclease-inactivated Cas protein is a nuclease-inactivated Cas9. (Item 11) 9. The expression cassette of any one of items 3 to 8, wherein the DNA-binding domain comprises a zinc finger domain. (Item 12) Item 12. The expression cassette according to Item 11, wherein the DNA-binding domain comprises 6 to 9 zinc finger domains. (Item 13) 13. The expression cassette of item 12, wherein the DNA-binding domain comprises six zinc fingers. (Item 14) 14. The expression cassette of item 13, wherein the DNA binding domain binds to a genomic region having 18 nucleotides. (Item 15) 13. The expression cassette of item 12, wherein the DNA-binding domain comprises nine zinc fingers. (Item 16) 16. The expression cassette of item 15, wherein the DNA binding domain binds to a genomic region having 27 nucleotides. (Item 17) 13. The expression cassette of any one of items 3 to 8 or 11 to 12, wherein the DNA-binding domain comprises a sequence having at least 95% sequence identity to any of SEQ ID NOs: 135, 371, 372 or 376. (Item 18) 18. The expression cassette of item 17, wherein the DNA binding domain comprises a sequence having any one of SEQ ID NOs: 135, 371, 372, or 376. (Item 19) 19. The expression cassette of any one of items 3 to 8 or 11 to 18, wherein the DNA-binding domain is derived from human EGR1 or human EGR3. (Item 20) 20. The expression cassette of any one of items 4 to 8 or 11 to 19, wherein the transcription activation domain comprises a VPR, VP64, CITED2 or CITED4 sequence, or a functional fragment thereof. (Item 21) 21. The expression cassette of any one of items 4 to 8 or 11 to 20, wherein the transcription activation domain comprises a human CITED2 or CITED4 sequence, or a functional fragment thereof. (Item 22) 22. The expression cassette of any one of items 1 to 21, further comprising a regulatory element that drives expression of the transcription factor at a higher level in PV neurons than in other cell types. (Item 23) 23. The expression cassette of item 22, wherein the regulatory element comprises a sequence having any one of SEQ ID NOs: 183, 184, 185, or 417. (Item 24) 24. The expression cassette of item 23, wherein the regulatory element comprises a sequence having SEQ ID NO: 183 or 185. (Item 25) 25. The expression cassette of any one of items 1 to 24, which is part of a viral vector. (Item 26) 26. The expression cassette of item 25, wherein the viral vector is an AAV virus. (Item 27) 27. The expression cassette of item 26, wherein the AAV virus is an AAV9 virus or an scAAV9 virus. (Item 28) 28. A method for increasing expression of SCN1A in a cell by administering the expression cassette of any one of items 1 to 27. (Item 29) 29. The method of item 28, wherein the cell is a PV neuron. (Item 30) 30. The method of any one of items 28 or 29, wherein the cell is in a subject. (Item 31) 31. The method of claim 30, wherein the subject is a mammal. (Item 32) 32. The method of claim 31, wherein the subject is a human. (Item 33) 33. The method of any one of items 28 to 32, wherein increasing the expression of SCN1A treats the disease, disorder or condition. (Item 34) 34. The method of claim 33, wherein the disorder is a central nervous system disorder. (Item 35) 35. The method of claim 34, wherein the disorder is Dravet syndrome. (Item 36) 36. The method of any one of items 34 or 35, wherein the symptom of the central nervous system disorder is neuronal hyperactivity. (Item 37) 37. The method of claim 36, wherein treating the central nervous system disorder comprises reducing neuronal hyperactivity. (Item 38) 36. The method of any one of items 34 or 35, wherein the symptom of the central nervous system disorder is seizures. (Item 39) 39. The method of claim 38, wherein treating the central nervous system disorder comprises reducing seizure frequency. (Item 40) 40. The method of any one of items 38 or 39, wherein treating the central nervous system disorder comprises reducing the severity of seizures. (Item 41) An expression cassette comprising a sequence encoding a non-naturally occurring transcription factor that increases expression of a GRN gene in a cell. (Item 42) 42. The expression cassette according to item 41, wherein the transcription factor binds to a genomic region having 18 to 27 nucleotides. (Item 43) 43. The expression cassette of any one of items 41 or 42, wherein the transcription factor comprises a DNA-binding domain. (Item 44) 44. The expression cassette of any one of items 41 to 43, wherein the transcription factor comprises a DNA-binding domain and a transcription activation domain. (Item 45) 45. The expression cassette of any one of items 43 or 44, wherein the DNA-binding domain comprises a guide RNA and a nuclease-inactivating Cas protein. (Item 46) 46. ​​The expression cassette of claim 45, wherein the nuclease-inactivated Cas protein is a nuclease-inactivated Cas9. (Item 47) 45. The expression cassette of any one of items 43 or 44, wherein the DNA-binding domain comprises a zinc finger domain. (Item 48) 48. The expression cassette according to Item 47, wherein the DNA-binding domain comprises 6 to 9 zinc finger domains. (Item 49) 49. The expression cassette of item 48, wherein the DNA-binding domain comprises six zinc fingers. (Item 50) 50. The expression cassette of item 49, wherein the DNA binding domain binds to a genomic region having 18 nucleotides. (Item 51) 49. The expression cassette of item 48, wherein the DNA-binding domain comprises nine zinc fingers. (Item 52) 52. The expression cassette of item 51, wherein the DNA binding domain binds to a genomic region having 27 nucleotides. (Item 53) 49. The expression cassette of any one of items 43 to 44 or 47 to 48, wherein the DNA-binding domain comprises a sequence having at least 95% sequence identity to any of SEQ ID NOs: 171 or 412 to 416. (Item 54) 54. The expression cassette according to Item 53, wherein the DNA-binding domain comprises a sequence having any one of SEQ ID NOs: 171 and 412 to 416. (Item 55) 55. The expression cassette of any one of items 43 to 44 or 47 to 54, wherein the DNA binding domain is derived from human EGR1 or human EGR3. (Item 56) 56. The expression cassette of any one of items 44 or 47 to 55, wherein the transcription activation domain comprises a VPR, VP64, CITED2 or CITED4 sequence, or a functional fragment thereof. (Item 57) 57. The expression cassette of any one of items 41 to 56, further comprising a regulatory element driving expression of the transcription factor. (Item 58) 58. The expression cassette of Item 57, wherein the regulatory element is a cell-type-selective regulatory element. (Item 59) 59. The expression cassette of any one of items 41 to 58, which is part of a viral vector. (Item 60) 60. The expression cassette of item 59, wherein the viral vector is an AAV virus. (Item 61) 61. The expression cassette of item 60, wherein the AAV virus is an AAV9 virus or a scAAV9 virus. (Item 62) A method for increasing expression of GRN in a cell by administering an expression cassette described in any one of items 41 to 61. (Item 63) An expression cassette encoding a non-naturally occurring DNA-binding protein comprising a DNA-binding domain that includes three or more zinc finger domains, wherein the DNA-binding protein increases or suppresses expression of an endogenous gene other than SCN1A by at least 5-fold and has 90% or greater sequence identity to its closest human counterpart. (Item 64) 64. The expression cassette of item 63, wherein the DNA-binding protein binds to a target site in the human genome to which the human counterpart protein does not naturally bind. (Item 65) 65. The expression cassette of item 63 or 64, wherein the human counterpart is EGR1. (Item 66) 65. The expression cassette of item 63 or 64, wherein the human counterpart is EGR3. (Item 67) 67. The expression cassette of any one of items 63 to 66, wherein the DNA binding protein comprises at least six zinc finger domains. (Item 68) 68. The expression cassette of item 67, wherein the DNA binding protein comprises nine zinc finger domains. (Item 69) 69. The expression cassette of any one of items 63 to 68, wherein the DNA-binding domain comprises at least one amino acid substitution at position −1, 2, 3 or 6 of the recognition helix of one or more zinc finger domains. (Item 70) 70. The expression cassette of any one of items 63 to 69, wherein the DNA-binding protein comprises one or more zinc finger domains derived from EGR1 or EGR3. (Item 71) 71. The expression cassette of any one of items 63 to 70, wherein the DNA binding protein further comprises a transcription effector domain. (Item 72) 72. The expression cassette of item 71, wherein the transcription effector domain is derived from CITED2 or CITED4. (Item 73) 73. The expression cassette of item 71 or 72, wherein the transcription effector domain is located C-terminal to the DNA-binding domain in the DNA-binding protein. (Item 74) 73. The expression cassette of item 71 or 72, wherein the effector domain is located N-terminal to the DNA-binding domain in the DNA-binding protein. (Item 75) 73. The expression cassette of claim 71 or 72, wherein the DNA-binding protein comprises an effector domain located N-terminally to the DNA-binding domain in the DNA-binding protein and an effector domain located C-terminally to the DNA-binding domain in the DNA-binding protein. (Item 76) 76. The expression cassette of any one of items 63 to 75, wherein the DNA binding protein recognizes a target binding site that is 18 bp or longer. (Item 77) 77. The expression cassette of item 76, wherein the DNA binding protein recognizes a 27 bp target binding site. (Item 78) 78. The expression cassette of any one of items 63 to 77, further comprising a cell type-selective regulatory element. (Item 79) Item 80. The expression cassette of any one of Items 63 to 78, in a viral vector. 80. The expression cassette of item 79, wherein the viral vector is AAV. (Item 81) Item 82. The expression cassette according to Item 80, wherein the AAV is AAV9 or scAAV9. 82. A method of treating a disease or condition, comprising administering a gene therapy comprising the expression cassette of any one of items 63 to 81. (Item 83) 83. The method of claim 82, wherein the DNA binding protein elicits a reduced or minimal immune response when expressed in a cell or in vivo. (Item 84) 84. The method of any one of paragraphs 82 or 83, wherein the cell is in a subject. (Item 85) 85. The method of item 84, wherein the subject is a mammal. (Item 86) 86. The method of item 85, wherein the subject is a human. [Brief explanation of the drawings]

[0025] [Figure 1] Figure 1 shows the effect of different regulatory elements on the expression of luciferase in 293T cells, measured by normalized luciferase activity.For example, the combination with the minimum CMV (minCMV) promoter drives the expression of luciferase at a level that is about 1.4 times higher than that driven by the minCMV promoter alone and about 60 times higher than that driven by the SCP promoter.In the same experiment, SEQ ID NO: 178 is linked with the minCMV promoter to drive the expression of luciferase at a level that is about 3.5 times higher than that driven by the minCMV promoter alone and about 140 times higher than that driven by the SCP promoter.

[0026] [Figure 2] FIG. 2 shows the size-normalized activity of each regulatory element (calculated by dividing the normalized luciferase activity shown in FIG. 1 by the length of the regulatory element in base pairs).

[0027] [Figure 3] Figure 3 shows selective expression of eGFP under the control of a GABAergic-selective regulatory element (RE), e.g., an RE selective for parvalbumin (PV) cells. AAVDJ vectors containing eGFP under the control of either the EF1α promoter or a GABAergic / PV-selective RE, e.g., SEQ ID NO: 183, including SEQ ID NO: 185, or SEQ ID NO: 184, were injected into mice expressing tdTomato (red fluorescent protein (RFP)) in GABAergic / PV cells. The top image shows the pattern of eGFP expressed from the construct containing the EF1α promoter (SEQ ID NO: 186), GABAergic / PV-selective RE (SEQ ID NO: 183 or SEQ ID NO: 184). The bottom image shows GABAergic / PV neurons, which are GAD2-positive. Cells expressing both eGFP and tdTomato can be identified by either overlaying the top image with the bottom image or by identifying fluorescence from the same location in both the top and bottom images. Examples of cells expressing both eGFP and tdTomato are indicated by arrows.

[0028] [Figure 4] Figure 4 shows quantification of the efficiency of expression of eGFP by the GABAergic / PV neurons in Figure 3 (e.g., when measured as the percentage of tdTomato-expressing cells that also express eGFP).

[0029] [Figure 5] Figure 5 shows quantification of the specificity of expression of eGFP by GABAergic / PV neurons in Figure 3 (e.g., when measured as the percentage of eGFP-expressing cells that also express tdTomato (or RFP+)).

[0030] [Figure 6]Figure 6 shows the upregulation of endogenous SCN1A using engineered transcription factors that bind to various regions on chromosome 2 (see GRCh38.p12). Data are presented as fold change in SCN1A expression relative to the control (EGFP-KASH) condition.

[0031] [Figure 7] FIG. 7 shows the relative expression of SCN1A presented as Log10 fold change relative to the eGFP control.

[0032] [Figure 8] Figure 8 shows the relative expression of endogenous SCN1A, TTC21B, SCN9A, SNCA, GRN, UTRN, and TTN transcripts presented as fold change over control.

[0033] [Figure 9] Figure 9A shows one embodiment of an eTF disclosed herein, in which the DNA-binding domain (DBD) of a naturally occurring human transcription factor (TF) has been modified, reprogrammed, or engineered to recognize a target binding site at or near a gene target of interest. The top diagram shows a naturally occurring transcription factor that lacks binding affinity for a target site (represented by a black bar) associated with an endogenous gene or gene target of interest. The bottom diagram shows an eTF in which the DBD comprises multiple zinc fingers (represented by cylinders) that have been modified, engineered, or reprogrammed to bind to a target site in a gene of interest, e.g., by doubling or tripling the zinc fingers of the naturally occurring DBD and / or making amino acid substitutions in the zinc fingers.

[0034] Figure 9B shows another embodiment of an eTF, or hybrid eTF, disclosed herein, comprising a DBD derived from a naturally occurring human TF fused to the transactivation domain (TAD) of a human coregulator protein or transcription cofactor, e.g., the TAD of a Cbp / p300-interacting transactivator protein such as CITED2 or CITED4. The top diagram shows a DBD derived from a human protein that has been engineered to specifically bind to a desired target site in the genome (black bar). The bottom diagram shows a DBD fused to a TAD, e.g., the TAD of CITED2, to form a hybrid eTF. Such a hybrid eTF drives expression of a target gene by binding to the target site sequence in the genome and modulating (e.g., activating) transcription of the gene target via the TAD in the eTF.

[0035] [Figure 10] Figure 10 shows the percentage of conservation (or sequence identity) between various transcription-modulating proteins and naturally occurring human proteins, as well as their sizes in base pairs (bp). In various embodiments, the eTFs of the present disclosure can be encoded by nucleic acid sequences of 1,000-1,800 bp and contain >90% global sequence identity (i.e., sequence identity across the full-length amino acid sequence of the eTF) to naturally occurring human proteins, thereby reducing immune responses or immunogenicity when delivered to cells or in vivo, e.g., via gene therapy. In comparison, dCas9-based transcription factors containing a non-activated Cas9 (dCas9) domain lack global sequence identity to endogenous human proteins and are larger in size. Conventionally constructed zinc finger proteins (ZFPs), or artificial or synthetic DNA-binding proteins containing in silico-designed zinc fingers, are smaller in size but can have lower sequence identity (approximately 55%) to endogenous human proteins.

[0036] [Figure 11]Figure 11 illustrates various approaches to modulating endogenous genes. In the first diagram, the transcriptional modulator protein comprises multiple synthetic zinc fingers (ZFs), such as in silico-designed ZFs, and includes a DBD fused to the transactivation domain (TAD) of VP64. In the second diagram, a wild-type TF occurs naturally but does not bind to a target site in a gene of interest. In the third diagram, an eTF comprises a naturally occurring TF whose DBD has been modified or reprogrammed to bind to an 18-nt target site at or near a gene of interest, e.g., where the DBD of the wild-type TF is replaced with a synthetic zinc finger. In the fourth diagram, an eTF is derived from a wild-type TF whose DBD has been engineered to recognize a 9-nt target binding site at or near a gene of interest. In the final figure, an eTF is derived from a wild-type TF in which the DBD of the wild-type TF is doubled and reprogrammed (e.g., via an amino acid substitution in the ZF) to form a six-zinc finger DBD that recognizes an 18-nt target binding site at or near a gene of interest. These various embodiments of eTFs can be assayed using a reporting activation assay in which the gene target is a report gene (e.g., EGFP). The eTFs in the first, third, fourth, and fifth figures result in expression of a reporter gene.

[0037] [Figure 12]Figure 12A shows a schematic diagram of an SCN1A activation assay in which expression of SCN1A in HEK293 cells is an indication of eTF binding to an SCN1A-specific target binding site and activation of SCN1A gene expression. The top diagram shows an eTF engineered to recognize a TRE binding site for an EGFP reporter gene, which fails to bind to the SCN1A target binding site and therefore fails to express SCN1A. The bottom diagram shows that an eTF derived from a naturally occurring human protein and containing at least 98% sequence identity to the human protein and a DBD engineered to bind to the 18-nt SCN1A target binding site results in SCN1A gene activation and therefore SCN1A expression.

[0038] Figure 12B shows the results of quantification of SCN1A activation assays with the exemplary eTFs shown in Figure 12A. An eTF (SEQ ID NO: 6) containing six zinc fingers with high global or overall sequence identity to EGR1 and whose DBD is engineered to recognize a specific binding site near or in the endogenous SCN1A gene resulted in 2.5-fold higher expression of SCN1A in transfected HEK293 cells compared to SEQ ID NO: 4, whose DBD is engineered to recognize the TRE binding site but not the SCN1A target binding site.

[0039] [Figure 13] Figure 13A shows a schematic diagram of SCN1A activation by two exemplary eTFs, each derived from a naturally occurring human protein. The top diagram shows an eTF in which the DBD of a naturally occurring human protein (e.g., EGR1 or EGR3) has been doubled to form a DBD with six zinc fingers and has been engineered or reprogrammed to bind to a target binding site near or in the endogenous SCN1A gene. The bottom diagram shows an eTF in which the DBD of a naturally occurring human protein has been tripled to form a DBD with nine zinc fingers and has been engineered or reprogrammed to bind to a target binding site near or in the endogenous SCN1A gene.

[0040] Figure 13B shows quantification of relative SCN1A expression activated by each of the eTFs shown in Figure 13A. In transfected HEK293 cells, an eTF (SEQ ID NO: 7) containing high sequence identity to EGR1 and a six-zinc finger DBD derived from EGR1 and engineered to recognize the SCN1A target binding site results in approximately 7-fold increased SCN1A expression compared to the empty vector control. An eTF (SEQ ID NO: 9) containing high sequence identity to EGR1 and a nine-zinc finger DBD derived from EGR1 and engineered to recognize the SCN1A target binding site results in approximately 20-fold increased SCN1A expression compared to the control.

[0041] [Figure 14] Figure 14 shows various embodiments of hybrid eTFs comprising a human DBD with high sequence identity to a naturally occurring human protein fused to a TAD of another protein, such as a viral activation domain, or to a TAD of another human protein, such as CITED2. In various embodiments, the local sequence identity between the DBD of the eTF and the DBD of the naturally occurring human protein is at least 85%. Such DBDs can be fused to various activation domains, such as a viral activation domain, e.g., VP64, which has 0% sequence identity to the human protein, or the CITED2 activation domain. Replacing the VP64 domain with the human CITED sequence increases the overall sequence identity of the hybrid eTF in the third column compared to the naturally occurring human protein.

[0042] [Figure 15] Figure 15 shows the relative expression of endogenous SCN1A in HERK293 cells using SCN1A-specific transcriptional activators (see Tables 33-34 in Example 6). Data are presented as fold change relative to the control condition and are shown on a Log10 scale.

[0043] [Figure 16]Figure 16A shows the relative expression of endogenous SCN1A in GABA neurons using an SCN1A-specific transcriptional activator (SEQ ID NO: 366). Data are presented as fold change relative to the control condition (CBA-EGFP).

[0044] Figure 16B shows the relative expression of endogenous SCN1A in GABA neurons using SCN1A-specific transcriptional activators (SEQ ID NO: 13 or SEQ ID NO: 15). Data are expressed as Logarithmic scale (Log2) as fold change relative to the control condition (CBA-EGFP). 10 is presented in.

[0045] [Figure 17] Figure 17 shows the relative expression of endogenous SCN1A and 40 nearest neighbor genes driven by an SCN1A-specific transcription factor (SEQ ID NO: 366). Data are presented as fold change relative to the control condition (CBA-EGFP-KASH) in Log10.

[0046] [Figure 18] Figures 18A and 18B show the expression of an SCN1A-specific transcription activator in vivo compared to a control expression cassette that expressed eGFP. Figure 18A shows the relative expression of the SCN1A gene in mice injected with either a control eGFP expression cassette or expression cassette A containing a transcription activator that binds to a target site resulting in upregulation of expression from the endogenous SCN1A gene. Figure 18B shows the change in SCN1A expression in terms of the average eGFP percentage. These experiments showed that the transcription activator in expression cassette A resulted in approximately 20-30% upregulation of SCN1A expression.

[0047] [Figure 19-1]Figures 19A, 19B, and 19C show upregulation of endogenous SCN1A in the hippocampus of wild-type mice using several SCN1A-specific transcription factors. Figure 19A: Expression cassette A; Figure 19B: Expression cassette B; and Figure 19C: SEQ ID NO: 365 or 366. SCN1A transcription factors were compared with control vector (EGFP-KASH) and CBA-EGFP-KASH. Data are presented as fold change relative to the EGFP-KASH condition. [Figure 19-2] Figures 19A, 19B, and 19C show upregulation of endogenous SCN1A in the hippocampus of wild-type mice using several SCN1A-specific transcription factors. Figure 19A: Expression cassette A; Figure 19B: Expression cassette B; and Figure 19C: SEQ ID NO: 365 or 366. SCN1A transcription factors were compared with control vector (EGFP-KASH) and CBA-EGFP-KASH. Data are presented as fold change relative to the EGFP-KASH condition.

[0048] [Figure 20-1] Figures 20A, 20B, 20C, 20D, and 20E show the effect of various SCN1A-specific transcription factors on hyperthermia seizures in a mouse model of Dravet syndrome compared to controls. P1 Scn1a+ / - mice were injected with either AAV9-EGFP or an AAV9 vector expressing an SCN1A-specific transcription factor (SEQ ID NOs: 305-309). P26-P28 injected mice underwent a hyperthermia-induced seizure assay to record the internal temperature at which they experienced tonic-clonic seizures. [Figure 20-2]Figures 20A, 20B, 20C, 20D, and 20E show the effect of various SCN1A-specific transcription factors on hyperthermia seizures in a mouse model of Dravet syndrome compared to controls. P1 Scn1a+ / - mice were injected with either AAV9-EGFP or an AAV9 vector expressing an SCN1A-specific transcription factor (SEQ ID NOs: 305-309). P26-P28 injected mice underwent a hyperthermia-induced seizure assay to record the internal temperature at which they experienced tonic-clonic seizures.

[0049] [Figure 21-1] Figures 21A, 21B, 21C, 21D, and 21E show survival rates in a mouse model of Dravet syndrome under various conditions. Figure 21A shows a comparison between wild-type and Scn1a+ / − mice in a survival assay. P1 Scn1a+ / − (N = 53) and Scn1a+ / + (N = 54) mice were injected with PBS. Mice were observed daily in their home cages, and the date of any deaths was recorded. There was a significant difference in survival rates between Scn1a+ / − and Scn1a+ / + animals (P < 0.0001). Figures 21B-E show the effect on survival of a mouse model of Dravet syndrome for mice treated with various SCN1A-specific transcription factors compared to controls. P1 Scn1a+ / - mice were injected with either PBS or an AAV9 vector expressing an SCN1A-specific transcription factor (SEQ ID NOs: 305-307, or 309). Mice were observed daily in their home cages, and the date of any deaths was recorded. [Figure 21-2]Figures 21A, 21B, 21C, 21D, and 21E show survival rates in a mouse model of Dravet syndrome under various conditions. Figure 21A shows a comparison between wild-type and Scn1a+ / − mice in a survival assay. P1 Scn1a+ / − (N = 53) and Scn1a+ / + (N = 54) mice were injected with PBS. Mice were observed daily in their home cages, and the date of any deaths was recorded. There was a significant difference in survival rates between Scn1a+ / − and Scn1a+ / + animals (P < 0.0001). Figures 21B-E show the effect on survival of a mouse model of Dravet syndrome for mice treated with various SCN1A-specific transcription factors compared to controls. P1 Scn1a+ / - mice were injected with either PBS or an AAV9 vector expressing an SCN1A-specific transcription factor (SEQ ID NOs: 305-307, or 309). Mice were observed daily in their home cages, and the date of any deaths was recorded.

[0050] [Figure 22] Figure 22 shows the relative Scn1A mRNA expression in different brain tissues after intraparenchymal delivery of an AAV9 vector encoding an SCN1A-specific transcription factor (SEQ ID NO: 305) into two cynomolgus macaques (cynomolgus macaques) at 1.2 x 1012 gc / animal, normalized to two untreated control animals. All animals were sacrificed 28 days after injection, and Scn1A mRNA was quantified in tissue samples by Taqman PCR. Data are reported as the normalized expression of target mRNA in different tissue sections from the brain. Similar results were recorded with different sets of primers / probes derived from the Scn1a gene.

[0051] [Figure 23] FIG. 23 shows upregulation of endogenous GRNs using engineered transcription factors that bind to various genomic regions.

[0052] [Figure 24]Figures 24A and 24B show in vitro experiments with various expression cassettes, each containing a transcriptional activator of GRN. Figure 24A shows the relative expression of Progranulin, presented as fold change over the control. Figure 24B shows an ELISA experiment with various transcriptional activators of Progranulin, expressed in terms of hPGRN (ng / mL) in the supernatant.

[0053] [Figure 25] Figure 25 shows the normalized luciferase activity of various regulatory elements (REs) in the expression of luciferase in 293T cells. For example, operably linking regulatory element SEQ ID NO: 178 to the minCMV promoter drove luciferase expression at levels approximately 3.5-fold higher than the minCMV promoter alone and approximately 140-fold higher than the SCP promoter.

[0054] [Figure 26] Figure 26 shows the levels of PGRN transcripts measured by qPCR in HEK293 cells transfected with plasmids expressing either EGFP or GRN-specific activators (SEQ ID NOs: 10, 16, 59, 60, 63, 64, 72, 73, 77, L, or 342). Data are presented as fold change relative to the control condition.

[0055] [Figure 27] Figure 27 shows the levels of PGRN protein secreted from HEK293 cells transfected with plasmids expressing either EGFP or GRN-specific activators (SEQ ID NO: 10 or 16) and measured by ELISA. Data are presented as fold change relative to control conditions.

[0056] [Figure 28]Figure 28 shows the level of PGRN transcript measured by qPCR in GABA neurons transfected with AAV vectors (serotype AAV-DJ) expressing either EGFP or GRN-specific activator (SEQ ID NO: 342) under the control of the CBA promoter. Data are presented as fold change relative to control conditions.

[0057] [Figure 29] Figure 29 shows the levels of PGRN protein secreted from GABA neurons infected with low (0.5E11 genome copies / well) and high (2E11 genome copies / well) doses of AAV vectors (serotype AAV-DJ) expressing either EGFP or GRN-specific activator (SEQ ID NO: 342) under the control of the CBA promoter, as measured by ELISA. Data are presented as fold changes relative to control conditions.

[0058] [Figure 30] Figure 30 shows plasma PGRN protein levels (measured in μg / mL) for mice treated with AAV9 containing expression cassettes J or G, each containing a transcriptional activator of GRN, at different doses compared to PBS controls.

[0059] [Figure 31-1]Figure 31A shows the results of quantification of reporter (EGFP) activation assays with various eTFs, expressed as relative EGFP expression compared to an empty vector control. An artificial transcription factor (SEQ ID NO: 51) containing a strong viral activation domain resulted in 70-fold increased relative EGFP expression. The unmodified human transcription factor EGR1 (SEQ ID NO: 1) did not activate the reporter gene compared to the control (empty vector). An eTF derived from EGR1 (SEQ ID NO: 2) containing six engineered zinc fingers resulted in 10-fold increased EGFP expression. An eTF derived from EGR1 containing three zinc fingers with at least one or more amino acid changes in the zinc fingers (SEQ ID NO: 3) resulted in 3-fold increased relative EGFP expression. Doubling the zinc fingers in the EGR1 finger (SEQ ID NO: 4) resulted in 15-fold increased relative EGFP expression.

[0060] [Figure 31-2] Figure 31B shows a fluorescent micrograph of the reporter (EGFP) activation assay quantified in Figure 31 A. Cells expressing EGFP have a pale gray appearance.

[0061] [Figure 32] Figure 32 shows various embodiments of eTFs derived from EGR3 and engineered to recognize target binding sites that result in expression of the EGFP reporter gene. An eTF (SEQ ID NO: 12) derived from the EGR3 protein and containing six zinc fingers engineered to recognize a TRE binding site in a reporter assay resulted in EGFP expression, as did an eTF (SEQ ID NO: 51) derived from EGR1 and containing a TAD derived from VP64. Both eTFs resulted in higher EGFP expression compared to a control containing a modified human DBD without the activation domain (encoded by SEQ ID NO: 187).

[0062] [Figure 33-1]Figure 33A shows a schematic diagram of various eTFs engineered to recognize TRE-binding sequences in an EGFP reporter activation assay. The first diagram shows a modified human DBD engineered to recognize the TRE-binding site but lacking the activation domain (SEQ ID NO: 50), which does not result in any activation of EGFP expression. In the second diagram, the modified human DBD is fused to the strong viral activation domain VP64 (SEQ ID NO: 95), which results in activation of EGFP expression. In the third and fourth diagrams, the modified human DBD is fused to the human activation domain of either CITED2 (SEQ ID NO: 96) or CITED4 (SEQ ID NO: 97), both of which activate EGFP expression.

[0063] Figure 33B shows quantification of relative EGFP expression for each eTF described in Figure 33A. SEQ ID NO: 50 resulted in a level of EGFP expression similar to the reporter-only control. Each of the eTFs having the sequences of SEQ ID NOs: 51-53 resulted in approximately 40-, 15-, and 10-fold increased relative EGFP expression, respectively, compared to the reporter-only control.

[0064] [Figure 33-2] FIG. 33C shows fluorescence micrographs for each eTF in the EGFP reporter activation assay shown in FIG. 33A and quantified in FIG. 33B.

[0065] [Figure 34] FIG. 34 provides an overview of various embodiments of eTFs with a wide range of transcriptional activity (or relative reporter expression) and varying degrees of sequence identity (or conservation) with naturally occurring human proteins.

[0066] [Figure 35]Figure 35A shows a schematic diagram of an Elispot assay for testing the immunogenicity of eTF disclosed herein. The Elispot assay is used to test whether T cells from normal, healthy donors can recognize and respond to peptides predicted to be immunogenic. T cells are mixed with antigen-presenting cells (e.g., dendritic cells or DCs) along with a pool of peptides to be tested in an Elispot well. If the T cells recognize the peptides as foreign, they become activated, proliferate, and secrete cytokines (e.g., interferon-gamma). After treatment with a color-developing reagent, interferon-gamma (IFNγ)-positive spots can be counted. A higher number of spots is associated with greater immunogenicity compared to a control (e.g., a known non-immunogenic peptide or buffer alone).

[0067] Figure 35B shows a close-up example of elispot results showing increased immunogenicity (right circle) compared to the control (left circle). IFN-gamma positive spots are shown as black spots.

[0068] [Figure 36] Figure 36 shows a vector containing an expression cassette comprising a non-naturally occurring transcriptional modulator, which comprises the DNA binding domain of a transcription factor operably linked to the activation domain of VP64 to increase expression of the SCN1A gene.

[0069] [Figure 37] Figure 37 shows an AAV vector containing an expression cassette, which includes a nuclease-inactivating dSaCa9 domain operably linked to a non-naturally occurring transcriptional modulator, e.g., the transcription activation domain of VP64. Such an expression cassette also includes a dSaCas9 transgene and an expressed guide RNA. Other elements shown include a nuclear localization signal (NLS), promoter, AAV ITRs, a polyA signal, and a selectable marker. DETAILED DESCRIPTION OF THE INVENTION

[0070] Detailed Description of the Disclosure Provided herein are engineered transcription factors, or eTFs, that are not naturally occurring and are designed to bind to genomic target sites and modulate the expression of an endogenous gene of interest. Such eTFs can be designed to upregulate or downregulate expression (RNA and / or protein expression) of the gene of interest.

[0071] In one aspect, the present application provides an eTF capable of upregulating expression of the sodium voltage-gated channel alpha subunit 1 (SCN1A) gene and increasing expression of its corresponding protein product Nav1.1, and methods of its use to treat diseases or disorders associated with defects in Nav1.1, such as Dravet syndrome.

[0072] In another aspect, the present application provides eTFs capable of upregulating expression of the progranulin (GRN) gene and increasing expression of the GRN protein, and methods of their use to treat diseases or disorders associated with defects in GRN, such as frontotemporal dementia (FTD).

[0073] In another aspect, the present application provides eTFs with high percent sequence identity to human proteins that can be designed to bind to genomic target sites and modulate (up-regulate or down-regulate) the expression of any gene of interest, including, for example, SCN1A or GRN. Such eTFs have little to no immunogenicity when administered to a subject, or have reduced immunogenicity compared to eTFs with lower percent identity to the human protein sequence. definition

[0074] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise. Furthermore, to the extent that the terms "including," "includes," "having," "has," "with," or variations thereof are used in either the detailed description and / or claims, such terms are intended to be inclusive in the same manner as the term "comprising."

[0075] The term "about" or "approximately" means within an acceptable range of error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within one standard deviation or within more than one standard deviation, in accordance with practice in the art. Alternatively, "about" can mean within a range of up to 20%, up to 15%, up to 10%, up to 5%, or up to 1% of a given value.

[0076] The terms "determining," "measuring," "evaluating," "assessing," "assaying," "analyzing," and their grammatical equivalents may be used interchangeably herein to refer to any form of measurement, including determining whether an element is present or absent (e.g., detecting). These terms may include both quantitative and / or qualitative determinations. Assessment may be relative or absolute.

[0077] The term "expression" refers to the process by which a nucleic acid sequence or polynucleotide is transcribed from a DNA template (e.g., into mRNA or other RNA transcript) and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. The transcript and the encoded polypeptide may be collectively referred to as a "gene product." If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

[0078] As used herein, "operably linked," "operably linked," "operably linked," or grammatical equivalents thereof, refers to the juxtaposition of genetic elements, e.g., promoters, enhancers, polyadenylation sequences, etc., in a relationship permitting them to operate in their expected manner. For example, a regulatory element, which may include a promoter and / or enhancer sequence, is operably linked to a coding region if the regulatory element helps initiate transcription of the coding sequence. Intervening residues may be present between the regulatory element and the coding region so long as this functional relationship is maintained.

[0079] As used herein, "vector" refers to a polymer or an association of polymers that contains or is associated with a polynucleotide and can be used to mediate the delivery of the polynucleotide to a cell. Examples of vectors include plasmids, viral vectors, liposomes, and other gene delivery vehicles. A vector generally contains a genetic element, such as a regulatory element, operatively linked to a gene to promote the expression of the gene in a target.

[0080] As used herein, "expression cassette" and "nucleic acid cassette" are used interchangeably to refer to a combination of nucleic acid sequences or elements that are expressed together or operably linked for expression. In some cases, an expression cassette refers to a combination of regulatory elements and the gene(s) to which they are operably linked for expression.

[0081] The term "AAV" is an abbreviation for adeno-associated virus and can be used to refer to the virus itself or its derivatives. The term covers all serotypes, subtypes, and both naturally occurring and recombinant forms, unless otherwise required. The abbreviation "rAAV" refers to recombinant adeno-associated virus, also known as recombinant AAV vector (or "rAAV vector"). The term "AAV" includes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, rh10, and their hybrids, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV. The genomic sequences of various AAV serotypes, as well as the sequences of native terminal repeats (TRs), Rep proteins, and capsid subunits, are known in the art. Such sequences can be found in the literature or in public databases such as GenBank. As used herein, "rAAV vector" refers to an AAV vector containing a polynucleotide sequence not of AAV origin (i.e., a polynucleotide heterologous to AAV), typically a sequence of interest for genetic transformation of a cell. Generally, the heterologous polynucleotide is flanked by at least one, and generally two, AAV inverted terminal repeats (ITRs). rAAV vectors can be either single-stranded (ssAAV) or self-complementary (scAAV). "AAV virus" or "AAV viral particle" refers to a viral particle composed of at least one AAV capsid protein and an encapsidated polynucleotide rAAV vector. When a particle contains a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, for example, a transgene delivered into a mammalian cell), it is typically referred to as an "rAAV vector particle" or simply an "rAAV particle." Thus, production of rAAV particles necessarily involves production of rAAV vectors, since such vectors are contained within the rAAV particles.

[0082] As used herein, the terms "treat," "treatment," "therapy," and the like refer to alleviating, delaying, or slowing the progression of a disease or disorder, preventing a disease or disorder, attenuating a disease or disorder, reducing the effects or symptoms of a disease or disorder, preventing the onset of a disease or disorder, inhibiting a disease or disorder, or ameliorating the onset of a disease or disorder. The methods of the present disclosure may be used in any mammal. Exemplary mammals include, but are not limited to, rats, cats, dogs, horses, cows, sheep, pigs, and more preferably humans. Therapeutic benefit includes eradication or amelioration of the underlying disorder being treated. Therapeutic benefit is also achieved by eradicating or ameliorating one or more physiological symptoms associated with the underlying disorder, such that an improvement is observed in the subject, even though the subject may still be afflicted with the underlying disorder. In some cases, for preventative benefit, a therapeutic agent may be administered to a subject at risk of developing a particular disease or reporting one or more physiological symptoms of the disease, even if the disease has not been diagnosed. The methods of the present disclosure may be used in any mammal. In some cases, treatment may result in a reduction or cessation of symptoms (e.g., a reduction in the frequency, duration, and / or severity of attacks). A prophylactic effect includes delaying or eliminating the appearance of the disease or condition, delaying or eliminating the onset of symptoms of the disease or condition, slowing, halting, or reversing the progression of the disease or condition, or any combination thereof.

[0083] The term "effective amount" or "therapeutically effective amount" refers to an amount of a composition described herein sufficient to affect the intended application, including but not limited to, disease treatment as defined below. The therapeutically effective amount can vary depending on the intended treatment application (in vivo), or the subject and disease state being treated, such as the subject's weight and age, the severity of the disease state, the mode of administration, etc., which can be easily determined by one skilled in the art. The term also applies to a dose that induces a specific response in target cells. The specific dose will vary depending on the particular composition selected, the administration regimen to be followed, whether it is administered in combination with other compounds, the timing of administration, the tissue to which it is administered, and the physical delivery system that carries it.

[0084] A "fragment" of a nucleotide or peptide sequence refers to a sequence that is distinct from the reference or "full-length" sequence.

[0085] A "variant" of a molecule refers to an allelic variation of such sequence, ie, a sequence substantially similar in structure and biological activity to either the entire molecule or a fragment thereof.

[0086] A "functional fragment" of a DNA or protein sequence refers to a fragment that retains a biological activity (either functional or structural) substantially similar to that of the full-length DNA or protein sequence. The biological activity of a DNA sequence can be its ability to affect expression in a manner known to be due to the full-length sequence.

[0087] The terms "subject" and "individual" are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. The methods described herein may be useful in human therapy, veterinary applications, and / or preclinical research in animal models of a disease or condition.

[0088] The term "in vivo" refers to an event that takes place in the body of a subject.

[0089] The term "in vitro" refers to an event that occurs outside the subject's body. For example, an in vitro assay encompasses any assay that is performed outside the subject. An in vitro assay encompasses cell-based assays in which live or dead cells are used. An in vitro assay also encompasses cell-free assays in which intact cells are not used.

[0090] Generally, "sequence identity" or "sequence homology," which can be used interchangeably, refers to the exact nucleotide-by-nucleotide or amino acid-by-amino acid correspondence between two polynucleotide or polypeptide sequences, respectively. Typically, the technique for determining sequence identity involves comparing two nucleotide or amino acid sequences and determining their percent identity. For example, sequence comparison for the purpose of assessing identity can be performed by any suitable alignment algorithm, including, but not limited to, the Needleman-Wunsch algorithm (see, for example, the EMBOSS Needle aligner available at www.ebi.ac.uk / Tools / psa / emboss_needle / , using default settings as needed), the BLAST algorithm (see, for example, the BLAST alignment tool available at blast.ncbi.nlm.nih.gov / Blast.cgi, using default settings as needed), and the Smith-Waterman algorithm (see, for example, the EMBOSS Water aligner available at www.ebi.ac.uk / Tools / psa / emboss_water / , using default settings as needed). Optimal alignment can be evaluated using any suitable parameters of the selected algorithm, including default parameters.The "percent identity" between two sequences, also known as "percent homology," can be calculated as the number of exact matches between two optimally aligned sequences, divided by the length of the reference sequence and multiplied by 100.Percent identity can also be determined by comparing sequence information using an advanced BLAST computer program, including version 2.2.9, available from the National Institutes of Health.The BLAST program is based on the alignment method of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990), and is described in Altschul, et al., J. Mol. Biol. 215:403-410 (1990); Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993); and Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997). Briefly, the BLAST program defines identity as the number of identical aligned symbols (i.e., nucleotides or amino acids) divided by the total number of symbols in the shorter of the two sequences. The program can be used to determine percent identity over the entire length of the sequences being compared. Default parameters are provided to optimize searches with short query sequences, for example, using the blastp program. The program also allows the use of a SEG filter to mask off segments of the query sequence as determined by the SEG program of Wootton and Federhen, Computers and Chemistry 17: 149-163 (1993). High sequence identity generally includes a range of approximately 80% to 100% sequence identity, and integer values ​​therebetween.

[0091] As used herein, "engineered" in reference to a protein refers to a non-naturally occurring protein, including, but not limited to, a protein derived from a naturally occurring protein, or where a naturally occurring protein has been modified or reprogrammed to have certain properties.

[0092] As used herein, "synthetic" and "artificial" are used interchangeably to refer to proteins or domains thereof that have low sequence identity (e.g., less than 50% sequence identity) to naturally occurring human proteins. For example, the VPR and VP64 domains are synthetic transactivation domains.

[0093] As used herein, "engineered transcription factor" or "eTF" refers to a non-naturally occurring DNA binding protein or a non-naturally occurring transcriptional modulator that has been modified or reprogrammed to bind to a specific target binding site and / or to contain an altered or replaced transcriptional effector domain.

[0094] As used herein, "DNA-binding domain" may be used to refer to one or more DNA-binding motifs, e.g., zinc finger or basic helix-loop-helix (bHLH) motifs, either individually or collectively as part of a DNA-binding protein.

[0095] The terms "transcription activation domain," "transcriptional activation domain," "transactivation domain," "trans-activating domain," and "TAD" are used interchangeably herein and refer to a domain of a protein that, together with a DNA-binding domain, can activate transcription from a promoter by contacting the transcription machinery (e.g., general transcription factors and / or RNA polymerase) either directly or through other proteins known as coactivators.

[0096] The terms "transcriptional repressor domain," "transcription repressor domain," and "TRD" are used interchangeably herein and refer to a domain of a protein that, together with a DNA-binding domain, can repress transcription from a promoter by contacting the transcriptional machinery (e.g., general transcription factors and / or RNA polymerase) either directly or through other proteins known as corepressors.

[0097] The term "GRCh38.p12" refers to the Genome Assembly with GenBank Assembly accession number GCA_000001405.27 and date 2017 / 12 / 21. Refers to Reference Consortium Human Build 38 Patch Release 12 (GRCh38.p12).

[0098] Unless otherwise indicated, all terms used herein have the same meaning as they would to a person skilled in the art, and the practice of the present invention employs conventional techniques of molecular biology, microbiology, and recombinant DNA technology within the knowledge of those skilled in the art. Engineered transcription factors (eTFs)

[0099] Transcription factor (TF) is a protein that binds to specific sequences in genome and controls gene expression. The engineered transcription factor or eTF provided herein is a non-naturally occurring protein that comprises a DNA binding domain (DBD) and at least one domain that is a transcription modulator, such as a transcription activation domain (TAD) or a transcription repressor domain (TRD). In one embodiment, eTF can comprise one DBD and one TAD (for example, TAD-DBD or DBD-TAD), and DBD and TAD can be derived from the same protein or different proteins. In another embodiment, an eTF may comprise one DBD and two TADs, where the DBD and TADs are derived from the same protein, the DBD is derived from a first protein and both TADs are derived from a second protein, the DBD and one TAD are derived from a first protein and the second TAD is derived from a second protein, or the DBD is derived from a first protein and one TAD is derived from a second protein and the second TAD is derived from a third protein (e.g., TAD1-DBD-TAD1 or TAD1-DBD-TAD2). In one embodiment, an eTF may comprise one DBD and one TRD (e.g., TRD-DBD or DBD-TRD), where the DBD and TRDs may be derived from the same protein or different proteins. In another embodiment, an eTF can comprise one DBD and two TRDs, where the DBD and TRDs are from the same protein, the DBD is from a first protein and both TRDs are from a second protein, the DBD and one TRD are from a first protein and the second TRD is from a second protein, or the DBD is from a first protein and one TRD is from a second protein and the second TRD is from a third protein (e.g., TRD1-DBD-TRD1 or TRD1-DBD-TRD2). In certain embodiments, the DBD can be a synthetic construct comprising domains from multiple proteins.

[0100] In certain embodiments, the DBD and TAD can be directly conjugated, for example, without an intervening amino acid sequence. In other embodiments, the DBD and TAD can be conjugated using a peptide linker. In certain embodiments, the DBD can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 60, 70, 75, 80, 90, or 100 amino acids, or 1 to 5, 1 to 10, 1 to 20, 1 to 30, 1 to 40, 1 to 50, 1 to 7 The DBD and TAD are conjugated to the TAD via a linker having 5, 1-100, 5-10, 5-20, 5-30, 5-40, 5-50, 5-75, 5-100, 10-20, 10-30, 10-40, 10-50, 10-75, 10-100, 20-30, 20-40, 20-50, 20-75, or 20-100 amino acids. In some cases, the DBD and TAD are conjugated via naturally occurring intervening residues found in the naturally occurring proteins from which the domains are derived. In other embodiments, the DBD and TAD are conjugated via a synthetic or exogenous linker sequence. Suitable linkers can be flexible, cleavable, non-cleavable, hydrophilic, and / or hydrophobic. In certain embodiments, the DBD and TAD can be fused together via a linker containing multiple glycine and / or serine residues. Examples of glycine / serine peptide linkers include [GS]n, [GGGS]n (SEQ ID NO: 436), [GGGGS]n (SEQ ID NO: 437), and [GGSG]n (SEQ ID NO: 438), where n is an integer equal to or greater than 1. In certain embodiments, a linker useful for conjugating a DBD and a TAD is GGSGGGSG (SEQ ID NO: 410).In certain embodiments, when a DBD is conjugated to two TADs, the first and second TADs may be conjugated to the DBD using the same or different linkers, or one TAD may be conjugated to the DBD using a linker and the other TAD may be conjugated directly to the DBD (e.g., without an intervening linker sequence), or both TADs may be conjugated directly to the DBD (e.g., without an intervening linker sequence).

[0101] The eTFs provided herein have different properties from naturally occurring transcription factors. In certain embodiments, the eTFs provided herein comprise a DBD derived from a naturally occurring protein, which has been modified so that the DBD binds to a different target site compared to the naturally occurring protein from which it is derived, so that the eTFs comprising such modified DBDs modulate the expression of different genes compared to the naturally occurring protein from which it is derived, and / or so that the eTFs comprising such modified DBDs modulate the expression of target genes differently (e.g., up-regulate vs. down-regulate) compared to the naturally occurring protein from which it is derived. In other embodiments, the eTFs provided herein comprise a TAD derived from a naturally occurring protein, which has been modified so that the eTFs comprising such modified TADs modulate the expression of different genes compared to the naturally occurring protein from which it is derived, and / or so that the eTFs comprising such modified TADs modulate the expression of target genes differently (e.g., up-regulate vs. down-regulate) compared to the naturally occurring protein from which it is derived. In certain embodiments, the eTFs provided herein comprise a DBD derived from a naturally occurring protein and a TAD derived from a naturally occurring protein (either the same protein or a different protein), wherein both the DBD and the TAD are modified. In such embodiments, the DBD may bind to a different target site compared to the naturally occurring protein from which it is derived, and eTFs comprising such modified DBDs and TADs modulate expression from different genes compared to the naturally occurring protein from which those domains are derived, and / or eTFs comprising such modified DBDs and TADs modulate target gene expression differently (e.g., up-regulate vs. down-regulate) compared to the naturally occurring protein from which the DBD and TAD domains are derived. DNA binding domain (DBD)

[0102] The eTFs provided herein can include any suitable DBD that binds to a target site of interest. In certain embodiments, the DBD can be a synthetically designed DBD. In other embodiments, the DBD can be derived from a naturally occurring protein. DBD families include basic helix-loop-helix (bHLH) (e.g., c-Myc), basic-leucine zipper (e.g., C / EBP), helix-turn-helix (e.g., Oct-1), and zinc finger (e.g., EGR3). These families exhibit a wide range of DNA binding specificities and gene targets. As contemplated herein, any one of the known human transcription factor proteins can serve as a protein platform for engineering and / or reprogramming the DBD to recognize a specific target site, resulting in modulation of the expression of an endogenous gene of interest. In exemplary embodiments, the DBDs provided herein include a zinc finger domain, a TALEN binding domain, or a gRNA / Cas complex.

[0103] The DBD provided herein can be designed to recognize any target site of interest. In exemplary embodiments, the DBD is engineered to recognize a target site that can modulate (e.g., up-regulate or down-regulate) expression from a gene of interest when bound by eTF. In exemplary embodiments, the DBD is designed to recognize a genomic location and modulate the expression of an endogenous gene when bound by eTF. The binding site that can modulate the expression of an endogenous gene of interest when bound by eTF can be located anywhere in the genome, resulting in the modulation of gene expression of the target gene. In various embodiments, the binding site may be located on a different chromosome than the gene of interest, on the same chromosome as the gene of interest, upstream of the transcription start site (TSS) of the gene of interest, downstream of the TSS of the gene of interest, proximal to the TSS of the gene of interest, distal to the gene of interest, within the coding region of the gene of interest, within an intron of the gene of interest, downstream of the poly A tail of the gene of interest, within a promoter sequence regulating the gene of interest, within an enhancer sequence regulating the gene of interest, or within a repressor sequence regulating the gene of interest.

[0104] The DBD can be designed to bind to a target binding site of any length, as long as it provides specific recognition of the target binding site sequence by the DBD, for example, with minimal or no off-target binding. In certain embodiments, the target binding site can modulate the expression of an endogenous gene of interest when bound by eTF at a level at least 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 75-fold, 100-fold, 150-fold, 200-fold, 250-fold, 500-fold, or more, relative to all other genes. In certain embodiments, the target binding site may modulate the expression of an endogenous gene of interest upon eTF binding at a level at least 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 75-fold, 100-fold, 150-fold, 200-fold, 250-fold, 500-fold, or more, compared to the 40 nearest neighboring genes (e.g., the 40 genes located closest on the chromosome, either upstream or downstream of the coding sequence of the gene of interest). In certain embodiments, the target binding site may be at least 5 bp, 10 bp, 15 bp, 20 bp, 25 bp, 30 bp, 35 bp, 40 bp, 45 bp, or 50 bp, or longer. The specific length of the binding site is determined by the type of DBD in the eTF. Generally, the longer the length of the binding site, the greater the specificity of binding and the modulation of gene expression (e.g., a longer binding site has fewer off-target effects). In certain embodiments, an eTF with a DBD that recognizes a longer target binding site has fewer off-target effects (e.g., modulation of expression of off-target genes, or genes other than the gene of interest, etc.) associated with non-specific binding compared to off-target effects observed with an eTF with a DBD that binds to a shorter target site. In some cases, the reduction in off-target binding is at least 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold lower compared to a comparable eTF with a DBD that recognizes a shorter target binding site.

[0105] In certain embodiments, the DBDs provided herein can be modified to have increased binding affinity, such that the TAD conjugated to the DBD can recruit more transcription factors and / or bind to longer target binding sites so that it can recruit such transcription factors for a longer period of time to have a greater effect on the expression level of the endogenous gene of interest. In certain embodiments, the eTF can be engineered so that the DBD stably binds to the target binding site and blocks transcription or the transcription machinery required to express the endogenous gene. In certain embodiments, the DBD can be modified to increase its specific binding (or on-target binding) to the desired target site and / or to decrease its non-specific or off-target binding.

[0106] In various embodiments, binding between the DBD or eTF and the target binding site can be determined using various methods. In certain embodiments, specific binding between the DBD or eTF and the target binding site can be determined using a mobility shift assay, a DNase protection assay, or any other in vitro method known in the art for assaying protein-DNA binding. In other embodiments, specific binding between the eTF and the target binding site can be determined using a functional assay, for example, by measuring gene expression (RNA or protein) when the eTF binds to the target binding site. For example, the target binding site can be placed upstream of a reporter gene (e.g., eGFP) or a target gene of interest on a vector contained in or integrated into the genome of a cell, where the cell expresses the eTF. A higher level of reporter gene (or target gene of interest) expression in the presence of the eTF compared to a control (e.g., no eTF or an eTF that recognizes a different target site) indicates that the DBD of the eTF binds to the target site. Suitable in vitro (e.g., non-cell-based) transcription and translation systems can also be used in a similar manner. In certain embodiments, an eTF that binds to a target site may have at least 2-fold, 3-fold, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 50-fold, 75-fold, 100-fold, 150-fold or more higher expression of a reporter gene or target gene of interest compared to a control (e.g., no eTF or an eTF that recognizes a different target site).

[0107] In other embodiments, the eTFs provided herein may contain a DBD that is from a zinc finger protein, derived from a zinc finger protein, or is a nuclease-inactivated zinc finger protein. The zinc finger contains one or more zinc ions (Zn) to stabilize folding. 2+Zinc finger (Znf) domains are small protein structural motifs characterized by the coordination of multiple nucleotides (nucleotides) that contact a DNA target site. The zinc finger (Znf) domain is a relatively small protein motif containing multiple finger-like protrusions that contact a DNA target site in tandem. The modular nature of zinc finger motifs allows numerous combinations of DNA sequences to bind with a high degree of affinity and specificity, making them ideally suited for engineering proteins that can be targeted to and bind to specific DNA sequences. Many engineered zinc finger arrays are based on the zinc finger domain of the mouse transcription factor Zif268. Zif268 has three individual zinc finger motifs that collectively bind to a 9-bp sequence with high affinity. A wide variety of zinc finger proteins have been identified and characterized into different types based on structure, as further described herein. Any such zinc finger proteins are useful in conjunction with the DBDs described herein.

[0108] Various methods are available for designing zinc finger proteins. For example, methods for designing zinc finger proteins to bind to target DNA sequences of interest have been described, see, for example, Liu Q, et al., Design of polydactyl zinc-finger proteins for unique addressing within complex genomes, Proc Natl Acad Sci USA. 94 (11): 5525-30 (1997); Wright DA et al., Standardized reagents and protocols for engineering zinc finger nucleases by modular assembly, Nat Protoc. Nat Protoc. 2006;1(3):1637-52; and CA Gersbach and T Gaj, Synthetic Zinc Finger Proteins: The Advent of Targeted Gene Regulation and Genome Modification Technologies, Am Chem Soc 47: 2309-2318 (2014). Additionally, various web-based tools are publicly available for designing zinc finger proteins to bind to DNA target sequences of interest; see, e.g., the Zinc Finger Nuclease Design Software Tools and Genome Engineering Data Analysis website from OmicX, available on the World Wide Web at omictools.com / zfns-category; and the Zinc Finger Tools design website from Scripps, available on the World Wide Web at scripps.edu / barbas / zfdesign / zfdesignhome.php.Additionally, various commercial services are available for designing zinc finger proteins to bind to desired DNA target sequences, see, for example, the commercial services or kits provided by Creative Biolabs (World Wide Web at creative-biolabs.com / Design-and-Synthesis-of-Artificial-Zinc-Finger-Proteins.html), the Zinc Finger Consortium Modular Assembly Kit available from Addgene (World Wide Web at addgene.org / kits / zfc-modular-assembly / ), or the CompoZr Custom ZFN Service from Sigma Aldrich (World Wide Web at sigmaaldrich.com / life-science / zinc-finger-nuclease-technology / custom-zfn.html).

[0109] In certain embodiments, the eTFs provided herein comprise a DBD that includes one or more zinc fingers, or are derived from the DBD of a zinc finger protein. In some cases, the DBD comprises multiple zinc fingers, with each zinc finger linked at its N-terminus, C-terminus, or both, to another zinc finger or another domain via an amino acid linker. In some cases, the DBDs provided herein comprise one or more zinc fingers from one or more of the zinc finger types listed in Table 5. In some cases, the DBDs provided herein comprise multiple zinc finger structures or motifs, or multiple zinc fingers having one or more of SEQ ID NOs: 115-130 or 141-164, or any combination thereof. In certain embodiments, the DBD comprises X-[ZF-X]n and / or [X-ZF]nX, where ZF is a zinc finger domain having any one of the motifs listed in Table 5 (e.g., any one of SEQ ID NOs: 39-49), X is an amino acid linker comprising 1 to 50 amino acids, and n is an integer between 1 and 15, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, each ZF can independently have the same or a different sequence from other ZF sequences in the DBD, and each linker X can independently have the same or a different sequence from other X sequences in the DBD. Each zinc finger can be linked at its C-terminus, N-terminus, or both to another sequence, zinc finger, or domain. In a DBD, each linker X can be identical in sequence, length, and / or properties (e.g., flexibility or charge), or can differ in sequence, length, and / or properties. In some cases, two or more linkers can be identical, while other linkers are different. In exemplary embodiments, a linker can be obtained or derived from a sequence connecting zinc fingers found in one or more naturally occurring zinc finger proteins provided in Table 5. In other embodiments, suitable linker sequences include, for example, linkers of five or more amino acids in length.For exemplary linker sequences of 6 or more amino acids in length, see also U.S. Patent Nos. 6,479,626; 6,903,185; and 7,153,949, each of which is hereby incorporated by reference in its entirety. The DBD proteins provided herein can include any combination of suitable linkers between the individual zinc fingers of the protein. The DBD proteins described herein can include any combination of suitable linkers between the individual zinc fingers of the protein.

[0110] In certain embodiments, the DBDs provided herein comprise one or more classical zinc fingers. A classical C2H2 zinc finger has two cysteine ​​residues in one chain and two histidine residues in another chain, coordinated by a zinc ion. A classical zinc finger domain has two β-sheets and one α-helix, which interacts with a DNA molecule to form the basis for DBD binding to a target site and may be referred to as the "recognition helix." In exemplary embodiments, the recognition helix of a zinc finger contains at least one amino acid substitution at position -1, 2, 3, or 6, thereby altering the binding specificity of the zinc finger domain. In other embodiments, the DBDs provided herein comprise one or more non-classical zinc fingers, such as C2-H2, C2-CH, and C2-C2.

[0111] In another embodiment, a DBD domain provided herein comprises a zinc finger motif having the following structure: LEPGEKP-[YKCPECGKSFS X HQRTH TGEKP]n-YKCPECGKSFS X HQRTH-TGKKTS (SEQ ID NO: 421), where n is an integer between 1 and 15, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, and each X is independently a recognition sequence (e.g., a recognition helix) capable of binding to a 3 bp target sequence. In exemplary embodiments, n is 3, 6, or 9. In particularly preferred embodiments, n is 6. In various embodiments, each X can independently have the same or a different amino acid sequence compared to other X sequences in the DBD. In an exemplary embodiment, each X is a sequence containing 7 amino acids designed to interact with a 3 bp target binding site of interest using the Zinc Finger Design Tool from Scripps on the World Wide Web at scripps.edu / barbas / zfdesign / zfdesignhome.php.

[0112] Because each zinc finger in a DBD recognizes 3 bp, the number of zinc fingers contained in the DBD informs the length of the binding site recognized by the DBD: for example, a DBD with one zinc finger recognizes a target binding site having 3 bp, a DBD with two zinc fingers recognizes a target binding site having 6 bp, a DBD with three zinc fingers recognizes a target binding site having 9 bp, a DBD with four zinc fingers recognizes a target binding site having 12 bp, a DBD with five zinc fingers recognizes a target binding site having 15 bp, a DBD with six zinc fingers recognizes a target binding site having 18 bp, a DBD with nine zinc fingers recognizes a target binding site having 27 bp, etc. Generally, DBDs that recognize longer target binding sites exhibit higher binding specificity (e.g., less off-target or nonspecific binding).

[0113] In other embodiments, the DBDs provided herein are derived from naturally occurring zinc finger proteins by making one or more amino acid substitutions in one or more of the recognition helices of the zinc finger domain to alter the binding specificity of the DBD (e.g., to alter the target site recognized by the DBD). The DBDs provided herein can be derived from any naturally occurring zinc finger protein. In various embodiments, such DBDs can be derived from zinc finger proteins of any species, e.g., mouse, rat, human, etc. In exemplary embodiments, the DBDs provided herein are derived from human zinc finger proteins. In certain embodiments, the DBDs provided herein are derived from naturally occurring proteins listed in Table 5. In exemplary embodiments, the DBD proteins provided herein are derived from human EGR zinc finger proteins, e.g., EGR1, EGR2, EGR3, or EGR4.

[0114] In certain embodiments, the DBDs provided herein are derived from naturally occurring proteins by modifying the DBD to increase the number of zinc finger domains in the DBD protein by repeating one or more zinc fingers in the DBD of the naturally occurring protein. In certain embodiments, such modifications include doubling, tripling, quadrupling, or further multiplying the zinc fingers in the DBD of the naturally occurring protein. In some cases, the number of zinc fingers from the DBD of a human protein is multiplied, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more copies of the same zinc finger motif are repeated in the DBD of the eTF. In some cases, the number of zinc finger sets from the DBD of a naturally occurring protein is increased, e.g., a set of three zinc fingers from the DBD of a naturally occurring protein is doubled to obtain an eTF with a DBD with six zinc fingers, tripled to obtain an eTF DBD with nine zinc fingers, or quadrupled to obtain an eTF DBD with twelve zinc fingers, etc. In some cases, a set of zinc fingers from the DBD of a naturally occurring protein is partially duplicated to form an eTF DBD with more zinc fingers, e.g., the DBD of the eTF comprises four zinc fingers, where these zinc fingers represent one copy of the first zinc finger, one copy of the second zinc finger, and two copies of the third zinc finger from the naturally occurring protein, for a total of four zinc fingers in the DBD of the eTF. Such DBDs are then further modified by making one or more amino acid substitutions in one or more of the recognition helices of the zinc finger domain to alter the binding specificity of the DBD (e.g., to alter the target site recognized by the DBD). In an exemplary embodiment, the DBD is derived from a naturally occurring human protein, e.g., a human EGR zinc finger protein, e.g., EGR1, EGR2, EGR3, or EGR4.

[0115] Human EGR1 and EGR3 are characterized by a three-finger C2H2 zinc finger DBD. The general binding rule for zinc fingers stipulates that all three fingers use the same amino acids in the alpha helix of each zinc finger to interact with their cognate DNA sequence with similar geometry to determine the specificity or recognition of the target binding site sequence. Such binding rules make it possible to modify the DBD of EGR1 or EGR3 to engineer a DBD that recognizes a desired target binding site. In some cases, the 7-amino acid DNA recognition helix in the zinc finger motif of EGR1 or EGR3 is modified according to published zinc finger design rules. In certain embodiments, each zinc finger in the three-finger DBD of EGR1 or EGR3 is modified, for example, by changing the sequence of one or more recognition helices and / or by increasing the number of zinc fingers in the DBD. In certain embodiments, EGR1 or EGR3 is reprogrammed to recognize a target binding site of at least 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, or more base pairs in a desired target site. In certain embodiments, such DBDs derived from ERG1 or EGR3 comprise at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more zinc fingers. In exemplary embodiments, one or more of the zinc fingers in the DBD comprises at least one amino acid substitution at position -1, 2, 3, or 6 of the recognition helix.

[0116] In various embodiments, an eTF comprising a DBD derived from EGR1 or EGR3 has a DNA binding specificity that differs from the binding specificity of naturally occurring EGR1 or EGR3, e.g., the DBD recognizes a target binding site having a sequence (SEQ ID NO: 373) that differs from the sequence of the binding site recognized by unmodified EGR1 or EGR3: (GCG(T / G)GGGCG).

[0117] In other embodiments, the eTF provided herein comprises a DBD that is a gRNA / Cas complex. CRISPR (clustered regularly interspaced short palindromic repeats) / Cas9 is a genome editing tool that enables site-specific genome targeting. The type II CRISPR / Cas system is a prokaryotic adaptive immune response system that uses non-coding RNA to guide Cas9 nuclease to induce site-specific DNA cleavage. The CRISPR / Cas9 system has been used to create a simple RNA-programmable method for mediating genome editing in mammalian cells. A single guide RNA (sgRNA) can be created to direct Cas9 nuclease to a specific genomic location where the gRNA / Cas9 complex then binds. The gRNA can be designed to bind to a desired target site using various methods and tools. For example, methods for designing gRNAs to bind to target DNA sequences of interest are described in Aach, et al. Flexible algorithm for identifying specific Cas9 targets in genomes. BioRxiv, Cold Spring Harbor Labs. doi: http: / / dx.doi.org / 10.1101 / 005074 (2014); Bae, et al. Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics. 30(10):1473-1475 (2014); Doench, JG et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotech 34, 184-191 (2016);Gratz, et al. Highly specific and efficient CRISPR / Cas9-catalyzed homology-directed repair in Drosophila. Genetics. 196(4):961-971 (2014);Heigwer, et al. E-CRISP: fast CRISPR target site identification. Nat Methods. 11(2):122-123 (2014);Ma, et al. A guide RNA sequence design platform for the CRISPR / Cas9 system for model organism genomes. Biomed Res Int. doi:http: / / doi.org / 10.1155 / 2013 / 270805 (2013);Montague, et al. CHOPCHOP: a CRISPR / Cas9 and TALEN web tool for genome editing. Nucleic Acids Res. 42(W1):W401-W407 (2014);Liu, et al. CRISPR-ERA: a comprehensive design tool for CRISPR-mediated gene editing, repression and activation. Bioinformatics. 31(22):3676-3678 (2015);Ran, et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature. 520(7546):186-191 (2015);Wu, et al. Target specificity of the CRISPR-Cas9 system. Quant Biol. 2(2):59-70 (2015); Xiao, et al. CasOT: a genome-wide Cas9 / gRNA off-target searching tool. Bioinformatics. 30(8):1180-1182 (2014); Zetsche, et al. Cpf1 is a single RNA-guided endonuclease of a Class 2 CRISPR-Cas System. Cell. 163(3):759-771 (2015). Additionally, various web-based tools are publicly available for designing gRNAs to bind to DNA target sequences of interest, see, e.g., the CRISPR gRNA Design tool available from AUTM on the World Wide Web at atum.bio / eCommerce / cas9 / input?multipleContacts=false; the CRISPRa / i gRNA design tool available from the Broad Institute on the World Wide Web at portals.broadinstitute.org / gpp / public / analysis-tools / sgrna-design-crisprai; the E-CRISP design tool available from the DKFZ German Cancer Research Center on the World Wide Web at e-crisp.org / E-CRISP / ; and the Knockout Guide Design tool available from Synthego on the World Wide Web at design.synthego.com / # / .Additionally, various commercial services are available for designing gRNAs to bind to DNA target sequences of interest, see, for example, the commercial services offered by IDT (world wide web at idtdna.com / site / order / designtool / index / CRISPR_SEQUENCE), ThermoFisher (world wide web at thermofisher.com / order / custom-oligo / crispr), and GenScript (world wide web at genscript.com / gRNA-design-tool.html).

[0118] In an exemplary embodiment, the DBD of the gRNA / Cas complex comprises a nuclease-deactivated Cas protein, i.e., dCas, such as dCas9, e.g., nuclease-deactivated Staphylococcus aureus (dSaCas9) or nuclease-deactivated Streptococcus pyogenes Cas9 (dSpCas9). The gRNA is provided as a sequence comprising a targeting region that targets the gRNA / Cas complex to a desired target site and a scaffold region that facilitates interaction with the Cas protein. Any suitable gRNA scaffold may be used in conjunction with the gRNAs provided herein. In an exemplary embodiment, the gRNA is a single gRNA, i.e., sgRNA, and comprises the following scaffold sequence: 5'-GTTTTAGTACTCTGGAAACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCTCGTCAACTTGTTGGCGAGA-3' (SEQ ID NO: 102). The targeting region of the guide RNA is attached to the 5' end of the scaffold sequence to form the complete sgRNA. In certain embodiments, gRNA and dCas protein can be expressed from the same expression cassette.In certain embodiments, U6 promoter is used to express gRNA.In other embodiments, gRNA can be expressed in cells that are engineered to stably express dCas-TAD protein, for example, by stably integrating dCas into genome or on a plasmid that is stably maintained extrachromosomally.

[0119] In other embodiments, the eTF provided herein may comprise a DBD derived from a TALEN, derived from a TALEN, or a nuclease-inactivated TALEN. Transcription activator-like effector nuclease (TALEN) is a restriction enzyme that contains a DBD and a nuclease domain that can be engineered to cleave specific sequences in DNA. TALENs are created by conjugating a TAL effector DNA binding domain to a DNA cleavage domain (e.g., a nuclease). Transcription activator-like effectors (TALEs) can be engineered to bind to desired target DNA sequences, thereby directing the nuclease domain to a specific location.

[0120] TAL effectors are bacterial proteins derived from Xanthomonas bacteria. The DNA-binding domain contains a repeated, highly conserved sequence of 33–34 amino acids with variable 12th and 13th amino acids. These two positions, termed the Repeat Variable Diresidue (RVD), are highly variable and show a strong correlation with specific nucleotide recognition. This direct link between amino acid sequence and DNA recognition allows for the engineering of DBDs that specifically target desired sequences by selecting combinations of repeat segments containing the appropriate RVD.

[0121] Various methods for designing TALEs are available. For example, methods for designing TALEs to bind to target DNA sequences of interest are described in T. Cermak et al., Nucleic Acids Research. 39 (12): e82 (2011); F. Zhang F et al., Nature Biotechnology. 29 (2): 149-53 (2011); R. Morbitzer et al., Nucleic Acids Research. 39 (13): 5790-9 (2011); T. Li et al., Nucleic Acids Research. 39 (14): 6315-25 (2011); R. Geissler et al., PLOS One. 6(5): e19509 (2011); and E. Weber et al., PLOS One. 6 (5): e19722 (2011). Additionally, various web-based tools are publicly available for designing TALEs to bind to DNA target sequences of interest, see, for example, E-Talen, available on the world wide web at e-talen.org / E-TALEN / TAL, and the Effector Nucleotide Targeter 2.0 tool, available on the world wide web at tale-nt.cac.cornell.edu / node / add / single-tale. Additionally, various commercially available services are available for designing TALEs to bind to DNA target sequences of interest, see, for example, the commercially available services provided by OmicX (world wide web at omictools.com / ), Addgene (world wide web at addgene.org / talen / guide / ) or ThermoFisher (world wide web at thermofisher.com / us / en / home / life-science / genome-editing / geneart-tals / tal-design-tool.html).Additionally, publicly available software programs (DNAWorks) can be used to design suitable oligonucleotides for the assembly of TALEs, see, e.g., D. Hoover D Methods in Molecular Biology. 852: 215-23 (2012). Transcriptional Modulation Domain

[0122] The eTFs provided herein may include any suitable domain capable of recruiting one or more protein factors (e.g., RNA polymerase II, CBP / p300, CREB, or KRAB) that can modulate transcription from a gene of interest or the level of gene expression from a gene of interest when the eTF binds to a target site via its DBD (e.g., a zinc finger DBD, a gRNA / Cas DBD, or a TALE DBD). In certain embodiments, such a domain recruits a protein factor that increases the level of transcription or gene expression of a gene of interest, and is a transcriptional activation domain (TAD). In other embodiments, such a domain recruits a protein factor that decreases the level of transcription or gene expression from a gene of interest, and is a transcriptional repressor domain (TRD). In certain embodiments, the transcriptional modulation domain (TAD or TRD) may be a synthetically designed domain. In other embodiments, the transcription modulation domain (TAD or TRD) can be derived from a naturally occurring protein, such as a transcription factor, transcription coactivator, transcription corepressor, or silencer protein. In various embodiments, the transcription modulation domain (TAD or TRD) can be derived from any species, such as a mouse, rat, monkey, virus, or human protein.

[0123] In an exemplary embodiment, a TAD suitable for use in the eTFs provided herein is derived from a viral protein. Exemplary TADs derived from viral proteins include, for example, the TAD domain of VP64 (SEQ ID NO: 95), VPR (SEQ ID NO: 114), VP16, VP128, p65, p300, or any functional fragment or variant thereof, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0124] In another exemplary embodiment, the TAD suitable for use in the eTFs provided herein is derived from a human protein. Exemplary TADs derived from human proteins include, for example, the TAD domain of CBP / p300-interacting transactivator 2 (CITED2) (SEQ ID NO: 96 or 98), CBP / p300-interacting transactivator 4 (CITED4) (SEQ ID NO: 97 or 100), EGR1 (SEQ ID NO: 1), or EGR3 (SEQ ID NO: 422), or any functional fragment or variant thereof, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0125] In another embodiment, a TRD suitable for use in an eTF provided herein comprises a KRAB domain capable of recruiting proteins that inhibit transcription, or any functional fragment or variant thereof, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0126] In certain embodiments, the zinc finger DBD is conjugated to a transcription modulation domain, such as a TAD or TRD, as described further below. In various embodiments, the zinc finger DBD can be conjugated to a TAD from a viral protein, such as VP64 or VPR, or a TAD from a human protein, such as CITED2 or CITED4. In certain embodiments, a zinc finger DBD from a human protein, such as EGR1 or EGR3, is conjugated to a TAD from a human protein, such as CITED2 or CITED4. In certain embodiments, a zinc finger DBD from a human protein, such as EGR1 or EGR3, is conjugated to a VP64 or VPR TAD. In certain embodiments, a synthetic zinc finger DBD, or a zinc finger DBD having less than 75% sequence identity to a human protein, such as EGR1 or EGR3, is conjugated to a TAD from a human protein, such as CITED2 or CITED4. In certain embodiments, a synthetic zinc finger DBD, or a zinc finger DBD with less than 75% sequence identity to a human protein, e.g., EGR1 or EGR3, is conjugated to a VP64 or VPR TAD.

[0127] In certain embodiments, the dCas protein is conjugated to a transcriptional modulation domain, such as a TAD or TRD, as described further below. In various embodiments, dCas9 can be conjugated to a TAD derived from a viral protein, such as VP64 or VPR, or a TAD derived from a human protein, such as CITED2 or CITED4. In an exemplary embodiment, dCas9 is conjugated to a VP64 or VPR TAD.

[0128] In certain embodiments, the TALE protein is conjugated to a transcription modulation domain, such as a TAD or TRD, as described further below. In various embodiments, the TALE may be conjugated to a TAD from a viral protein, such as VP64 or VPR, or a TAD from a human protein, such as CITED2 or CITED4. In an exemplary embodiment, the TALE is conjugated to a VP64 or VPR TAD. eTFs that upregulate SCN1A

[0129] In one aspect, the present application provides an eTF capable of upregulating the expression of the sodium voltage-gated channel alpha subunit 1 (SCN1A) gene and increasing the expression of its corresponding protein product, Nav1.1. The SCN1A gene belongs to a family of genes encoding subunits used to assemble sodium channels. These channels, which transport positively charged sodium ions into cells, play an important role in the cells' ability to generate and transmit electrical signals. The SCN1A gene encodes one part (alpha subunit) of the sodium channel called Nav1.1. These channels are primarily found in the brain, where they control the flow of sodium ions into cells. The Nav1.1 channel is involved in transmitting signals from one nerve cell (or neuron) to another. Some mutations in the SCN1A gene have been found to cause genetic epilepsy with febrile seizures plus (GEFS+), a seizure disorder with varying degrees of severity. These conditions include simple febrile (fever-related) seizures, which begin in infancy and usually cease by age 5, and febrile seizures plus (FS+). FS+ involves febrile seizures and other types of seizures, including those not associated with fever (afebrile seizures), which continue beyond childhood. The GEFS+ spectrum also includes other conditions that cause more severe seizures that may last longer and be difficult to control, such as Dravet syndrome (also known as severe myoclonic epilepsy of infancy, or SMEI). These recurrent seizures (epilepsies) can worsen over time and are often accompanied by a decline in brain function. Many other mutations have been associated with familial hemiplegic migraine, a form of migraine that runs in families, and at least one mutation has been associated with the effectiveness of certain anti-seizure medications. Thus, the eTFs provided herein that increase SCN1A expression can be used to treat various diseases or disorders associated with mutations in the Nav1.1 channel.

[0130] In certain embodiments, the eTFs disclosed herein that upregulate SCN1A are at least 9 bp, 12 bp, 15 bp, 18 bp, 21 bp, 24 bp, 27 bp, 30 bp, 33 bp, or 36 bp in size; longer than 9 bp, 12 bp, 15 bp, 18 bp, 21 bp, 24 bp, 27 bp, or 30 bp; or longer than 9-33 bp, 9-30 bp, 9-27 bp, 9-24 bp, 9-21 bp, 9-18 bp, 9-15 bp, 9-12 bp, 12-33 bp, 12-30 bp, The eTFs disclosed herein recognize a target binding site that is 12-27 bp, 12-24 bp, 12-21 bp, 12-18 bp, 12-15 bp, 15-33 bp, 15-30 bp, 15-27 bp, 15-24 bp, 15-21 bp, 15-18 bp, 18-33 bp, 18-30 bp, 18-27 bp, 18-24 bp, 18-21 bp, 21-33 bp, 21-30 bp, 21-27 bp, 21-24 bp, 24-33 bp, 24-30 bp, 24-27 bp, 27-33 bp, 27-30 bp, or 30-33 bp. In exemplary embodiments, the eTFs disclosed herein that upregulate SCN1A recognize a target binding site that is 18-27 bp, 18 bp, or 27 bp.

[0131] In certain embodiments, the eTF disclosed herein that upregulates SCN1A recognizes a target binding site located on chromosome 2. In certain embodiments, the eTF disclosed herein that upregulates SCN1A recognizes a target binding site located on chromosome 2 within 110 kb, 100 kb, 90 kb, 80 kb, 70 kb, 60 kb, 50 kb, 40 kb, 30 kb, 20 kb, 10 kb, 5 kb, 4 kb, 3 kb, 2 kb or 1 kb upstream or downstream of the TSS of SCN1A. In certain embodiments, the eTF disclosed herein that upregulates SCN1A recognizes a target binding site located on chromosome 2 within 110 kb upstream of the TSS of SCN1A. In certain embodiments, the eTF disclosed herein that upregulates SCN1A recognizes a target binding site located on chromosome 2 within 110 kb downstream of the TSS of SCN1A. In exemplary embodiments, such target binding sites are 18-27 bp, 18 bp or 27 bp.

[0132] In certain embodiments, eTFs disclosed herein that upregulate SCN1A recognize a target binding site located on chromosome 2 within positions 166179652-165989571, 166128050-166127958, 166155414-166140590, 166179652-1661777272, or 1659990246-165989592 (all referenced to GRCh38.p12). In exemplary embodiments, such target binding sites are 18-27 bp, 18 bp, or 27 bp.

[0133] In certain embodiments, an eTF disclosed herein that upregulates SCN1A recognizes a target binding site that is: (i) 18-27 bp, 18 bp, or 27 bp; (ii) 166178880, 166178871, 166177369, 166177362, 166177299, 16617 7299, 166155393, 166155264, 166149373, 166149176, 166149165, 166149118, 166148953, 166148565, 166142396, 166142391, 166142344, 166142239, 166141162, 166140 928, 166140590, 165990076, 165989684, 165989571, 166155255, 166155099, 166148843, 166148361, 166142219, 166141090, 165990246, 165990193, 166149168, 166127991, 166128002, 166128037 or 166128025 (all with reference to GRCh38.p12), and (iii) is capable of producing at least a 1.2-fold increase in expression of SCN1A when bound by an eTF disclosed herein.

[0134] In certain embodiments, an eTF disclosed herein that upregulates SCN1A (i) binds to a target site comprising or consisting of any of SEQ ID NOs: 35-37, 101, 105-111, 136, 195-211, 224-238, or 240-267, and (ii) is capable of producing at least a 1.2-fold increase in expression of SCN1A when bound by an eTF disclosed herein.

[0135] In certain embodiments, an eTF disclosed herein that upregulates SCN1A recognizes a target binding site that: (i) is 18-27 bp, 18 bp, or 27 bp; (ii) overlaps with a location on chromosome 2 selected from 166155255, 166155099, 166148843, 166148361, 166142219, 166141090, 165990246, 165990193, 166149168, 166127991, 166128002, 166128037, or 166128025 (all with reference to GRCh38.p12); and (iii) is capable of producing at least a 2-fold increase in expression of SCN1A when bound by an eTF disclosed herein.

[0136] In certain embodiments, an eTF disclosed herein that upregulates SCN1A (i) binds to a target site comprising or consisting of any of SEQ ID NOs: 35-36, 108-109, 136, 209-210, 226, 228, 233, 236, or 247-248, and (ii) is capable of producing at least a 2-fold increase in expression of SCN1A when bound by an eTF disclosed herein.

[0137] In certain embodiments, an eTF disclosed herein that upregulates SCN1A recognizes a target binding site that: (i) is 18-27 bp, 18 bp, or 27 bp, and (ii) overlaps with a location on chromosome 2 selected from 166149168, 166127991, 166128002, 166128037, or 166128025 (all with reference to GRCh38.p12), and (iii) is capable of producing at least a 5-fold increase in expression of SCN1A when bound by an eTF disclosed herein.

[0138] In certain embodiments, an eTF disclosed herein that upregulates SCN1A (i) binds to a target site comprising or consisting of any of SEQ ID NOs: 35, 36, 108, 109, or 136, and (ii) is capable of producing at least a 5-fold increase in the expression of SCN1A when bound by an eTF disclosed herein.

[0139] In certain embodiments, an eTF disclosed herein that upregulates SCN1A recognizes a target binding site that: (i) is 18-27 bp, 18 bp, or 27 bp, (ii) overlaps with a location on chromosome 2 selected from 166128002, 166128037, or 166128025 (all with reference to GRCh38.p12), and (iii) is capable of producing at least a 15-fold increase in expression of SCN1A when bound by an eTF disclosed herein.

[0140] In certain embodiments, an eTF disclosed herein that upregulates SCN1A (i) binds to a target site comprising or consisting of any of SEQ ID NOs: 36, 108, or 109, and (ii) is capable of producing at least a 15-fold increase in expression of SCN1A when bound by an eTF disclosed herein.

[0141] In certain embodiments, an eTF disclosed herein that upregulates SCN1A recognizes a target binding site that: (i) is 18-27 bp, 18 bp, or 27 bp, (ii) overlaps with a location on chromosome 2 selected from 166128037 or 166128025 (all with reference to GRCh38.p12), and (iii) is capable of producing at least a 20-fold increase in expression of SCN1A when bound by an eTF disclosed herein.

[0142] In certain embodiments, an eTF disclosed herein that upregulates SCN1A (i) binds to a target site comprising or consisting of either SEQ ID NO: 36 or 109, and (ii) is capable of producing at least a 20-fold increase in expression of SCN1A when bound by an eTF disclosed herein.

[0143] In certain embodiments, an eTF disclosed herein that upregulates SCN1A recognizes a target binding site that is: (i) 18-27 bp, 18 bp, or 27 bp, (ii) overlaps with a location on chromosome 2 at position 166128025, and (iii) is capable of producing at least a 25-fold increase in expression of SCN1A when bound by an eTF disclosed herein.

[0144] In certain embodiments, an eTF disclosed herein that upregulates SCN1A (i) binds to a target site comprising or consisting of SEQ ID NO: 36, and (ii) is capable of producing at least a 25-fold increase in expression of SCN1A when bound by an eTF disclosed herein.

[0145] In certain embodiments, an eTF disclosed herein that upregulates SCN1A (i) recognizes a target binding site that is 18-27 bp, 18 bp, or 27 bp, and (ii) binds to a genomic region that is within at least 1 kb, 750 bp, 500 bp, 400 bp, 300 bp, 200 bp, 100 bp, or 50 bp of a genomic location having the sequence of any one of SEQ ID NOs: 35-37, 101, 105-111, 136, 195-211, 224-238, or 240-267. In certain embodiments, the target binding site is capable of producing at least a 1.2-fold, 2-fold, 5-fold, 15-fold, 20-fold, or 25-fold increase in expression of SCN1A when bound by an eTF disclosed herein.

[0146] In certain embodiments, an eTF disclosed herein that upregulates SCN1A (i) recognizes a target binding site that is 18-27 bp, 18 bp, or 27 bp, and (ii) binds to a genomic region that at least partially overlaps with a genomic location having the sequence of any one of SEQ ID NOs: 35-37, 101, 105-111, 136, 195-211, 224-238, or 240-267. In certain embodiments, the target binding site is capable of producing at least a 1.2-fold, 2-fold, 5-fold, 15-fold, 20-fold, or 25-fold increase in expression of SCN1A when bound by an eTF disclosed herein.

[0147] In certain embodiments, an eTF disclosed herein that upregulates SCN1A recognizes a target binding site having any one of the following sequences: SEQ ID NOs: 35-37, 101, 105-111, 136, 195-211, 224-238, or 240-267. In certain embodiments, the target binding site is capable of producing at least a 1.2-fold, 2-fold, 5-fold, 15-fold, 20-fold, or 25-fold increase in expression of SCN1A when bound by an eTF disclosed herein.

[0148] In certain embodiments, eTFs disclosed herein that upregulate SCN1A result in at least a 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 50-fold, 100-fold or greater upregulation of SCN1A expression (SCN1A RNA and / or Nav1.1 protein) in a cell or in vivo, or at least a 50%, 60%, 70%, 75%, 80%, 90%, 100%, 125%, 150%, 200%, 250%, 300%, 400%, or 500% or greater upregulation, compared to a control (e.g., no eTF or an eTF that does not recognize the target site). In various embodiments, upregulation of SCN1A expression can be detected using PCR, Western blot, or immunoassay.

[0149] In certain embodiments, an eTF disclosed herein that upregulates SCN1A binds to a target site that is capable of increasing SCN1A expression by at least 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 8-fold, 10-fold, 12-fold, 15-fold, 18-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 75-fold, 100-fold or more, or by at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 100%, 125%, 150%, 200%, 250%, 300%, 400%, or 500% or more relative to a control in a transcription activation assay. An exemplary SCN1A transcription activation assay is provided herein in Example 3. Briefly, HEK293 cells are transfected with a plasmid carrying eTF or a control eGFP reporter construct. 48 hours after transfection, cells are harvested, RNA is isolated and reverse transcribed, and the resulting cDNA samples are analyzed by qPCR (e.g., using primers with SEQ ID NOs: 190 and 191) to quantify the level of endogenous SCN1A transcripts. GAPDH can be used as a reference gene to determine the relative level of SCN1A expression.

[0150] In certain embodiments, eTFs disclosed herein that upregulate SCN1A have minimal off-target effects, e.g., off-target effects associated with non-specific binding, such as modulation of expression of off-target genes or genes other than SCN1A. In one embodiment, eTFs disclosed herein that upregulate SCN1A specifically upregulate SCN1A compared to a control by at least 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, or 50-fold greater than the expression caused by the eTF for one or more off-target genes compared to a control. In exemplary embodiments, an eTF disclosed herein that upregulates SCN1A is a gene encoding the 40 nearest neighboring genes (e.g., the 40 nearest genes located in the coding sequence of SCN1A on chromosome 2) produced by the eTF compared to a control, such as PLA2R1, ITGB6, RBMS1, TANK, PSMD14, TBR1, SLC4A10, DPP4, FAP, IFIH1, GCA, FIGN, GRB14, COBLL1, SLC38A11, SCN3A, SCN2A, , CSRNP3, GALNT3, TTC21B, SCN9A, SCN7A, B3GALT1, STK39, CERS6, NOSTRIN, SPC25, ABCB11, DHRS9, BBS5, KLHL41, FASTKD1, PPIG, CCDC173, PHOSPHO2, KLHL23, SSB, METTL5, UBR3, and MYO3B, specifically upregulating transcription from the SCN1A gene compared to a control by at least 15-fold greater than transcription of the SCN1A gene (see Table 33). In various embodiments, upregulation of transcription from the SCN1A gene can be detected using PCR methods.

[0151] In certain embodiments, the eTFs disclosed herein that upregulate SCN1A are used to treat Scn1a in Dravet syndrome. tm1keaIt is possible to reduce the frequency of seizures in a hyperthermic seizure (HTS) assay in a mouse model. In certain embodiments, the eTF disclosed herein can reduce the frequency of seizures at 42.6°C in an HTS assay by at least 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold or more, or by at least 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more, compared to a control (e.g., PBS treatment or treatment with an AAV vector comprising a sequence encoding eGFP). In certain embodiments, the eTFs disclosed herein can reduce the frequency of seizures at 42.6°C in an HTS assay such that at least 60%, 62%, 65%, 70%, 75%, 76%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the mice run in the assay are seizure-free at 42.6°C. An exemplary HTS assay is described herein in Example 11. Briefly, litters of pups born to male Scn1a+ / − mice crossed with female C57B1 / 6J mice can be administered an AAV9 vector encoding an SCN1A-upregulating eTF provided herein or a control vector encoding eGFP via bilateral ICV at P1. Mice may be administered approximately 1.0E10-5.0E12 gc / mouse. HTS assays are performed in SCN1A heterozygous and SCN1A wild-type mice on a mixed 129Stac x C57BL / 6 background at P26-P28 by increasing the mouse's temperature (under controlled conditions with temperature monitoring) by approximately 0.5°C every 2 minutes until the onset of the first tonic-clonic seizure accompanied by loss of posture or until a body temperature of 43°C is reached. Mice are considered seizure-free if no seizures accompanied by loss of posture are detected throughout the course of the experiment.

[0152] In certain embodiments, the eTFs disclosed herein that upregulate SCN1A are used in mice that are heterozygous for SCN1A, e.g., Scn1a tm1kea It is possible to increase the survival of mouse strains. In certain embodiments, the eTFs disclosed herein can increase the survival rate of SCN1A heterozygous mice at P100 by at least 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold or more, or by at least 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more, compared to a control (e.g., PBS treatment or treatment with an AAV vector comprising a sequence encoding eGFP). In certain embodiments, the eTFs disclosed herein can increase the survival rate of SCN1A heterozygous mice at P100, such that at least 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the mice run in the assay are still alive at P100. An exemplary survival assay is described herein in Example 12. Briefly, litters of pups born to male Scn1a+ / − mice crossed with female C57B1 / 6J mice can be administered AAV9 vectors via bilateral ICV at P1. Mice can be administered approximately 1.0E10 to 5.0E12 gc / mouse. The number of mice surviving to P100 is determined.

[0153] In certain embodiments, the eTFs disclosed herein that upregulate SCN1A have a high percent identity to one or more human proteins (as described further below). In certain embodiments, such eTFs have at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% overall sequence identity to one or more human proteins. In certain embodiments, such eTFs exhibit reduced immunogenicity compared to eTFs with a lower overall percent sequence identity to one or more human proteins. In various embodiments, the reduced immunogenicity can be measured using an elispot assay, immunoassay, or in silico method. In certain embodiments, such eTFs can include a DBD derived from human EGR1 or EGR3 and a TAD derived from human EGR1, EGR3, CITED2, or CITED4.

[0154] In certain embodiments, an eTF disclosed herein that upregulates SCN1A comprises a DNA-binding domain having one or more zinc finger domains that comprise a recognition helix comprising any one of SEQ ID NOs: 115-130. In certain embodiments, an eTF disclosed herein that upregulates SCN1A comprises a DNA-binding domain having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 zinc finger domains, each zinc finger domain independently comprising a recognition helix comprising any one of SEQ ID NOs: 115-130. In certain embodiments, an eTF disclosed herein that upregulates SCN1A comprises a DNA-binding domain having six zinc finger domains, each zinc finger domain independently comprising a recognition helix comprising any one of SEQ ID NOs: 115-130. In certain embodiments, an eTF disclosed herein that upregulates SCN1A comprises a DNA-binding domain having nine zinc finger domains, each zinc finger domain independently comprising a recognition helix comprising any one of SEQ ID NOs: 115-130. In exemplary embodiments, such an eTF comprises a DNA-binding domain having SEQ ID NO: 421, wherein each X is independently selected from any one of SEQ ID NOs: 115-130, and n is 6 or 9.

[0155] In certain embodiments, an eTF disclosed herein that upregulates SCN1A comprises a DNA-binding domain having any one of the following: (i) a sequence comprising RSDNLVR x REDNLHT x RSDELVR x QSGNLTE x TSGHLVR x QNSTLTE (SEQ ID NO: 135), where x can be a linker of 1 to 50 amino acids; (ii) a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 135; or (ii) a functional fragment of (i) or (ii). In certain embodiments, such an eTF further comprises one or more TADs selected from VP64, VPR, CITED2, or CITED4. In one embodiment, such an eTF comprises a VPR TAD domain conjugated to the C-terminus of the DBD. In certain embodiments, such an eTF comprises a CITED2 TAD conjugated to the N-terminus, C-terminus, or N- and C-terminus of the DBD. In certain embodiments, such an eTF comprises a CITED4 TAD conjugated to the N-terminus, C-terminus, or N- and C-terminus of the DBD. In certain embodiments, such an eTF is capable of binding to a target site having SEQ ID NO: 256 and is capable of upregulating expression of SCN1A by at least 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold or more compared to a control, or by at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 100%, 125%, 150%, 200%, 250%, 300%, 400%, or 500% or more compared to a control.

[0156] In certain embodiments, an eTF disclosed herein that upregulates SCN1A comprises a DNA-binding domain having any one of the following: (i) a sequence comprising RSDNLVR x HRTTLTN x REDNLHT x TSHSLTE x QSSSLVR x REDNLHT (SEQ ID NO: 371), where x can be a linker of 1 to 50 amino acids; (ii) a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 371; or (ii) a functional fragment of (i) or (ii). In certain embodiments, such an eTF further comprises one or more TADs selected from VP64, VPR, CITED2, or CITED4. In one embodiment, such an eTF comprises a VPR TAD domain conjugated to the C-terminus of the DBD. In certain embodiments, such an eTF comprises a CITED2 TAD conjugated to the N-terminus, C-terminus, or N- and C-terminus of the DBD. In certain embodiments, such an eTF comprises a CITED4 TAD conjugated to the N-terminus, C-terminus, or N- and C-terminus of the DBD. In certain embodiments, such an eTF is capable of binding to a target site having SEQ ID NO: 264 and is capable of upregulating expression of SCN1A by at least 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold or more compared to a control, or by at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 100%, 125%, 150%, 200%, 250%, 300%, 400%, or 500% or more compared to a control.

[0157] In certain embodiments, an eTF disclosed herein that upregulates SCN1A comprises a DNA-binding domain having any one of the following: (i) a sequence comprising RRDELNV x RSDHLTN x RSDDLVR x RSDNLVR x HRTTLTN x REDNLHT x TSHSLTE x QSSSLVR x REDNLHT (SEQ ID NO: 372); (ii) a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 372; or (ii) a functional fragment of (i) or (ii). In certain embodiments, such an eTF further comprises one or more TADs selected from VP64, VPR, CITED2, or CITED4. In one embodiment, such an eTF comprises a VPR TAD domain conjugated to the C-terminus of the DBD. In certain embodiments, such an eTF comprises a CITED2 TAD conjugated to the N-terminus, C-terminus, or N- and C-terminus of the DBD. In certain embodiments, such an eTF comprises a CITED4 TAD conjugated to the N-terminus, C-terminus, or N- and C-terminus of the DBD. In certain embodiments, such an eTF is capable of binding to a target site having SEQ ID NO: 37 and is capable of upregulating expression of SCN1A by at least 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold or more compared to a control, or by at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 100%, 125%, 150%, 200%, 250%, 300%, 400%, or 500% or more compared to a control.

[0158] In certain embodiments, an eTF disclosed herein that upregulates SCN1A comprises a DNA-binding domain having any one of the following: (i) a sequence comprising DPGALVR x RSDNLVR x QSGDLRR x THLDLIR x TSGNLVR x RSDNLVR (SEQ ID NO: 376); (ii) a sequence having at least 89%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 376; or (ii) a functional fragment of (i) or (ii). In certain embodiments, such an eTF further comprises one or more TADs selected from VP64, VPR, CITED2, or CITED4. In one embodiment, such an eTF comprises a VPR TAD domain conjugated to the C-terminus of the DBD. In certain embodiments, such an eTF comprises a CITED2 TAD conjugated to the N-terminus, C-terminus, or N- and C-terminus of the DBD. In certain embodiments, such an eTF comprises a CITED4 TAD conjugated to the N-terminus, C-terminus, or N- and C-terminus of the DBD. In certain embodiments, such an eTF is capable of binding to a target site having SEQ ID NO: 136 and is capable of upregulating expression of SCN1A by at least 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold or more compared to a control, or by at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 100%, 125%, 150%, 200%, 250%, 300%, 400%, or 500% or more compared to a control.

[0159] In certain embodiments, the eTFs disclosed herein that upregulate SCN1A comprise any one of the following: (i) a sequence comprising any one of SEQ ID NOs: 6-9, 13-15, 57-58, 61-62, 67-71, 74-75, 268-282, 295-299, 305-325, or 364-366; (ii) a sequence comprising any one of SEQ ID NOs: 22-25, 29-31, 84-85, 88, 90-92, 131-135, 371-372, 373-374, 375-376, 377-378, 378-379, 380-381, 382-383, 384-385, 386-387, 388-389, 390-391, 400-401, 401-402, 402-403, 403-404, 404-405, 405-406, 406-407, 407-408, 410-411, 412-413, 414-415, 416-417, 418-419, 420-421, 422-423, 424-425, 426-427, 428-429, 430-431, 432-433, 434-435, 436-437, 438-439, 440-441, 442-443, 444-445, 446- (iii) a sequence comprising any one of sequences 76, 391-409 or 423-435; (iii) a sequence comprising at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any of the sequences in (i) or (ii); or (iv) a functional fragment or variant of any of the sequences in (i), (ii) or (iii). In exemplary embodiments, such eTFs are capable of upregulating SCN1A expression by at least 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold or more compared to a control, or by at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 100%, 125%, 150%, 200%, 250%, 300%, 400%, or 500% or more compared to a control.

[0160] In certain embodiments, an eTF disclosed herein that upregulates SCN1A comprises any one of the following: (i) a sequence comprising any one of SEQ ID NOs: 268-282, 305-312, or 365-366; (ii) a sequence comprising any one of SEQ ID NOs: 131-135, 371-372, 376, 391-409, or 423-426; (iii) a sequence comprising at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the sequences in (i) or (ii); or (iv) a functional fragment or variant of any of the sequences in (i), (ii), or (iii). In exemplary embodiments, such eTFs are capable of upregulating SCN1A expression by at least 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold or more compared to a control, or by at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 100%, 125%, 150%, 200%, 250%, 300%, 400%, or 500% or more compared to a control.

[0161] In certain embodiments, an eTF disclosed herein that upregulates SCN1A comprises any one of the following: (i) a sequence comprising any one of SEQ ID NOs: 6-9, 13-15, 57-58, 61-62, 67-71, 74-75, 295-299, 313-325, or 364; (ii) a sequence comprising any one of SEQ ID NOs: 22-25, 29-31, 84-85, 88, 90-92, or 427-435; (iii) a sequence comprising at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the sequences in (i) or (ii); or (iv) a functional fragment or variant of any of the sequences in (i), (ii), or (iii). In exemplary embodiments, such eTFs are capable of upregulating SCN1A expression by at least 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold or more compared to a control, or by at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 100%, 125%, 150%, 200%, 250%, 300%, 400%, or 500% or more compared to a control. In exemplary embodiments, such eTFs have at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% overall sequence identity to one or more human proteins. In certain embodiments, such eTFs exhibit reduced immunogenicity compared to eTFs having a lower overall percent sequence identity to one or more human proteins. In various embodiments, the reduction in immunogenicity may be measured using an elispot assay, an immunoassay, or an in silico method.

[0162] In certain embodiments, an eTF disclosed herein that upregulates SCN1A comprises any one of the following: (i) a sequence comprising SEQ ID NO: 305; (ii) a sequence comprising SEQ ID NO: 423; (iii) a sequence comprising at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of (i) or (ii); or (iv) a functional fragment or variant of any of the sequences of (i), (ii), or (iii). In exemplary embodiments, such an eTF comprises SEQ ID NO: 135 and binds to a target site having SEQ ID NO: 256. In exemplary embodiments, such eTFs are capable of upregulating SCN1A expression by at least 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold or more compared to a control, or by at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 100%, 125%, 150%, 200%, 250%, 300%, 400%, or 500% or more compared to a control.

[0163] In certain embodiments, an eTF disclosed herein that upregulates SCN1A comprises any one of the following: (i) a sequence comprising SEQ ID NO: 306; (ii) a sequence comprising SEQ ID NO: 424; (iii) a sequence comprising at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of (i) or (ii); or (iv) a functional fragment or variant of any of the sequences of (i), (ii), or (iii). In exemplary embodiments, such an eTF comprises SEQ ID NO: 135 and binds to a target site having SEQ ID NO: 256. In exemplary embodiments, such eTFs are capable of upregulating SCN1A expression by at least 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold or more compared to a control, or by at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 100%, 125%, 150%, 200%, 250%, 300%, 400%, or 500% or more compared to a control.

[0164] In certain embodiments, an eTF disclosed herein that upregulates SCN1A comprises any one of the following: (i) a sequence comprising SEQ ID NO: 308; (ii) a sequence comprising SEQ ID NO: 425; (iii) a sequence comprising at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of (i) or (ii); or (iv) a functional fragment or variant of any of the sequences of (i), (ii), or (iii). In exemplary embodiments, such an eTF comprises SEQ ID NO: 135 and binds to a target site having SEQ ID NO: 256. In exemplary embodiments, such eTFs are capable of upregulating SCN1A expression by at least 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold or more compared to a control, or by at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 100%, 125%, 150%, 200%, 250%, 300%, 400%, or 500% or more compared to a control.

[0165] In certain embodiments, an eTF disclosed herein that upregulates SCN1A comprises any one of the following: (i) a sequence comprising SEQ ID NO: 313; (ii) a sequence comprising SEQ ID NO: 427; (iii) a sequence comprising at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of (i) or (ii); or (iv) a functional fragment or variant of any of the sequences of (i), (ii), or (iii). In exemplary embodiments, such an eTF comprises SEQ ID NO: 135 and binds to a target site having SEQ ID NO: 256. In exemplary embodiments, such eTFs are capable of upregulating SCN1A expression by at least 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold or more compared to a control, or by at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 100%, 125%, 150%, 200%, 250%, 300%, 400%, or 500% or more compared to a control. In exemplary embodiments, such eTFs have at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% overall sequence identity to one or more human proteins. In certain embodiments, such eTFs exhibit reduced immunogenicity compared to eTFs having a lower overall percent sequence identity to one or more human proteins. In various embodiments, the reduction in immunogenicity may be measured using an elispot assay, an immunoassay, or an in silico method.

[0166] In certain embodiments, the eTF disclosed herein that upregulates SCN1A comprises any one of the following: (i) a sequence comprising SEQ ID NO: 316; (ii) a sequence comprising SEQ ID NO: 430; (iii) a sequence comprising at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of (i) or (ii); or (iv) a functional fragment or variant of any of the sequences of (i), (ii), or (iii). In exemplary embodiments, such an eTF comprises SEQ ID NO: 135 and binds to a target site having SEQ ID NO: 256. In exemplary embodiments, such eTFs are capable of upregulating SCN1A expression by at least 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold or more compared to a control, or by at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 100%, 125%, 150%, 200%, 250%, 300%, 400%, or 500% or more compared to a control. In exemplary embodiments, such eTFs have at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% overall sequence identity to one or more human proteins. In certain embodiments, such eTFs exhibit reduced immunogenicity compared to eTFs having a lower overall percent sequence identity to one or more human proteins. In various embodiments, the reduction in immunogenicity may be measured using an elispot assay, an immunoassay, or an in silico method.

[0167] In certain embodiments, an eTF disclosed herein that upregulates SCN1A comprises any one of the following: (i) a sequence comprising SEQ ID NO: 317; (ii) a sequence comprising SEQ ID NO: 431; (iii) a sequence comprising at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of (i) or (ii); or (iv) a functional fragment or variant of any of the sequences of (i), (ii), or (iii). In exemplary embodiments, such an eTF comprises SEQ ID NO: 371 and binds to a target site having SEQ ID NO: 264. In exemplary embodiments, such eTFs are capable of upregulating SCN1A expression by at least 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold or more compared to a control, or by at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 100%, 125%, 150%, 200%, 250%, 300%, 400%, or 500% or more compared to a control. In exemplary embodiments, such eTFs have at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% overall sequence identity to one or more human proteins. In certain embodiments, such eTFs exhibit reduced immunogenicity compared to eTFs having a lower overall percent sequence identity to one or more human proteins. In various embodiments, the reduction in immunogenicity may be measured using an elispot assay, an immunoassay, or an in silico method.

[0168] In certain embodiments, an eTF disclosed herein that upregulates SCN1A comprises any one of the following: (i) a sequence comprising SEQ ID NO: 315; (ii) a sequence comprising SEQ ID NO: 429; (iii) a sequence comprising at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of (i) or (ii); or (iv) a functional fragment or variant of any of the sequences of (i), (ii), or (iii). In exemplary embodiments, such an eTF comprises SEQ ID NO: 135 and binds to a target site having SEQ ID NO: 256. In exemplary embodiments, such eTFs are capable of upregulating SCN1A expression by at least 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold or more compared to a control, or by at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 100%, 125%, 150%, 200%, 250%, 300%, 400%, or 500% or more compared to a control. In exemplary embodiments, such eTFs have at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% overall sequence identity to one or more human proteins. In certain embodiments, such eTFs exhibit reduced immunogenicity compared to eTFs having a lower overall percent sequence identity to one or more human proteins. In various embodiments, the reduction in immunogenicity may be measured using an elispot assay, an immunoassay, or an in silico method.

[0169] In certain embodiments, an eTF disclosed herein that upregulates SCN1A comprises any one of the following: (i) a sequence comprising SEQ ID NO: 319; (ii) a sequence comprising SEQ ID NO: 433; (iii) a sequence comprising at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of (i) or (ii); or (iv) a functional fragment or variant of any of the sequences of (i), (ii), or (iii). In exemplary embodiments, such an eTF comprises SEQ ID NO: 371 and binds to a target site having SEQ ID NO: 264. In exemplary embodiments, such eTFs are capable of upregulating SCN1A expression by at least 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold or more compared to a control, or by at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 100%, 125%, 150%, 200%, 250%, 300%, 400%, or 500% or more compared to a control. In exemplary embodiments, such eTFs have at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% overall sequence identity to one or more human proteins. In certain embodiments, such eTFs exhibit reduced immunogenicity compared to eTFs having a lower overall percent sequence identity to one or more human proteins. In various embodiments, the reduction in immunogenicity may be measured using an elispot assay, an immunoassay, or an in silico method.

[0170] In certain embodiments, an eTF disclosed herein that upregulates SCN1A comprises any one of the following: (i) a sequence comprising SEQ ID NO: 318; (ii) a sequence comprising SEQ ID NO: 432; (iii) a sequence comprising at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of (i) or (ii); or (iv) a functional fragment or variant of any of the sequences of (i), (ii), or (iii). In exemplary embodiments, such an eTF comprises SEQ ID NO: 371 and binds to a target site having SEQ ID NO: 264. In exemplary embodiments, such eTFs are capable of upregulating SCN1A expression by at least 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold or more compared to a control, or by at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 100%, 125%, 150%, 200%, 250%, 300%, 400%, or 500% or more compared to a control. In exemplary embodiments, such eTFs have at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% overall sequence identity to one or more human proteins. In certain embodiments, such eTFs exhibit reduced immunogenicity compared to eTFs having a lower overall percent sequence identity to one or more human proteins. In various embodiments, the reduction in immunogenicity may be measured using an elispot assay, an immunoassay, or an in silico method.

[0171] In certain embodiments, an eTF disclosed herein that upregulates SCN1A comprises any one of the following: (i) a sequence comprising SEQ ID NO: 314; (ii) a sequence comprising SEQ ID NO: 428; (iii) a sequence comprising at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of (i) or (ii); or (iv) a functional fragment or variant of any of the sequences of (i), (ii), or (iii). In exemplary embodiments, such an eTF comprises SEQ ID NO: 135 and binds to a target site having SEQ ID NO: 256. In exemplary embodiments, such eTFs are capable of upregulating SCN1A expression by at least 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold or more compared to a control, or by at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 100%, 125%, 150%, 200%, 250%, 300%, 400%, or 500% or more compared to a control. In exemplary embodiments, such eTFs have at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% overall sequence identity to one or more human proteins. In certain embodiments, such eTFs exhibit reduced immunogenicity compared to eTFs having a lower overall percent sequence identity to one or more human proteins. In various embodiments, the reduction in immunogenicity may be measured using an elispot assay, an immunoassay, or an in silico method.

[0172] In certain embodiments, an eTF disclosed herein that upregulates SCN1A comprises any one of the following: (i) a sequence comprising SEQ ID NO: 440; (ii) a sequence comprising SEQ ID NO: 441; (iii) a sequence comprising at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of (i) or (ii); or (iv) a functional fragment or variant of any of the sequences of (i), (ii), or (iii). In exemplary embodiments, such an eTF comprises SEQ ID NO: 135 and binds to a target site having SEQ ID NO: 256. In exemplary embodiments, such eTFs are capable of upregulating SCN1A expression by at least 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold or more compared to a control, or by at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 100%, 125%, 150%, 200%, 250%, 300%, 400%, or 500% or more compared to a control. In exemplary embodiments, such eTFs have at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% overall sequence identity to one or more human proteins. In certain embodiments, such eTFs exhibit reduced immunogenicity compared to eTFs having a lower overall percent sequence identity to one or more human proteins. In various embodiments, the reduction in immunogenicity may be measured using an elispot assay, an immunoassay, or an in silico method.

[0173] In certain embodiments, an eTF disclosed herein that upregulates SCN1A comprises any one of the following: (i) a sequence comprising SEQ ID NO: 325; (ii) a sequence comprising SEQ ID NO: 435; (iii) a sequence comprising at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of (i) or (ii); or (iv) a functional fragment or variant of any of the sequences of (i), (ii), or (iii). In exemplary embodiments, such an eTF comprises SEQ ID NO: 371 and binds to a target site having SEQ ID NO: 264. In exemplary embodiments, such eTFs are capable of upregulating SCN1A expression by at least 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold or more compared to a control, or by at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 100%, 125%, 150%, 200%, 250%, 300%, 400%, or 500% or more compared to a control. In exemplary embodiments, such eTFs have at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% overall sequence identity to one or more human proteins. In certain embodiments, such eTFs exhibit reduced immunogenicity compared to eTFs having a lower overall percent sequence identity to one or more human proteins. In various embodiments, the reduction in immunogenicity may be measured using an elispot assay, an immunoassay, or an in silico method.

[0174] In certain embodiments, an eTF disclosed herein that upregulates SCN1A comprises a DBD that includes a gRNA / Cas complex, wherein the gRNA comprises a targeting sequence comprising any one of SEQ ID NOs: 105-111, 113, 195-211, 224-238, or 240-251. The targeting sequence of the gRNA is attached to the 5' end of a scaffold sequence having the following sequence: 5'-GTTTTAGTACTCTGGAAACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCTCGTCAACTTGTTGGCGAGA-3' (SEQ ID NO: 102). In an exemplary embodiment, the Cas protein is a nuclease-inactivated Cas9 protein. In certain embodiments, such eTFs further comprise one or more TADs conjugated to the Cas protein, wherein the TADs are selected from VP64, VPR, CITED2, or CITED4. In one embodiment, such eTFs comprise a VPR TAD domain conjugated to the C-terminus of the Cas protein. In certain embodiments, such eTFs comprise a CITED2 TAD conjugated to the N-terminus, C-terminus, or N- and C-terminus of a Cas protein. In certain embodiments, such eTFs comprise a CITED4 TAD conjugated to the N-terminus, C-terminus, or N- and C-terminus of a Cas protein. In exemplary embodiments, such eTFs are capable of upregulating SCN1A expression by at least 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, or more, compared to a control. eTF upregulates progranulin (GRN)

[0175] In another aspect, the present application provides an eTF that can upregulate the expression from the progranulin (GRN) gene and increase the expression of GRN protein. Progranulin (GRN or PGRN), also known as granulin precursor, is a secreted cysteine-rich glycoprotein with growth factor-like properties. GRN is located on chromosome 17q21.31 (NCBI reference sequence: NC_000017.11). Granulin plays a role in angiogenesis, wound repair, cell proliferation and inflammation. Mutations in the progranulin gene and / or defects in secreted progranulin are associated with various neurodegenerative and metabolic diseases, such as frontotemporal lobar degeneration; frontotemporal degeneration or frontotemporal dementia, an early-onset neurodegenerative disease associated with partial progranulin deficiency; progressive non-fluent aphasia; semantic dementia; Parkinson's disease; Alzheimer's disease; and neuronal ceroid lipofuscinosis with complete progranulin deficiency.Mutations and / or defects in progranulin are also associated with atherosclerosis, a progressive disease characterized by inflammation and thickening of arterial walls due to lipid accumulation and cell proliferation.Therefore, the eTF provided herein that increases the expression of GRN can be used to treat various diseases or disorders associated with mutations in GRN.

[0176] In certain embodiments, the eTFs disclosed herein that upregulate GRNs are at least 9 bp, 12 bp, 15 bp, 18 bp, 21 bp, 24 bp, 27 bp, 30 bp, 33 bp, or 36 bp in size; longer than 9 bp, 12 bp, 15 bp, 18 bp, 21 bp, 24 bp, 27 bp, or 30 bp; or longer than 9-33 bp, 9-30 bp, 9-27 bp, 9-24 bp, 9-21 bp, 9-18 bp, 9-15 bp, 9-12 bp, 12-33 bp, 12-30 bp, 1 The eTFs disclosed herein recognize a target binding site that is 2-27 bp, 12-24 bp, 12-21 bp, 12-18 bp, 12-15 bp, 15-33 bp, 15-30 bp, 15-27 bp, 15-24 bp, 15-21 bp, 15-18 bp, 18-33 bp, 18-30 bp, 18-27 bp, 18-24 bp, 18-21 bp, 21-33 bp, 21-30 bp, 21-27 bp, 21-24 bp, 24-33 bp, 24-30 bp, 24-27 bp, 27-33 bp, 27-30 bp, or 30-33 bp. In exemplary embodiments, the eTFs disclosed herein that upregulate GRN recognize a target binding site that is 18-27 bp, 18 bp, or 27 bp.

[0177] In certain embodiments, the eTFs disclosed herein that upregulate GRN recognize a target binding site located on chromosome 17. In certain embodiments, the eTFs disclosed herein that upregulate GRN recognize a target binding site located on chromosome 17 within 110 kb, 100 kb, 90 kb, 80 kb, 70 kb, 60 kb, 50 kb, 40 kb, 30 kb, 20 kb, 10 kb, 5 kb, 4 kb, 3 kb, 2 kb, or 1 kb upstream or downstream of the TSS of GRN. In exemplary embodiments, such target binding sites are 18-27 bp, 18 bp, or 27 bp.

[0178] In certain embodiments, the eTFs disclosed herein that upregulate GRN recognize a target binding site located on chromosome 17 within positions 44,344,963-44,345,178 (with reference to GRCh38.p12). In exemplary embodiments, such target binding site is 18-27 bp, 18 bp, or 27 bp.

[0179] In certain embodiments, an eTF disclosed herein that upregulates GRN (i) recognizes a target binding site that is 18-27 bp, 18 bp, or 27 bp, and (ii) binds to a genomic region that is within at least 1 kb, 750 bp, 500 bp, 400 bp, 300 bp, 200 bp, 100 bp, or 50 bp of a genomic location having the sequence of any one of SEQ ID NOs: 38, 113, or 330-336. In certain embodiments, the target binding site is capable of producing at least a 1.2-fold, 2-fold, 5-fold, 15-fold, 20-fold, or 25-fold increase in expression of GRN when bound by an eTF disclosed herein.

[0180] In certain embodiments, an eTF disclosed herein that upregulates GRN (i) recognizes a target binding site that is 18-27 bp, 18 bp, or 27 bp, and (ii) binds to a genomic region that at least partially overlaps with a genomic location having the sequence of any one of SEQ ID NOs: 38, 113, or 330-336. In certain embodiments, the target binding site is capable of producing at least a 1.2-fold, 2-fold, 5-fold, 15-fold, 20-fold, or 25-fold increase in SCN1A expression when bound by an eTF disclosed herein.

[0181] In certain embodiments, an eTF disclosed herein that upregulates GRN recognizes a target binding site having any one of the following sequences: SEQ ID NOs: 38, 113, 330-336. In certain embodiments, the target binding site is capable of producing at least a 1.2-fold, 2-fold, 5-fold, 15-fold, 20-fold, or 25-fold increase in SCN1A expression when bound by an eTF disclosed herein.

[0182] In certain embodiments, the eTFs disclosed herein that upregulate GRN result in at least a 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 50-fold, 100-fold or greater upregulation of GRN expression (GRN RNA and / or protein) in cells or in vivo, or a 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 100%, 125%, 150%, 200%, 250%, 300%, 400%, or 500% or greater upregulation of GRN expression compared to a control (e.g., no eTF or an eTF that does not recognize the target site). In various embodiments, upregulation of GRN expression can be detected using PCR, Western blot, or immunoassay.

[0183] In certain embodiments, the eTF disclosed herein that upregulates GRN has minimal off-target effects, such as off-target effects associated with non-specific binding, such as the modulation of the expression of off-target genes or genes other than GRN.In one embodiment, the eTF disclosed herein that upregulates GRN specifically upregulates GRN at least 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold or 50-fold greater than the expression caused by eTF for one or more off-target genes compared to a control.In exemplary embodiments, the eTF disclosed herein that upregulates GRN specifically upregulates the transcription from the GRN gene compared to a control at least 5-fold, 10-fold, 15-fold, 20-fold or greater than the transcription of the 10, 20, 30, 40 or 50 nearest neighboring genes (for example, the 10, 20, 30, 40 or 50 nearest genes located in the coding sequence of GRN on chromosome 17) caused by eTF compared to a control. In various embodiments, upregulation of transcription from the GRN gene can be determined using PCR methods.

[0184] In certain embodiments, the eTFs disclosed herein that upregulate GRN have a high percent identity to one or more human proteins (as described further below). In certain embodiments, such eTFs have at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% overall sequence identity to one or more human proteins. In certain embodiments, such eTFs exhibit reduced immunogenicity compared to eTFs with a lower overall percent sequence identity to one or more human proteins. In various embodiments, the reduced immunogenicity can be measured using an elispot assay, immunoassay, or in silico method. In certain embodiments, such eTFs can include a DBD derived from human EGR1 or EGR3 and a TAD derived from human EGR1, EGR3, CITED2, or CITED4.

[0185] In certain embodiments, an eTF disclosed herein that upregulates GRN comprises a DNA-binding domain having one or more zinc finger domains that comprise a recognition helix comprising any one of SEQ ID NOs: 141-164. In certain embodiments, an eTF disclosed herein that upregulates GRN comprises a DNA-binding domain having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 zinc finger domains, each zinc finger domain independently comprising a recognition helix comprising any one of SEQ ID NOs: 141-164. In certain embodiments, an eTF disclosed herein that upregulates GRN comprises a DNA-binding domain having six zinc finger domains, each zinc finger domain independently comprising a recognition helix comprising any one of SEQ ID NOs: 141-164. In certain embodiments, an eTF disclosed herein that upregulates GRN comprises a DNA-binding domain having nine zinc finger domains, each zinc finger domain independently comprising a recognition helix comprising any one of SEQ ID NOs: 141-164. In exemplary embodiments, such an eTF comprises a DNA-binding domain having SEQ ID NO: 421, wherein each X is independently selected from any one of SEQ ID NOs: 141-164, and n is 6 or 9.

[0186] In certain embodiments, the eTF disclosed herein that upregulates GRN comprises a DNA-binding domain having any one of the following: (i) a sequence comprising RNDTLTE x DPGALVR x TSGELVR x RSDNLVR x TSGELVR x TKNSLTE (SEQ ID NO: 171); (ii) a sequence having at least 89%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 171; or (ii) a functional fragment of (i) or (ii). In certain embodiments, such an eTF further comprises one or more TADs selected from VP64, VPR, CITED2, or CITED4. In one embodiment, such an eTF comprises a VPR TAD domain conjugated to the C-terminus of the DBD. In certain embodiments, such an eTF comprises a CITED2 TAD conjugated to the N-terminus, C-terminus, or N- and C-terminus of the DBD. In certain embodiments, such eTFs comprise a CITED4 TAD conjugated to the N-terminus, C-terminus, or N- and C-terminus of the DBD. In certain embodiments, such eTFs are capable of binding to a target site having SEQ ID NO: 331 and are capable of upregulating expression of GRN by at least 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, or more compared to a control, or by at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 100%, 125%, 150%, 200%, 250%, 300%, 400%, or 500% or more compared to a control.

[0187] In certain embodiments, an eTF disclosed herein that upregulates GRN comprises a DNA-binding domain having any one of the following: (i) a sequence comprising RSDNLVR x DPGHLVR x RSDHLTT x RSDELVR x RSDKLVR x TTGNLTV (SEQ ID NO: 412); (ii) a sequence having at least 89%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 412; or (ii) a functional fragment of (i) or (ii). In certain embodiments, such an eTF further comprises one or more TADs selected from VP64, VPR, CITED2, or CITED4. In one embodiment, such an eTF comprises a VPR TAD domain conjugated to the C-terminus of the DBD. In certain embodiments, such an eTF comprises a CITED2 TAD conjugated to the N-terminus, C-terminus, or N- and C-terminus of the DBD. In certain embodiments, such eTFs comprise a CITED4 TAD conjugated to the N-terminus, C-terminus, or N- and C-terminus of the DBD. In certain embodiments, such eTFs are capable of binding to a target site having SEQ ID NO: 332 and are capable of upregulating the expression of GRN by at least 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, or more compared to a control, or by at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 100%, 125%, 150%, 200%, 250%, 300%, 400%, or 500% or more compared to a control.

[0188] In certain embodiments, the eTF disclosed herein that upregulates GRN comprises a DNA-binding domain having any one of the following: (i) a sequence comprising RSDHLTT x RSDELVR x RSDKLVR x TTGNLTV x QLAHLRA x TKNSLTE (SEQ ID NO: 413); (ii) a sequence having at least 89%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 413; or (ii) a functional fragment of (i) or (ii). In certain embodiments, such an eTF further comprises one or more TADs selected from VP64, VPR, CITED2, or CITED4. In one embodiment, such an eTF comprises a VPR TAD domain conjugated to the C-terminus of the DBD. In certain embodiments, such an eTF comprises a CITED2 TAD conjugated to the N-terminus, C-terminus, or N- and C-terminus of the DBD. In certain embodiments, such eTFs comprise a CITED4 TAD conjugated to the N-terminus, C-terminus, or N- and C-terminus of the DBD. In certain embodiments, such eTFs are capable of binding to a target site having SEQ ID NO: 333 and are capable of upregulating expression of GRN by at least 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, or more compared to a control, or by at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 100%, 125%, 150%, 200%, 250%, 300%, 400%, or 500% or more compared to a control.

[0189] In certain embodiments, the eTF disclosed herein that upregulates GRN comprises a DNA-binding domain having any one of the following: (i) a sequence comprising SPADLTR x DSGNLRV x QLAHLRA x QRANLRA x REDNLHT x RSDNLVR (SEQ ID NO: 414); (ii) a sequence having at least 89%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 414; or (ii) a functional fragment of (i) or (ii). In certain embodiments, such an eTF further comprises one or more TADs selected from VP64, VPR, CITED2, or CITED4. In one embodiment, such an eTF comprises a VPR TAD domain conjugated to the C-terminus of the DBD. In certain embodiments, such an eTF comprises a CITED2 TAD conjugated to the N-terminus, C-terminus, or N- and C-terminus of the DBD. In certain embodiments, such an eTF comprises a CITED4 TAD conjugated to the N-terminus, C-terminus, or N- and C-terminus of the DBD. In certain embodiments, such an eTF is capable of binding to a target site having SEQ ID NO: 38 and is capable of upregulating expression of GRN by at least 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold or more compared to a control, or by at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 100%, 125%, 150%, 200%, 250%, 300%, 400%, or 500% or more compared to a control.

[0190] In certain embodiments, an eTF disclosed herein that upregulates GRN comprises a DNA-binding domain having any one of the following: (i) a sequence comprising TSHSLTE x HKNALQN x ERSHLRE x SKKALTE x QRANLRA x RKDNLKN x QSSNLVR x QSSSLVR x QAGHLAS (SEQ ID NO: 415); (ii) a sequence having at least 89%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 415; or (ii) a functional fragment of (i) or (ii). In certain embodiments, such an eTF further comprises one or more TADs selected from VP64, VPR, CITED2, or CITED4. In one embodiment, such an eTF comprises a VPR TAD domain conjugated to the C-terminus of the DBD. In certain embodiments, such an eTF comprises a CITED2 TAD domain conjugated to the N-terminus, C-terminus, or N- and C-terminus of the DBD. In certain embodiments, the eTF comprises a CITED4 TAD conjugated to the N-terminus, C-terminus, or N-terminus and C-terminus of the DBD. In certain embodiments, the eTF can bind to a target site having SEQ ID NO: 334 and can upregulate the expression of GRN by at least 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold or more compared to a control, or by at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 100%, 125%, 150%, 200%, 250%, 300%, 400%, or 500% or more compared to a control.

[0191] In certain embodiments, the eTF disclosed herein that upregulates GRN comprises a DNA-binding domain having any one of the following: (i) a sequence comprising QSGDLRR x SPADLTR x DSGNLRV x QLAHLRA x QRANLRA x REDNLHT x RSDNLVR (SEQ ID NO: 416); (ii) a sequence having at least 89%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 416; or (ii) a functional fragment of (i) or (ii). In certain embodiments, such an eTF further comprises one or more TADs selected from VP64, VPR, CITED2, or CITED4. In one embodiment, such an eTF comprises a VPR TAD domain conjugated to the C-terminus of the DBD. In certain embodiments, such an eTF comprises a CITED2 TAD conjugated to the N-terminus, C-terminus, or N- and C-terminus of the DBD. In certain embodiments, such eTFs comprise a CITED4 TAD conjugated to the N-terminus, C-terminus, or N- and C-terminus of the DBD. In certain embodiments, such eTFs are capable of binding to a target site having SEQ ID NO: 335 and are capable of upregulating the expression of GRN by at least 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold or more compared to a control, or by at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 100%, 125%, 150%, 200%, 250%, 300%, 400%, or 500% or more compared to a control.

[0192] In certain embodiments, the eTFs disclosed herein that upregulate GRNs comprise any one of the following: (i) a sequence comprising any one of SEQ ID NOs: 10, 16, 59-60, 63-64, 72-73, 76-77, or 337-350; (ii) a sequence comprising any one of SEQ ID NOs: 26, 32, 86-89, 93, 165-171, 377-390, or 412-416; (iii) a sequence comprising at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the sequences in (i) or (ii); or (iv) a functional fragment or variant of any of the sequences in (i), (ii), or (iii). In exemplary embodiments, such eTFs are capable of upregulating GRN expression by at least 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold or more compared to a control, or by at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 100%, 125%, 150%, 200%, 250%, 300%, 400%, or 500% or more compared to a control.

[0193] In certain embodiments, an eTF disclosed herein that upregulates GRN comprises a DBD that includes a gRNA / Cas complex, wherein the gRNA comprises a targeting sequence comprising SEQ ID NO: 113. The targeting sequence of the gRNA is attached to the 5' end of a scaffold sequence having the following sequence: 5'-GTTTTAGTACTCTGGAAACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCTCGTCAACTTGTTGGCGAGA-3' (SEQ ID NO: 102). In exemplary embodiments, the Cas protein is a nuclease-inactivated Cas9 protein. In certain embodiments, such an eTF further comprises one or more TADs conjugated to the Cas protein, wherein the TADs are selected from VP64, VPR, CITED2, or CITED4. In one embodiment, such an eTF comprises a VPR TAD domain conjugated to the C-terminus of the Cas protein. In certain embodiments, such an eTF comprises a CITED2 TAD conjugated to the N-terminus, C-terminus, or N- and C-terminus of the Cas protein. In certain embodiments, such eTFs comprise a CITED4 TAD conjugated to the N-terminus, C-terminus, or N- and C-terminus of a Cas protein. In exemplary embodiments, such eTFs are capable of upregulating GRN expression by at least 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, or more compared to a control, or by at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 100%, 125%, 150%, 200%, 250%, 300%, 400%, or 500% or more compared to a control. eTF highly homologous to human proteins

[0194] In another aspect, the present application provides eTFs with high percent sequence identity to one or more human proteins that can be designed to bind to genomic target sites and modulate (up-regulate or down-regulate) the expression of any gene of interest, including, for example, SCN1A or GRN. Such eTFs have little to no immunogenicity when administered to a subject, or have reduced immunogenicity compared to eTFs with lower percent identity to the human protein sequence.

[0195] In certain embodiments, the eTFs provided herein have at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to one or more human proteins. When the eTFs provided herein contain one DBD and one TAD derived from the same protein, percent identity to the human protein can be determined by dividing the total number of amino acid residues in the eTF that match the human protein from which it is derived (e.g., EGR1 or EGR3) by the total number of amino acid residues in the eTF. If a provided eTF contains one DBD from one human protein and one TAD derived from a different human protein, the percent identity to human can be determined by calculating the percent identity to human of each domain separately and summing the two together, for example, (i) calculating the total number of amino acid residues in the DBD that match the human protein from which it is derived (e.g., EGR1 or EGR3) divided by the total number of amino acid residues in the eTF; (ii) calculating the total number of amino acid residues in the TAD that match the human protein from which it is derived (e.g., CITED2 or CITED4) divided by the total number of amino acid residues in the eTF; and (iii) summing the sums of (i) and (ii). In such embodiments, the domains are divided as follows: a first domain extending from the N-terminus of the eTF to the start of the coding sequence for the second domain, and a second domain extending from the start of the coding sequence for the second domain to the C-terminus of the eTF (e.g., for an eTF with the configuration NLS-DBD-linker-NLS-TAD, the first domain is the NLS-DBD-linker and the second domain is the NLS-TAD).When an eTF provided herein comprises one DBD from a human protein and two TADs derived from one or more different human proteins, the percent identity to human can be determined by calculating the percent identity to human of each domain separately and summing all three together, for example, (i) calculating the total number of amino acid residues in the DBD that match the human protein from which it is derived (e.g., EGR1 or EGR3) divided by the total number of amino acid residues in the eTF; (ii) calculating the total number of amino acid residues in a first TAD that match the human protein from which it is derived (e.g., CITED2 or CITED4) divided by the total number of amino acid residues in the eTF; (iii) calculating the total number of amino acid residues in a second TAD that match the human protein from which it is derived (e.g., CITED2 or CITED4) divided by the total number of amino acid residues in the eTF; and (iv) summing the totals of (i), (ii), and (iii). In such embodiments, these domains are divided as follows: a first domain extending from the N-terminus of the eTF to the start of the coding sequence for the second domain, a second domain extending from the start of the coding sequence for the second domain to the start of the coding sequence for the third domain, and a third domain extending from the start of the coding sequence for the third domain to the C-terminus of the eTF (e.g., for an eTF with the configuration NLS-TAD1-linker-NLS-DBD-linker-NLS-TAD2, the first domain is NLS-TAD1-linker, the second domain is NLS-DBD-linker, and the third domain is NLS-TAD2). Percent identity to one or more human proteins described in this section can be determined using the percent identity output obtained using standard protein BLAST tools available from NCBI (e.g., the blastp suite alignment tool using the blastp (protein → protein) algorithm with default parameters), available on the World Wide Web from the NCBI website at blast.ncbi.nlm.nih.gov / .

[0196] In certain embodiments, the eTFs provided herein have the advantage of eliciting little, minimal, or no harmful immune responses in human subjects due to their high degree of sequence identity with naturally occurring human proteins. In certain embodiments, the eTFs provided herein exhibit reduced immunogenicity, for example, at least 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50-fold or greater reduction in immunogenicity, compared to the immunogenicity observed with eTFs that have a lower percent identity with one or more human proteins, for example, eTFs that have less than 50%, 55%, 65%, or 70% sequence identity with one or more human proteins. In some cases, the reduction in immunogenicity can be measured using an elispot assay, immunoassay, or in silico method. Gene therapies with low or minimal immunogenicity have several advantages, including improved patient tolerance, reduced dosages required to achieve therapeutic efficacy, prolonged therapeutic efficacy after a single administration, the ability to administer multiple or multiple doses if needed, sustained therapeutic efficacy for a longer period per administration, increased stability, and / or increased efficacy of the gene therapy.

[0197] In certain embodiments, eTFs provided herein that have a high percent sequence identity to one or more human proteins comprise a DBD and a TAD derived from one or more naturally occurring human proteins. In certain embodiments, such eTFs can comprise a DBD derived from any naturally occurring human protein that contains a DBD. In exemplary embodiments, eTFs provided herein that have a high percent sequence identity to one or more human proteins comprise a DBD derived from a naturally occurring zinc finger protein, such as any one of the zinc finger proteins listed in Table 7. In certain embodiments, eTFs provided herein that have a high percent sequence identity to one or more human proteins comprise a DBD derived from a human EGR protein, such as EGR1, EGR2, EGR3, or EGR4. In exemplary embodiments, eTFs provided herein that have a high percent sequence identity to one or more human proteins comprise a DBD derived from human EGR1 or EGR3. In various embodiments, eTFs provided herein with high percent sequence identity to one or more human proteins comprise a DBD derived from a human zinc finger protein, where minimal amino acid changes (e.g., 1, 2, 3, 4, 5, 6, 7, or 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 2-3, 2-4, 2-5, 2-6, 2-7, 3-4, 3-5, 36, or 3-7 amino acid changes) have been made in one or more zinc finger domains of the DBD to alter the binding specificity of the DBD to recognize a target binding site of interest. Such sequence modifications are preferably made in the recognition helices of the zinc finger domains of the DBD, while the remainder of the human zinc finger DBD or protein (including the TAD) remains unmodified to preserve as high a sequence identity as possible to naturally occurring human proteins.

[0198] In certain embodiments, the eTFs provided herein that share a high percent sequence identity with one or more human proteins comprise one or more transcription modulation domains (e.g., TADs or TRDs) derived from a human protein conjugated to a DBD derived from the human protein. In various embodiments, the transcription modulation domain can be derived from any naturally occurring human protein that has a domain capable of recruiting one or more protein factors (e.g., RNA polymerase II, coactivator proteins, or corepressor proteins) that can modulate transcription from a gene of interest when the eTF binds to a target site via the DBD, or one or more protein factors that can modulate the level of gene expression from the gene of interest. In exemplary embodiments, the TAD is derived from a human EGR protein, such as human EGR1, EGR2, EGR3, or EGR4, or a human cited protein, such as human CITED2 or CITED4 protein. In exemplary embodiments, the eTFs provided herein that share a high percent sequence identity with one or more human proteins comprise a TAD derived from human EGR1 or EGR3 protein. In another exemplary embodiment, an eTF provided herein that has a high percent sequence identity to one or more human proteins comprises a TAD derived from a human CITED2 or CITED4 protein.

[0199] In one embodiment, an eTF provided herein that has a high percent sequence identity to one or more human proteins may comprise one human DBD (hDBD) and one human TAD (hTAD) (e.g., hTAD-hDBD or hDBD-hTAD), where the hDBD and hTAD may be derived from the same human protein or different human proteins. In another embodiment, an eTF provided herein having a high percent sequence identity to one or more human proteins may comprise one hDBD and two hTADs, wherein the hDBD and hTADs are from the same human protein, the hDBD is from a first human protein and both hTADs are from a second human protein, the hDBD and one hTAD are from a first human protein and the second hTAD is from a second human protein, or the hDBD is from a first human protein and one hTAD is from a second human protein and the second hTAD is from a third human protein (e.g., hTAD1-hDBD-hTAD1 or hTAD1-hDBD-hTAD2).

[0200] In exemplary embodiments, eTFs provided herein that have high percent sequence identity to one or more human proteins include any one of the following configurations: (i) one hDBD and one hTAD, both derived from human EGR1; (ii) one hDBD and one hTAD, both derived from human EGR3; (iii) one hDBD from human EGR1 and one hTAD from CITED2 (e.g., hEGR1 DBD-hCITED2 TAD or hCITED2 TAD-hEGR1 DBD); (iv) one hDBD from human EGR1 and one hTAD from human CITED4 (e.g., hEGR1 DBD-hCITED4 TAD or hCITED4 TAD-hEGR1 DBD); (v) one hDBD from human EGR3 and one hTAD from CITED2 (e.g., hEGR3 DBD-hCITED2 TAD or hCITED2 TAD-hEGR3 (vi) one hDBD derived from human EGR3 and one hTAD derived from human CITED4 (e.g., hEGR3 DBD-hCITED4 TAD or hCITED4 TAD-hEGR3 DBD); (vii) one hDBD derived from human EGR1 and two hTADs derived from CITED2 (e.g., hCITED2 TAD-hEGR1 DBD-hCITED2 TAD); (viii) one hDBD derived from human EGR1 and two hTADs derived from human CITED4 (e.g., hCITED4 TAD-hEGR1 DBD-hCITED4 TAD); (ix) one hDBD derived from human EGR3 and two hTADs derived from human CITED2 (e.g., hCITED2 TAD-hEGR3 DBD-hCITED2 TAD); (x) one hDBD derived from human EGR3 and two hTADs derived from human CITED4 (e.g., hCITED4 TAD-hEGR3 DBD-hCITED4 TAD);(xi) one hDBD derived from human EGR1, a first hTAD derived from human CITED2, and a second hTAD derived from human CITED4 (e.g., hCITED2 TAD-hEGR1 DBD-hCITED4 TAD or hCITED4 TAD-hEGR1 DBD-hCITED2 TAD); or (xii) one hDBD derived from human EGR3, a first hTAD derived from human CITED2, and a second hTAD derived from human CITED4 (e.g., hCITED2 TAD-hEGR3 DBD-hCITED4 TAD or hCITED4 TAD-hEGR3 DBD-hCITED2 TAD).

[0201] In another embodiment, an eTF provided herein having a high percent sequence identity to one or more human proteins may comprise one hDBD and one hTRD (e.g., hTRD-hDBD or hDBD-hTRD), where the hDBD and hTRD may be derived from the same protein or different proteins. In another embodiment, an eTF provided herein having a high percent sequence identity to one or more human proteins may comprise one hDBD and two hTRDs, wherein the hDBD and hTRD are from the same human protein, the hDBD is from a first human protein and both hTRDs are from a second human protein, the hDBD and one hTRD are from a first human protein and the second hTRD is from a second human protein, or the hDBD is from a first human protein and one hTRD is from a second human protein and the second hTRD is from a third human protein (e.g., hTRD1-hDBD-hTRD1 or hTRD1-hDBD-hTRD2).

[0202] In certain embodiments, eTFs provided herein that have high percent sequence identity to one or more human proteins include any one of the following: (i) a sequence comprising any one of SEQ ID NOs: 6-10, 13-16, 57-64, 67-77, 295-299, 313-325, 345-350, 364, 436; (ii) a sequence comprising any one of SEQ ID NOs: 22-26, 29-32, 84-93, 385-390, 406-410, 412-414, 416-418, 418-420, 418-422, 418-424, 418-426, 418-428, 418-429, 420-430, 420-431, 420-432, 420-433, 420-434, 420-435, 420-436, 420-437, 420-438, 420-439, 430-431, 431-432, 432-433, 432-434, 432-436 ... (iii) a sequence comprising at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any of the sequences in (i) or (ii); or (iv) a functional fragment or variant of any of the sequences in (i), (ii) or (iii). In exemplary embodiments, such eTFs are capable of upregulating SCN1A or GRN expression by at least 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold or more compared to a control, or by at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 100%, 125%, 150%, 200%, 250%, 300%, 400%, or 500% or more compared to a control. In exemplary embodiments, such eTFs have at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% overall sequence identity to one or more human proteins. In certain embodiments, such eTFs exhibit reduced immunogenicity compared to eTFs having a lower overall percent sequence identity to one or more human proteins. In various embodiments, the reduction in immunogenicity may be measured using an elispot assay, an immunoassay, or an in silico method.

[0203] In certain embodiments, eTFs provided herein that have a high percent sequence identity to one or more human proteins may further comprise one or more amino acid sequences or domains, such as a nuclear localization signal or linker, in addition to the DBD and TAD domains. Furthermore, polynucleotides encoding eTFs provided herein that have a high percent sequence identity to one or more human proteins may further comprise one or more nucleic acid sequences, such as a promoter, enhancer, polyA tail, etc., in addition to the coding sequence of the eTF. In such embodiments, one or more of the additional amino acid and / or nucleic acid sequences is preferably a human sequence, is derived from a human sequence, or has at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a human protein. Polynucleotides

[0204] In another aspect, the application provides a polynucleotide encoding any of the eTFs disclosed herein.

[0205] In certain embodiments, the present application provides a polynucleotide comprising any one of the following: (i) a nucleic acid sequence encoding an eTF comprising any one of SEQ ID NOs: 6-10, 13-16, 57-64, 67-77, 268-282, 305-325, 337-350, 364, 295-299, or 365-366, or a variant or functional fragment thereof; (ii) a nucleic acid sequence encoding a functional fragment of an eTF having any one of SEQ ID NOs: 6-10, 13-16, 57-64, 67-77, 268-282, 305-325, 337-350, 364, 295-299, or 365-366. or (iii) a nucleic acid encoding an eTF having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an eTF having any one of SEQ ID NOs: 6-10, 13-16, 57-64, 67-77, 112, 268-282, 305-325, 337-350, 364, 295-299, 365-366, or a variant or functional fragment thereof, wherein the eTF is capable of upregulating SCN1A or GRN.

[0206] In certain embodiments, the application provides a polynucleotide comprising any one of the following: (i) a nucleic acid sequence encoding a DBD comprising any one of SEQ ID NOs: 22-26, 29-32, 84-93, 131-135, 165-171, 377-409, or 423-435, or a variant or functional fragment thereof; (ii) a nucleic acid encoding a functional fragment of a DBD having any one of SEQ ID NOs: 22-26, 29-32, 84-93, 131-135, 165-171, 377-409, or 423-435; or (iii) a nucleic acid encoding a functional fragment of a DBD having any one of SEQ ID NOs: 22-26, 29-32, 84-93, 112, 131-135, 165-171, 377-409, or 423-435. 95%, 96%, 97%, 98%, 99% or more sequence identity to a DBD having any one of SEQ ID NOs: 22-26, 29-32, 84-93, 112, 131-135, 165-171, 377-409 or 432-435, or a variant or functional fragment thereof, wherein the eTF is capable of binding to a target site bound by any one of SEQ ID NOs: 22-26, 29-32, 84-93, 112, 131-135, 165-171, 377-409 or 432-435.

[0207] In certain embodiments, the present application provides a polynucleotide encoding an eTF that upregulates endogenous SCN1A, wherein the polynucleotide comprises any one of the following: (i) a nucleic acid sequence encoding an eTF comprising any one of SEQ ID NOs: 6-9, 13-15, 57-58, 61-62, 67-71, 74-75, 268-282, 295-299, 305-325, or 364-366; (ii) a nucleic acid sequence encoding an eTF comprising any one of SEQ ID NOs: 6-9, 13-15, 57-58, 61-62, 67-71, 74-75, 268-282, 295-299, 305-325, or 364-366. or (iii) a nucleic acid encoding an eTF having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an eTF having any one of SEQ ID NOs: 6-9, 13-15, 57-58, 61-62, 67-71, 74-75, 268-282, 295-299, 305-325 or 364-366, wherein the eTF is capable of upregulating SCN1A.

[0208] In certain embodiments, the present application provides a polynucleotide encoding a DBD that binds to a genomic target site capable of upregulating endogenous SCN1A when bound by an eTF disclosed herein, wherein the polynucleotide comprises any one of the following: (i) a nucleic acid sequence encoding a DBD comprising any one of SEQ ID NOs: 22-25, 29-31, 84-85, 88, 90-92, 131-135, 371-372, 376, 391-409, or 423-435; (ii) a nucleic acid encoding a functional fragment of a DBD having any one of SEQ ID NOs: 22-25, 29-31, 84-85, 88, 90-92, 131-135, 371-372, 376, 391-409, or 423-435; or (iii) a nucleic acid encoding an eTF having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a DBD having any one of SEQ ID NOs: 22-25, 29-31, 84-85, 88, 90-92, 131-135, 371-372, 376, 391-409 or 423-435, wherein the DBD is capable of binding to a target site to which any one of SEQ ID NOs: 22-25, 29-31, 84-85, 88, 90-92, 131-135, 371-372, 376, 391-409 or 423-435 binds.

[0209] In certain embodiments, the present application provides a polynucleotide encoding a DBD that binds to a genomic target site capable of upregulating endogenous SCN1A when bound by an eTF disclosed herein, wherein the polynucleotide comprises any one of the following: (i) a nucleic acid sequence encoding a DBD comprising any one of SEQ ID NOs: 135, 371, 372, or 376; (ii) a nucleic acid encoding a functional fragment of a DBD having any one of SEQ ID NOs: 135, 371, 372, or 376; or (iii) a nucleic acid sequence encoding a functional fragment of a DBD having any one of SEQ ID NOs: 135, 371, 372, or 376. 90-92, 131-135, 371-372, 376 or 391-409.

[0210] In certain embodiments, the present application provides a polynucleotide encoding an eTF that upregulates an endogenous GRN, wherein the polynucleotide comprises any one of the following: (i) a nucleic acid sequence encoding an eTF comprising any one of SEQ ID NOs: 10, 16, 59-60, 63, 64, 72-73, 76, 77, or 337-350; (ii) a nucleic acid sequence encoding a functional fragment of an eTF having any one of SEQ ID NOs: 10, 16, 59-60, 63, 64, 72-73, 76, 77, or 337-350. or (iii) a nucleic acid encoding an eTF having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an eTF having any one of SEQ ID NOs: 10, 16, 59-60, 63, 64, 72-73, 76, 77, or 337-350, wherein the eTF is capable of upregulating a GRN.

[0211] In certain embodiments, the present application provides a polynucleotide encoding a DBD that binds to a genomic target site capable of upregulating an endogenous GRN when bound by an eTF disclosed herein, wherein the polynucleotide comprises any one of the following: (i) a nucleic acid sequence encoding a DBD comprising any one of SEQ ID NOs: 26, 32, 86-89, 93, 165-171, or 377-390; (ii) a nucleic acid encoding a functional fragment of a DBD having any one of SEQ ID NOs: 26, 32, 86-89, 93, 165-171, or 377-390. or (iii) a nucleic acid encoding an eTF having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a DBD having any one of SEQ ID NOs: 26, 32, 86-89, 93, 165-171 or 377-390, wherein the DBD is capable of binding to a target site bound by any one of SEQ ID NOs: 26, 32, 86-89, 93, 165-171 or 377-390.

[0212] In certain embodiments, the present application provides a polynucleotide encoding an eTF capable of regulating endogenous SCN1A, wherein the polynucleotide comprises any one of the following: (i) a nucleic acid sequence having any of SEQ ID NOs: 353-363 or 454; (ii) a nucleic acid sequence having any one of SEQ ID NOs: 442-453; (iii) a nucleic acid having a functional fragment of any one of the sequences of (i) or (ii); or (iv) a nucleic acid having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of the sequences of (i), (ii), or (iii), wherein the polynucleotide encodes an eTF capable of upregulating SCN1A.

[0213] In certain embodiments, the present application provides a polynucleotide encoding an eTF capable of regulating endogenous SCN1A, wherein the polynucleotide comprises any one of the following: (i) a nucleic acid sequence having SEQ ID NO: 353; (ii) a nucleic acid sequence having SEQ ID NO: 442; (iii) a nucleic acid having a functional fragment of any one of the sequences in (i) or (ii); or (iv) a nucleic acid having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of the sequences in (i), (ii), or (iii). In exemplary embodiments, such a polynucleotide encodes an eTF having SEQ ID NO: 305, or a functional fragment or variant thereof capable of upregulating SCN1A.

[0214] In certain embodiments, the present application provides a polynucleotide encoding an eTF capable of regulating endogenous SCN1A, wherein the polynucleotide comprises any one of the following: (i) a nucleic acid sequence having SEQ ID NO: 354; (ii) a nucleic acid sequence having SEQ ID NO: 443; (iii) a nucleic acid having a functional fragment of any one of the sequences in (i) or (ii); or (iv) a nucleic acid having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of the sequences in (i), (ii), or (iii). In exemplary embodiments, such a polynucleotide encodes an eTF having SEQ ID NO: 306, or a functional fragment or variant thereof capable of upregulating SCN1A.

[0215] In certain embodiments, the present application provides a polynucleotide encoding an eTF capable of regulating endogenous SCN1A, wherein the polynucleotide comprises any one of the following: (i) a nucleic acid sequence having SEQ ID NO: 355; (ii) a nucleic acid sequence having SEQ ID NO: 444; (iii) a nucleic acid having a functional fragment of any one of the sequences in (i) or (ii); or (iv) a nucleic acid having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of the sequences in (i), (ii), or (iii). In exemplary embodiments, such a polynucleotide encodes an eTF having SEQ ID NO: 308, or a functional fragment or variant thereof capable of upregulating SCN1A.

[0216] In certain embodiments, the present application provides a polynucleotide encoding an eTF capable of regulating endogenous SCN1A, wherein the polynucleotide comprises any one of the following: (i) a nucleic acid sequence having SEQ ID NO: 356; (ii) a nucleic acid sequence having SEQ ID NO: 445; (iii) a nucleic acid having a functional fragment of any one of the sequences in (i) or (ii); or (iv) a nucleic acid having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of the sequences in (i), (ii), or (iii). In exemplary embodiments, such a polynucleotide encodes an eTF having SEQ ID NO: 313, or a functional fragment or variant thereof capable of upregulating SCN1A.

[0217] In certain embodiments, the present application provides a polynucleotide encoding an eTF capable of regulating endogenous SCN1A, wherein the polynucleotide comprises any one of the following: (i) a nucleic acid sequence having SEQ ID NO: 357; (ii) a nucleic acid sequence having SEQ ID NO: 451; (iii) a nucleic acid having a functional fragment of any one of the sequences in (i) or (ii); or (iv) a nucleic acid having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of the sequences in (i), (ii), or (iii). In exemplary embodiments, such a polynucleotide encodes an eTF having SEQ ID NO: 314, or a functional fragment or variant thereof capable of upregulating SCN1A.

[0218] In certain embodiments, the present application provides a polynucleotide encoding an eTF capable of regulating endogenous SCN1A, wherein the polynucleotide comprises any one of the following: (i) a nucleic acid sequence having SEQ ID NO: 358; (ii) a nucleic acid sequence having SEQ ID NO: 448; (iii) a nucleic acid having a functional fragment of any one of the sequences in (i) or (ii); or (iv) a nucleic acid having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of the sequences in (i), (ii), or (iii). In exemplary embodiments, such a polynucleotide encodes an eTF having SEQ ID NO: 315, or a functional fragment or variant thereof capable of upregulating SCN1A.

[0219] In certain embodiments, the present application provides a polynucleotide encoding an eTF capable of regulating endogenous SCN1A, wherein the polynucleotide comprises any one of the following: (i) a nucleic acid sequence having SEQ ID NO: 359; (ii) a nucleic acid sequence having SEQ ID NO: 446; (iii) a nucleic acid having a functional fragment of any one of the sequences in (i) or (ii); or (iv) a nucleic acid having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of the sequences in (i), (ii), or (iii). In exemplary embodiments, such a polynucleotide encodes an eTF having SEQ ID NO: 316, or a functional fragment or variant thereof capable of upregulating SCN1A.

[0220] In certain embodiments, the present application provides a polynucleotide encoding an eTF capable of regulating endogenous SCN1A, wherein the polynucleotide comprises any one of the following: (i) a nucleic acid sequence having SEQ ID NO: 360; (ii) a nucleic acid sequence having SEQ ID NO: 447; (iii) a nucleic acid having a functional fragment of any one of the sequences in (i) or (ii); or (iv) a nucleic acid having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of the sequences in (i), (ii), or (iii). In exemplary embodiments, such a polynucleotide encodes an eTF having SEQ ID NO: 317, or a functional fragment or variant thereof capable of upregulating SCN1A.

[0221] In certain embodiments, the present application provides a polynucleotide encoding an eTF capable of regulating endogenous SCN1A, wherein the polynucleotide comprises any one of the following: (i) a nucleic acid sequence having SEQ ID NO: 361; (ii) a nucleic acid sequence having SEQ ID NO: 450; (iii) a nucleic acid having a functional fragment of any one of the sequences in (i) or (ii); or (iv) a nucleic acid having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of the sequences in (i), (ii), or (iii). In exemplary embodiments, such a polynucleotide encodes an eTF having SEQ ID NO: 318, or a functional fragment or variant thereof capable of upregulating SCN1A.

[0222] In certain embodiments, the present application provides a polynucleotide encoding an eTF capable of regulating endogenous SCN1A, wherein the polynucleotide comprises any one of the following: (i) a nucleic acid sequence having SEQ ID NO: 362; (ii) a nucleic acid sequence having SEQ ID NO: 449; (iii) a nucleic acid having a functional fragment of any one of the sequences in (i) or (ii); or (iv) a nucleic acid having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of the sequences in (i), (ii), or (iii). In exemplary embodiments, such a polynucleotide encodes an eTF having SEQ ID NO: 319, or a functional fragment or variant thereof capable of upregulating SCN1A.

[0223] In certain embodiments, the present application provides a polynucleotide encoding an eTF capable of regulating endogenous SCN1A, wherein the polynucleotide comprises any one of the following: (i) a nucleic acid sequence having SEQ ID NO: 454; (ii) a nucleic acid sequence having SEQ ID NO: 452; (iii) a nucleic acid having a functional fragment of any one of the sequences in (i) or (ii); or (iv) a nucleic acid having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of the sequences in (i), (ii), or (iii). In exemplary embodiments, such a polynucleotide encodes an eTF having SEQ ID NO: 440, or a functional fragment or variant thereof capable of upregulating SCN1A.

[0224] In certain embodiments, the present application provides a polynucleotide encoding an eTF capable of regulating endogenous SCN1A, wherein the polynucleotide comprises any one of the following: (i) a nucleic acid sequence having SEQ ID NO: 363; (ii) a nucleic acid sequence having SEQ ID NO: 453; (iii) a nucleic acid having a functional fragment of any one of the sequences in (i) or (ii); or (iv) a nucleic acid having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of the sequences in (i), (ii), or (iii). In exemplary embodiments, such a polynucleotide encodes an eTF having SEQ ID NO: 325, or a functional fragment or variant thereof capable of upregulating SCN1A. Expression cassette

[0225] In another aspect, the application provides an expression cassette comprising a polynucleotide encoding an eTF provided herein and one or more regulatory elements.

[0226] In certain embodiments, a polynucleotide encoding an eTF disclosed herein is part of an expression cassette that includes one or more regulatory elements in addition to the eTF-encoding sequence. In an exemplary embodiment, a polynucleotide encoding an eTF disclosed herein is part of an expression cassette that includes a promoter positioned upstream of the eTF-encoding sequence so as to drive expression of the eTF in a cell.

[0227] In certain embodiments, the expression cassette disclosed herein comprises a polynucleotide encoding an eTF and a constitutive promoter located upstream of the eTF-encoding sequence, capable of driving expression of the eTF in cells. Examples of constitutive promoters include the GAD2 promoter, human synapsin promoter, CBA promoter, CMV promoter, minCMV promoter, TATA box, super core promoter, or EF1α promoter, or a combination thereof.

[0228] In certain embodiments, the expression cassettes disclosed herein comprise a polynucleotide encoding eTF and a short promoter capable of driving expression of eTF in a cell. In certain embodiments, short promoters suitable for use with the nucleic acid molecules described herein include those having a length of 500 bp, 450 bp, 400 bp, 350 bp, 300 bp, 250 bp, 225 bp, 200 bp, 175 bp, 150 bp, 145 bp, 140 bp, 135 bp, 130 bp, 125 bp, 120 bp, 115 bp, 110 bp, 105 bp, 100 bp, 95 bp, 90 bp, Less than 85bp, 80bp, or 75bp, or approximately 80-300bp, 80-275bp, 80-250bp, 80-200bp, 80-150bp, 80-125bp, 80-120bp, 80-115bp, 80-110bp, 80-105bp, 80-100bp, 85-300bp, 85-275bp, 85-250bp, 85-200bp, 85-150bp, 85-125bp p, 85~120bp, 85~115bp, 85~110bp, 85~105bp, 85~100bp, 90~300bp, 90~275bp, 90~250bp, 90~200bp, 90~1 50bp, 90~125bp, 90~120bp, 90~115bp, 90~110bp, 90~105bp, 90~100bp, 95~300bp, 95~275bp, 95~250bp, 95 100-110bp, or 100-105bp. In exemplary embodiments, short promoters suitable for use in accordance with the expression cassettes described herein comprise about 100-120bp, about 117bp, or about 100bp.

[0229] In certain embodiments, the expression cassettes disclosed herein comprise a short promoter comprising or consisting of any one of the following, operably linked to a polynucleotide encoding any one of the eTFs disclosed herein: (i) SEQ ID NO: 178; (ii) a variant or functional fragment thereof; or (iii) a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of (i) or (ii). Other examples of short promoter sequences can be found in PCT Publication No. WO2018 / 213786.

[0230] In certain embodiments, the expression cassettes disclosed herein comprise a polynucleotide encoding an eTF and a cell-type-selective promoter positioned upstream of the eTF-encoding sequence, such that the expression of the eTF can be selectively driven in a cell of interest. In certain embodiments, the cell-type-selective promoter can be selective for (e.g., selectively drive expression in) any cell type of interest, such as cardiac cells, liver cells, muscle cells, bone cells, neurons, or a subpopulation thereof. In exemplary embodiments, the expression cassettes disclosed herein comprise a PV-selective regulatory element (e.g., a promoter, an enhancer, and / or a promoter and an enhancer) positioned upstream of the eTF-encoding polynucleotide and the eTF-encoding sequence, such that the expression of the eTF can be selectively driven in PV cells. A PV-selective regulatory element refers to a regulatory element that specifically modulates gene expression in PV neurons. In certain embodiments, the PV-selective regulatory element enhances expression in PV neurons compared to one or more other CNS cell types. In certain embodiments, the PV-selective regulatory element represses transcription and / or translation processes in off-target cell types.

[0231] In certain embodiments, the PV-selective regulatory element provided herein causes selective gene expression in PV cells compared to off-target cell types.In some cases, the off-target cell types include, but are not limited to, excitatory neurons, non-PV CNS cell types, and non-neuronal CNS cell types.In certain embodiments, the PV-selective regulatory element causes selective gene expression in PV neurons over at least 1, 2, 3, 4, 5 or more non-PV CNS cell types. In some examples, the non-PV CNS cells are excitatory neurons, dopaminergic neurons, astrocytes, microglia, motor neurons, vascular cells, or non-GABAergic neurons (e.g., cells that do not express one or more of GAD2, GAD1, NKX2.1, DLX1, DLX5, SST, and VIP), non-PV neurons (e.g., GABAergic neurons that do not express parvalbumin), or other CNS cells (e.g., CNS cell types that have never expressed PV, GAD2, GAD1, NKX2.1, DLX1, DLX5, SST, or VIP). In some cases, the PV-selective regulatory elements provided herein result in increased selectivity in gene expression in PV neurons compared to non-PV GABAergic cells. In some cases, cell types are distinguished by having different cell markers, morphology, phenotype, genotype, function, and / or any other means for classifying cell types.

[0232] The selectivity of expression driven by a PV-selective regulatory element can be measured in several ways. In one embodiment, the selectivity of gene expression in PV cells over non-PV cells can be measured by comparing the number of PV cells expressing detectable levels of transcripts from a gene operably linked to a PV-selective regulatory element to the total number of cells expressing that gene (e.g., the ratio of PV cells expressing that gene to total cells (PV + non-PV cells)). For example, selectivity for PV neurons can be determined using an immunohistochemistry-based colocalization assay that uses a gene encoding a fluorescent protein (e.g., eGFP) operably linked to a PV-selective regulatory element and an antibody that identifies PV cells (e.g., an anti-PV antibody that specifically interacts with PV neurons) linked to a second fluorescent label (e.g., red fluorescent protein) to measure gene expression. The selectivity of expression in PV cells can be calculated by dividing the number of cells expressing both PV and eGFP (e.g., PV cells) by the total number of cells expressing eGFP (e.g., PV cells and non-PV cells) and multiplying by 100 to convert to a percentage. The higher the percentage of PV cells that express the transgene, the more selective the regulatory element is for PV cells. In certain embodiments, the PV-selective regulatory elements provided herein can be highly selective for expression in PV cells. For example, the PV-selective regulatory elements provided herein can exhibit a selectivity of about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than about 99% for PV neurons (e.g., PV neurons / total cells x 100).

[0233] In some cases, the PV-selective regulatory elements provided herein are short. In some cases, the size of the PV-selective regulatory element is compatible with the cloning capacity of a vector, such as a viral vector or rAAV, such that the combined size of the transgene and one or more PV-selective regulatory elements does not exceed the cloning capacity of the vector. In some cases, the PV-selective regulatory element has a length of at most about 2050 bp, 2000 bp, 1900 bp, 1800 bp, 1700 bp, 1600 bp, 1500 bp, 1400 bp, 1300 bp, 1200 bp, 1100 bp, 1000 bp, 900 bp, 800 bp, 700 bp, 600 bp, 500 bp, 400 bp, 300 bp, 200 bp, or 100 bp. In some cases, the PV-selective regulatory element is between about 500-600 bp, 500-700 bp, 500-800 bp, 500-900 bp, 500-1000 bp, or 500-1500 bp.

[0234] In certain embodiments, the PV-selective regulatory elements provided herein comprise or consist of any one of the following: (i) SEQ ID NOs: 183-185 and 417; (ii) a variant, functional fragment, or combination thereof; or (iii) a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of (i) or (ii). In some cases, the regulatory element comprises any one of SEQ ID NOs: 183-185 and 417. Other examples of PV-selective regulatory elements can be found in PCT Publication No. WO2018 / 187363.

[0235] In an exemplary embodiment, the present application provides a method for the production of SCN1A under the control of a PV-selective regulatory element.

[0010] Provided are expression cassettes comprising a nucleic acid sequence encoding an eTF. In certain embodiments, the present application provides an expression cassette comprising a nucleic acid sequence encoding an SCN1A eTF comprising a DBD having any one of the following sequences: SEQ ID NO: 135, 371-372, or 376, under the control of a PV-selective regulatory element having any one of SEQ ID NOs: 183-185, or 417. In certain embodiments, the present application provides an expression cassette comprising a nucleic acid sequence encoding an SCN1A eTF comprising any one of the following sequences: SEQ ID NOs: 6-9, 13-15, 57-58, 61-62, 67-71, 74-75, 268-282, 295-299, 305-325, or 364-366, under the control of a PV-selective regulatory element having any one of SEQ ID NOs: 183-185, or 417. In certain embodiments, the present application provides an expression cassette comprising a nucleic acid sequence comprising any one of the following sequences under the control of a PV-selective regulatory element having any one of SEQ ID NOs: 183-185, or 417: SEQ ID NOs: 353-363. In certain embodiments, the present application provides an expression cassette comprising a nucleic acid sequence encoding an SCN1A eTF comprising a DBD having any one of the following sequences under the control of a PV-selective regulatory element having any one of SEQ ID NOs: 183 or 185: SEQ ID NOs: 135, 371, 372, or 376. In certain embodiments, the present application provides an expression cassette comprising a nucleic acid sequence encoding an SCN1A eTF comprising any one of the following sequences under the control of a PV-selective regulatory element having any one of SEQ ID NOs: 183 or 185: SEQ ID NOs: 6-9, 13-15, 57-58, 61-62, 67-71, 74-75, 268-282, 295-299, 305-325, or 364-366. In certain embodiments, the present application provides an expression cassette comprising a nucleic acid sequence comprising any one of the following sequences: SEQ ID NOs: 353-363, under the control of a PV-selective regulatory element having any one of SEQ ID NOs: 183 or 185.

[0236] In certain embodiments, the present application provides a method for the production of a GRN under the control of a cell-type selective regulatory element. Provided is an expression cassette comprising a nucleic acid sequence encoding eTF. In certain embodiments, the cell type-selective regulatory element can drive the expression of eTF, which upregulates GRN at a higher level in cells selected from the group consisting of central nervous system cells, frontal cortex cells, glial cells, microglial cells, Purkinje cells, pyramidal cells (e.g., Betz cells), motor neurons, cerebral cortical neurons and striatal cells, compared to other cell types (e.g., non-CNS cells).

[0237] In certain embodiments, the expression cassettes provided herein may contain, in addition to a promoter, one or more additional regulatory elements, such as sequences associated with transcription initiation or termination, enhancer sequences, and efficient RNA processing signals. Exemplary regulatory elements include, for example, introns, enhancers, UTRs, stability elements, WPRE sequences, Kozak consensus sequences, post-translational response elements, microRNA binding sites, or polyadenylation (polyA) sequences, or combinations thereof. Regulatory elements can function to modulate gene expression at the transcriptional, post-transcriptional, or translational stages of gene expression. At the RNA level, regulation can occur at the level of translation (e.g., stability elements that stabilize mRNA for translation), RNA cleavage, RNA splicing, and / or transcription termination. In various embodiments, regulatory elements can recruit transcription factors to the coding region that increase gene expression selectivity in the cell type of interest, increase the rate at which RNA transcripts are produced, increase the stability of the produced RNA, and / or increase the rate of protein synthesis from RNA transcripts.

[0238] In certain embodiments, the expression cassettes described herein further comprise a polyA sequence. Suitable polyA sequences include, for example, an artificial polyA (PA75) having a length of approximately 75 bp (see, for example, WO2018 / 126116), bovine growth hormone polyA, SV40 early polyA signal, SV40 late polyA signal, rabbit beta globin polyA, HSV thymidine kinase polyA, protamine gene polyA, adenovirus 5 EIb polyA, growth hormone polyA, or PBGD polyA. In an exemplary embodiment, the polyA sequence suitable for use in the expression cassettes provided herein is hGH polyA (SEQ ID NO: 327) or synthetic polyA (SEQ ID NO: 326). Typically, the polyA sequence is located downstream of the polynucleotide encoding eTF in the expression cassettes described herein.

[0239] In certain embodiments, the expression cassettes provided herein further comprise one or more nucleic acid sequences encoding one or more nuclear localization signals (NLS). Any NLS peptide that promotes the import of the protein to which it is bound into the cell nucleus can be used. Examples of NLS include, for example, the SV40 large T antigen NLS, the nucleoplasmin NLS, the EGL-13 NLS, the c-Myc NLS, and the TUS protein NLS. See, for example, C. Dingwall et al., J. Cell Biol. 107: 841-9 (1988); J. P. Makkerh See, e.g., M. Ray et al., Curr Biol. 6: 1025-7 (1996); and M. Ray et al., Bioconjug. Chem. 26: 1004-7 (2015). The NLS may be located anywhere on the eTF protein sequence, but in preferred embodiments, it is conjugated to the N-terminus of the eTF or to a domain of the eTF. In exemplary embodiments, the nucleic acid cassettes provided herein encode eTFs having an NLS fused to the N-terminus of the eTF. In other embodiments, the nucleic acid cassettes provided herein encode eTFs having a first NLS fused to the N-terminus of the eTF and a second NLS located between the DBD and TAD domains of the eTF. Expression vector

[0240] In certain embodiments, the expression cassette described herein can be incorporated into an expression vector.The expression vector can be used to deliver the expression cassette to target cells via transfection or transduction.The vector can be an integrating or non-integrating vector, which refers to the vector's ability to integrate the expression cassette or transgene into the genome of host cells.Examples of expression vectors include, but are not limited to: (a) non-viral vectors, such as nucleic acid vectors including linear oligonucleotides and circular plasmids; artificial chromosomes, such as human artificial chromosomes (HAC), yeast artificial chromosomes (YAC) and bacterial artificial chromosomes (BAC or PAC); episomal vectors; transposons (e.g., PiggyBac); and (b) viral vectors, such as retroviral vectors, lentiviral vectors, adenoviral vectors and adeno-associated viral vectors.

[0241] Expression vectors can be linear oligonucleotides or circular plasmids and can be delivered to cells via various transfection methods, including physical and chemical methods. Physical methods generally refer to delivery methods that use physical forces to overcome cell membrane barriers to facilitate intracellular delivery of genetic material. Examples of physical methods include the use of needles, ballistic DNA, electroporation, sonoporation, photoporation, magnetofection, and hydroporation. Chemical methods generally refer to methods in which chemical carriers deliver nucleic acid molecules to cells, which can include inorganic particles, lipid-based vectors, polymer-based vectors, and peptide-based vectors.

[0242] In some embodiments, expression vectors are administered to target cells using cationic lipids (e.g., cationic liposomes). Various types of lipids have been investigated for gene delivery, such as lipid nanoemulsions (e.g., which are dispersions of one immiscible liquid in another immiscible liquid stabilized by an emulsifier) ​​or solid lipid nanoparticles.

[0243] In some embodiments, the expression vector is administered to the target cell using a peptide-based delivery vehicle. Peptide-based delivery vehicles may have the advantages of protecting the delivered genetic material, targeting specific cell receptors, disrupting endosomal membranes, and delivering the genetic material into the nucleus. In some embodiments, the expression vector is administered to the target cell using a polymer-based delivery vehicle. The polymer-based delivery vehicle may comprise natural proteins, peptides, and / or polysaccharides, or synthetic polymers. In one embodiment, the polymer-based delivery vehicle comprises polyethyleneimine (PEI). PEI can condense DNA into positively charged particles that bind to anionic cell surface residues and are brought into the cell via endocytosis. In other embodiments, the polymer-based delivery vehicle may comprise poly-L-lysine (PLL), poly(DL-lactic acid) (PLA), poly(DL-lactide-co-glycoside) (PLGA), polyornithine, polyarginine, histones, protamine, dendrimers, chitosan, synthetic amino derivatives of dextran, and / or cationic acrylic polymers. In certain embodiments, the polymer-based delivery vehicle may comprise a mixture of polymers, such as PEG and PLL.

[0244] In certain embodiments, expression vector can be a viral vector suitable for gene therapy.The preferred features of viral gene therapy vector or gene delivery vector can include: the ability to reproduce reproducibly and stably and be purified to high titer; the ability to mediate targeted delivery (for example, the ability to specifically deliver transgene to target tissue or organ without wide vector dissemination elsewhere); and the ability to mediate gene delivery and transgene expression without inducing harmful side effects.

[0245] Several types of viruses, such as the non-pathogenic parvoviruses called adeno-associated viruses, have been engineered for gene therapy by utilizing the viral infection pathway but avoiding replication and subsequent expression of viral genes that could lead to toxicity. Such viral vectors can be obtained by deleting all or part of the coding region from the viral genome, but leaving intact sequences (e.g., terminal repeat sequences) that may be necessary for function, such as packaging of the vector genome into a viral capsid or integration of the vector nucleic acid (e.g., DNA) into host chromatin.

[0246] In various embodiments, suitable viral vectors include retroviruses (e.g., types A, B, C, and D viruses), adenoviruses, parvoviruses (e.g., adeno-associated viruses, or AAVs), coronaviruses, negative-strand RNA viruses, such as orthomyxoviruses (e.g., influenza viruses), rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), paramyxoviruses (e.g., measles and Sendai viruses), positive-strand RNA viruses, such as picornaviruses and alphaviruses, as well as double-stranded DNA viruses, including adenoviruses, herpesviruses (e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxviruses (e.g., vaccinia, fowlpox, and canarypox). Examples of retroviruses include avian leukosis sarcoma virus, human T-lymphotropic virus type 1 (HTLV-1), bovine leukemia virus (BLV), lentiviruses, and spumaviruses. Other viruses include, for example, Norwalk virus, togavirus, flavivirus, reovirus, papovavirus, hepadnavirus, and hepatitis virus. Viral vectors can be classified into two groups—integrating and non-integrating—according to their ability to integrate into the host genome. Oncoretroviruses and lentiviruses can integrate into host cell chromatin, while adenoviruses, adeno-associated viruses, and herpesviruses predominantly persist as extrachromosomal episomes in the cell nucleus.

[0247] In certain embodiments, a suitable viral vector is a retroviral vector. Retrovirus refers to viruses of the Retroviridae family. Examples of retroviruses include oncoretroviruses, such as murine leukemia virus (MLV), and lentiviruses, such as human immunodeficiency virus 1 (HIV-1). The retroviral genome is a single-stranded (ss) RNA and contains various genes that can be provided in cis or trans. For example, a retroviral genome may contain cis-acting sequences, such as two long terminal repeats (LTRs), along with elements for gene expression, reverse transcription, and integration into the host chromosome. Other components include a packaging signal (psi or ψ) for specific RNA packaging into newly formed virions and a polypurine tract (PPT), which is the site of initiation of plus-strand DNA synthesis during reverse transcription. In addition, a retroviral genome may contain gag, pol, and env genes. The gag gene encodes structural proteins, the pol gene encodes an enzyme that associates with ssRNA and performs reverse transcription of viral RNA into DNA, and the env gene encodes the viral envelope. Generally, gag, pol and env are provided in trans for viral replication and packaging.

[0248] In certain embodiments, the retroviral vector provided herein may be a lentiviral vector. At least five serogroups or serotypes of lentiviruses have been recognized. Viruses of different serotypes can differentially infect certain cell types and / or hosts. Lentiviruses include, for example, primate retroviruses and non-primate retroviruses. Primate retroviruses include HIV and simian immunodeficiency virus (SIV). Non-primate retroviruses include feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), and visna virus. Lentiviruses or lentivectors may be capable of transducing quiescent cells. Similar to oncoretroviral vectors, the design of lentivectors may be based on the separation of cis-acting and trans-acting sequences.

[0249] In certain embodiments, the present application provides an expression vector designed for delivery by an optimized therapeutic retroviral vector. The retroviral vector can be a lentivirus that includes a left (5') LTR; a sequence that assists viral packaging and / or nuclear import; a promoter; optionally one or more additional regulatory elements (such as an enhancer or polyA sequence); optionally a lentiviral reverse response element (RRE); a construct that includes a PV-selective regulatory element operably linked to a sequence encoding eTF; optionally an insulator; and a right (3') retroviral LTR.

[0250] In an exemplary embodiment, the viral vector provided herein is an adeno-associated virus (AAV). AAV is a small, replication-defective, non-enveloped animal virus that infects humans and some other primate species. AAV is known not to cause human disease and induces a mild immune response. AAV vectors can also infect both dividing and quiescent cells without being integrated into the host cell genome.

[0251] The AAV genome consists of a linear, single-stranded DNA approximately 4.7 kb in length. The genome consists of two open reading frames (ORFs) flanked by approximately 145 bp inverted terminal repeat (ITR) sequences. The ITRs consist of a nucleotide sequence at the 5' end (5'ITR) containing a palindromic sequence and a nucleotide sequence located at the 3' end (3'ITR). These ITRs function in cis by folding back through complementary base pairing to form a T-shaped hairpin structure that serves as a primer during the initiation of DNA replication for second-strand synthesis. The two open reading frames encode the rep and cap genes, which are involved in virion replication and packaging. In an exemplary embodiment, the AAV vectors provided herein do not contain either the rep or cap genes. Such genes can be provided in trans to produce virions, as further described below.

[0252] In certain embodiments, the AAV vector may comprise a stuffer nucleic acid. In some embodiments, the stuffer nucleic acid may encode a green fluorescent protein or an antibiotic resistance gene such as kanamycin or ampicillin. In certain embodiments, the stuffer nucleic acid may be located outside the ITR sequence (e.g., relative to the eTF transgene sequence and regulatory sequence located between the 5'ITR sequence and the 3'ITR sequence).

[0253] There are various serotypes of AAV, including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and AAV13. These serotypes differ in their tropism, or the type of cells they infect. AAV can contain genomes and capsids from multiple serotypes (e.g., pseudotypes). For example, AAV can contain a serotype 2 genome (e.g., ITRs) packaged in a capsid from serotype 5 or serotype 9. Pseudotypes can improve transduction efficiency as well as alter tropism.

[0254] In some cases, AAV serotypes that can cross the blood-brain barrier or infect cells of the CNS are preferred. In some cases, AAV9 or a variant thereof is used to deliver an expression cassette of the present disclosure that includes a PV-selective regulatory element operably linked to a transgene encoding an eTF that upregulates SCN1A.

[0255] In an exemplary embodiment, the present application provides an expression vector designed for delivery by AAV. The AAV can be any serotype, for example, AAV1, AAV2, AAV3, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-DJ, or a chimeric, hybrid, or variant AAV. The AAV can also be a self-complementary AAV (scAAV). In certain embodiments, the expression vector designed for delivery by AAV comprises a 5'ITR and a 3'ITR. In certain embodiments, the expression vector designed for delivery by AAV comprises a 5'ITR, a promoter, a transgene encoding an eTF, and a 3'ITR. In certain embodiments, the expression vector designed for delivery by AAV comprises a 5'ITR, an enhancer, a promoter, a transgene encoding an eTF, a polyA sequence, and a 3'ITR. Exemplary AAV expression vectors are shown in Figures 36 and 37. host cell

[0256] In another aspect, the present invention relates to a host cell comprising an expression cassette or expression vector encoding the eTF of the present invention. The host cell may be a bacterial cell, yeast cell, insect cell, or mammalian cell. In an exemplary embodiment, the host cell refers to any cell line that is susceptible to infection by the virus of interest and is suitable for in vitro culture.

[0257] In certain embodiments, the host cells provided herein may be used for ex vivo gene therapy. In such embodiments, the cells are transfected with a nucleic acid molecule or expression vector containing a sequence encoding the eTF of the present invention and subsequently transplanted into a patient or subject. The transplanted cells may be of autologous, allogeneic, or xenogeneic origin. For clinical use, cell isolation is generally performed under Good Manufacturing Practice (GMP) conditions. Prior to transplantation, the quality of the cells and the absence of microorganisms or other contaminants are typically checked, and preconditioning, such as by radiation and / or immunosuppressive treatment, may be performed. Furthermore, the host cells may be transplanted together with growth factors to stimulate cell proliferation and / or differentiation.

[0258] In certain embodiments, host cell can be used for ex vivo gene therapy.Preferably, this cell is eukaryotic cell, for example, mammalian cell, including but not limited to human, non-human primate, for example, ape; chimpanzee; monkey and orangutan, domesticated animal, including dog and cat, and livestock, for example, horse, cow, pig, sheep and goat, or other mammalian species, including but not limited to mouse, rat, guinea pig, rabbit, hamster, etc. Those skilled in the art can select more suitable cell according to the patient or subject to be transplanted.

[0259] In certain embodiments, the host cells provided herein may be cells with self-renewal and pluripotency properties, such as stem cells or induced pluripotent stem cells. The stem cells are preferably mesenchymal stem cells. Mesenchymal stem cells (MSCs) can differentiate into at least one of osteoblasts, chondrocytes, adipocytes, or myocytes and can be isolated from any type of tissue. Generally, MSCs are isolated from bone marrow, adipose tissue, umbilical cord, or peripheral blood. Methods for obtaining them are well known to those skilled in the art. Induced pluripotent stem cells (also known as iPS cells or iPSCs) are a type of pluripotent stem cells that can be generated directly from adult cells. Yamanaka et al. induced iPS cells by transferring Oct3 / 4, Sox2, Klf4, and c-Myc genes into mouse and human fibroblasts and allowing the cells to express these genes (WO2007 / 069666). Thomson et al. subsequently produced human iPS cells using Nanog and Lin28 in place of Klf4 and c-Myc (WO2008 / 118820).

[0260] In an exemplary embodiment, the host cell provided herein is a packaging cell. The cell can be an adherent cell or a suspension cell. The packaging cell and the helper vector or virus or DNA construct(s) together provide all the missing functions required for complete replication and packaging of the viral vector in trans.

[0261] Preferably, the packaging cells are eukaryotic cells, such as mammalian cells, including monkey, human, canine, and rodent cells. Examples of human cells include PER.C6 cells (WO01 / 38362), MRC-5 (ATCC CCL-171), WI-38 (ATCC CCL-75), HEK-293 cells (ATCC CRL-1573), HeLa cells (ATCC CCL2), and fetal rhesus lung cells (ATCC CL-160). Examples of non-human primate cells include Vero cells (ATCC CCL81), COS-1 cells (ATCC CRL-1650), or COS-7 cells (ATCC CRL-1651). Examples of canine cells include MDCK cells (ATCC CCL-34). Examples of rodent cells include hamster cells, such as BHK21-F, HKCC, or CHO cells.

[0262] As an alternative to mammalian sources, cell lines for use in the present invention can be derived from avian sources, such as chickens, ducks, geese, quails, or pheasants. Examples of avian cell lines include avian embryonic stem cells (WO01 / 85938 and WO03 / 076601), immortalized duck retinal cells (WO2005 / 042728), and avian embryonic stem cell-derived cells, including chicken cells (WO2006 / 108846) or duck cells, such as the EB66 cell line (WO2008 / 129058 and WO2008 / 142124).

[0263] In another embodiment, the host cell is an insect cell, such as an SF9 cell (ATCC CRL-1711), an Sf21 cell (IPLB-Sf21), an MG1 cell (BTI-TN-MG1), or a High Five™ cell (BTI-TN-5B1-4).

[0264] In certain embodiments, the host cells provided herein comprising a recombinant AAV vector / genome of the invention (e.g., comprising a sequence encoding eTF) may further comprise one or more additional nucleic acid constructs, such as (i) a nucleic acid construct encoding the rep and cap genes but lacking ITR sequences (e.g., an AAV helper plasmid); and / or (ii) a nucleic acid construct (e.g., a plasmid) providing adenoviral functions necessary for AAV replication. In exemplary embodiments, the host cells provided herein comprise: i) an expression vector (i.e., a recombinant AAV genome) comprising a sequence encoding the eTF of the invention; ii) a nucleic acid construct encoding the AAV rep and cap genes lacking ITR sequences; and iii) a nucleic acid construct comprising adenoviral helper genes (described further below).

[0265] In certain embodiments, the rep gene, cap gene, and adenoviral helper genes can be combined on a single plasmid (Blouin V et al. J Gene Med. 2004; 6(suppl): S223-S228; Grimm D. et al. Hum. Gene Ther. 2003; 7: 839-850). Thus, in another exemplary embodiment, the host cell provided herein comprises: i) an expression vector (i.e., a recombinant AAV genome) comprising a sequence encoding the eTF of the present invention; and ii) a plasmid encoding the AAV rep and cap genes without the ITR sequences and further comprising adenovirus helper genes.

[0266] In another embodiment, the host cell provided herein comprises: a) an expression vector (i.e., a recombinant AAV genome) comprising a sequence encoding the eTF of the present invention; b) a plasmid encoding the AAV rep and cap genes without ITR sequences; and c) a plasmid comprising RNA of the adenovirus helper genes E2a, E4, and VA; wherein the co-transfection is performed in cells that constitutively express and transcomplement the adenovirus E1 gene, preferably mammalian cells, such as HEK-293 cells (ATCC CRL-1573).

[0267] In certain embodiments, suitable host cells for large-scale production of AAV vectors are insect cells that can be infected with a combination of recombinant baculoviruses (Urabe et al. Hum. Gene Ther. 2002; 13: 1935-1943). For example, SF9 cells can be co-infected with three baculovirus vectors, each expressing the AAV rep, AAV cap, and AAV vector to be packaged. These recombinant baculovirus vectors provide the viral helper gene functions required for viral replication and / or packaging.

[0268] Further guidance for the construction and production of virions for gene therapy according to the present invention can be found in Viral Vectors for Gene Therapy, Methods and Protocols. Series: Methods in Molecular Biology, Vol. 737. Merten and Al-Rubeai (Eds.); 2011 Humana Press (Springer); Gene Therapy. M. Giacca. 2010 Springer-Verlag; Heilbronn R. and Weger S. Viral Vectors for Gene Transfer: Current Status of Gene Therapeutics. In: Drug Delivery, Handbook of Experimental Pharmacology 197; M. Schafer-Korting (Ed.). 2010 Springer-Verlag; pp. 143-170; Adeno-Associated Virus: Methods and Protocols. RO Snyder and P. Moulliier (Eds.). 2011 Humana Press (Springer); Bunning H. et al. Recent developments in adeno-associated virus technology. J. Gene Med. 2008; 10:717-733; and Adenovirus: Methods and Protocols. M. Chillon and A. Bosch (Eds.); Third Edition. 2014 Humana Press (Springer). Virions and methods for producing virions

[0269] In certain embodiments, the present application provides a viral particle comprising a viral vector comprising a sequence encoding the eTF of the present invention. The terms "viral particle" and "virion" are used interchangeably herein and refer to an infectious, typically replication-deficient viral particle comprising a viral genome (e.g., a viral expression vector) packaged within a capsid, and in some cases, for example, for retroviruses, within a lipid envelope surrounding the capsid. "Capsid" refers to the structure into which the viral genome is packaged. A capsid consists of several oligomeric structural subunits made of proteins. For example, AAV has an icosahedral capsid formed by the interaction of three capsid proteins: VP1, VP2, and VP3. In one embodiment, the virion provided herein is a recombinant AAV virion, i.e., a rAAV virion, obtained by packaging an AAV vector comprising a PV-selective regulatory element operably linked to a sequence encoding the eTF described herein into a protein shell.

[0270] In certain embodiments, the recombinant AAV virions provided herein can be prepared by encapsidating an AAV genome from a specific AAV serotype into a viral particle formed by the native Cap protein corresponding to the same specific serotype of AAV. In other embodiments, the AAV viral particles provided herein comprise a viral vector containing the ITR(s) of a given AAV serotype packaged in a protein from a different serotype. For example, see Bunning H et al. J Gene Med 2008; 10: 717-733. For example, viral vectors with ITRs from a given AAV serotype can be packaged into: a) viral particles composed of capsid proteins from the same or different AAV serotypes (e.g., AAV2 ITRs and AAV9 capsid proteins; AAV2 ITRs and AAV8 capsid proteins; etc.); b) mosaic viral particles composed of a mixture of capsid proteins from different AAV serotypes or variants (e.g., AAV2 ITRs together with AAV1 and AAV9 capsid proteins); c) between different AAV serotypes or variants. a) chimeric viral particles composed of capsid proteins truncated by domain swapping in (e.g., AAV2 ITRs together with AAV8 capsid protein with AAV9 domains); or b) targeted viral particles engineered to exhibit selective binding domains that enable stringent interaction with target cell-specific receptors (e.g., AAV5 ITRs together with AAV9 capsid protein genetically truncated by insertion of a peptide ligand; or AAV9 capsid protein non-genetically modified by coupling of a peptide ligand to the capsid surface).

[0271] Those skilled in the art will understand that the AAV virions provided herein can contain capsid proteins of any AAV serotype. In one embodiment, the viral particle contains capsid proteins from an AAV serotype selected from the group consisting of AAV1, AAV2, AAV5, AAV8 and AAV9, which are more suitable for delivery to the CNS (M. Hocquemiller et al., Hum Gene Ther 27(7): 478-496 (2016)). In a specific embodiment, the viral particle contains an expression cassette of the present invention, wherein the 5'ITR and 3'ITR sequences of the expression cassette are those of the AAV2 serotype, and the capsid protein is that of the AAV9 serotype.

[0272] Numerous methods are known in the art for producing rAAV virions, including transfection, stable cell line production, and infectious hybrid virus production systems, including adenovirus-AAV hybrids, herpesvirus-AAV hybrids (Conway, J.E. et al., (1997) J. Virology 71(11):8780-8789), and baculovirus-AAV hybrids. All rAAV production cultures for the production of rAAV viral particles require: 1) suitable host cells, including, for example, a human-derived cell line, such as HeLa, A549, or 293 cells, or an insect-derived cell line, such as SF-9, in the case of a baculovirus production system; 2) appropriate helper virus functions, provided by wild-type or mutant adenovirus (e.g., a temperature-sensitive adenovirus), herpesvirus, baculovirus, or a plasmid construct providing helper functions; 3) AAV rep and cap genes and gene products; 4) a transgene (e.g., a promoter operably linked to a sequence encoding an eTF as described herein) flanked by AAV ITR sequences; and 5) appropriate media and media components to support rAAV production.

[0273] In various embodiments, the host cells described herein comprise the following three components: (1) rep and cap genes, (2) genes providing helper functions, and (3) a transgene (e.g., a promoter operably linked to a sequence encoding an eTF described herein flanked by ITRs). The AAV rep gene, AAV cap gene, and genes providing helper functions can be introduced into a cell by incorporating these genes into a vector, such as a plasmid, and introducing the vector into a host cell. The rep, cap, and helper function genes can be incorporated into the same or different plasmids. In a preferred embodiment, the AAV rep and cap genes are incorporated into one plasmid, and the genes providing helper functions are incorporated into another plasmid. The various plasmids (e.g., containing the AAV rep and cap genes, helper functions, or transgenes) for creating a host cell for virion production can be introduced into a cell by using any suitable method well known in the art. Examples of transfection methods include, but are not limited to, calcium phosphate co-precipitation, DEAE-dextran, polybrene, electroporation, microinjection, liposome-mediated fusion, lipofection, retroviral infection and biolistic transfection.In certain embodiments, the plasmids that provide rep gene and cap gene, helper function and transgene (for example, promoter operably linked to the sequence encoding eTF flanked by ITRs as disclosed herein) can be simultaneously introduced into cells.In another embodiment, the plasmids that provide rep gene and cap gene and helper function can be introduced into cells before or after the introduction of the plasmid containing transgene.In an exemplary embodiment, cells are co-transfected with three plasmids (e.g., a triple transfection method): (1) a plasmid containing a transgene (e.g., a promoter operably linked to a sequence encoding an eTF disclosed herein flanked by ITRs), (2) a plasmid containing the AAV rep and cap genes, and (3) a plasmid containing genes providing helper functions. Exemplary host cells can be 293, A549, or HeLa cells.

[0274] In other embodiments, one or more of (1) the AAV rep and cap genes, (2) genes providing helper functions, and (3) the transgene may be carried by the packaging cell episomally and / or integrated into the genome of the packaging cell. In one embodiment, the host cell may be a packaging cell in which the AAV rep and cap genes and the helper functions are stably maintained in the host cell, and the host cell is transiently transfected with a plasmid containing the transgene (e.g., a promoter operably linked to a sequence encoding an eTF disclosed herein flanked by ITRs). In another embodiment, the host cell is a packaging cell in which the AAV rep and cap genes are stably maintained in the host cell, and the host cell is transiently transfected with a plasmid containing the transgene (e.g., a promoter operably linked to a sequence encoding an eTF disclosed herein flanked by ITRs) and a plasmid containing the helper functions. In another embodiment, the host cell can be a packaging cell in which helper functions are stably maintained in the host cell, and the host cell is transiently transfected with a plasmid containing a transgene (e.g., a promoter operably linked to a sequence encoding an eTF as disclosed herein flanked by ITRs) and a plasmid containing the rep and cap genes. In another embodiment, the host cell can be a producer cell line stably transfected with the rep and cap genes, helper functions, and transgene sequences (e.g., a promoter operably linked to a sequence encoding an eTF as disclosed herein flanked by ITRs). Exemplary packaging and producer cells can be derived from 293, A549, or HeLa cells.

[0275] In another embodiment, the producer cell line is an insect cell line (typically Sf9 cells) infected with a baculovirus expression vector that provides the Rep and Cap proteins. This system does not require adenovirus helper genes (Ayuso E, et al. al., Curr. Gene Ther. 2010, 10:423-436).

[0276] The term "cap protein," as used herein, refers to a polypeptide having at least one functional activity of a native AAV Cap protein (e.g., VP1, VP2, VP3). Examples of functional activities of a cap protein include the ability to induce capsid formation, promote the accumulation of single-stranded DNA, promote packaging of AAV DNA into capsids (i.e., encapsidation), bind to a cellular receptor, and promote virion entry into a host cell. In principle, any Cap protein can be used in the context of the present invention.

[0277] It has been reported that cap proteins have an effect on the host tropism, cell, tissue, or organ specificity, receptor usage, infection efficiency, and immunogenicity of AAV viruses. Therefore, AAV caps for use in rAAV can be selected, for example, taking into consideration the subject's species (e.g., human or non-human), the subject's immunological status, the subject's suitability for long-term or short-term treatment, or specific therapeutic applications (e.g., treatment of specific diseases or disorders, or delivery to specific cells, tissues, or organs). In certain embodiments, the cap protein is derived from an AAV from the group consisting of AAV1, AAV2, AAV5, AAV8, and AAV9 serotypes. In an exemplary embodiment, the cap protein is derived from AAV9.

[0278] In some embodiments, the AAV Cap for use in the methods of the present invention can be generated by mutagenesis (i.e., by insertion, deletion, or substitution) of one of the above-described AAV caps or their encoding nucleic acids. There is at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% or higher similarity to one or more of the caps.

[0279] In some embodiments, the AAV cap is a chimera comprising domains from two, three, four, or more of the above-mentioned AAV caps. In some embodiments, the AAV cap is a mosaic of VP1, VP2, and VP3 monomers from two or three different AAVs or recombinant AAVs. In some embodiments, the rAAV composition comprises more than one of the above-mentioned caps.

[0280] In some embodiments, AAV caps for use in rAAV virions are engineered to contain heterologous sequences or other modifications. For example, peptide or protein sequences that confer selective targeting or immune evasion can be engineered into the cap protein. Alternatively, or in addition, the cap can be chemically modified to pegylate (i.e., PEGylate) the surface of the rAAV, thereby promoting immune evasion. The cap protein can also be mutagenized (e.g., to eliminate its natural receptor binding or to mask immunogenic epitopes).

[0281] The term "rep protein," as used herein, refers to a polypeptide having at least one functional activity of a native AAV rep protein (e.g., rep 40, 52, 68, 78). Examples of functional activities of the rep protein include promoting DNA replication through recognition, binding, and nicking of AAV DNA replication origins, as well as any activity associated with the physiological function of the protein, including DNA helicase activity. Additional functions include modulation of transcription from an AAV (or other heterologous) promoter and site-specific integration of AAV DNA into a host chromosome. In certain embodiments, the AAV rep gene may be derived from an AAV serotype selected from the group consisting of serotypes AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAVrhlO; more preferably, from an AAV serotype selected from the group consisting of AAV1, AAV2, AAV5, AAV8, and AAV9.

[0282] In some embodiments, AAV rep proteins for use in the methods of the invention can be generated by mutagenesis (i.e., by insertion, deletion, or substitution) of one of the above-mentioned AAV rep or their encoding nucleic acids, hi some embodiments, the AAV rep has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% or more similarity to one or more of the above-mentioned AAV rep.

[0283] The phrase "helper function" or "helper gene" as used herein refers to viral proteins on which AAV depends for replication. Helper functions include proteins required for AAV replication, including, but not limited to, proteins involved in AAV gene transcription activation, stage-specific AAV mRNA splicing, AAV DNA replication, cap expression product synthesis, and AAV capsid assembly. Viral accessory functions can be derived from any known helper virus, such as adenovirus, herpesvirus (other than herpes simplex virus type 1), and vaccinia virus. Helper functions include, but are not limited to, adenovirus E1, E2a, VA, and E4, or herpesvirus UL5, ULB, UL52, and UL29, and herpesvirus polymerase. In a preferred embodiment, the proteins on which AAV depends for replication are derived from adenovirus.

[0284] In some embodiments, viral proteins on which AAV depends for replication for use in the methods of the invention may be generated by mutagenesis (i.e., by insertion, deletion, or substitution) of one of the above-mentioned viral proteins or their encoding nucleic acids, hi some embodiments, the viral protein has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% or more similarity to one or more of the above-mentioned viral proteins.

[0285] Methods for assaying the function of the cap, rep and viral proteins on which AAV depends for replication are well known in the art.

[0286] Host cells for expressing a transgene of interest (e.g., a promoter operably linked to a sequence encoding eTF) can be grown under conditions suitable for the assembly of AAV virions. In certain embodiments, host cells are grown for an appropriate period of time to promote the assembly of AAV virions and their release into the medium. Generally, cells can be grown for about 24 hours, about 36 hours, about 48 hours, about 72 hours, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or up to about 10 days. After about 10 days (or sooner, depending on the culture conditions and the specific host cells used), the level of production generally decreases significantly. Generally, the time of culture is measured in terms of virus production. For example, in the case of AAV, virus production generally begins when helper virus functions are provided in suitable host cells as described herein. Generally, cells are harvested about 48 to about 100 hours after helper virus infection (or the initiation of virus production), preferably about 48 to about 96 hours, preferably about 72 to about 96 hours, preferably about 68 to about 72 hours.

[0287] rAAV production cultures can be grown under a variety of conditions (over a wide temperature range, for various periods of time, etc.) appropriate for the particular host cells being utilized. rAAV production cultures include attachment-dependent cultures that can be cultured in suitable attachment-dependent vessels, such as roller bottles, hollow fiber filters, microcarriers, and packed-bed or fluidized-bed bioreactors. rAAV vector production cultures can also include suspension-adapted host cells, such as HeLa, 293, and SF-9 cells, which can be cultured in a variety of ways, including, for example, spinner flasks, stirred-tank bioreactors, and disposable systems, such as the Wave bag system.

[0288] Suitable media known in the art can be used for the production of rAAV virions. These media include, but are not limited to, media produced by Hyclone Laboratories and JRH, including modified Eagle's medium (MEM) and Dulbecco's modified Eagle's medium (DMEM), each of which is hereby incorporated by reference in its entirety. In certain embodiments, rAAV production culture media can be supplemented with serum or serum-derived recombinant proteins at levels of 0.5% to 20% (v / v or w / v). Alternatively, rAAV vectors can be produced in serum-free conditions, which may also be referred to as animal-derived product-free media.

[0289] After culturing host cells to allow AAV virion production, the resulting virions can be collected and purified.In certain embodiments, AAV virions can be obtained from the host cells of production culture by (1) lysis of host cells, and / or (2) from the culture medium of these cells after a certain period of time, preferably 72 hours after transfection.rAAV virions can be collected from the spent medium from production culture, provided that the cells are cultured under conditions that cause the release of rAAV virions from intact cells into the medium (see, for example, U.S. Patent No. 6,566,118).Suitable methods for lysing cells are also known in the art, and include, for example, multiple freeze / thaw cycles, sonication, microfluidization, and treatment with chemicals, such as detergents and / or proteases.

[0290] After recovery, the rAAV virions can be purified. As used herein, the term "purified" includes preparations of rAAV virions that lack at least some of the other components that may also be present in the location where the rAAV virions naturally occur or from which they were originally prepared. Thus, for example, purified rAAV virions can be prepared using isolation techniques to enrich them from a source mixture, such as a culture lysate or production culture supernatant. Enrichment can be measured in various ways, such as by the percentage of DNase-resistant particles (DRPs) or genome copies (gc) present in the solution, or by infectivity, or can be measured relative to contaminants present in the source mixture, including second, potentially interfering substances, such as production culture contaminants or in-process contaminants, including helper viruses, media components, etc.

[0291] In certain embodiments, rAAV production culture harvests may be clarified to remove host cell debris, hi some embodiments, production culture harvests may be clarified using a variety of standard techniques, such as centrifugation or filtration through filters with a pore size of 0.2 μm or larger (e.g., cellulose acetate filters or a series of depth filters).

[0292] In certain embodiments, the rAAV production culture harvest is further treated with Benzonase™ to digest any high molecular weight DNA present in the production culture. In some embodiments, Benzonase™ digestion is carried out under standard conditions, e.g., at a final concentration of 1-2.5 units / ml of Benzonase™ at temperatures ranging from ambient to 37°C for a period of 30 minutes to several hours.

[0293] In certain embodiments, rAAV virions can be isolated or purified using one or more of the following purification steps: equilibrium centrifugation; flow-through anion exchange filtration; tangential flow filtration (TFF) to concentrate rAAV particles; rAAV capture by apatite chromatography; heat inactivation of helper virus; rAAV capture by hydrophobic interaction chromatography; buffer exchange by size exclusion chromatography (SEC); nanofiltration; and rAAV capture by anion exchange chromatography, cation exchange chromatography, or affinity chromatography. These steps can be used alone, in various combinations, or in different orders. Methods for purifying rAAV particles can be found, for example, in Xiao et al., (1998) Journal of Virology 72:2224-2232; U.S. Patent Nos. 6,989,264 and 8,137,948; and WO2010 / 148143.

[0294] In certain embodiments, purified AAV virions can be dialyzed against PBS, filtered, and stored at −80° C. The titer of the viral genome can be determined by quantitative PCR using linearized plasmid DNA as a standard curve (e.g., Lockheed Martin, NY). M, et al., Hum. Gene Ther. 2010; 21:1273-1285). Pharmaceutical Composition

[0295] In certain embodiments, the present application provides a composition comprising a sequence encoding eTF and a pharmaceutically acceptable carrier. In other embodiments, the present application provides a virion comprising a sequence encoding eTF and a pharmaceutically acceptable carrier. In exemplary embodiments, such a composition is suitable for gene therapy applications. The pharmaceutical composition is preferably sterile and stable under the conditions of manufacture and storage. A sterile solution can be achieved, for example, by filtration through a sterile filtration membrane.

[0296] Acceptable carriers and excipients in pharmaceutical compositions are preferably non-toxic to recipients at the dosages and concentrations used.Acceptable carriers and excipients may include buffers such as phosphate, citrate, HEPES, and TAE, antioxidants such as ascorbic acid and methionine, preservatives such as hexamethonium chloride, octadecyldimethylbenzyl ammonium chloride, resorcinol, and benzalkonium chloride, proteins such as human serum albumin, gelatin, dextran, and immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, histidine, and lysine, and carbohydrates such as glucose, mannose, sucrose, and sorbitol.The pharmaceutical composition of the present disclosure can be administered parenterally in the form of an injectable formulation.Pharmaceutical compositions for injection can be formulated using a sterile solution or any pharmaceutically acceptable liquid as a vehicle. Pharmaceutically acceptable vehicles include, but are not limited to, sterile water and physiological saline.

[0297] The pharmaceutical compositions of the present disclosure can be prepared in microcapsules, such as hydroxymethylcellulose or gelatin microcapsules and polymethylmethacrylate microcapsules.The pharmaceutical compositions of the present disclosure can also be prepared in other drug delivery systems, such as liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules.The pharmaceutical compositions for gene therapy can be in an acceptable diluent or can comprise a sustained-release matrix in which the gene delivery vehicle is embedded.

[0298] The pharmaceutical compositions provided herein may be formulated for parenteral, subcutaneous, intravenous, intramuscular, intraarterial, intraparenchymal, intrathecal, intracisternal, intraventricular, or intraperitoneal administration. Pharmaceutical compositions may also be formulated for or administered via nasal, spray, oral, aerosol, rectal, or vaginal administration. In one embodiment, the pharmaceutical compositions provided herein are administered to the CNS or cerebrospinal fluid (CSF), i.e., by intraparenchymal, intrathecal, intracisternal, or intraventricular injection. Tissue targets may be specific, such as the CNS, or may be a combination of several tissues, e.g., muscle and CNS tissues. Exemplary tissues or other targets may include the liver, skeletal muscle, cardiac muscle, fat deposits, kidney, lung, vascular endothelium, epithelium, hematopoietic cells, CNS, and / or CSF. In a preferred embodiment, the pharmaceutical compositions provided herein comprising eTFs that upregulate SCN1A or GRN are administered to the CNS or CSF by intraparenchymal, intrathecal, intracisternal, or intracerebroventricular injection. One or more of these methods can be used to administer the pharmaceutical compositions of the present disclosure.

[0299] In certain embodiments, the pharmaceutical compositions provided herein comprise an "effective amount" or "therapeutically effective amount." As used herein, such an amount refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result, e.g., increasing the level of SCN1A expression and / or reducing the frequency and / or duration of seizures, or increasing the level of GRN expression and / or treating a GRN-related disease or disorder, e.g., FTD.

[0300] The dosage of the pharmaceutical composition of the present disclosure depends on factors including the route of administration, the disease being treated, and the subject's physical characteristics (e.g., age, weight, overall health). The dosage can be adjusted to provide an optimal therapeutic response. Typically, the dosage can be an amount that effectively treats the disease without inducing significant toxicity. In one embodiment, the AAV vectors provided herein are administered in a dose of 5×10 11 ~1×10 14 In a more specific embodiment, the AAV vector may be administered to a patient for treatment of an SCN1A deficiency (including, e.g., Dravet syndrome) or a GRN deficiency (including, e.g., FTD) in an amount or dose in the range of gc / kg (genome copies per kilogram of patient body weight (gc / kg)). 11 gc / kg ~ approx. 3×10 13 gc / kg or approximately 1 x 10 12 ~Approx. 1×10 14 gc / kg or approximately 1 x 10 12 ~Approx. 1×10 13 An amount within the range of gc / kg, or approximately 5 × 10 11 gc / kg, 1 × 10 12 gc / kg, 1.5 × 10 12 gc / kg, 2.0 × 10 12 gc / kg, 2.5 × 10 12 gc / kg, 3 × 10 12 gc / kg, 3.5 × 10 12 gc / kg, 4 × 10 12 gc / kg, 4.5 × 10 12 gc / kg, 5 × 10 12gc / kg, 5.5 × 10 12 gc / kg, 6 × 10 12 gc / kg, 6.5 × 10 12 gc / kg, 7 × 10 12 gc / kg, 7.5 × 10 12 gc / kg, 8 × 10 12 gc / kg, 8.5 × 10 12 gc / kg, 9 × 10 12 gc / kg or 9.5 x 10 12 The dose is administered in gc / kg. gc / kg can be determined, for example, by qPCR or digital droplet PCR (ddPCR) (see, for example, M. Locke et al., J. Med. Soc. 2014). (See, e.g., et al, Hum Gene Ther Methods. 2014 Apr;25(2):115-25). In another embodiment, the AAV vectors provided herein are administered at a concentration of 1 x 10 9 ~1×10 11 The vector may be administered to a patient for treatment of SCN1A deficiency (including, for example, Dravet syndrome) in an amount or dose in the range of 1×10 iu / kg (infectious units (iu) of vector per kg of subject or patient body weight). In certain embodiments, the pharmaceutical composition may be formed into unit doses as needed. Such a single dosage unit may be about 1×10 9 gc ~ approx. 1×10 15 It may contain gc.

[0301] The pharmaceutical compositions of the present disclosure can be administered to a subject in need thereof, for example, one or more times (e.g., 1 to 10 or more times) daily, weekly, monthly, twice a year, yearly, or as medically necessary. In exemplary embodiments, a single administration is sufficient. In one embodiment, a pharmaceutical composition comprising an expression cassette encoding an eTF that upregulates SCN1A or GRN is suitable for use in a human subject and is administered by intraparenchymal, intrathecal, intracisternal, or intracerebroventricular injection. In one embodiment, the pharmaceutical composition is delivered via a peripheral vein by bolus injection. In other embodiments, the pharmaceutical composition is delivered via a peripheral vein by infusion over about 10 minutes (±5 minutes), about 20 minutes (±5 minutes), about 30 minutes (±5 minutes), about 60 minutes (±5 minutes), or about 90 minutes (±10 minutes).

[0302] In another aspect, the present application further provides kits comprising the nucleic acid molecules, vectors, host cells, virions, or pharmaceutical compositions described herein in one or more containers. The kits may include instructions or packaging materials describing how to administer the nucleic acid molecules, vectors, host cells, or virions contained within the kit to a patient. The containers of the kits may be of any suitable material, such as glass, plastic, metal, etc., and of any suitable size, shape, or configuration. In certain embodiments, the kits may include one or more ampoules or syringes containing the nucleic acid molecules, vectors, host cells, virions, or pharmaceutical compositions in a suitable liquid or solution form. Treatment method

[0303] In various embodiments, the present application provides methods for using the eTFs disclosed herein. In certain embodiments, the present application provides methods for administering an expression cassette, expression vector, or viral particle comprising a polynucleotide encoding the eTFs disclosed herein to modulate the expression of a gene of interest in a cell. In certain embodiments, the eTFs can upregulate the expression of a gene of interest. In other embodiments, the eTFs can downregulate the expression of a gene of interest. In various embodiments, the eTFs can be used to modulate the expression of a gene of interest in a cell in vitro, in vivo, or ex vivo.

[0304] In certain embodiments, the present application provides a method for treating a disease or disorder associated with a gene of interest by administering to a subject in need thereof an expression cassette, expression vector, or viral particle comprising a polynucleotide encoding an eTF that modulates expression of the gene of interest. In exemplary embodiments, the disease or disorder is associated with haploinsufficiency of the gene of interest, and the expression cassette, expression vector, or viral particle comprises a polynucleotide encoding an eTF that upregulates expression of the gene of interest. In certain embodiments, the method of treating a disease or disorder comprises administering an expression cassette, expression vector, or viral particle comprising a polynucleotide encoding an eTF that modulates expression of an endogenous gene of interest associated with such disease or disorder, such that overexpression or underexpression of such gene is corrected by standard medical care to fall within the level range of healthy individuals or within a defined normal range. In certain embodiments, the methods disclosed herein are used to treat a disease or disorder associated with an endogenous gene that contains one or more mutations that result in aberrant expression of the gene. In some cases, such endogenous genes are genes that are too large to be delivered as a transgene in gene therapy, or that are poorly expressed when delivered as a transgene.

[0305] In certain embodiments, the present application provides methods for ameliorating symptoms associated with a disease or disorder by administering to a subject in need thereof an expression cassette, expression vector, or viral particle comprising a polynucleotide encoding an eTF that modulates the expression of a gene associated with such disease or disorder.

[0306] In an exemplary embodiment, the present application provides a method for treating a disease, disorder, or symptom associated with a mutation in SCN1A, a defect in Nav1.1, and / or reduced activity of Nav1.1, by administering to a subject in need thereof an expression cassette, expression vector, or viral particle containing a polynucleotide encoding an eTF that upregulates the expression of the SCN1A gene or its protein product, Nav1.1. Voltage-gated sodium ion channels are important for the generation and propagation of action potentials in striated muscle and nervous tissue. Voltage-gated sodium ion channels are heteromeric complexes consisting of one large, central pore-forming glycosylated alpha subunit and two smaller auxiliary beta subunits. The large alpha subunit, Nav1.1, encoded by the SCN1A gene has been associated with various diseases or disorders, such as Dravet syndrome. Nav1.1 is expressed in neurons and can assemble with various beta subunits, including Navβ1, expressed by the SCN1B gene.

[0307] In certain embodiments, the present application provides methods for treating diseases associated with mutations in SCN1A or reduced activity of Nav1.1 using eTFs that upregulate expression of the endogenous SCN1A gene. Diseases and disorders associated with SCN1A mutations include, but are not limited to, Dravet syndrome, Ohtahara syndrome, epilepsy, early infantile epileptic encephalopathy 6 (EIEE6), familial febrile seizures 3A (FEB3A), intractable childhood epilepsy with generalized tonic-clonic seizures, and other conditions. seizures ) (ICEGTC), familial hemiplegic migraine (migraine, familial hemiplegic type 3 (FHM3), Panayiotopoulos syndrome, familial atrial fibrillation type 13 (ATFB13), generalized epilepsy with febrile seizures plus type 1 SCN1A mutations or abnormalities have also been associated with seizure disorders, epilepsy, autism, familial hemiplegic migraine type 3 (FHM3), genetic epilepsy with febrile seizures plus (GEFS+), and the effectiveness of certain antiepileptic medications. For example, the ICS5N+5G>A mutation in SCN1A is associated with the maximum safe dose of the antiepileptic drugs phenytoin and carbamazepine.

[0308] In certain embodiments, the present application provides a method for treating a subject with or at risk of developing Dravet syndrome by administering an expression cassette, expression vector, or viral particle containing a polynucleotide encoding an eTF that upregulates SCN1A. Dravet syndrome is characterized by prolonged febrile and non-febrile seizures within the first year of a child's life. The disease progresses to other seizure types, such as myoclonic and partial seizures, psychomotor retardation, and ataxia. It is characterized by cognitive dysfunction, behavioral disorders, and motor disorders. Behavioral disorders often include hyperactivity and impulsivity, and in rarer cases, autistic-like behavior. Dravet syndrome is also associated with sleep disorders, including somnolence and insomnia. In many patients, Dravet syndrome is caused by genetic mutations that result in the production of nonfunctional proteins. Treating disorders associated with genetic causes presents many challenges. Therefore, most existing treatments have focused on preventive medical management of seizures and other symptoms.

[0309] In 70–90% of patients, Dravet syndrome is caused by a nonsense mutation in the SCN1A gene, which results in a premature stop codon and therefore a nonfunctional protein. Typically, a missense mutation in either the S5 or S6 segment of the sodium channel pore results in loss of channel function and the development of Dravet syndrome. Heterozygous inheritance of an SCN1A mutation is all that is required to produce a defective sodium channel; patients with Dravet syndrome still have one normal copy of the gene. Therefore, the disease is characterized as one of haploinsufficiency, and increasing the expression of a functional copy of SCN1A can restore normal levels of Nav1.1 production.

[0310] Symptoms associated with Dravet syndrome include seizures, memory impairment, developmental delay, poor muscle tone and / or cognitive problems. Treatment with the expression cassette, expression vector, or viral particle described herein can result in the improvement of one or more symptoms, for example, a reduction in the number, duration, and / or intensity of seizures. Administration of the gene therapy described herein to a subject at risk of developing Dravet syndrome can prevent the onset or slow the progression of one or more symptoms of Dravet syndrome.

[0311] In certain embodiments, treatment with an expression cassette, expression vector, or viral particle comprising a polynucleotide encoding an eTF that upregulates SCN1A as described herein reduces the duration and / or frequency of seizures, e.g., seizures associated with Dravet syndrome, by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, %, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more reduction.

[0312] In some Alzheimer's patients, the production of amyloid beta (Aβ), which involves many peptides and proteases, can affect neuronal excitability, leading to seizures and downregulation of the Nav1.1 sodium channel in PV neurons. In another embodiment, the present application provides a method for treating a subject suffering from Alzheimer's disease by administering an expression cassette, expression vector, or viral particle described herein, which contains a polynucleotide encoding an eTF that upregulates SCN1A. Symptoms associated with Alzheimer's disease include short-term memory loss, cognitive difficulties, seizures, and difficulties with language, executive function, perception (agnosia), and motor execution (apraxia). Treatment with an expression cassette, expression vector, or viral particle containing a polynucleotide encoding an eTF that upregulates SCN1A can result in the improvement of one or more Alzheimer's disease symptoms, such as a reduction in the progression of memory loss or the prevention of one or more symptoms. In some cases, treatment can result in the modification of high gamma power brain activity. Treatment may result in at least a 10%, 20%, 30%, 40%, 50%, 60%, 70% or greater reduction in seizure frequency and / or seizure severity, or a reduction in high gamma power activity, compared to no treatment. In some cases, treatment may result in an improvement in cognitive function. Learning and / or memory may be improved by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or greater than 100%, compared to no treatment or before treatment with a polynucleotide encoding an eTF that upregulates SCN1A as disclosed herein.

[0313] In some cases, treatment with an expression cassette, expression vector, or viral particle comprising a polynucleotide encoding an eTF that upregulates SCN1A reduces high gamma power activity (e.g., high gamma power activity associated with Alzheimer's disease) by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or more compared to an untreated control or compared to pre-treatment levels. , 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% reduction.

[0314] Parkinsonism refers to a collection of signs and symptoms found in Parkinson's disease (PD), including slowness (bradykinesia), stiffness (rigidity), tremor and imbalance (postural instability).In some cases, administering to a subject at risk of developing Parkinson's disease or a subject suffering from Parkinson's disease, an expression cassette, an expression vector or a viral particle comprising a polynucleotide encoding the eTF described herein that upregulates SCN1A can prevent the onset of one or more symptoms, or slow down the progression of Parkinson's disease by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% compared with no treatment.

[0315] In certain embodiments, the present application provides a method that can be used to treat a subject at risk of developing a disease. The subject may be known to be susceptible to a disease, such as a neurological disease, or a disease associated with epilepsy, seizures, and / or encephalopathy. The subject may be susceptible to a disease due to genetic events or known risk factors. For example, the subject may have a mutation in SCN1A associated with Dravet syndrome. In some cases, the subject may be susceptible to a disease such as Alzheimer's disease due to the subject's age. In some cases, the subject may have an insufficient amount of SCN1A protein, and treating the SCN1A-related disease involves administering an expression cassette, expression vector, or viral particle comprising a polynucleotide encoding an eTF that upregulates endogenous SCN1A, as described herein.

[0316] In certain embodiments, the treatment using the expression cassette, expression vector or viral particle comprising the polynucleotide encoding the eTF that upregulates endogenous SCN1A provided herein can reduce or halt the symptoms associated with Dravet syndrome or other SCN1A-related diseases or disorders.For example, the treatment can improve learning, memory, cognitive function and / or motor function; can reduce the frequency and / or duration of seizures; and / or reduce temperature sensitivity (or increase the temperature threshold that induces seizures).

[0317] In another exemplary embodiment, the present application provides a method for treating a disease or disorder associated with a mutation in the GRN gene or a deficiency or reduced activity in the GRN protein by administering to a subject in need thereof an expression cassette, expression vector, or viral particle comprising a polynucleotide encoding an eTF that upregulates expression of the GRN gene or protein product.

[0318] Progranulin is a secreted cysteine-rich glycoprotein with growth factor-like properties and belongs to the granulin protein family. Granulins (e.g., granulins A-G and paragranulin) play roles in angiogenesis, wound repair, cell proliferation, and inflammation. Mutations in the progranulin gene or defects in secreted progranulin are associated with various neurodegenerative and metabolic diseases. Deficiency of secreted granulin in the central nervous system (CNS) can lead to neurodegeneration, including frontotemporal lobar degeneration (FTLD); frontotemporal degeneration or frontotemporal dementia (FTD), an early-onset neurodegenerative disease associated with partial progranulin deficiency; progressive non-fluent aphasia (PNFA); semantic dementia; Parkinson's disease; Alzheimer's disease; and neuronal ceroid lipofuscinosis (NCL), which is associated with complete deficiency of progranulin. FTD refers to a group of complex neurodegenerative disorders characterized by progressive deterioration of the frontotemporal lobes, resulting in changes in behavior and / or speech, e.g., loss of motor coordination, social dysfunction, memory loss, and muscle tremors.

[0319] Generally, patients with one or more pathogenic mutations in one copy of the progranulin gene develop FTD, a subtype of FTLD. Clinical symptoms of FTLD include behavioral frontotemporal dementia (FTD), characterized by atrophy of the prefrontal and / or anterior temporal lobes, social and executive dysfunction, semantic dementia, and primary progressive non-fluent aphasia with early language impairment. FTD is an adult-onset behavioral disorder followed by frontal lobe dementia, parkinsonism, and muscle atrophy.

[0320] Progranulin has also been linked to metabolic diseases and is recognized as an adipokine involved in diet-induced obesity and insulin resistance. Mutations or defects in progranulin have been associated with atherosclerosis, a progressive disease characterized by a strong inflammatory component and thickening of the arterial wall due to lipid accumulation and cellular proliferation.

[0321] Mutations in GRNs can include nonsense mutations, splice site mutations, insertions and deletions that result in shifts in the normal reading frame, and various point mutations. Nonsense, splice site, and frameshift mutations can lead to haploinsufficiency due to nonsense-mediated mRNA decay or nuclear degradation of the transcript. Missense mutations have been observed in some patients with sporadic FTLD, Alzheimer's disease, and amyotrophic lateral sclerosis (ALS). Various mutations in GRNs associated with diseases or disorders are known, including mutations in the GRN coding region, such as -8+5G>C; -8+3A>T; 2T>C, 3G>A, 26CA(A9D); 63_64insC; 90_91insCTGC; 102ΔC; 138+1G>A; 154ΔA; 234_235ΔAG; 243ΔC; 361ΔG; 373C>T; 380_381ΔCT; 384_387ΔTAGT; 388_391ΔCAGT; 463-1G>A; 468_474ΔCTGCTGT; 675_676ΔCA; 708+1G>A; 707+1G>C; 709-2A>G; 759_760ΔTG; 813_816ΔCACT; 835_835+1insCTGA; 836-1G>C; 909ΔC; 910_911insTG; 911G>A; 933+1G>A; 942C>A; 998ΔG; 1095_1096ΔCT; 1145ΔC; 1157G>A; 1201C>T; 1231_1232ΔGT; 1232_1233insGT; 1252C>T; 1395_1296insC; 1402C>T; 1414-15_1590Δ; 1477C>T; or combinations thereof. See Eriksen JL, Mackenzie IR. Progranulin: normal function and role in neurodegeneration. J Neurochem. 2008 Jan;104(2):287-97. Currently, there is no cure for such GRN-related neurodegenerative and metabolic diseases. Treatment options that target GRNs, such as therapies that increase progranulin levels and / or function in vivo, are needed.

[0322] In certain embodiments, the expression cassette, expression vector or viral particle comprising the polynucleotide that encodes the eTF that upregulates endogenous GRN expression described herein can be used to treat the subject in need thereof, and the subject has any one or more of the GRN mutations listed above.In some cases, the subject in need thereof comprises haploinsufficiency in GRN or GRN defect.In some cases, the subject in need thereof has or is at risk of FTD, Alzheimer's disease, Parkinson's disease and / or atherosclerosis.

[0323] In certain embodiments, an expression cassette, expression vector, or viral particle comprising a polynucleotide encoding an eTF that upregulates endogenous GRN expression as described herein can be administered to a subject with FTD, Alzheimer's disease, Parkinson's disease, and / or atherosclerosis, or a subject at risk of developing FTD, Alzheimer's disease, Parkinson's disease, and / or atherosclerosis.In another example, treatment can be administered to a subject suffering from FTD, Alzheimer's disease, Parkinson's disease, and / or atherosclerosis.Treatment with an expression cassette, expression vector, or viral particle comprising a polynucleotide encoding an eTF that upregulates endogenous GRN expression as described herein can result in the improvement of one or more symptoms associated with FTD, Alzheimer's disease, Parkinson's disease, and / or atherosclerosis, for example, a reduction in one or more symptoms associated with behavioral and / or speech changes caused by FTD, or a reduction in the thickness of arterial walls in atherosclerosis. Other symptoms associated with GRN-related central nervous system disorders include, for example, the presence of Lewy bodies, haploinsufficiency of progranulin (GRN), lack of social interaction, lysosomal abnormalities, loss of motor coordination, and muscle tremors.In certain embodiments, treatment can cause at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70% recovery of GRN expression level or function compared with wild-type GRN.In certain embodiments, treatment can cause at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, or more than 100% improvement in cognitive function, for example, speech and / or memory improvement, compared with before treatment. In certain embodiments, treatment with an expression cassette, expression vector, or viral particle comprising a polynucleotide encoding an eTF that upregulates endogenous GRN expression as described herein can delay or halt the progression of symptoms associated with FTD, Alzheimer's disease, Parkinson's disease, and / or atherosclerosis.In some cases, treatment with an expression cassette, expression vector or viral particle comprising a polynucleotide encoding an eTF that upregulates endogenous GRN expression as described herein can increase cell repair in the CNS, frontal cortex or striatum by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100% compared with no treatment, or can reverse cell damage in the CNS, frontal cortex or striatum by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100%.In some cases, treating GRN-related central nervous system disorders includes reducing the frequency and / or severity of muscle tremors.

[0324] In certain embodiments, expression cassettes, expression vectors, or viral particles comprising polynucleotides encoding eTFs that upregulate endogenous GRN expression as described herein can be used to treat subjects diagnosed with diseases, such as neurological or neurodegenerative diseases or metabolic diseases associated with GRN.The subjects can be patients suffering from FTD, Alzheimer's disease, Parkinson's disease, and / or atherosclerosis.In some embodiments, the subjects are patients with FTD.The subjects can be patients suffering from GRN-related diseases or conditions, such as dementia or metabolic diseases.In some cases, genetic testing can be used to screen patients for mutations in the GRN gene.If a patient has a pathogenic mutation in GRN, expression cassettes, expression vectors, or viral particles comprising polynucleotides encoding eTFs that upregulate endogenous GRN expression as described herein can be used to prevent, alleviate, or treat the progression of neurodegenerative or metabolic diseases, such as FTD, Alzheimer's disease, Parkinson's disease, and / or atherosclerosis.

[0325] In certain embodiments, the subject treated with the expression cassette, expression vector or viral particle comprising the polynucleotide encoding the eTF that upregulates endogenous GRN expression described herein is a subject diagnosed with a mutation or genetic abnormality in GRN. Such a mutation can be homozygous or heterozygous, or an abnormal insertion, deletion or substitution of one or more bases in the GRN gene, which affects the expression, secretion, solubility, activity and / or proteolytic cleavage of the protein in vivo.

[0326] In certain embodiments, expression cassettes, expression vectors, or viral particles comprising polynucleotides encoding eTFs that upregulate endogenous GRN expression as described herein can be used to treat subjects with or at risk of neurodegenerative or metabolic diseases associated with GRN mutations and / or defects, such as FTLD, FTD, progressive non-fluent aphasia, semantic dementia, Parkinson's disease, Alzheimer's disease, NCL, diabetes, or atherosclerosis, to increase GRN expression and / or function in vivo to treat, prevent, or reduce the effects of the disease. In some cases, expression cassettes, expression vectors, or viral particles comprising polynucleotides encoding eTFs that upregulate endogenous GRN expression as described herein can be used to treat, alleviate, reduce, or manage symptoms of such diseases, such as speech dysfunction, social deficits, impaired motor skills or coordination, muscle atrophy, muscle tremors, neurological atrophy, memory loss, parkinsonism, and / or lysosomal abnormalities. In some cases, such compositions are used to reduce inflammation, reduce cell death, reduce neuronal atrophy, reduce motor neuron atrophy, and / or increase cell repair in the CNS, or particularly in the frontal cortex or striatum.

[0327] In certain embodiments, the expression cassette, expression vector, or viral particle comprising the polynucleotide encoding the eTF described herein that upregulates endogenous GRN expression can be used to treat subjects at risk of developing neurodegenerative or metabolic diseases.Subjects can be known to be prone to diseases, such as neurological or metabolic diseases, through various screening or diagnostic methods, including methods for detecting genetic mutations in GRN or blood tests for secreted GRN protein levels.Subjects can be prone to diseases due to genetic events or known risk factors.For example, subjects can have mutations in GRN associated with FTD, Alzheimer's disease, Parkinson's disease, and / or atherosclerosis.In some cases, subjects can have insufficient amounts of GRN protein or its isoform.For example, subjects can be known to have insufficient amounts of GRN protein.

[0328] The terms "subject" and "individual" are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. The methods described herein may be useful in human therapy, veterinary applications, and / or preclinical studies in animal models of a disease or condition. In various embodiments, the subject that can be treated according to the methods described herein is a mammal, such as a mouse, rat, hamster, guinea pig, gerbil, cow, sheep, pig, goat, donkey, horse, dog, cat, llama, monkey (e.g., macaque, e.g., rhesus monkey or cynomolgus monkey), or human. In an exemplary embodiment, the subject is a human.

[0329] The following table provides the sequences disclosed herein. Table 1: Genomic loci and protein sequences of SCN1A and GRN [Table 1-1] [Table 1-2] Table 2: Sequences of eTFs with high sequence identity to human EGR1 / EGR3 [Table 2-1] [Table 2-2] [Table 2-3] Table 3: DBD sequences of the eTFs disclosed in Table 2 [Table 3-1] [Table 3-2] Table 4: Target site sequences recognized by the eTFs disclosed in Table 2 [Table 4-1] [Table 4-2] Table 5: Different types of zinc finger structures and proteins for generating eTFs [Table 5-1] [Table 5-2] Table 6: Examples of full-length amino acid sequences of eTFs [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5] [Table 6-6] Table 7: DBD sequences of the eTFs disclosed in Table 6 [Table 7-1] [Table 7-2] Table 8: Amino acid sequences of exemplary TADs that can be fused to any DBD of an eTF disclosed herein [Table 8] Table 9: Percent sequence identity between TADs and protein platforms [Table 9-1] [Table 9-2] Table 10: Examples of eTFs and their target site sequences [Table 10] Table 11: Examples of dSaCas9 protein sequences and guide RNAs for targeting target sites capable of upregulating the endogenous SCN1A gene [Table 11] Table 12: Examples of zinc fingers that can be combined in an eTF to modulate the SCN1A gene [Table 12-1] [Table 12-2] Table 13: Examples of DBDs of eTFs for modulating SCN1A [Table 13-1] [Table 13-2] [Table 13-3] Table 14: Examples of DNA-binding domains containing multiple zinc fingers that modulate SCN1A [Table 14] Table 15: Example target site sequences for modulating SCN1A [Table 15-1] [Table 15-2] Table 16: Exemplary eTF nucleic acids disclosed herein that can upregulate SCN1A expression array [Table 16-1] [Table 16-2] [Table 16-3] [Table 16-4] [Table 16-5] [Table 16-6] [Table 16-7] [Table 16-8] [Table 16-9] [Table 16-10] [Table 16-11] [Table 16-12] [Table 16-13] [Table 16-14] Table 17: Examples of eTFs that upregulate the expression of SCN1A [Table 17] Table 18: Sequences of eTFs that upregulate the expression of SCN1A [Table 18-1] [Table 18-2] [Table 18-3] [Table 18-4] [Table 18-5] [Table 18-6] Table 19: PolyA sequences disclosed herein [Table 19] Table 20: Examples of zinc fingers for modulating expression of endogenous GRN genes [Table 20] Table 21: Examples of DBDs of eTFs that modulate the expression of GRNs, including multiple ZFs [Table 21-1] [Table 21-2] Table 22: Examples of target site sequences for modulating the expression of GRNs [Table 22] Table 23: Examples of eTFs that modulate the expression of GRNs [Table 23-1] [Table 23-2] Table 24: Examples of regulatory elements (REs) disclosed herein [Table 24-1] [Table 24-2] [Table 24-3] Table 25: Additional sequences disclosed herein [Table 25-1] [Table 25-2] [Table 25-3] [Table 25-4] Table 26: SCN1A genomic regions targeted by gRNAs and dCas proteins [Table 26] Table 27: Examples of SCN1A genomic regions targeted by zinc finger eTFs [Table 27] Table 28: Amino acid sequences of eTFs that recognize SCN1A target sites [Table 28] Table 29: Amino acid sequences of DBDs of eTFs [Table 29] Table 30: GRN target regions and eTFs for upregulating expression of GRN [Table 30-1] [Table 30-2] [Table 30-3] [Table 30-4] Table 31: Exemplary DBDs of eTFs for upregulating GRN expression [Table 31-1] [Table 31-2] [Example]

[0330] The following examples are included to further describe some aspects of the disclosure and should not be used to limit the scope of the invention. Example 1 Increased gene expression in HEK293T cells

[0331] HEK293T cells were transfected with plasmid DNA containing the luciferase gene under the control of one of several different regulatory elements: a promoterless control, SCP, CMV, SEQ ID NO:178 operably linked to minCMV, SEQ ID NO:182 operably linked to minCMV, and CAG. Normalized luciferase values ​​from each construct are shown in Figure 1. Size-normalized activity values ​​from each construct are shown in Figure 2. Constructs with the minCMV promoter linked to the regulatory element of SEQ ID NO:178 or 182 drove higher levels of luciferase expression than minCMV alone and SCP alone.

[0332] This experiment showed that SEQ ID NOs: 178 and 182 are REs that drive high gene expression in cells. Such REs can be added to the expression cassettes disclosed herein, including non-naturally occurring transcriptional activators of SCN1A, to increase the expression of the transcriptional activator in cells, resulting in increased expression of the endogenous SCN1A gene. In some cases, one or more REs selected from SEQ ID NOs: 178 and / or 182 are operably linked to a transcriptional activator that increases SCN1A expression in cells. Such regulatory elements can be added to the expression cassette upstream and / or downstream of the transcriptional activator in the expression cassette. Example 2 Selectivity for parvalbumin neurons

[0333] Selectivity for parvalbuminergic neurons, a type of GABAergic neuron, was determined using fluorescence imaging. AAV vectors containing eGFP operably linked to either a control promoter (EF1α) or a PV-selective RE, and an AAV vector containing the CRE-dependent tdTomato, were co-injected into GAD2-IRES-CRE mice (Jackson Labs).

[0334] Mice were injected with 1.5 μL of AAV vector (5 12 ~1 13A 1000-µL (gc / ml) solution was infused bilaterally into the dorsal and ventral hippocampi at a rate of 0.3 µL / min with a 4-minute rest period following the injection. Mice were anesthetized using isoflurane (2%, 800 mL / min O2) for the injection. Bupivacaine / epinephrine was used for local analgesia, and carprofen was used for intraoperative and postoperative analgesia. Animals were positioned in a stereotaxic frame (Kopf Instruments, USA) using the following coordinates for the dorsal hippocampus (AP-2.0 mm from the dura, ±1.5 mm lateral, DV-1.4 mm) and ventral hippocampus (AP-3.1 mm from the dura, ±2.8 mm lateral, DV-3.8 mm). A Hamilton syringe (model number 80308, 10 µL syringe and corresponding 30-gauge blunt-tip needle) was used with a stereotaxic micromanipulator to designate and drill a burr hole. The drill was used only to penetrate the bone. After drilling, the infusion cannula was lowered to the desired depth within the brain. The injection settings for the Ultra Micro Pump III (World Precision Instruments) with a Micro 4 controller were: injection volume: 1.5 μL, infusion rate: 0.3 μL / min. The needle was gradually lowered (over approximately 1 minute) to DV-0.1 mm under the injection coordinates (-1.5 mm for the dorsal hippocampus and -3.9 mm for the ventral hippocampus, respectively, then elevated to DV-1.4 or -3.8). Before injection, the needle was allowed to equilibrate for 1 minute. Upon completion of delivery, the needle was left in place for 4 minutes and then withdrawn over approximately 1 minute. After all four injections were completed, the skin incision was closed with sutures. Treated mice underwent daily health checks for the remainder of the study and were weighed weekly to monitor body weight.

[0335] For tissue collection, mice were euthanized via an overdose of isoflurane and perfused with 4% paraformaldehyde (PFA). Fragments of brain tissue containing the hippocampus were harvested and placed in 4% PFA at 4°C for at least 12 hours. The brain tissue was then dehydrated in 30...

Claims

1. 1. A polynucleotide comprising a nucleic acid sequence encoding a non-naturally occurring DNA binding protein, said non-naturally occurring DNA binding protein comprising a DNA binding domain, said DNA binding domain being a zinc finger DNA binding domain comprising SEQ ID NO: 165, and said DNA binding domain binds to a target site comprising SEQ ID NO:

331.

2. 1. A polynucleotide comprising a nucleic acid sequence encoding a non-naturally occurring DNA binding protein, said non-naturally occurring DNA binding protein comprising a DNA binding domain, said DNA binding domain being a zinc finger DNA binding domain comprising SEQ ID NO: 166, and said DNA binding domain binds to a target site comprising SEQ ID NO:

332.

3. 1. A polynucleotide comprising a nucleic acid sequence encoding a non-naturally occurring DNA binding protein, said non-naturally occurring DNA binding protein comprising a DNA binding domain, said DNA binding domain being a zinc finger DNA binding domain comprising SEQ ID NO: 167, and said DNA binding domain binds to a target site comprising SEQ ID NO:

333.

4. 1. A polynucleotide comprising a nucleic acid sequence encoding a non-naturally occurring DNA binding protein, said non-naturally occurring DNA binding protein comprising a DNA binding domain, said DNA binding domain being a zinc finger DNA binding domain comprising SEQ ID NO: 169, and said DNA binding domain binds to a target site comprising SEQ ID NO:

334.

5. 1. A polynucleotide comprising a nucleic acid sequence encoding a non-naturally occurring DNA binding protein, said non-naturally occurring DNA binding protein comprising a DNA binding domain, said DNA binding domain being a zinc finger DNA binding domain comprising SEQ ID NO: 170, and said DNA binding domain binds to a target site comprising SEQ ID NO:

335.

6. The polynucleotide of any one of claims 1 to 5, wherein the non-naturally occurring DNA binding protein further comprises a transcription activation domain (TAD), a nuclear localization signal (NLS), or both.

7. 7. The polynucleotide of claim 6, wherein the non-naturally occurring DNA binding protein further comprises the TAD and the NLS, and wherein the non-naturally occurring DNA binding protein is capable of upregulating endogenous Progranulin (PGRN) expression in a cell.

8. 7. The polynucleotide of claim 6, wherein the TAD is selected from the group consisting of VP64, VPR, VP16, VP128, p65, p300, CITED2, CITED4, EGR1 and EGR3.

9. The polynucleotide of claim 8, wherein the TAD comprises a sequence selected from the group consisting of SEQ ID NO:95, SEQ ID NO:114, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, and SEQ ID NO:

100.

10. 7. The polynucleotide of claim 6, wherein the non-naturally occurring DNA binding protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO:338 and SEQ ID NO:

344.

11. 7. An expression cassette comprising the polynucleotide of claim 6, wherein the expression cassette comprises a regulatory element operably linked to a nucleic acid sequence encoding a non-naturally occurring DNA binding protein.

12. The expression cassette of claim 11, wherein the regulatory element is selected from the group consisting of an intron, an enhancer, a promoter, a UTR, a stability element, a WPRE sequence, a Kozak consensus sequence, a post-translational response element, a microRNA binding site, a polyadenylation (polyA) sequence, or any combination thereof.

13. 13. The expression cassette of claim 12, wherein the regulatory element comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO:178, SEQ ID NO:179, SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:326, and SEQ ID NO:

327.

14. 13. The expression cassette of claim 12, wherein the non-naturally occurring DNA binding protein further comprises the TAD and the NLS, and wherein the non-naturally occurring DNA binding protein is capable of upregulating endogenous Progranulin (PGRN) expression in a cell.

15. 13. The expression cassette of claim 12, wherein the TAD comprises a sequence selected from the group consisting of SEQ ID NO:95, SEQ ID NO:114, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, and SEQ ID NO:

100.

16. 13. The expression cassette of claim 12, wherein the non-naturally occurring DNA binding protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO:338 and SEQ ID NO:

344.

17. 17. An expression vector comprising the expression cassette according to any one of claims 11 to 16, wherein the expression vector is a plasmid, a viral vector or a liposome.

18. 18. The expression vector of claim 17, wherein the expression vector is a viral vector, the viral vector being an adeno-associated viral (AAV) vector.

19. 19. The expression vector of claim 18, wherein the AAV vector is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, rhlO, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, ovine AAV, scAAV, scAAV1, scAAV2, scAAV5, scAAV8, scAAV9, and hybrids thereof.

20. A pharmaceutical composition comprising an expression vector according to any one of claims 17 to 19 and one or more pharma- ceutically acceptable excipients.

21. 20. An in vitro method for increasing the expression level of GRN in a cell, comprising contacting the cell with a polynucleotide according to any one of claims 1 to 10, an expression cassette according to any one of claims 11 to 16, or an expression vector according to any one of claims 17 to 19.

22. A composition for increasing the expression level of GRN in a cell, comprising a polynucleotide according to any one of claims 1 to 10, an expression cassette according to any one of claims 11 to 16, or an expression vector according to any one of claims 17 to 19.

23. A composition for modulating expression of GRN and treating a disease or disorder, comprising a polynucleotide according to any one of claims 1 to 10, an expression cassette according to any one of claims 11 to 16, or an expression vector according to any one of claims 17 to 19.

24. A composition for treating a central nervous system disorder, comprising a polynucleotide according to any one of claims 1 to 10, an expression cassette according to any one of claims 11 to 16, or an expression vector according to any one of claims 17 to 19.

25. A composition for treating frontotemporal degeneration (FTD), Parkinson's disease, Alzheimer's disease or atherosclerosis, comprising a polynucleotide according to any one of claims 1 to 10, an expression cassette according to any one of claims 11 to 16, or an expression vector according to any one of claims 17 to 19.

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