Methods and compositions for modulating frataxin expression and treating friedreich ataxia

Abstract

To provide methods and compositions for modulating frataxin (FXN) expression for the purpose of treating Friedreich ataxia (FRDA).SOLUTION: Provided herein is a modulating agent comprising a targeting moiety that binds to an expression control element of the frataxin (FXN) gene and an effector moiety comprising an epigenetic modifying moiety capable of modulating, e.g., increasing, expression of FXN. Also provided are compositions capable of modulating, e.g., increasing, expression of the FXN gene. Without wishing to be bound by theory, it is thought that a modulating agent comprising a genomic sequence element (e.g., an expression control element) that is contained within the FXN gene or operably linked thereto and an effector portion (e.g., an epigenetic modifying portion) capable of modulating (e.g., increasing) expression of FXN may be useful for modulating, e.g., increasing, FXN expression.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 62 / 904,391 (filed September 23, 2019), the contents of which are incorporated herein by reference. [Background technology]

[0002] Friedreich's ataxia (FRDA) is a fatal autosomal recessive neurodegenerative disease primarily caused by a homozygous GAA repeat expansion mutation in the first intron of the frataxin (FXN) gene, which leads to inhibition of FXN transcription and reduced FXN protein expression. Pathological hallmarks of FRDA include degeneration of large sensory neurons in the dorsal root ganglia (DRG), degenerative atrophy of the spinal cord, hypertrophic cardiomyopathy, and diabetes. There is a need for methods and compositions that modulate, e.g., increase, the expression of FXN in patients suffering from FRDA and / or related conditions. Summary of the Invention [Means for solving the problem]

[0003] The present disclosure provides, in part, compositions for regulating, e.g., increasing, expression of the frataxin (FXN) gene. Without wishing to be bound by theory, it is believed that a modulator comprising: a genomic sequence element (e.g., an expression control element) contained within or operably linked to the FXN gene; and an effector moiety (e.g., an epigenetic modification moiety) capable of regulating (e.g., increasing) expression of FXN may be useful for regulating, e.g., increasing expression of FXN.

[0004] Thus, in some aspects, the present disclosure relates to a modulator comprising a targeting moiety that binds to an expression control element of the frataxin (FXN) gene and an effector moiety that comprises an epigenetic modification moiety that can regulate, e.g., increase, expression of FXN. In another aspect, the present disclosure relates to a modulator comprising, in part, a targeting moiety that binds to an expression control element of the frataxin (FXN) gene, a first effector moiety that can regulate, e.g., increase, expression of FXN, and a second effector moiety that can regulate, e.g., increase, expression of FXN, wherein the first and second effector moieties are different moieties. In another aspect, the present disclosure relates to a modulator comprising, in part, a targeting moiety that binds to an expression control element of the frataxin (FXN) gene, the targeting moiety comprising a zinc finger molecule, and an effector moiety that can regulate, e.g., increase, expression of FXN.

[0005] In another aspect, the disclosure pertains, in part, to nucleic acid molecules encoding a modulating agent, the modulating agent comprising a targeting moiety that binds to an expression control element of the frataxin (FXN) gene and an effector moiety that comprises an epigenetic modification moiety that can modulate, e.g., increase, expression of FXN, wherein the nucleic acid molecule is linear and non-viral. In another aspect, the disclosure pertains, in part, to nucleic acid molecules (contained within or comprising viral nucleic acid, e.g., nucleic acid molecules contained within or comprising viral vectors) that encode a modulating agent as described herein.

[0006] In another aspect, the present disclosure relates, in part, to a recombinant RNA encoding a modulator, the modulator comprising a targeting portion that binds to an expression control element of the frataxin (FXN) gene and an effector portion that can modulate, e.g., increase, the expression of FXN.

[0007] In another aspect, the disclosure pertains, in part, to viral vectors that include the nucleic acids or recombinant RNA described herein.

[0008] In another aspect, the present disclosure relates, in part, to a nanoparticle (e.g., a lipid nanoparticle (LNP)) comprising a nucleic acid, e.g., recombinant RNA, encoding a modulator, wherein the modulator comprises a targeting moiety that binds to an expression control element of the frataxin (FXN) gene and an effector moiety that can modulate, e.g., increase, expression of FXN.

[0009] The present disclosure further provides, in part, methods for regulating, e.g., increasing, expression of the frataxin (FXN) gene, for example, in a patient in need thereof (e.g., a patient with FRDA). Without wishing to be bound by theory, it is believed that administering a modulating agent comprising a targeting moiety that directs the modulating agent to a genomic sequence element (e.g., an expression control element) contained within or operably linked to the FXN gene; and an effector moiety (e.g., comprising an epigenetic modification moiety) that can modulate (e.g., increase) expression of FXN may be modulated, e.g., increased, and / or FXN protein levels may be increased in a patient in need thereof.

[0010] Thus, in some aspects, the present disclosure relates, in part, to a method for increasing expression of the frataxin (FXN) gene in a cell, which comprises contacting the cell with a modulator (the modulator comprising: a targeting moiety that binds to an expression control element of the frataxin (FXN) gene; and an effector moiety that can modulate, e.g., increase, expression of FXN), thereby increasing FXN expression in the cell, wherein FXN expression is increased for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks (and optionally permanently). In another aspect, the disclosure relates, in part, to a method of increasing expression of the frataxin (FXN) gene in a cell, comprising contacting the cell with a modulating agent (the modulating agent comprising: a targeting moiety that binds to an expression control element of the frataxin (FXN) gene; and an effector moiety that can modulate, e.g., increase, expression of FXN), thereby increasing FXN expression in the cell, wherein the cell contains at least 44 copies of an FXN allele with a GAA expansion, and after treatment with the modulating agent, the FXN allele is expressed at a level that is 1.5x (i.e., 1.5-fold) the expression level of a similar cell not contacted with the modulating agent. In another aspect, the disclosure relates, in part, to a method of increasing expression of frataxin (FXN) in a cell, comprising contacting the cell with a modulating agent described herein.

[0011] The present disclosure provides human cells comprising one or two frataxin (FXN) alleles with at least 44 copies of a GAA expansion, wherein the FXN alleles are expressed at a level higher than the level of FXN expression in cells that have not been treated with a modulator (e.g., a modulator described herein) capable of modulating FXN expression. Without wishing to be bound by theory, it is believed that cells treated with a modulator described herein may exhibit, for example, increased FXN expression for a period of time that exceeds the time the modulator is / was present in the cells. In some embodiments, the FXN allele is expressed at a level that is at least 1.5x (i.e., 1.5-fold), 1.6x, 1.7x, 1.8x, 1.9x, 2x, 2.1x, 2.2x, 2.3x, 2.4x, 2.5x, 2.6x, 2.7x, 2.8x, 2.9x, 3x, 3.1x, 3.2x, 3.3x, 3.4x, 3.5x, 3.6x, 3.7x, 3.8x, 3.9x, 4x, 4.1x, 4.2x, 4.4x, 4.5x, 4.6x, 4.7x, 4.8x, 4.9x, or 5x the reference level, wherein the reference level is the level of FXN expression in a cell that is not treated with a modulator capable of modulating FXN expression (e.g., a modulator described herein). In some embodiments, the cell is a muscle cell (e.g., a cardiac muscle cell, e.g., a cardiomyocyte) or a neuron (e.g., a cell of the central nervous system, a cell of the spinal cord, e.g., a cell of the dorsal root ganglion (DRG)). In some embodiments, the neuron is a glutamatergic neuron.

[0012] Further features of any of the aforementioned methods or compositions include one or more of the embodiments listed below.

[0013] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the embodiments recited below.

[0014] All publications, patent applications, patents, and other references (e.g., sequence database reference numbers) cited herein are incorporated by reference in their entirety. For example, all GenBank, Unigene, and Entrez sequences referenced herein, e.g., in any table herein, are incorporated by reference. Unless otherwise indicated, sequence accession numbers listed herein, including all tables herein, refer to database entries current as of September 23, 2019. When a gene or protein has multiple sequence accession numbers, all sequence variants are encompassed.

[0015] Enumeration of Embodiments 1. The following: a targeting moiety that binds to an expression control element of the frataxin (FXN) gene; an effector moiety comprising an epigenetic modifying moiety capable of modulating, e.g., increasing, expression of FXN; A regulator comprising: 2. The following: a targeting moiety that binds to an expression control element of the frataxin (FXN) gene; a first effector moiety capable of modulating, e.g., increasing, the expression of FXN; a second effector moiety capable of modulating, e.g., increasing, the expression of FXN; wherein said first and second effector moieties are different moieties. 3. The following: a targeting moiety that binds to an expression control element of the frataxin (FXN) gene, the targeting moiety comprising a zinc finger molecule; an effector moiety capable of modulating, e.g., increasing, the expression of FXN; A regulator comprising: 4. A nucleic acid molecule encoding a modulating agent, said modulating agent being: a targeting moiety that binds to an expression control element of the frataxin (FXN) gene; and an effector moiety capable of modulating, e.g., increasing, expression of FXN, wherein the nucleic acid molecule is linear and non-viral. 5. A nucleic acid encoding a regulator according to any one of embodiments 1 to 4. 6. A recombinant RNA encoding a regulator, said regulator being: a targeting moiety that binds to an expression control element of the frataxin (FXN) gene; an effector moiety capable of modulating, e.g., increasing, the expression of FXN; A recombinant RNA comprising: 7. Nanoparticles (e.g., lipid nanoparticles (LNPs)) comprising a nucleic acid, e.g., recombinant RNA, encoding a modulating agent, wherein the modulating agent is: a targeting moiety that binds to an expression control element of the frataxin (FXN) gene; and an effector moiety capable of modulating, e.g., increasing, the expression of FXN. 8. A viral vector comprising the nucleic acid or recombinant RNA molecule according to any one of embodiments 4 to 6. 9. A method for increasing frataxin (FXN) expression in a cell, comprising: contacting a cell with a modulating agent, thereby increasing FXN expression in said cell, said modulating agent being: a targeting moiety that binds to an expression control element of the frataxin (FXN) gene; an effector moiety capable of modulating, e.g., increasing, expression of FXN; A method wherein FXN expression is increased for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks (and optionally permanently). 10. A method for increasing frataxin (FXN) expression in a cell, comprising: contacting a cell with a modulating agent, thereby increasing FXN expression in said cell, said modulating agent being: a targeting moiety that binds to an expression control element of the frataxin (FXN) gene; an effector moiety capable of modulating, e.g., increasing, expression of FXN; wherein the cells contain at least 44 copies of an FXN allele with a GAA expansion, and after treatment with a modulator, the FXN allele is expressed at a level at least 1.5x (i.e., 1.5 times) the expression level of a similar cell not contacted with the modulator. 11. A method for increasing frataxin (FXN) expression in a cell, comprising: contacting a cell with a modulator, nucleic acid, recombinant RNA, nanoparticle, or viral vector of any of embodiments 1 to 8; This increases FXN expression in the cells. A method comprising: 12. The following: a targeting moiety that binds to an expression control element of the frataxin (FXN) gene, wherein the expression control element does not include the promoter or transcription site of FXN; an effector moiety capable of modulating, e.g., increasing, the expression of FXN; A regulator comprising: 13. The following: a targeting moiety that binds to a nucleic acid sequence of an expression control element of the frataxin (FXN) gene, wherein the position of the nucleic acid element closest to the TSS is i) about 150 bases upstream of the TSS; or ii) about 50 bases downstream of the TSS; an effector moiety capable of modulating, e.g., increasing, the expression of FXN; A regulator comprising: 14. The following: At least 44 copies of the frataxin (FXN) allele with a GAA expansion In human cells comprising the FXN allele is expressed at a level of at least 1.5x (i.e., 1.5-fold), 1.6x, 1.7x, 1.8x, 1.9x, 2x, 2.1x, 2.2x, 2.3x, 2.4x, 2.5x, 2.6x, 2.7x, 2.8x, 2.9x, 3x, 3.1x, 3.2x, 3.3x, 3.4x, 3.5x, 3.6x, 3.7x, 3.8x, 3.9x, 4x, 4.1x, 4.2x, 4.4x, 4.5x, 4.6x, 4.7x, 4.8x, 4.9x, or 5x the reference level, wherein the reference level is the level of FXN expression in a cell that has not been treated with a modulator capable of modulating FXN expression (e.g., a modulator as described in any preceding claim), The cell is a human cell, which is a muscle cell, a nerve cell, or a cell of the dorsal root ganglion. 15. The following: Two frataxin (FXN) alleles, each with at least 44 copies of the GAA expansion In human cells comprising each allele is expressed at a level of at least 1.5x (i.e., 1.5-fold), 1.6x, 1.7x, 1.8x, 1.9x, 2x, 2.1x, 2.2x, 2.3x, 2.4x, 2.5x, 2.6x, 2.7x, 2.8x, 2.9x, 3x, 3.1x, 3.2x, 3.3x, 3.4x, 3.5x, 3.6x, 3.7x, 3.8x, 3.9x, 4x, 4.1x, 4.2x, 4.4x, 4.5x, 4.6x, 4.7x, 4.8x, 4.9x, or 5x the reference level, wherein the reference level is the level of FXN expression in a cell that has not been treated with a modulator capable of modulating FXN expression (e.g., a modulator as described in any preceding claim); The human cell is a muscle cell, a nerve cell, or a cell of the dorsal root ganglion. 16. The modulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method of any of embodiments 1-13, wherein said effector moiety comprises an epigenetic modification moiety. 17. The modulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method of any of embodiments 1 to 13 or 16, wherein the epigenetic modification moiety comprises a histone methyltransferase, a DNA demethylase, a histone acetyltransferase, or a functional fragment or variant of any of them. 18. The modulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method of any of embodiments 1-13, 16, or 17, wherein said effector moiety comprises a DNA demethylase or a functional fragment or variant thereof, such as a protein selected from TET1, TET2, TET3, or TDG, or a functional fragment or variant of any of them. 19. The modulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method of any of embodiments 1-13 or 16-18, wherein said effector moiety comprises a histone methyltransferase or a functional fragment or variant thereof, such as a protein selected from DOT1L, PRDM9, PRMT1, PRMT2, PRMT3, PRMT4, PRMT5, NSD1, NSD2, NSD3, or a functional fragment or variant of any of them. 20. The modulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method of any of embodiments 1-13 or 16-19, wherein said effector moiety comprises a protein selected from histone acetyltransferase or a functional fragment or variant thereof, e.g., p300, CREB-binding protein (CBP), or a functional fragment or variant of any of them. 21. The modulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method of any of embodiments 1-13 or 16-20, wherein the effector moiety comprises a protein selected from a transcriptional activator or a functional fragment or variant thereof, such as VP16, VP64, VP160, or VPR. 22. The modulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method of any of embodiments 1-13 or 16-21, wherein said effector moiety comprises VPR or a functional fragment or variant thereof. 23. The modulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method of any of embodiments 1-13 or 16-22, wherein said effector moiety comprises p300 or a functional fragment or variant thereof. 24. The modulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method of any of embodiments 1-13 or 16-23, wherein said effector moiety comprises p65 or a functional fragment or variant thereof. 25. The modulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method of any of embodiments 1-13 or 16-24, wherein said effector moiety comprises RTA or a functional fragment or variant thereof. 26. The modulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method of any of embodiments 1-13 or 16-25, wherein the effector moiety comprises one, two, or all of a DNA demethylase, an acetyltransferase, or a transcriptional activator, or functional fragments of any thereof. 27. The modulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method of any of embodiments 1-13 or 16-26, wherein said targeting moiety comprises a Cas9 molecule. 28. The Cas9 molecule is capable of inhibiting Streptococcus (e.g., S. pyogenes or S. thermophilus), Francisella (e.g., F. novicida), Staphylococcus (e.g., S. aureus), Acidaminococcus (e.g., Acidaminococcus species), or any of the following: 28. The modulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method of embodiment 27, comprising a Cas9 protein from a species of Neisseria (e.g., N. meningitidis), Cryptococcus, Corynebacterium, Haemophilus, Eubacterium, Pasteurella, Prevotella, Veillonella, or Marinobacter. 29. The modulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method of embodiment 27 or 28, wherein said Cas9 molecule comprises a Cas9 protein that substantially lacks nuclease activity, e.g., dCas9, e.g., comprising an inactive RuvC and / or HNH domain. 30. The modulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method of any of embodiments 27-29, wherein the Cas9 molecule comprises (e.g., is non-covalently associated with) a gRNA, e.g., an sgRNA, and wherein the gRNA binds to an expression control element. 31. The modulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method of embodiment 30, wherein the gRNA comprises a nucleic acid selected from any of SEQ ID NOs: 4-26, or a sequence having at least 80, 85, 90, 95, or 99% identity to any of SEQ ID NOs: 4-26. 32. The modulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method of any of embodiments 1-13 or 16-26, wherein said targeting moiety comprises a TAL effector molecule. 33. The modulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method of embodiment 32, wherein the TAL effector molecule comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 TAL effector DNA-binding domains (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 repeating variable diresidues (RVDs)). 34. The modulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method of any of embodiments 1-13, or 16-26, wherein the targeting moiety comprises a zinc finger molecule. 35. A regulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method described in any of embodiments 1 to 13 or 16 to 34, wherein the expression control element comprises an enhancer or promoter or a portion thereof operably linked to the FXN gene. 36. A regulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method described in any of embodiments 1 to 13 or 16 to 35, wherein the expression control element comprises, in whole or in part, an anchor sequence operably linked to an anchor sequence-mediated junction comprising the FXN gene. 37. A regulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method described in any of embodiments 1 to 13 or 16 to 36, wherein the targeting moiety binds to a nucleic acid sequence comprising the transcription start site (TSS) of the FXN gene. 38. The targeting moiety is located at 500, 490, 480, 470, 460, 450, 440, 430, 420, 410, 400, 390, 380, 370, 360, 350, 340, 330, 320, 310, 300, 290, 280, 270, 260, 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 115, 125, 130, 145, 150, 160, 175, 170, 180, 190, 195, 190, 200, 200, 210, 210, 220, 230, 240, 250, 260, 270, 280, 290, 290, 280, 290, 260, 250, 260, 270, 280, 29 ... 38. The modulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method of any of embodiments 1-13 or 16-37, wherein the modulator binds to a nucleic acid sequence that is 0, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide or less upstream or downstream (and optionally at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, or 90 nucleotides upstream or downstream). 39. A modulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method described in any of embodiments 1 to 13, or 16 to 37, wherein the targeting moiety binds to a nucleic acid sequence approximately 50 to 150, 50 to 70, 70 to 90, 90 to 110, 110 to 130, or 130 to 150 nucleotides upstream of the transcription start site (TSS) of the FXN gene. 40. The modulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method of any of embodiments 1-13 or 16-39, wherein the targeting moiety comprises a Zn finger molecule containing 2, 3, 4, 5, or 6 Zn finger proteins. 41. The modulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method of any of embodiments 1-13 or 16-40, wherein said targeting moiety binds to a nucleic acid sequence selected from the sequences depicted by the genomic coordinates in Table 3. 42. The targeting moiety comprises a Cas9 molecule, e.g., a dCas9 molecule, and the effector moiety comprises p300 or a functional fragment or variant thereof. The modulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method of any of embodiments 1-13, 16-31, 35-39, or 41. 43. The targeting moiety comprises a Cas9 molecule, e.g., a dCas9 molecule, and the effector moiety comprises VP64 or a functional fragment or variant thereof. The modulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method of any of embodiments 1-13, 16-31, 35-39, or 41. 44. The targeting moiety comprises an enzymatically inactive Cas nuclease, e.g., a dCas9 molecule, and the effector moiety comprises p300 or a functional fragment or variant thereof. The modulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method of any of embodiments 1-13, 16-31, 35-39, or 41. 45. The targeting moiety comprises an enzymatically inactive Cas nuclease, e.g., a dCas9 molecule, and the effector moiety comprises VP64 or a functional fragment or variant thereof. The modulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method of any of embodiments 1-13, 16-31, 35-39, or 41. 46. ​​The targeting moiety comprises an enzymatically inactive Cas nuclease, e.g., a dCas9 molecule, and the effector moiety comprises VPR or a functional fragment or variant thereof. The modulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method of any of embodiments 1-13, 16-31, 35-39, or 41. 47. The targeting moiety comprises an enzymatically inactive Cas nuclease, e.g., a dCas9 molecule, and the effector moiety comprises VP64 or a functional fragment or variant thereof, p65 or a functional fragment or variant thereof, and RTA or a functional fragment or variant thereof. The modulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method of any of embodiments 1-13, 16-31, 35-39, or 41. 48. The targeting moiety comprises a TAL effector molecule (e.g., the TAL effector molecule binds upstream, e.g., about 50-150 nucleotides upstream, e.g., about 100 nucleotides upstream, of the FXN gene TSS), and the effector moiety comprises VPR or a functional fragment or variant thereof. The modulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method of any of embodiments 1-13, 16-31, 35-39, or 41. 49. The targeting moiety comprises a TAL effector molecule (e.g., the TAL effector molecule binds upstream, e.g., about 50-150 nucleotides upstream, e.g., about 100 nucleotides upstream, of the FXN gene TSS), and the effector moiety comprises VP64 or a functional fragment or variant thereof, p65 or a functional fragment or variant thereof, and RTA or a functional fragment or variant thereof; The modulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method of any of embodiments 1-13, 16-31, 35-39, or 41. 50. The targeting moiety comprises a zinc finger molecule (e.g., the zinc finger molecule binds upstream, e.g., about 50 to 150 nucleotides upstream, e.g., about 100 nucleotides upstream, of the FXN gene TSS), and the effector moiety comprises VPR or a functional fragment or variant thereof. The modulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method of any of embodiments 1-13, 16-31, 35-39, or 41. 51. The targeting moiety comprises a zinc finger molecule (e.g., the zinc finger molecule binds upstream, e.g., about 50 to 150 nucleotides upstream, e.g., about 100 nucleotides upstream, of the FXN gene TSS), and the effector moiety comprises VP64 or a functional fragment or variant thereof, p65 or a functional fragment or variant thereof, and RTA or a functional fragment or variant thereof; The modulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method of any of embodiments 1-13, 16-31, 35-39, or 41. 52. The modulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method of any of embodiments 1-13 or 16-51, wherein said modulator comprises or is a fusion molecule. 53. The modulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method of any of embodiments 1-13 or 16-52, wherein the modulator comprises an amino acid sequence selected from any of SEQ ID NOs: 304-309, or an amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98, or 99% identity thereto. 54. The modulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method of embodiment 52 or 53, wherein the fusion molecule comprises a targeting moiety and an effector moiety covalently linked, e.g., by a peptide bond, e.g., as part of a single polypeptide chain. 55. The modulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method of any of embodiments 1-13 or 16-54, wherein the modulator comprises or is a conjugate. 56. The modulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method of embodiment 55, wherein said conjugate comprises a targeting moiety and an effector moiety covalently linked, for example, by a non-peptide bond. 57. The modulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method of any of embodiments 1-13 or 16-56, wherein the modulator further comprises an additional moiety. 58. The modulating agent, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method of embodiment 57, wherein said additional moiety comprises a purification tag (e.g., a moiety that aids in the purification of the modulating agent), a bioavailability or pharmacokinetic moiety (e.g., a moiety that increases the bioavailability or modulates the pharmacokinetic properties of the modulating agent), a solubility moiety (e.g., a moiety that increases the solubility, e.g., physiological solubility, of the modulating agent), a detection moiety (e.g., a moiety that aids in the detection and / or quantitation of the presence or level of the modulating agent, e.g., a fluorescent moiety or fluorescent dye), a multimerization moiety (e.g., a moiety that promotes multimerization (e.g., dimerization, trimerization, or tetramerization) of the modulating agent), or an association moiety (e.g., a moiety that enables the modulating agent to associate with a structure, e.g., a membrane or a laboratory test device (e.g., a plate or tube wall)). 59. A complex comprising a regulator according to any one of embodiments 1 to 3, 12, 13, or 16 to 58 and a nucleic acid sequence having an expression control sequence for the FXN gene. 60. A cell comprising the modulator, nucleic acid, recombinant RNA, nanoparticle, or viral vector of any of embodiments 1-8, 12, 13, or 16-58. 61. A cell comprising a nucleic acid encoding the modulator of any of embodiments 1-3, 12, 13, or 16-58. 62. A method of delivering a regulator, nucleic acid, recombinant RNA, nanoparticle, or viral vector according to any one of embodiments 1-8, 12, 13, or 16-58 to a cell, comprising contacting the cell with the regulator, nucleic acid, recombinant RNA, nanoparticle, or viral vector, thereby delivering the regulator, nucleic acid, recombinant RNA, nanoparticle, or viral vector to the cell. 63. The method of embodiment 62, further comprising contacting the cell with one or more (e.g., two or three) gRNAs that bind to expression control elements of the FXN gene or DNA encoding the gRNA. 64. A method for regulating, for example, increasing, transcription of the frataxin (FXN) gene, comprising: contacting a cell with a modulator, nucleic acid, recombinant RNA, nanoparticle, or viral vector of any of embodiments 1-8, 12, 13, or 16-58; This allows the expression of the FXN gene to be regulated, for example, increased. A method comprising: 65. The method of any of embodiments 62-64, wherein the contacting is carried out in vivo, in vitro, or ex vivo. 66. A method of treating a patient with Friedreich's ataxia (FRDA), comprising administering: administering to the patient a modulator, nucleic acid, recombinant RNA, nanoparticle, or viral vector according to any one of embodiments 1 to 8, 12, 13, or 16 to 58; This allows the patient to be treated. A method comprising: 67. The method of embodiment 66, wherein administering comprises intravenous or intrathecal administration. 68. The method comprises measuring the level of FXN in blood (e.g., whole blood) relative to the level of FXN in blood (e.g., whole blood) in the absence of the modulator, nucleic acid, recombinant RNA, nanoparticle, or viral vector (e.g., as described in Deutsch et al. or Oglesbee et al. 68. The method of any of embodiments 62-67, wherein the IL-16 expression level (as measured by the method of any of embodiments 62-67) is increased by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 300, or 400%. 69. The method of any of embodiments 62-68, wherein the method reduces or eliminates at least one symptom of FDRA, such as a symptom selected from the following: ataxia, dysarthria, muscle weakness, spasticity (e.g., leg spasticity), scoliosis, bladder dysfunction, reflex impairment, loss of position and / or vibration sense, cardiomyopathy, or diabetes. 70. The method of embodiment 68 or 69, wherein the level of FXN in blood (e.g., whole blood) is increased for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or at least 1, 2, 3, 4, or 5 years. 71. The method of any of embodiments 62-69, wherein the method increases FXN levels in blood (e.g., whole blood) for at least 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, or 96 hours. 72. A method for increasing frataxin (FXN) expression in a cell, comprising: contacting a modulating agent of any of the preceding embodiments with a cell; This increases the expression of the FXN gene in the cells. Including, wherein FXN expression is increased for at least 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, or 96 hours (and optionally permanently).

[0016] definition a, an, the: As used herein, "a", "a" and "the" include plural referents unless the context clearly requires otherwise.

[0017] Agent: As used herein, the term "agent" refers to any chemical class of compound or entity, including, for example, polypeptides, nucleic acids, sugars, lipids, small molecules, metals, or combinations or complexes thereof. As will be clear to one of ordinary skill in the art from the context, in some embodiments, the term may also be used to refer to an entity that is or includes a cell or organism, or a fraction, extract, or component thereof. Alternatively, or additionally, as will be understood by one of ordinary skill in the art from the context, in some embodiments, the term may also be used to refer to a natural product, in that it occurs in nature and / or is obtained from nature. In some embodiments, also as will be understood by one of ordinary skill in the art from the context, the term may also refer to one or more entities that are man-made, in that they have been artificially designed, engineered, and / or produced by man and / or do not exist in nature. In some embodiments, an agent may be used in isolated or pure form; in some embodiments, an agent may be used in unpurified form. In some embodiments, potential agents may be provided as a collection or library, and, for example, they may be screened to identify or characterize active agents therein. In some embodiments, the term "agent" can refer to a compound or entity that is or includes a polymer; in some embodiments, the term can refer to a compound that includes one or more polymer moieties. In some embodiments, the term "agent" can refer to a compound or entity that is not a polymer and / or that is substantially free of all polymers and / or one or more specific polymer moieties. In some embodiments, the term can refer to a compound or entity that lacks or is substantially free of all polymer moieties.

[0018] Anchor sequence: As used herein, the term "anchor sequence" refers to a sequence recognized by a junction nucleation polypeptide (e.g., a nucleation polypeptide) and sufficient to bind to form an anchor sequence-mediated junction. In some embodiments, the anchor sequence comprises one or more CTCF binding motifs. In some embodiments, the anchor sequence is not located within a gene coding region. In some embodiments, the anchor sequence is located within an intergenic region. In some embodiments, the anchor sequence is not located within an enhancer or promoter. In some embodiments, the anchor sequence is located at least 400 bp, at least 450 bp, at least 500 bp, at least 550 bp, at least 600 bp, at least 650 bp, at least 700 bp, at least 750 bp, at least 800 bp, at least 850 bp, at least 900 bp, at least 950 bp, or at least 1 kb away from any transcription start site. In some embodiments, the anchor sequence is located in a region not associated with genomic imprinting, monoallelic expression, and / or monoallelic epigenetic marks. In some embodiments of the present disclosure, techniques are provided that can specifically target a particular anchor sequence or sequences without targeting other anchor sequences (e.g., sequences that may contain junction nucleation polypeptide (e.g., CTCF) binding motifs in other contexts); such targeting anchor sequences are sometimes referred to as "target anchor sequences." In some embodiments, the sequence and / or activity of the target anchor sequence is modulated, but the sequence and / or activity of one or more other anchor sequences that may be present in the same system as the targeting anchor sequence (e.g., within the same cell and / or in some embodiments, on the same nucleic acid molecule, e.g., the same chromosome) is not modulated.

[0019] Anchor sequence-mediated junction: As used herein, the term "anchor sequence-mediated junction" (also abbreviated ASMC) refers to a DNA structure that arises from and / or is maintained by the physical interaction or binding of at least two anchor sequences within the DNA by one or more proteins, such as nucleation polypeptides, or one or more proteins and / or nucleic acid entities (e.g., RNA or DNA), which bind to the anchor sequences, allowing for spatial proximity and functional linkage of the anchor sequences.

[0020] Associated with: As used herein, two events or entities are "associated" with one another when the presence, level, function, and / or form of one correlates with that of the other. For example, in some embodiments, a particular entity (e.g., a polypeptide, gene signature, metabolite, microorganism, etc.) is considered to be associated with a particular disease, disorder, or condition when its presence, level, function, and / or form correlates with the incidence of and / or susceptibility to the disease, disorder, or condition. In some embodiments, two or more entities are physically "associated" with one another if they directly or indirectly interact to bring and / or remain in physical proximity to one another. In some embodiments, two or more entities that are physically associated with one another are covalently bound to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently bound to one another, but are non-covalently bound by, for example, hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof. In some embodiments, a target gene is "associated" with an anchor sequence-mediated junction if modulation (e.g., disruption) of the anchor sequence-mediated junction causes alteration of expression (e.g., transcription) of the target gene. For example, in some embodiments, modulation (e.g., disruption) of the anchor sequence-mediated junction alters expression of the target gene by associating or disassociating an enhancing or silencing / repressor sequence with the target gene. In some embodiments, a target gene is associated with an ASMC if the target gene, or a portion thereof, is located within an ASMC.

[0021] Domain: As used herein, the term "domain" refers to a section or portion of an entity. In some embodiments, a "domain" is associated with a particular structural and / or functional characteristic of the entity, and when the domain is physically separated from the rest of its parent entity, it substantially or completely retains said particular structural and / or functional characteristic. Alternatively, or in addition, in some embodiments, a domain may be or comprise a portion of an entity that, when separated from the (parent) entity and linked to another (recipient) entity, substantially retains and / or confers on the recipient entity one or more structural and / or functional characteristics that characterized it in the parent entity. In some embodiments, a domain is or comprises a section or portion of a molecule (e.g., a small molecule, carbohydrate, lipid, nucleic acid, polypeptide, etc.). In some embodiments, a domain is or comprises a section of a polypeptide. In some such embodiments, the domain is characterized by particular structural elements (e.g., particular amino acid sequences or sequence motifs, α-helical characteristics, β-sheet characteristics, coiled-coil characteristics, random coil characteristics, etc.) and / or particular functional characteristics (e.g., binding activity, enzymatic activity, folding activity, signaling activity, etc.).

[0022] Effector moiety: As used herein, the term "effector moiety" refers to a domain that, upon localization to an appropriate site within the nucleus of a cell, can alter the expression of a target gene (e.g., FXN). In some embodiments, the effector moiety recruits components of the transcription machinery. In some embodiments, the effector moiety inhibits the recruitment of transcription factor or repressor components. In some embodiments, the effector moiety comprises an epigenetic modifying moiety (e.g., epigenetically modifies a target DNA sequence).

[0023] Epigenetic modification moiety: As used herein, "epigenetic modification moiety" refers to: i) the structure, e.g., two-dimensional structure, of chromatin; and / or ii) a domain that alters an epigenetic marker (e.g., one or more of DNA methylation, histone methylation, histone acetylation, histone sumoylation, histone phosphorylation, and RNA-associated silencing) when the epigenetic modification moiety is properly localized to a nucleic acid (e.g., by a targeting moiety). In some embodiments, the epigenetic modification moiety comprises an enzyme, or a functional fragment or variant thereof, that affects (e.g., increases or decreases the level of) one or more epigenetic markers. In some embodiments, the epigenetic modification moiety comprises a DNA methyltransferase, a histone methyltransferase, a CREB-binding protein (CBP), or a functional fragment of any of these.

[0024] Expression control sequence: As used herein, the term "expression control sequence" refers to a nucleic acid sequence that increases or decreases the transcription of a gene, including, but not limited to, promoters and enhancers. An "enhancing sequence" refers to a subtype of expression control sequence that increases the likelihood of gene transcription. A "silencing or repressor sequence" refers to a subtype of expression control sequence that decreases the likelihood of gene transcription.

[0025] Fusion molecule: As used herein, the term "fusion molecule" refers to a compound comprising two or more moieties that are covalently linked, e.g., a targeting moiety and an effector moiety. A fusion molecule and its moieties may comprise any combination of polypeptides, nucleic acids, glycans, small molecules, or other components described herein (e.g., a targeting moiety may comprise a nucleic acid and an effector moiety may comprise a polypeptide). In some embodiments, a fusion molecule is a fusion protein comprising one or more polypeptide domains covalently linked, e.g., via a peptide bond. In some embodiments, a fusion molecule is a conjugate molecule comprising a targeting moiety and an effector moiety, which are linked by a covalent bond other than a peptide bond or a phosphodiester bond (e.g., a targeting moiety comprising a nucleic acid and an effector moiety comprising a polypeptide, linked by a covalent bond other than a peptide bond or a phosphodiester bond). In some embodiments, a modulating agent is or comprises a fusion molecule.

[0026] Genome complex: As used herein, the term "genome complex" refers to a complex that brings together two genome sequence elements spaced apart from one another on one or more chromosomes through the interaction of multiple proteins and / or other components (possibly including the genome sequence elements). In some embodiments, the genome sequence elements are anchor sequences to which one or more protein components of the complex bind. In some embodiments, the genome complex may be an anchor sequence-mediated junction (ASMC). In some embodiments, the genome complex comprises one or more ASMCs. In some embodiments, the genome sequence elements may be or may include anchor sequences (e.g., CTCF binding motifs). In some embodiments, the genome sequence elements include at least one or both of a promoter and / or enhancer. In some embodiments, genome complex formation is nucleated at the genome sequence elements and / or by binding of one or more protein components to the genome sequence elements. As will be appreciated by those skilled in the art, in some embodiments, colocalization (e.g., coupling) of genomic sites through complex formation alters DNA topology at or near (e.g., in some embodiments, between) genomic sequence elements. In some embodiments, the genomic complexes described herein are nucleated by nucleation polypeptides, such as, for example, CTCF and / or cohesin. In some embodiments, the genomic complexes described herein may include, for example, one or more of the following: CTCF, cohesin, non-coding RNA (e.g., enhancer RNA (eRNA)), transcription machinery proteins (e.g., RNA polymerase, one or more transcription factors selected from the group consisting of, for example, TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, etc.), transcriptional regulators (e.g., Mediator, P300, enhancer-binding proteins, repressor-binding proteins, histone modifiers, etc.).In some embodiments, the genome complexes described herein comprise one or more polypeptide components and / or one or more nucleic acid components (e.g., one or more RNA components), which, in some embodiments, can interact with each other and / or with one or more genome sequence elements (e.g., anchor sequences, promoter sequences, regulatory sequences (e.g., enhancer sequences)) to trap a stretch of genomic DNA in a topological configuration that it would not adopt absent complex formation.

[0027] Moiety: As used herein, the term "moiety" refers to a known chemical group or entity having a particular structure and / or activity, as described herein.

[0028] Modulator: As used herein, the term "modulator" refers to an agent that comprises one or more targeting moieties and one or more effector moieties and is capable of altering (e.g., increasing or decreasing) the expression of a target gene, e.g., FXN.

[0029] Nucleating Polypeptide: As used herein, the term "nucleating polypeptide" or "junction nucleating polypeptide" refers to a protein that binds directly or indirectly to an anchor sequence, which may interact with one or more junction nucleating polypeptides (which may interact with the anchor sequence or another nucleic acid) to form a dimer (or higher-order structure) consisting of two or more such junction nucleating polypeptides, which may or may not be identical to one another. When junction nucleating polypeptides bound to different anchor sequences associate with one another, thereby maintaining the different anchor sequences in physical proximity to one another, the resulting structure is an anchor sequence-mediated junction. That is, the close physical proximity of a nucleating polypeptide-anchor sequence that interacts with another nucleating polypeptide-anchor sequence creates an anchor sequence-mediated junction (in some cases, a DNA loop) that begins and ends at the anchor sequence. As will be readily understood by those of skill in the art upon reading this specification, terms such as "nucleating polypeptide," "nucleating molecule," "nucleating protein," and "junction nucleating protein" may, in some cases, be used to refer to a junction nucleating polypeptide. Similarly, as will be readily understood by those of skill in the art upon reading this specification, an assembled collection of two or more junction nucleating polypeptides (which may, in some embodiments, include multiple copies of the same agent and / or, in some embodiments, one or more of multiple different agents) may also be referred to as a "complex," "dimer," "multimer," etc.

[0030] Operably linked: As used herein, the phrase "operably linked" refers to a juxtaposition wherein the described components are in a relationship permitting them to function in their intended manner. An expression control sequence "operably linked" to a functional element, e.g., a gene, is joined such that expression and / or activity of the functional element, e.g., the gene, is achieved under conditions compatible with the expression control sequences. In some embodiments, an "operably linked" expression control sequence is contiguous (e.g., shared) with a coding element, e.g., a gene, of interest; in some embodiments, an operably linked expression control sequence acts in trans with or otherwise at a distance from a functional element, e.g., a gene, of interest. In some embodiments, operably linked means that two nucleic acid sequences are contained on the same nucleic acid molecule. In other embodiments, operably linked can also mean that two nucleic acid sequences are close to each other on the same nucleic acid molecule, e.g., within 1000, 500, 100, 50, or 10 base pairs of each other, or adjacent to each other.

[0031] Pharmaceutical composition: As used herein, the term "pharmaceutical composition" refers to an active agent (e.g., a modulator, e.g., an inhibitor) formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dose suitable for administration in a treatment regimen that, when administered to a relevant population, exhibits a statistically significant probability of achieving the intended therapeutic effect. In some embodiments, the pharmaceutical composition may be specially formulated for administration in solid or liquid form, including oral administration, e.g., drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., buccal, sublingual, and those intended for systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, e.g., subcutaneous, intramuscular, intravenous, or epidural injection (e.g., as a sterile solution or suspension), or sustained release formulations; topical application, e.g., as a cream, ointment, or controlled release patch or spray applied to the skin, lungs, or oral cavity; vaginal or rectal, e.g., as a pessary, cream, or foam; sublingual; intraocular; transdermal; or designed for nasal, pulmonary, and / or other mucosal surfaces.

[0032] Proximity: As used herein, "proximity" refers to the proximity of two sites, e.g., nucleic acid sites, such that binding of an expression repressor at the first site and / or modification of the first site by an expression repressor domain produces the same or substantially the same effect as binding and / or modification of the other site. For example, a DNA targeting moiety can bind to a first site that is proximal to an enhancer (second site), and the repressor bound to the DNA targeting moiety can epigenetically modify the first site such that the effect of the enhancer on expression of the target gene is modified substantially in the same manner as if the second site (enhancer sequence) had been bound and / or modified. In some embodiments, a site proximal to a target gene (e.g., an exon, intron, or splice site within the target gene), proximal to a transcriptional regulatory sequence operably linked to the target gene, or proximal to an anchor sequence is less than 5000, 4000, 3000, 2000, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 50, or 25 base pairs from the target gene (e.g., an exon, intron, or splice site within the target gene), transcriptional regulatory sequence, or anchor sequence (and optionally at least 20, 25, 50, 100, 200, or 300 base pairs from the target gene (e.g., an exon, intron, or splice site within the target gene), transcriptional regulatory sequence, or anchor sequence).

[0033] Specific: As used herein, the term "specific" refers to an agent that has activity and is understood by those of skill in the art to mean that the agent distinguishes between a potential target entity or condition. For example, in some embodiments, an agent is said to "specifically" bind to a target if it preferentially binds to that target in the presence of one or more competing targets. In some embodiments, the specific interaction depends on the presence of a particular structural feature of the target entity (e.g., an epitope, cleft, binding site). It should be understood that specificity need not be absolute. In some embodiments, specificity is assessed relative to the specificity of the binding agent for one or more potential target entities (e.g., competitors). In some embodiments, specificity is assessed relative to the specificity of a reference specific binding agent. In some embodiments, specificity is assessed relative to the specificity of a reference non-specific binding agent. In some embodiments, the agent or entity does not detectably bind to another competing target under conditions of binding to the target entity. In some embodiments, a binding agent binds to its target entity with a higher on rate, a lower off rate, increased affinity, reduced dissociation, and / or increased stability compared to another competing target.

[0034] Substantially: As used herein, the term "substantially" refers to the qualitative condition of exhibiting the full or nearly full extent or degree of a desired characteristic or property. Those skilled in the art will understand that biological and chemical phenomena rarely, if ever, reach completion and / or proceed to perfection, or achieve or avoid absolute results. Thus, the term "substantially" may be used in some embodiments herein to capture the possible lack of perfection inherent in many biological and chemical phenomena.

[0035] Target: An agent or entity is considered to "target" another agent or entity in accordance with the present disclosure if it specifically binds to the targeting agent or entity under conditions in which they contact each other. In some embodiments, for example, an antibody (or antigen-binding fragment thereof) targets its cognate epitope or antigen. In some embodiments, a nucleic acid having a specific sequence targets a nucleic acid of a substantially complementary sequence. In some embodiments, binding to a target is direct binding; in some embodiments, binding to a target can be indirect binding. In some embodiments, a modulating agent targets a genome complex, e.g., ASMC, for example, by binding to a component (e.g., a polypeptide, nucleic acid, and / or genome sequence element) of the genome complex, e.g., ASMC.

[0036] Target gene: As used herein, the term "target gene" refers to a gene that is targeted for regulation, e.g., regulation of a gene or regulation of an epigenetic marker associated with said gene. In some embodiments, the target gene is part of a targeted genome complex (e.g., a gene that has at least a portion of its genome sequence as part of a target genome complex, within an anchor sequence-mediated junction), and this genome complex is targeted by one or more modulatory agents described herein. In some embodiments, the target gene is regulated by the genomic sequence of the target gene that is directly contacted by a modulatory agent described herein. In some embodiments, the target gene is regulated by one or more components of a genome complex, which is the part that is contacted by a modulatory agent described herein. In some embodiments, the target gene is external to the target genome complex, e.g., it is a gene that encodes a component of the target genome complex (e.g., a subunit of a transcription factor). In some embodiments, the target gene is associated with a genome complex described herein.

[0037] Targeting moiety: As used herein, the term "targeting moiety" means an agent or entity that specifically targets, e.g., binds to, a genomic sequence element (e.g., an expression control sequence or an anchor sequence) that is adjacent to and / or operably linked to a target gene (e.g., FXN).

[0038] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" refers to an amount of a substance (e.g., a therapeutic agent, composition, and / or formulation) that, when administered as part of a treatment regimen, elicits a desired biological response. In some embodiments, a therapeutically effective amount of a substance is an amount that, when administered to a subject suffering from or susceptible to a disease, disorder, and / or condition, is sufficient to treat, diagnose, prevent, and / or delay the onset of the disease, disorder, and / or condition. As will be understood by one of skill in the art, the effective amount of a substance can vary depending on factors such as the desired biological endpoint, the substance to be delivered, and the target cell or tissue. For example, in some embodiments, an effective amount of a compound in a formulation for treating a disease, disorder, and / or condition is an amount that ameliorates, alleviates, reduces, inhibits, prevents, delays the onset of, reduces the severity of, and / or reduces the incidence of, one or more symptoms or characteristics of the disease, disorder, and / or condition. In some embodiments, a therapeutically effective amount is administered in a single dose; in some embodiments, multiple doses are required to deliver a therapeutically effective amount. In an embodiment of the present invention, for example, the following items are provided: (Item 1) below: a targeting moiety that binds to an expression control element of the frataxin (FXN) gene; an effector moiety comprising an epigenetic modifying moiety capable of increasing expression of FXN; A regulator comprising: (Item 2) below: a targeting moiety that binds to an expression control element of the frataxin (FXN) gene; a first effector moiety capable of increasing the expression of FXN; a second effector moiety capable of increasing expression of FXN; A regulator comprising: A modulator wherein said first and second effector moieties are different moieties. (Item 3) below: a targeting moiety that binds to an expression control element of the frataxin (FXN) gene, the targeting moiety comprising a zinc finger molecule; an effector moiety capable of increasing the expression of FXN; A regulator comprising: (Item 4) A nucleic acid encoding the regulator according to any one of items 1 to 3. (Item 5) 1. A recombinant RNA encoding a modulating agent, wherein said modulating agent is: a targeting moiety that binds to an expression control element of the frataxin (FXN) gene; an effector moiety capable of increasing the expression of FXN; A recombinant RNA comprising: (Item 6) A nanoparticle (e.g., a lipid nanoparticle (LNP)) comprising a nucleic acid, e.g., recombinant RNA, encoding a modulating agent, wherein the modulating agent is: a targeting moiety that binds to an expression control element of the frataxin (FXN) gene; an effector moiety capable of increasing the expression of FXN; Nanoparticles comprising: (Item 7) 1. A method for increasing frataxin (FXN) expression in a cell, comprising: contacting a cell with a modulating agent, thereby increasing FXN expression in said cell, said modulating agent being: a targeting moiety that binds to an expression control element of the frataxin (FXN) gene; an effector moiety capable of increasing expression of FXN, FXN expression is increased for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks (and optionally permanently); or The method, wherein the cells contain at least 44 copies of an FXN allele having a GAA expansion, and after treatment with the modulating agent, the FXN allele is expressed at a level at least 1.5x (i.e., 1.5 times) the expression level of similar cells not contacted with the modulating agent. (Item 8) 1. A method for increasing frataxin (FXN) expression in a cell, comprising: contacting a cell with a modulator, nucleic acid, recombinant RNA, nanoparticle, or viral vector according to any one of items 1 to 7; This increases FXN expression in the cells. A method comprising: (Item 9) 9. The regulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method according to any one of items 1 to 8, wherein the effector moiety comprises an epigenetic modification moiety. (Item 10) 10. The regulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method according to any one of items 1 to 9, wherein the epigenetic modification moiety comprises a DNA methyltransferase, a histone methyltransferase, a DNA demethylase, a histone acetyltransferase, or a functional fragment or variant of any of them. (Item 11) 11. The modulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method according to any one of items 1 to 10, wherein the effector moiety comprises a DNA demethylase or a functional fragment or variant thereof, for example a protein selected from TET1, TET2, TET3, or TDG, or a functional variant or fragment of any of them. (Item 12) 12. The modulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method according to any one of items 1 to 11, wherein the effector moiety comprises a histone methyltransferase or a functional fragment or variant thereof, for example a protein selected from DOT1L, PRDM9, PRMT1, PRMT2, PRMT3, PRMT4, PRMT5, NSD1, NSD2, NSD3, or a functional variant or fragment of any of them. (Item 13) 13. The modulator, nucleic acid, recombinant RNA, nanoparticle, viral vector or method according to any one of items 1 to 12, wherein the effector moiety comprises a protein selected from histone acetyltransferase or a functional fragment or variant thereof, for example p300, CREB-binding protein (CBP), or a functional fragment or variant of either thereof. (Item 14) 14. The modulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method according to any one of items 1 to 13, wherein the effector moiety comprises a protein selected from a transcriptional activator or a functional fragment or variant thereof, such as VP16, VP64, VP160, or VPR. (Item 15) 15. The modulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method of any one of items 1 to 14, wherein the targeting moiety comprises a Cas9 molecule. (Item 16) The Cas9 molecule can be used to identify and / or characterize Streptococcus (e.g., S. pyogenes or S. thermophilus), Francisella (e.g., F. novicida), Staphylococcus (e.g., S. aureus), Acidaminococcus (e.g., Acidaminococcus species), or any of the following: 16. The modulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method of paragraph 15, comprising a Cas9 protein from a species of Clostridium moniliforme (e.g., Clostridium moniliforme), ... (Item 17) 17. The modulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method of paragraph 15 or 16, wherein the Cas9 molecule comprises a Cas9 protein that substantially lacks nuclease activity, e.g., dCas9, e.g., comprising an inactive RuvC and / or HNH domain. (Item 18) 18. The modulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method of any one of items 15 to 17, wherein the Cas9 molecule comprises (e.g., is non-covalently associated with) a gRNA, e.g., an sgRNA, and the gRNA binds to the expression control element. (Item 19) 19. The modulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method of Item 18, wherein the gRNA comprises a nucleic acid sequence selected from any of SEQ ID NOs: 4 to 26, or a sequence having at least 80, 85, 90, 95, or 99% identity to any of SEQ ID NOs: 4 to 26. (Item 20) 15. The modulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method of any one of items 1 to 14, wherein the targeting moiety comprises a TAL effector molecule. (Item 21) 15. The modulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method of any one of items 1 to 14, wherein the targeting moiety comprises a Zn finger molecule. (Item 22) 15. The modulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method of any one of items 1 to 14, wherein the targeting moiety comprises a Zn finger molecule having 2, 3, 4, 5, or 6 Zn finger proteins. (Item 23) 23. The regulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method according to any one of items 1 to 22, wherein the expression control element comprises an enhancer or promoter or a portion thereof operably linked to the FXN gene. (Item 24) The targeting portion is located within 500, 490, 480, 470, 460, 450, 440, 430, 420, 410, 400, 390, 380, 370, 360, 350, 340, 330, 320, 310, 300, 290, 280, 270, 260, 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 115, 125, 130, 145, 150, 165, 170, 180, 195, 190, 200, 210, 210, 220, 230, 240, 250, 265, 275, 280, 295, 295, 300, 310, 320, 330, 340, 350, 365, 375, 380, 395, 400, 410, 420, 430, 440, 450, 460, 470, 460, 480, 490, 480, 490, 480, 470, 460, 450, 440, 430, 420, 410, 400, 490, 480, 470, 460, 450, 24. The modulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method of any one of paragraphs 1 to 23, which binds to a nucleic acid sequence that is less than 1, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide upstream or downstream (and optionally at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, or 90 nucleotides upstream or downstream). (Item 25) 25. The modulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method of any one of items 1 to 24, wherein the targeting moiety binds to a nucleic acid sequence selected from the sequences depicted by the genomic coordinates in Table 3. (Item 26) the targeting moiety comprises a Cas9 molecule, e.g., a dCas9 molecule, and the effector moiety comprises p300 or a functional fragment or variant thereof; 26. The regulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method according to any one of items 1 to 25. (Item 27) the targeting moiety comprises a Cas9 molecule, e.g., a dCas9 molecule, and the effector moiety comprises VP64 or a functional fragment or variant thereof; 26. The regulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method according to any one of items 1 to 25. (Item 28) the targeting moiety comprises an enzymatically inactive Cas nuclease, e.g., a dCas9 molecule, and the effector moiety comprises VP64 or a functional fragment or variant thereof, p65 or a functional fragment or variant thereof, and RTA or a functional fragment or variant thereof; 26. The regulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method according to any one of items 1 to 25. (Item 29) the targeting moiety comprises a TAL effector molecule (e.g., the TAL effector molecule binds upstream, e.g., about 50-150 nucleotides upstream, e.g., about 100 nucleotides upstream, of the FXN gene TSS), and the effector moiety comprises VPR or a functional fragment or variant thereof; 26. The regulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method according to any one of items 1 to 25. (Item 30) the targeting moiety comprises a TAL effector molecule (e.g., the TAL effector molecule binds upstream, e.g., about 50-150 nucleotides upstream, e.g., about 100 nucleotides upstream, of the FXN gene TSS), and the effector moiety comprises VP64 or a functional fragment or variant thereof, p65 or a functional fragment or variant thereof, and RTA or a functional fragment or variant thereof; 26. The regulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method according to any one of items 1 to 25. (Item 31) the targeting moiety comprises a zinc finger molecule (e.g., the zinc finger molecule binds upstream, e.g., about 50 to 150 nucleotides upstream, e.g., about 100 nucleotides upstream, of the FXN gene TSS), and the effector moiety comprises VPR or a functional fragment or variant thereof; 26. The regulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method according to any one of items 1 to 25. (Item 32) the targeting moiety comprises a zinc finger molecule (e.g., the zinc finger molecule binds upstream, e.g., about 50 to 150 nucleotides upstream, e.g., about 100 nucleotides upstream, of the FXN gene TSS); and the effector moiety comprises VP64 or a functional fragment or variant thereof, p65 or a functional fragment or variant thereof, and RTA or a functional fragment or variant thereof; 26. The regulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method according to any one of items 1 to 25. (Item 33) 33. The modulating agent, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method according to any one of items 1 to 32, wherein the modulating agent comprises or is a fusion molecule. (Item 34) 34. The modulator, nucleic acid, recombinant RNA, nanoparticle, viral vector, or method of any one of items 1 to 25 or 33, wherein the modulator comprises an amino acid sequence selected from any of SEQ ID NOs: 304 to 309, or an amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98, or 99% identity thereto. (Item 35) A complex comprising the regulator according to any one of items 1 to 3 or 9 to 34 and a nucleic acid sequence having an expression control sequence for the FXN gene. (Item 36) A cell comprising the regulator, nucleic acid, recombinant RNA, nanoparticle, or viral vector according to any one of items 1 to 6 or 9 to 34. (Item 37) A cell comprising a nucleic acid encoding the regulator according to any one of Items 1 to 3 or 9 to 34. (Item 38) A method for delivering the regulator, nucleic acid, recombinant RNA, nanoparticle, or viral vector according to any one of items 1 to 6 or 9 to 34 to a cell, comprising: contacting the cell with the modulator, nucleic acid, recombinant RNA, nanoparticle, or viral vector; This allows delivery of the modulator, nucleic acid, recombinant RNA, nanoparticle, or viral vector to the cell. A method comprising: (Item 39) 1. A method of modulating, e.g., increasing, transcription of a frataxin (FXN) gene, comprising: contacting a cell with the regulator, nucleic acid, recombinant RNA, nanoparticle, or viral vector according to any one of items 1 to 6 or 9 to 34; This allows the expression of the FXN gene to be regulated, for example, increased. A method comprising: (Item 40) 1. A method of treating a patient with Friedreich's ataxia (FRDA), comprising administering to a patient: administering to the patient the regulator, nucleic acid, recombinant RNA, nanoparticle, or viral vector according to any one of items 1 to 6 or 9 to 34; This allows the patient to be treated. A method comprising: (Item 41) The method of any one of items 38 to 40, wherein the method increases FXN levels in blood (e.g., whole blood) by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 300, or 400% compared to FXN levels in blood (e.g., whole blood) in the absence of the modulator, nucleic acid, recombinant RNA, nanoparticle, or viral vector. (Item 42) 42. The method of any one of items 38 to 41, wherein the method reduces or eliminates at least one symptom of FDRA, for example, a symptom selected from the following: ataxia, dysarthria, muscle weakness, spasticity (e.g., leg spasticity), scoliosis, bladder dysfunction, reflex impairment, loss of position and / or vibration sense, cardiomyopathy, or diabetes. (Item 43) 43. The method of claim 41 or 42, wherein the level of FXN in blood (e.g., whole blood) is increased for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or at least 1, 2, 3, 4, or 5 years. (Item 44) 44. The method of any one of items 38 to 43, wherein the method increases FXN levels in blood (e.g., whole blood) for at least 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, or 96 hours.

[0039] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication incorporating the color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief explanation of the drawings]

[0040] [Figure 1] Figure 1A shows Western blots for FXN in control primary fibroblasts (HDFn-normal) and fibroblasts from a FRDA patient (GM04078). Figure 1B shows a graph of FXN protein expression measured by ELISA in HDFn and GM04078 cells. Figure 1C shows a graph of aconitase in HDFn and GM04078 cells. [Figure 2] Figure 2A shows a graph of FXN gene expression relative to the HPRT1 reference gene in untreated GM04078 cells (Ctrl), GM04078 cells treated with a regulator comprising a non-targeting sgRNA (NT), or GM04078 cells treated for 48 hours with a regulator comprising one of several pools of sgRNAs targeting the FXN gene. Figure 2B shows a graph of FXN gene expression relative to the GAPDH reference gene in untreated GM04078 cells (Ctrl), GM04078 cells treated with a regulator comprising a non-targeting sgRNA (NT), or GM04078 cells treated for 48 hours with a regulator comprising one of several pools of sgRNAs targeting the FXN gene. [Figure 3]Figure 3A shows a graph of FXN gene expression relative to HPRT1 in GM04078 cells treated with a regulator comprising a non-targeting sgRNA (NT) or with a regulator comprising one of several pools of sgRNAs 48 hours after LNP transfection or 72 hours after transfection. Figure 3B shows a Western blot for FXN showing FXN levels in untreated GM04078 cells (Ctrl), GM04078 cells treated with a regulator comprising a non-targeting sgRNA (NT), or with a regulator comprising one of several pools of sgRNAs 48 hours after LNP transfection or 72 hours after transfection. [Figure 4] Figure 4A shows a graph of FXN gene expression relative to the HPRT1 reference gene in GM04078 cells and HDFn cell lines treated with modulators 48 hours after LNP transfection. Figure 4B shows a graph of FXN gene expression relative to the GAPDH reference gene in GM04078 cells and HDFn cell lines treated with modulators 48 hours after LNP transfection. Ctrl: Untreated (Ctrl); NT: Treated with modulators containing a non-targeting sgRNA; Pool 1 or Pool 4: Treated with modulators containing either sgRNA Pool 1 or Pool 4. [Figure 5] A graph of aconitase activity as rate (nmol / min / mg) is shown for untreated GM04078 control cells, GM04078 cells treated with a modulator containing a non-targeting sgRNA (NT), and GM04078 cells treated with a modulator containing one of several pools of sgRNA (Pool 1 or Pool 4). [Figure 6] A schematic representation of the FXN gene, the repeat region, and the location of the sgRNAs included in the sgRNA pool is shown. [Figure 7] A timeline of cell inoculation and processing for RNA analysis (top) and a schematic diagram of the regions of genomic DNA targeted by an exemplary pool of sgRNAs (bottom) are shown. [Figure 8]Graphs of FXN gene expression in WTiPSC-derived cardiomyocytes (iCardiomyocytes) and WTiPSC-derived glutamatergic cortical neurons (iNeurons) after treatment with exemplary fusion molecules (left: dCas9-VPR and right: dCas9-p300). [Figure 9] 1 shows a graph of relative FXN expression as measured by RNA levels in fibroblasts from FRDA patients treated with a modulator comprising a TAL effector molecule or a targeting molecule comprising dCas9 and an effector molecule comprising VPR. [Figure 10] 1 shows a graph of relative FXN expression as measured by protein levels in fibroblasts from FRDA patients treated with a modulator comprising a TAL effector molecule or a targeting molecule comprising dCas9 and an effector molecule comprising VPR. [Figure 11] Graph of relative FXN expression as measured by RNA levels in mice injected with a modulator comprising a fusion molecule containing dCas9-VPR at increasing post-injection time points. [Figure 12] Graph of relative FXN expression as measured by protein levels in mice injected with a modulator comprising a fusion molecule containing dCas9-VPR at increasing post-injection time points. [Figure 13] A graph of relative FXN expression as measured by RNA levels in cells from FRDA patients or normal patients is shown at various time points after treatment, where cells were treated with a modulatory agent, including a fusion molecule containing dCas9-VPR. [Figure 14] A graph of relative FXN expression, as measured by RNA levels, in cells from FRDA patients or normal patients is shown at various time points after treatment, where cells were treated with a modulator, including a fusion molecule containing dCas9-p300. [Figure 15] Graphs of FXN expression, as measured by protein levels, in iPSC cardiomyocytes derived from FRDA and normal patients treated with modulators containing fusion molecules containing either dCas9-VPR or dCas9-p300 are shown at increasing post-injection time points. DETAILED DESCRIPTION OF THE INVENTION

[0041] Provided herein are compositions and methods for modulating, e.g., increasing, frataxin (FXN) expression, e.g., in a subject in need thereof. FRDA is associated with an autosomal GAA repeat expansion in the FXN gene, which reduces the level of FXN protein expression. Without wishing to be bound by theory, it is believed that increasing the level of FXN protein in a subject suffering from FRDA (e.g., generally, or in one or more specific target tissues) can reduce or eliminate the symptoms of FRDA. The present disclosure provides, in part, a modulatory agent comprising a targeting moiety that binds to a genomic sequence element (e.g., an expression control element) operably linked to a target gene (e.g., FXN) and an effector moiety that, when localized by the targeting moiety, can modulate expression of the target gene. In some embodiments, the modulatory agent disclosed herein specifically binds via the targeting moiety to an expression control element (e.g., a promoter or enhancer) operably linked to the FXN gene, and the effector moiety modulates expression of FXN.

[0042] The present disclosure further provides nucleic acids encoding the modulating agents and methods and compositions for delivering the nucleic acids, as well as methods for increasing FXN expression in cells using the modulating agents described herein.

[0043] regulator As described herein, the present disclosure provides techniques for modulating (e.g., increasing) expression of a target gene, e.g., FXN, by contacting a cell with a modulating agent described herein. In some embodiments, the modulating agent comprises a targeting moiety and an effector moiety. In some embodiments, the modulating agent comprises a targeting moiety and one effector moiety. In some embodiments, the modulating agent comprises a targeting moiety and multiple effector moieties (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more effector domains (and optionally, less than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 effector domains)).

[0044] Generally, a modulatory agent described herein binds (e.g., via a targeting moiety) to a genomic sequence element that is proximate to and / or operably linked to a target gene (e.g., FXN). In some embodiments, binding of the modulatory agent to the genomic sequence element modulates (e.g., increases) expression of the target gene (e.g., FXN). For example, binding of a modulatory agent comprising an effector moiety that recruits or inhibits the recruitment of a component of the transcription machinery to the genomic sequence element may modulate (e.g., increase) expression of the target gene (e.g., FXN). As another example, binding of a modulatory agent comprising an effector moiety (e.g., an epigenetic modification moiety) with enzymatic activity may modulate (e.g., increase) expression of the target gene (e.g., FXN) through localization of the enzymatic activity of the effector moiety. As yet another example, both binding of the modulatory agent to the genomic sequence element and localization of the enzymatic activity of the modulatory agent may contribute to the resulting modulation (e.g., increase) of expression of the target gene (e.g., FXN).

[0045] In some embodiments, a modulator increases expression of a target gene (e.g., FXN) by promoting transcription of the target gene. The modulator can recruit components of the transcription machinery to the target gene or an expression control sequence operably linked to the target gene. The modulator can inhibit the interaction of an inhibitor of transcription with the target gene or an expression control sequence operably linked to the target gene.

[0046] In some embodiments, increasing expression comprises increasing the level of mRNA encoded by the target gene (e.g., FXN). In some embodiments, increasing expression comprises increasing the level of protein encoded by the target gene (e.g., FXN). In some embodiments, increasing expression comprises increasing both the level of mRNA and protein encoded by the target gene. In some embodiments, expression of a target gene (e.g., FXN) in a cell contacted with or containing a modulating agent is at least 1.05x (i.e., 1.05-fold), 1.1x, 1.15x, 1.2x, 1.25x, 1.3x, 1.35x, 1.4x, 1.45x, 1.5x, 1.55x, 1.6x, 1.65x, 1.7x, 1.75x, 1.8x, 1.85x, 1.9x, 1.95x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, or 100x greater than the level of expression of the target gene in a similar cell not contacted with or not containing the modulating agent. The expression of target genes can be assayed by methods well known to those skilled in the art, including RT-PCR, ELISA, or Western blot.Subjects, such as patients, patients with FRDA can be evaluated by assessing blood (e.g., whole blood) levels of FXN, for example, using any of the following methods: Oglesbee et al.Clin Chem.2013 Oct;59(10):1461-9.doi:10.1373 / clinchem.2013.207472 or Deutsch et al.J Neurol Neurosurg Psychiatry.2014 Sep;85(9):994-1002.doi:10.1136 / jnnp-2013-306788 (the contents of which are incorporated herein by reference in their entirety).

[0047] The modulating agents of the present disclosure can be used to increase expression of a target gene (e.g., FXN) in a cell over a period of time. In some embodiments, expression of the target gene in a cell contacted with or containing a modulating agent is appreciably increased for 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, or 24 hours, or at least 1, 2, 3, 4, 5, 6, 7, 10, or 14 days, or at least 1, 2, 3, 4, or 5 weeks, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or at least 1, 2, 3, 4, or 5 years (e.g., indefinitely). Optionally, expression of the target gene in cells contacted with or containing the modulating agent is appreciably increased over a period of 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 year or less.

[0048] A modulator may comprise multiple effector moieties, where each effector moiety has a different functionality than the other effector moieties. For example, a modulator may comprise two effector moieties, where a first effector moiety has transcriptional activator functionality and a second effector moiety has DNA demethylase functionality. In some embodiments, a modulator comprises effector moieties whose functionality is complementary to each other with respect to increasing expression of a target gene (e.g., FXN), e.g., where the functionality allows for enhanced expression and, optionally, when present individually, does not enhance expression or only negligibly enhances expression. In some embodiments, a modulator comprises multiple effector moieties, where each effector moiety complements each other, e.g., where each effector moiety increases expression of a target gene (e.g., FXN).

[0049] In some embodiments, a modulating agent comprises a combination of effector moieties whose functionality synergizes with each other with respect to increasing expression of a target gene (e.g., FXN). Without wishing to be bound by theory, it is believed that epigenetic modifications to a genomic locus are additive, in that multiple transcriptionally activating epigenetic markers (e.g., multiple different types of epigenetic markers and / or overproduction of a given type) together promote expression more efficiently (e.g., greater increase in expression and / or longer duration of expression) than individual modifications alone. In some embodiments, a modulating agent comprises multiple effector moieties, where each effector moiety synergizes with each of the other effector moieties, e.g., each effector moiety increases expression of a target gene (e.g., FXN). In some embodiments, a modulating agent (comprising multiple effector moieties that synergize with each other) is more effective at promoting expression of a target gene (e.g., FXN) than a modulating agent comprising individual effector moieties. In some embodiments, a modulator comprising multiple effector moieties is at least 1.05x (i.e., 1.05-fold), 1.1x, 1.15x, 1.2x, 1.25x, 1.3x, 1.35x, 1.4x, 1.45x, 1.5x, 1.55x, 1.6x, 1.65x, 1.7x, 1.75x, 1.8x, 1.85x, 1.9x, 1.95x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, or 100x more effective at increasing expression of a target gene (e.g., FXN) than a modulator comprising individual effector moieties.

[0050] In some embodiments, the modulating agent modulates (e.g., increases) expression of a target gene (e.g., FXN) by altering one or more epigenetic markers associated with the target gene or an expression control sequence operably linked to the target gene. In some embodiments, the alteration comprises increasing the level of an epigenetic marker associated with the target gene or an expression control sequence operably linked to the target gene. In some embodiments, the alteration comprises decreasing the level of an epigenetic marker associated with the target gene or an expression control sequence operably linked to the target gene. Epigenetic markers include, but are not limited to, DNA methylation, histone methylation, and histone deacetylation.

[0051] In some embodiments, altering the level of the epigenetic marker increases the level of the epigenetic marker associated with the target gene or an expression control sequence operably linked to the target gene by at least 1.05 times (i.e., 1.05x), 1.1x, 1.15x, 1.2x, 1.25x, 1.3x, 1.35x, 1.4x, 1.45x, 1.5x, 1.55x, 1.6x, 1.65x, 1.7x, 1.75x, 1.8x, 1.85x, 1.9x, 1.95x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, or 100x increase. In some embodiments, altering the level of the epigenetic marker increases the level of the epigenetic marker associated with the target gene or an expression control sequence operably linked to the target gene by at least 1.05 times (i.e., 1.05x), 1.1x, 1.15x, 1.2x, 1.25x, 1.3x, 1.35x, 1.4x, 1.45x, 1.5x, 1.55x, 1.6x, 1.65x, 1.7x, 1.75x, 1.8x, 1.85x, 1.9x, 1.95x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, or 100x reduction. The levels of epigenetic markers can be assayed by methods well known to those skilled in the art, including whole genome bisulfite sequencing, reduced representation bisulfite sequencing, bisulfite amplicon sequencing, methylation arrays, pyrosequencing, ChIP-seq, or ChIP-qPCR.

[0052] The modulating agents of the present disclosure can be used to alter the level of an epigenetic marker associated with a target gene or an expression control sequence operably linked to the target gene in a cell over a period of time. In some embodiments, the level of an epigenetic marker associated with a target gene or an expression control sequence operably linked to the target gene in a cell contacted with or containing a modulating agent is appreciably increased over 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, or 24 hours, or at least 1, 2, 3, 4, 5, 6, 7, 10, or 14 days, or at least 1, 2, 3, 4, or 5 weeks, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or at least 1, 2, 3, 4, or 5 years (e.g., indefinitely). In some embodiments, the level of an epigenetic marker associated with a target gene or an expression control sequence operably linked to a target gene in a cell contacted with or containing a modulating agent is appreciably reduced over 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, or 24 hours, or at least 1, 2, 3, 4, 5, 6, 7, 10, or 14 days, or at least 1, 2, 3, 4, or 5 weeks, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or at least 1, 2, 3, 4, or 5 years (e.g., indefinitely). Optionally, the level of an epigenetic marker associated with the target gene or an expression control sequence operably linked to the target gene in cells contacted with or containing the modulating agent is appreciably increased or decreased over a period of 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 year or less.

[0053] The modulating agent may be or comprise a fusion molecule, hi some embodiments, the fusion molecule comprises a targeting moiety and an effector moiety covalently linked to each other, e.g., by a peptide bond.

[0054] In some embodiments, the modulating agent, e.g., the targeting portion of a fusion molecule, comprises no more than 100, 90, 80, 70, 60, 50, 40, 30, or 20 nucleotides (and optionally at least 10, 20, 30, 40, 50, 60, 70, 80, or 90 nucleotides). In some embodiments, the modulator, e.g., effector portion of a fusion molecule, comprises no more than 2000, 1900, 1800, 1700, 1600, 1500, 1400, 1300, 1200, 1100, 1000, 900, 800, 700, 600, 500, 400, 300, 200, or 100 amino acids (and optionally at least 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, or 1900 amino acids). In some embodiments, the modulator, e.g., the effector portion of the fusion molecule, has a nucleotide sequence of 100-2000, 100-1900, 100-1800, 100-1700, 100-1600, 100-1500, 100-1400, 100-1300, 100-1200, 100-1100, 100-1000, 100-900, 100-800, 100-700 , 100~600, 100~500, 100~400, 100~300, 100~200, 200~2000, 200~1900, 200~1800, 200~1700, 200~1600, 200~1500, 200~1400, 200~1300, 200~1200, 200~1100, 200~1000, 200~900, 200~800 , 200~700, 200~600, 200~500, 200~400, 200~300, 300~2000, 300~1900, 300~1800, 300~1700, 300~1600, 300~1500, 300~1400, 300~1300, 300~1200, 300~1100, 300~1000, 300~900, 300~800 , 300~700, 300~600, 300~500, 300~400, 400~2000, 400~1900, 400~1800, 400~1700, 400~1600, 400~1500, 400~1400, 400~1300, 400~1200, 400~1100, 400~1000, 400~900, 400~800, 400~700,400~600、400~500、500~2000、500~1900、500~1800、500~1700、500~1600、500~1500、500~1400、500~1300、500~1200、500~1100、500~1000、500~900、500~800、500~700、500~600、600~2000、600~1900、600~1800、600~1700、600~1600、600~1500、600~1400、600~1300、600~1200、600~1100、600~1000、600~900、600~800、600~700、700~2000、700~1900、700~1800、700~1700、700~1600、700~1500、700~1400、700~1300、700~1200、700~1100、700~1000、700~900、700~800、800~2000、800~1900、800~1800、800~1700、800~1600、800~1500、800~1400、800~1300、800~1200、800~1100、800~1000、800~900、900~2000、900~1900、900~1800、900~1700、900~1600、900~1500、900~1400、900~1300、900~1200、900~1100、900~1000、1000~2000、1000~1900、1000~1800、1000~1700、1000~1600、1000~1500、1000~1400、1000~1300、1000~1200、1000~1100、1100~2000、1100~1900、1100~1800、1100~1700、1100~1600、1100~1500、1100~1400、1100~1300、1100~1200、1200~2000、1200~1900、1200~1800、1200~1700、1200~1600、1200~1500、1200~1400、1200~1300、1300~2000、1300~1900、1300~1800、1300~1700、1300~1600、1300~1500、1300~1400、1400~2000、1400~1900、1400~1800、1400~1700、1400~1600、1400~1500、1500~2000、Contains 1500 to 1900, 1500 to 1800, 1500 to 1700, 1500 to 1600, 1600 to 2000, 1600 to 1900, 1600 to 1800, 1600 to 1700, 1700 to 2000, 1700 to 1900, 1700 to 1800, 1800 to 2000, 1800 to 1900, or 1900 to 2000 amino acids.

[0055] The modulating agent may comprise a nucleic acid, e.g., one or more nucleic acids. The term "nucleic acid" refers to any compound that is or can be incorporated into an oligonucleotide chain. In some embodiments, a nucleic acid is a compound and / or substance that is or can be incorporated into an oligonucleotide chain via a phosphodiester bond. As will be clear from the context, in some embodiments, "nucleic acid" refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides); in some embodiments, "nucleic acid" refers to an oligonucleotide chain comprising individual nucleic acid residues. In some embodiments, "nucleic acid" is or comprises RNA; in some embodiments, "nucleic acid" is or comprises DNA. In some embodiments, a nucleic acid is or comprises less than 50% ribonucleotides and is referred to herein as ribonucleic acid (RNA). In some embodiments, a nucleic acid is, comprises, or consists of one or more naturally occurring nucleic acid residues. In some embodiments, a nucleic acid is, comprises, or consists of one or more nucleic acid analogs. In some embodiments, a nucleic acid analog differs from a nucleic acid in that it does not utilize a phosphodiester backbone. For example, in some embodiments, the nucleic acid is, comprises, or consists of one or more "peptide nucleic acids," which are known in the art and have peptide bonds rather than phosphodiester linkages in the backbone, and are considered within the scope of the present invention. Alternatively, or in addition, in some embodiments, the nucleic acid has one or more phosphorothioate and / or 5'-N-phosphoramidite linkages rather than phosphodiester linkages. In some embodiments, the nucleic acid is, comprises, or consists of one or more naturally occurring nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine).In some embodiments, the nucleic acid is, comprises, or consists of one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, methylated bases, intervening bases, and combinations thereof). In some embodiments, a nucleic acid comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) compared to those present in naturally occurring nucleic acids. In some embodiments, a nucleic acid has a nucleotide sequence that encodes a functional gene product such as RNA or a protein. In some embodiments, a nucleic acid comprises one or more introns. In some embodiments, a nucleic acid is prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), reproduction in a recombinant cell or system, and chemical synthesis. As used herein, "recombinant" when used to refer to a nucleic acid refers to any nucleic acid that does not occur in nature. In some embodiments, the nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues in length.In some embodiments, the nucleic acid can have a length of about 2 to about 5,000 nt, about 10 to about 100 nt, about 50 to about 150 nt, about 100 to about 200 nt, about 150 to about 250 nt, about 200 to about 300 nt, about 250 to about 350 nt, about 300 to about 500 nt, about 10 to about 1,000 nt, about 50 to about 1,000 nt, about 100 to about 1,000 nt, about 1,000 to about 2,000 nt, about 2,000 to about 3,000 nt, about 3,000 to about 4,000 nt, about 4,000 to about 5,000 nt, or any range therebetween. In some embodiments, the nucleic acid is partially or completely single-stranded; in some embodiments, the nucleic acid is partially or completely double-stranded. In some embodiments, the nucleic acid has a nucleotide sequence that encodes a polypeptide or includes at least one element that is the complement of a sequence encoding a polypeptide. In some embodiments, the nucleic acid has enzymatic activity.

[0056] In some embodiments, the targeting moiety comprises or is a nucleic acid. In some embodiments, the effector moiety comprises or is a nucleic acid. In some embodiments, the nucleic acid comprised in the moiety may be or contain DNA, RNA, and / or an artificial or synthetic nucleic acid or a nucleic acid analog or mimic. For example, in some embodiments, the nucleic acid may be or include one or more of the following: genomic DNA (gDNA), complementary DNA (cDNA), peptide nucleic acid (PNA), peptide-oligonucleotide conjugate, locked nucleic acid (LNA), bridged nucleic acid (BNA), polyamide, triplex-forming oligonucleotide, antisense oligonucleotide, tRNA, mRNA, rRNA, miRNA, gRNA, siRNA or other RNAi molecule (e.g., one that targets a non-coding RNA described herein and / or one that targets the expression product of a specific gene associated with a targeted genome complex described herein), etc. Nucleic acid sequences suitable for use in modulating agents can include modified oligonucleotides (e.g., chemical modifications, such as modifications that alter the backbone linkages, sugar moieties, and / or nucleobases) and / or artificial nucleic acids. In some embodiments, nucleic acid sequences include, but are not limited to, genomic DNA, cDNA, peptide nucleic acid (PNA) or peptide-oligonucleotide conjugates, locked nucleic acid (LNA), bridged nucleic acid (BNA), polyamide, triplex-forming oligonucleotides, modified DNA, antisense DNA oligonucleotides, tRNA, mRNA, rRNA, modified RNA, miRNA, gRNA, and siRNA or other RNA or DNA molecules. In some embodiments, the nucleic acid may include one or more residues that are not naturally occurring DNA or RNA residues, may include one or more linkages that are not phosphodiester bonds (e.g., may be phosphorothioate linkages), and / or may include one or more modifications, such as, for example, 2'O modifications, e.g., 2'-OMeP.A variety of nucleic acid structures useful for preparing synthetic nucleic acids are known in the art (see, e.g., WO 2017 / 0628621 and WO 2014 / 012081), and one of skill in the art will understand that these may be used in accordance with the present disclosure.

[0057] Some examples of nucleic acids include, but are not limited to, nucleic acids that hybridize with an endogenous target gene, e.g., FXN (e.g., gRNA or antisense ssDNA described elsewhere herein), nucleic acids that hybridize with an exogenous nucleic acid such as viral DNA or RNA, nucleic acids that hybridize with RNA, nucleic acids that interfere with gene transcription, nucleic acids that interfere with RNA translation, nucleic acids that stabilize RNA or destabilize RNA, such as by targeting it for degradation, nucleic acids that interfere with DNA or RNA binding factors by interfering with their expression or their function, and nucleic acids that bind to and regulate the function of an intracellular protein or protein complex.

[0058] In some embodiments, the modulating agent comprises one or more nucleoside analogs. In some embodiments, the nucleic acid sequence may comprise one or more nucleoside analogs in addition to or as a substitute for one or more naturally occurring nucleosides, for example, purines or pyrimidines, such as adenine, cytosine, guanine, thymine, and uracil. In some embodiments, the nucleic acid sequence comprises one or more nucleoside analogs. Nucleoside analogs include, but are not limited to, nucleoside analogs such as 5-fluorouracil; 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 4-methylbenzimidazole, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, , dihydrouridine, β-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosylqueosine, 5' -Methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid(v), wybutoxocine, pseudouracil, queusine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid(v), 5-methyl-2-thiouracil , 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w, 2,6-diaminopurine, 3-nitropyrrole, inosine, thiouridine, queusine, wyosine, diaminopurine, isoguanine, isocytosine, diaminopyrimidine, 2,4-difluorotoluene, isoquinoline, pyrrolo[2,3-β]pyridine, and any other capable of base pairing with a purine or pyrimidine side chain.

[0059] Targeting section A targeting moiety refers to an agent or entity that specifically targets, e.g., binds to, a genomic sequence element (e.g., an expression control sequence or anchor sequence) that is proximal to and / or operably linked to a target gene (e.g., FXN). In some embodiments, a targeting moiety targets, e.g., binds to, a component of a genomic complex (e.g., ASMC). In some embodiments, a targeting moiety targets, e.g., binds to, an expression control sequence (e.g., a promoter or enhancer) operably linked to FXN. In some embodiments, a targeting moiety targets, e.g., binds to, a target gene (e.g., FXN) or a portion of a target gene. The target of a targeting moiety is sometimes referred to as its targeting component. A targeting component may be any genomic sequence element operably linked to a target gene or the target gene itself, including, but not limited to, a promoter, enhancer, anchor sequence, exon, intron, UTR coding sequence, splice site, or transcription start site.

[0060] In some embodiments, the interaction between the targeting moiety and its targeting component interferes with one or more other interactions that the targeting component would otherwise form. In some embodiments, the binding between the targeting moiety and the targeting component prevents the targeting component from interacting with another transcription factor, genome complex component, or genome sequence element. In some embodiments, the binding between the targeting moiety and the targeting component reduces the binding affinity of the targeting component for another transcription factor, genome complex component, or genome sequence element. In some embodiments, the K Dis increased by at least 1.05× (i.e., 1.05-fold), 1.1×, 1.2×, 1.3×, 1.4×, 1.5×, 1.6×, 1.7×, 1.8×, 1.9×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 20×, 50×, or 100× (and optionally, 20×, 10×, 9×, 8×, 7×, 6×, 5×, 4×, 3×, 2×, 1.9×, 1.8×, 1.7×, 1.6×, 1.5×, 1.4×, 1.3×, 1.2×, or 1.1× or less) in the presence of a modulator that includes a targeting moiety compared to the absence of a modulator that includes a targeting moiety. D Changes in expression are determined using ChIP-Seq or ChIP-qPCR.

[0061] In some embodiments, binding between the targeting moiety and the targeting component alters, e.g., reduces, the level of a genomic complex (e.g., ASMC) comprising the targeting component. In some embodiments, the level of a genomic complex (e.g., ASMC) comprising the targeting component is reduced by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% (and optionally up to 100, 90, 80, 70, 60, 50, 40, 30, or 20%) in the presence of a modulating agent comprising a targeting moiety compared to the absence of the modulating agent. In some embodiments, binding between the targeting moiety and the targeting component alters, e.g., reduces, the occupancy of a genomic complex (e.g., ASMC) at a genomic sequence element (e.g., a target gene or an expression control sequence operably linked thereto). In some embodiments, occupancy is reduced by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% (and optionally up to 100, 90, 80, 70, 60, 50, 40, 30, or 20%) in the presence of a modulating agent comprising a targeting moiety relative to the absence of said modulating agent. Genome complex formation, changes in affinity of the targeting moiety for other complex components, and / or changes in topology of genomic DNA affected by the genome can be assessed using, for example, HiChIP, ChIAPET, 4C, or 3C, e.g., HiChIP.

[0062] In some embodiments, binding of the targeting moiety to the targeting component alters, e.g., reduces, occupancy of a genome complex (e.g., ASMC) at a genome sequence element (e.g., a gene, promoter, or enhancer, e.g., associated with a genome or transcription complex). In some embodiments, binding of the targeting moiety to the targeting component reduces occupancy of a genome complex (e.g., ASMC) at a genome sequence element by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% (and optionally, up to 100, 90, 80, 70, 60, 50, 40, 30, or 20%) in the presence of a modulating agent comprising a targeting moiety compared to the absence of the modulating agent. In some embodiments, occupancy refers to the frequency with which an element can be found bound to another element, as determined, for example, by HiC, ChIP, immunoprecipitation, or other binding measurement assays known in the art. In some embodiments, occupancy may be determined using an integrity index (eg, a change in the integrity index may correspond to a change in occupancy).

[0063] In some embodiments, binding of the targeting moiety to the targeting component alters, e.g., reduces, the occupancy of the targeting component in / at a genomic complex (e.g., ASMC). In some embodiments, binding of the targeting moiety to the targeting component reduces the occupancy of the targeting component in / at a genomic complex (e.g., ASMC) by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% (and optionally up to 100, 90, 80, 70, 60, 50, 40, 30, or 20%) in the presence of a modulating agent comprising a targeting moiety compared to the absence of the modulating agent.

[0064] In some embodiments, binding of the targeting moiety to the targeting component alters (e.g., increases) expression of a target gene (e.g., FXN) associated with and / or operably linked to the targeting component. In some embodiments, binding of the targeting moiety to the targeting component alters (e.g., increases) expression of a target gene (e.g., FXN) associated with a genome complex (e.g., ASMC) that includes the targeting component. In some embodiments, expression of the target gene is increased by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000% (and optionally up to 1000, 900, 800, 700, 600, 500, 400, 300, or 200%) in the presence of a modulating agent that includes a targeting moiety compared to the absence of the modulating agent.

[0065] In some embodiments, the targeting moiety is designed and / or administered such that it specifically targets, e.g., binds to, a particular genomic sequence element (e.g., a particular genomic complex (e.g., ASMC) that contains said genomic sequence element) relative to other genomic sequences that may be present in the same system (e.g., cell, tissue, etc.). In some embodiments, the targeting moiety comprises a nucleic acid sequence that is complementary to a targeting component, e.g., an expression control sequence, an anchor sequence, or a target gene (e.g., FXN). In some embodiments, the targeting moiety comprises a nucleic acid sequence that is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% complementary to the targeting component.

[0066] In some embodiments, the targeting moiety may be or include a CRISPR / Cas molecule, a TAL effector molecule, a Zn finger molecule, or a nucleic acid molecule.

[0067] CRISPR / Cas molecule In some embodiments, the targeting moiety is or includes a CRISPR / Cas molecule, which includes a protein involved in the clustered regularly interspaced short palindromic repeats (CRISPR) system, e.g., a Cas protein, and optionally a guide RNA, e.g., a single guide RNA (sgRNA).

[0068] The CRISPR system is an adaptive defense system first discovered in bacteria and archaea. CRISPR systems use RNA-guided nucleases called CRISPR-associated or "Cas" endonucleases (e.g., Cas9 or Cpf1) to cleave foreign DNA. For example, in a typical CRISPR / Cas system, the endonuclease is directed to the target nuclease sequence (e.g., the site in the genome where the sequence is to be edited) by a sequence-specific, non-coding "guide RNA" that targets a single- or double-stranded DNA sequence. Three classes (I-III) of CRISPR systems have been identified. Class II CRISPR systems use a single Cas endonuclease (rather than multiple Cas proteins). Class II CRISPR systems include a type II Cas endonuclease, e.g., Cas9, a CRISPR RNA ("crRNA"), and a trans-acting crRNA ("tracrRNA"). The crRNA includes a "guide RNA," which is typically an approximately 20-nucleotide RNA sequence that corresponds to the target DNA sequence. The crRNA also contains a region that binds to the tracrRNA to form a partially double-stranded structure, which is cleaved by RNase III to yield a crRNA / tracrRNA hybrid. The crRNA / tracrRNA hybrid then directs the Cas9 endonuclease to recognize and cleave the target DNA sequence. The target DNA sequence must generally be adjacent to a "protospacer adjacent motif" ("PAM") specific to a given Cas endonuclease; however, PAM sequences occur throughout a given genome.CRISPR endonucleases identified from diverse prokaryotic species have unique PAM sequence requirements; example PAM sequences include 5'-NGG (Streptococcus pyogenes), 5'-NNAGAA (Streptococcus thermophilus CRISPR1), 5'-NGGNG (Streptococcus thermophilus CRISPR3), and 5'-NNNGATT (Neisseria meningiditis). Some endoglucanases, such as Cas9 endonuclease, bind to G-rich PAM sites, e.g., 5'-NGG, and perform blunt-end cleavage of target DNA three nucleotides upstream (5') of the PAM site. Another class II CRISPR system contains a V-type endonuclease, Cpf1, which is smaller than Cas9; examples include AsCpf1 (from Acidaminococcus sp.) and LbCpf1 (from Lachnospiraceae sp.). Cpf1-associated CRISPR arrays are processed into mature crRNA without the need for tracrRNA; in other words, the Cpf1 system only requires Cpf1 nuclease and crRNA to cleave the target DNA sequence. Cpf1 endonuclease binds to T-rich PAM sites, such as 5'-TTN. Cpf1 also recognizes the 5'-CTA PAM motif. Cpf1 cleaves target DNA by introducing offset or staggered double-stranded breaks with 4- or 5-nucleotide 5' overhangs, e.g., cleaving the target DNA with a 5-nucleotide offset or staggered cut located 18 nucleotides downstream (3') from the PAM on the coding strand and 23 nucleotides downstream from the PAM on the complementary strand; the 5-nucleotide overhangs created by such offset cuts allow for more precise genome editing by DNA insertion via homologous recombination compared to insertion with blunt-cut DNA.See, for example, Zetsche et al. (2015) Cell, 163:759-771.

[0069] A wide variety of CRISPR-associated (Cas) genes or proteins can be used in the techniques provided herein, and the choice of Cas protein can vary depending on the specific requirements of the method. Specific examples of Cas proteins include Class II systems, including Cas1, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Cpf1, C2C1, or C2C3. In some embodiments, the Cas protein, e.g., the Cas9 protein, can be derived from any of a variety of prokaryotic species. In some embodiments, a particular Cas protein, e.g., a particular Cas9 protein, is selected to recognize a specific protospacer adjacent motif (PAM) sequence. In some embodiments, the DNA targeting moiety includes a sequence-targeting polypeptide, e.g., a Cas protein, e.g., Cas9. In certain embodiments, the Cas protein, e.g., the Cas9 protein, can be obtained from bacteria or archaea or synthesized using known methods. In certain embodiments, the Cas protein may be derived from a Gram-positive or Gram-negative bacterium.In certain embodiments, the Cas protein is selected from the group consisting of Streptococcus (e.g., S. pyogenes or S. thermophilus), Francisella (e.g., F. novicida), Staphylococcus (e.g., S. aureus), Acidaminococcus (e.g., Acidaminococcus spp.), and the like. sp. BV3L6), Neisseria (e.g., N. meningitidis), Cryptococcus, Corynebacterium, Haemophilus, Eubacterium, Pasteurella, Prevotella, Veillonella, or Marinobacter.

[0070] In some embodiments, Cas proteins require a protospacer adjacent motif (PAM) to be present in or adjacent to the target DNA sequence for the Cas protein to bind and / or function. In some embodiments, the PAM is or includes, from 5' to 3', NGG, YG, NNGRRT, NNNRRT, NGA, TYCV, TATV, NTTN, or NNNGATT, where N represents any nucleotide, Y represents C or T, R represents A or G, and V represents A, C, or G. In some embodiments, the Cas protein is a protein listed in Table 1. In some embodiments, the Cas protein includes one or more mutations that alter its PAM. In some embodiments, the Cas protein includes E1369R, E1449H, and R1556A mutations, or similar substitutions for the amino acids corresponding to said positions. In some embodiments, the Cas protein comprises E782K, N968K, and R1015H mutations, or analogous substitutions at the amino acids corresponding to said positions. In some embodiments, the Cas protein comprises D1135V, R1335Q, and T1337R mutations, or analogous substitutions at the amino acids corresponding to said positions. In some embodiments, the Cas protein comprises S542R and K607R mutations, or analogous substitutions at the amino acids corresponding to said positions. In some embodiments, the Cas protein comprises S542R, K548V, and N552R mutations, or analogous substitutions at the amino acids corresponding to said positions.

[0071] [Table 1]

[0072] In some embodiments, the Cas protein is modified to inactivate the nuclease (e.g., nuclease-deleted Cas9). While wild-type Cas9 generates double-strand breaks (DSBs) at specific DNA sequences targeted by the gRNA, several CRISPR endonucleases are available with modified functionality, for example: "nickase" versions of Cas9 generate only single-strand breaks; catalytically inactive Cas9 ("dCas9") does not cleave the target DNA. In some embodiments, binding of dCas9 to a DNA sequence interferes with transcription at that site due to steric hindrance. In some embodiments, the targeting moiety is or includes a catalytically inactive Cas9, e.g., dCas9. Many catalytically inactive Cas9 proteins are known in the art. In some embodiments, dCas9 includes mutations in each endonuclease domain of the Cas protein, e.g., D10A and H840A mutations.

[0073] In some embodiments, the targeting moiety may include a gRNA or a Cas molecule linked (e.g., covalently) to it. A gRNA is a short, synthetic RNA composed of a "scaffold" sequence required for Cas-protein binding and a user-defined, approximately 20-nucleotide targeting sequence for the genomic target. In practice, guide RNA sequences are generally designed to have a length between 17 and 24 nucleotides (e.g., 19, 20, or 21 nucleotides) and are complementary to the targeting nucleic acid sequence. Custom gRNA generators and algorithms are commercially available for use in designing effective guide RNAs. Gene editing has also been achieved using chimeric "single guide RNAs" ("sgRNAs"), engineered (synthetic) single RNA molecules that mimic the naturally occurring crRNA-tracrRNA complex and contain both a tracrRNA (for binding to a nuclease) and at least one crRNA (to guide the nuclease to the sequence targeted for editing). Chimeric editing sgRNAs have also been demonstrated to be effective for use with Cas proteins; see, e.g., Hendel et al. (2015) Nature Biotechnol., 985-991.

[0074] In some embodiments, the gRNA comprises a nucleic acid sequence complementary to a DNA sequence associated with the target gene. In some embodiments, the DNA sequence is, comprises, or overlaps with an expression control element operably linked to the target gene. In some embodiments, the DNA sequence is, comprises, or overlaps with a genomic sequence listed in Table 3. In some embodiments, the gRNA comprises a nucleic acid sequence that is at least 80, 85, 90, 95, 99, or 100% complementary to a genomic sequence listed in Table 3. In some embodiments, the gRNA comprises a nucleic acid sequence selected from SEQ ID NOs: 4-26, or a sequence having at least 80, 85, 90, 95, or 99% identity to a sequence selected from SEQ ID NOs: 4-26. In some embodiments, a gRNA used in conjunction with a targeting moiety comprising a Cas molecule is an sgRNA.

[0075] TAL effector molecules In some embodiments, the targeting moiety is or comprises a TAL effector molecule, e.g., a TAL effector molecule that specifically binds to a DNA sequence, comprising multiple effector domains or fragments thereof, and optionally one or more additional portions of a naturally occurring TAL effector (e.g., the N- and / or C-termini of multiple TAL effector domains).

[0076] TALEs are naturally occurring proteins secreted by many pathogenic bacterial species, such as the plant pathogen Xanthomonas, that regulate gene expression in host plants, promoting bacterial colonization and survival. The specific binding of TAL effectors is based on a central repeat domain (repeat variable dinucleotide, RVD domain), which typically consists of tandemly arranged, nearly identical repeats of 33 or 34 amino acids.

[0077] Members of the TAL effector family differ primarily in the number and order of their repeats. The number of repeats ranges from 1.5 to 33.5, with the C-terminal repeats typically being short (e.g., approximately 20 amino acids) and commonly referred to as "half-repeats." Each repeat in a TAL effector is characterized by a one-to-one base pair correlation (one repeat recognizes one base pair in the target gene sequence), with different repeat types exhibiting different base pair specificities. Generally, the fewer the number of repeats, the weaker the protein-DNA interaction. A number of 6.5 repeats has been found to be sufficient to activate transcription of a reporter gene (Scholze et al., 2010).

[0078] Repeat-to-repeat variation occurs primarily at amino acid positions 12 and 13, which are therefore termed "hypervariable," and this accounts for the specificity of the interaction with target DNA promoter sequences, as shown in Table 2, which lists exemplary repeat variable dinucleotides (RVDs) and their correspondence with nucleobase targets.

[0079] [Table 2]

[0080] Therefore, it is possible to modify the repeats of TAL effectors to target specific DNA sequences. Further studies have revealed that RVD NK can target G. TAL effector target sites also tend to contain a T flanking the 5' base targeted by the first repeat, although the exact mechanism of this recognition is unknown. To date, over 113 TAL effector sequences have been identified. Non-limiting examples of TAL effectors from Xanthomonas include Hax2, Hax3, Hax4, AvrXa7, AvrXa10, and AvrBs3.

[0081] Thus, the TAL effector domain of the TAL effector molecule of the invention can be used to inhibit the growth of any bacterial species (e.g., Xanthomonas species, such as African strains of Xanthomonas oryzae pv. Oryzae (Yu et al. 2011), Xanthomonas campestris pv. raphani strain 756C, and Xanthomonas oryzae pv. oryzicola strain BLS256 (Bogdanove et al. 2013)). al. 2011). As used herein, a TAL effector domain according to the present invention comprises an RVD domain and flanking sequences (sequences on the N-terminus and / or C-terminus of the RVD) also derived from a naturally occurring TAL effector. It may contain more or fewer repeats than the RVD of a naturally occurring TAL effector. The TAL effector molecules of the present invention are designed to target a given DNA sequence based on the aforementioned codes and other codes known in the art. TAL effector domains (e.g., repeats (monomers or The number of TAL effector domains (e.g., repeats) and their sequences are selected based on the desired DNA target sequence. For example, TAL effector domains, e.g., repeats, may be removed or added to tailor to a particular target sequence. In one embodiment, a TAL effector molecule of the invention comprises between 6.5 and 33.5 TAL effector domains, e.g., repeats. In one embodiment, a TAL effector molecule of the invention comprises between 8 and 33.5 TAL effector domains, e.g., repeats, for example, between 10 and 25 TAL effector domains, e.g., repeats, for example, between 10 and 14 TAL effector domains, e.g., repeats.

[0082] In some embodiments, a TAL effector molecule comprises a TAL effector domain that corresponds to a perfect match with the DNA target sequence. In some embodiments, mismatches between the repeat and the target base pair on the DNA target sequence are tolerated only if they allow the expression repression system, e.g., the expression repressor, comprising the TAL effector molecule, to function. Generally, TALE binding is inversely correlated with the number of mismatches. In some embodiments, the TAL effector molecule of the expression repressor of the present invention contains no more than seven, six, five, four, three, two, or one mismatch with the target DNA sequence, and optionally no mismatches. Without wishing to be bound by theory, generally, the fewer TAL effector domains in the TAL effector molecule, the fewer mismatches can be tolerated, which still allows the expression repression system, e.g., the expression repressor, comprising the TAL effector molecule, to function. Binding affinity is believed to depend on the total number of matching repeat-DNA combinations. For example, a TAL effector molecule with 25 or more TAL effector domains can tolerate up to 7 mismatches.

[0083] In addition to the TAL effector domain, the TAL effector molecules of the present invention may contain additional sequences derived from naturally occurring TAL effectors. The length of the C-terminal and / or N-terminal sequences included on either side of the TAL effector domain portion of the TAL effector molecule can vary and can be selected by those skilled in the art based on, for example, the work of Zhang et al. (2011). Zhang et al. characterized several C-terminal and N-terminal truncation mutants in Hax3-derived TAL effector-based proteins and identified key elements that contribute to optimal binding to target sequences and thus transcriptional activation. Generally, transcriptional activity was found to be inversely correlated with the length of the N-terminus. Regarding the C-terminus, a key element was identified in the DNA-binding residues within the first 68 amino acids of the Hax3 sequence. Thus, in some embodiments, the first 68 amino acids on the C-terminal side of the TAL effector domain of a naturally occurring TAL effector are included in the TAL effector molecule of the expression repressor of the present invention. Thus, in one embodiment, the TAL effector molecule of the present invention comprises: 1) one or more TAL effector domains derived from a naturally occurring TAL effector; 2) at least 70, 80, 90, 100, 110, 120, 130, 140, 150, 170, 180, 190, 200, 220, 230, 240, 250, 260, 270, 280 or more amino acids derived from a naturally occurring TAL effector N-terminal to the TAL effector domain; and / or 3) at least 68, 80, 90, 100, 110, 120, 130, 140, 150, 170, 180, 190, 200, 220, 230, 240, 250, 260 or more amino acids C-terminal to the TAL effector domain.

[0084] Zn finger molecules In some embodiments, the targeting moiety is or comprises a zinc finger molecule. The zinc finger molecule comprises a zinc finger protein, such as a naturally occurring or engineered zinc finger protein, or a fragment thereof.

[0085] In some embodiments, the zinc finger molecule comprises a non-naturally occurring zinc finger protein engineered to bind to a selected target DNA sequence (see, e.g., Beerli, et al. (2002) Nature Biotechnol. 20:135-141; Pabo, et al. (2001) Ann. Rev. Biochem. 70:313-340; Isalan, et al. (2001) Nature Biotechnol. 19:656-660; Segal, et al. (2001) Curr. Opin. Biotechnol. 12:632-637; Choo, et al. al. (2000) Curr.Opin.Struct.Biol.10:411-416; US Patent No. 6,453,242; US Patent No. 6,534,261; US ​​Patent No. 6,59 Specification No. 9,692; Specification No. 6,503,717; Specification No. 6,689,558; Specification No. 7,030,215; Specification No. 6,794,136; Specification No. 7,067,3 Nos. 17, 7,262,054, 7,070,934, 7,361,635, 7,253,273; and U.S. Patent Application Publication Nos. 2005 / 0064474; 2007 / 0218528; 2005 / 0267061 (all of which are incorporated by reference herein in their entireties).

[0086] Engineered zinc finger proteins may have new binding specificities compared to naturally occurring zinc finger proteins. Engineering methods include, but are not limited to, rational design and various types of selection. Rational design, for example, includes the use of triplet (quadruplet) nucleotide sequences and individual zinc finger amino acid sequences, where each triplet or quadruplet nucleotide sequence is linked to one or more amino acid sequences of zinc fingers that bind to the specific triplet or quadruplet sequence. See, for example, U.S. Patent Nos. 6,453,242 and 6,534,261 (the entire contents of which are incorporated herein by reference).

[0087] Exemplary selection methods, including phage display and two-hybrid systems, are disclosed in U.S. Patent Nos. 5,789,538; 5,925,523; 6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759; and 6,242,568; as well as WO 98 / 37186; WO 98 / 53057; WO 00 / 27878; and WO 01 / 88197 and British Patent No. 2,338,237. In addition, enhanced binding specificity for zinc finger proteins is described, for example, in WO 02 / 077227.

[0088] Furthermore, as disclosed in the above and other references, zinc finger domains and / or multi-fingered zinc finger proteins can be linked to one another using any suitable linker sequence, including, for example, linkers five or more amino acids in length. See also U.S. Pat. Nos. 6,479,626; 6,903,185; and 7,153,949 for exemplary linker sequences six or more amino acids in length. The proteins described herein can also include any combination of suitable linkers between the individual zinc fingers of the protein. Additionally, enhanced binding specificity for zinc finger binding domains is described, for example, in co-owned International Publication No. WO 02 / 077227.

[0089] Zinc finger proteins and methods for the design and construction of fusion proteins (and polynucleotides encoding same) are well known to those of skill in the art and are described in detail in U.S. Patent Nos. 6,140,0815; 789,538; 6,453,242; 6,534,261; 5,925,523; 6,007,988; 6,013,453; and 6,200,759; WO 95 / 19431. Brochure; Brochure No. 96 / 06166; Brochure No. 98 / 53057; Brochure No. 98 / 54311; Brochure No. 00 / 27878; Brochure No. 01 / 60970; Brochure No. 01 / 88197; Brochure No. 02 / 099084; Brochure No. 98 / 53058; Brochure No. 98 / 53059; Brochure No. 98 / 53060; Brochure No. 02 / 016536; and Brochure No. 03 / 016496.

[0090] Furthermore, as disclosed above and in other references, zinc finger proteins and / or multi-finger zinc finger proteins can be linked to each other using any suitable linker sequence, such as a linker five or more amino acids in length, similar to, for example, a fusion protein. See U.S. Patent Nos. 6,479,626; 6,903,185; and 7,153,949 for exemplary linker sequences six or more amino acids in length. The zinc finger molecules described herein can include any combination of suitable linkers between the individual zinc finger proteins and / or multi-finger zinc finger proteins of the zinc finger molecule.

[0091] In certain embodiments, the DNA targeting moiety comprises a zinc finger molecule comprising an engineered zinc finger protein that binds (sequence-specifically) to a target DNA sequence. In some embodiments, the zinc finger molecule comprises one zinc finger protein or a fragment thereof. In other embodiments, the zinc finger molecule comprises multiple zinc finger proteins (or fragments thereof), for example, 2, 3, 4, 5, 6 or more zinc finger proteins (and optionally, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 or fewer finger proteins). In some embodiments, the zinc finger molecule comprises at least three zinc finger proteins. In some embodiments, the zinc finger molecule comprises 4, 5, or 6 fingers. In some embodiments, the zinc finger molecule comprises 8, 9, 10, 11, or 12 fingers. In some embodiments, a zinc finger molecule comprising three zinc finger proteins recognizes a target DNA sequence comprising 9 or 10 nucleotides. In some embodiments, a zinc finger molecule comprising four zinc finger proteins recognizes a target DNA sequence comprising 12 to 14 nucleotides, and in some embodiments, a zinc finger molecule comprising six zinc finger proteins recognizes a target DNA sequence comprising 18 to 21 nucleotides.

[0092] In some embodiments, the zinc finger molecule comprises a two-handed zinc finger protein. A two-handed zinc finger protein is a protein in which two clusters of zinc finger proteins are separated by intervening amino acids, such that the two zinc finger domains bind to two discontinuous target sites. An example of a two-handed zinc finger binding protein is SIP1, in which a cluster of four zinc finger proteins is located at the amino terminus of the protein and a cluster of three zinc finger proteins is located at the carboxyl terminus (see Remade, et al. (1999) EMBO Journal, 18(18):5073-5084). Each cluster of zinc fingers in these proteins can bind to a unique target sequence, and the spacing between the two target sequences can include many nucleotides.

[0093] In some embodiments, the targeting moiety is or includes a DNA-binding domain derived from a nuclease. For example, recognition sequences for homing endonucleases and meganucleases such as I-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII, and I-TevIII are known. U.S. Patent Nos. 5,420,032; 6,833,252; Belfort, et al. (1997) Nucleic Acids Res., 25:3379-3388; Dujon, et al. (1989) Gene 82:115-118; Perler, et al. (1994) Nucleic Acids Res. 22:1125-1127; Jasin (1996) Trends Genet. 12:224-228; Gimble, et al. (1996) J. Mol. Biol. 263:163-180; Argast, et al. (1998) J. Mol. Biol. 280:345-353 and the New England Biolabs catalog. Additionally, the DNA binding specificity of homing endonucleases and meganucleases can be engineered to bind non-natural target sites. See, e.g., Chevalier, et al. (2002) Molec. Cell 10:895-905; Epinat, et al. (2003) Nucleic Acids Res. 31:2952-2962; Ashworth, et al. (2006) Nature 441:656-659; Paques, et al. (2007) Current Gene Therapy 7:49-66; U.S. Patent Application Publication No. 2007 / 0117128.

[0094] Target sequence The targeting moiety targets, e.g., binds to, a genomic sequence element that is adjacent to and / or operably linked to a target gene (e.g., FXN). In some embodiments, the genomic sequence element is or comprises an expression control sequence. In some embodiments, the genomic sequence element is or comprises an anchor sequence. In some embodiments, the genomic sequence element is or comprises a target gene (e.g., FXN) or a portion of a target gene. In some embodiments, the targeting moiety binds to a target sequence that is contained within or partially contained in a genomic sequence element. In some embodiments, the targeting moiety binds to a target sequence that is at least 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, or 35 bases in length (and optionally, up to 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, or 20 bases in length). In some embodiments, the targeting moiety binds to a target sequence that is 10-30, 15-30, 15-25, 18-24, 19-23, 20-23, 21-23, or 22-23 bases in length. In some embodiments, the target sequence is 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, or 40 bases in length.

[0095] Anchor sequence Generally, an anchor sequence is a genome sequence element to which a genome complex component, e.g., a nucleation polypeptide, specifically binds. In one embodiment, binding to the anchor sequence nucleates genome complex (e.g., ASMC) formation.

[0096] An anchor sequence-mediated junction (ASMC) comprises multiple anchor sequences, e.g., two or more anchor sequences. In some embodiments, the anchor sequences can be manipulated or altered to modulate (e.g., disrupt) naturally occurring genomic complexes (e.g., ASMCs) or to form new genomic complexes (e.g., ASMCs) (e.g., to form non-native genomic complexes (e.g., ASMCs) with exogenous or altered anchor sequences). Such alterations can, for example, alter the topological structure of DNA, thereby modulating gene expression by, for example, modulating the ability of target genes to interact with gene regulatory and control factors (e.g., expression control sequences, e.g., promoter, enhancer, or repressor sequences).

[0097] In some embodiments, the chromatin structure is modified by substituting, adding, or deleting one or more nucleotides within the anchor sequence. In some embodiments, the chromatin structure is modified by substituting, adding, or deleting one or more nucleotides within the anchor sequence of the anchor sequence-mediated junction.

[0098] In some embodiments, the anchor sequence comprises a nucleation polypeptide binding motif, for example, a CTCF-binding motif: N(T / C / G)N(G / A / T)CC(A / T / G)(C / G)(C / T / A)AG(G / A)(G / T)GG(C / A / T)(G / A)(C / G)(C / T / A)(G / A / C)(SEQ ID NO: 1) (N is any nucleotide) Includes:

[0099] The CTCF-binding motif may also be in the reverse orientation, e.g. (G / A / C)(C / T / A)(C / G)(G / A)(C / A / T)GG(G / T)(G / A)GA(C / T / A)(C / G)(A / T / G)CC(G / A / T)N(T / C / G)N(SEQ ID NO: 2) may be.

[0100] In some embodiments, the anchor sequence comprises SEQ ID NO:1 or SEQ ID NO:2, or a sequence that is at least 75%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to either SEQ ID NO:1 or SEQ ID NO:2.

[0101] In some embodiments, the anchor sequence-mediated junction comprises at least one first anchor sequence and at least one second anchor sequence, e.g., in some embodiments, the first anchor sequence and the second anchor sequence may each comprise a nucleation polypeptide binding motif, e.g., each comprises a CTCF binding motif.

[0102] In some embodiments, the first anchor sequence and the second anchor sequence comprise different sequences, e.g., the first anchor sequence comprises a CTCF binding motif and the second anchor sequence comprises an anchor sequence other than a CTCF binding motif. In one embodiment, each anchor sequence comprises a nucleating polypeptide binding motif and one or more flanking nucleotides on either or both sides of the nucleating polypeptide binding motif.

[0103] Two CTCF binding motifs (e.g., consecutive or non-consecutive CTCF binding motifs) capable of forming ASMCs can be present in the genome in any orientation, e.g., in the same orientation (tandem), either 5'-3' (left tandem, e.g., two CTCF binding motifs comprising SEQ ID NO: 1) or 3'-5' (right tandem, e.g., two CTCF binding motifs comprising SEQ ID NO: 2), or in a convergent orientation, where one CTCF binding motif comprises SEQ ID NO: 1 and the other comprises SEQ ID NO: 2. CTCFBSDB 2.0: Database For CTCF binding motifs and genome organization (http: / / insulatordb.uthsc.edu / ), CTCF binding motifs associated with target genes can be identified.

[0104] In some embodiments, the anchor sequence comprises a CTCF binding motif associated with a target gene (eg, FXN), where the target gene is associated with a disease, disorder and / or condition (eg, FRDA).

[0105] In some embodiments, the methods of the disclosure include modulating, e.g., disrupting, a genome complex (e.g., ASMC) by, e.g., modifying chromatin structure, substituting, adding, or deleting one or more nucleotides within an anchor sequence, e.g., a nucleation polypeptide binding motif. One or more nucleotides can also be specifically targeted, e.g., targetedly altered, for substitution, addition, or deletion within an anchor sequence, e.g., a nucleation polypeptide binding motif.

[0106] In some embodiments, a genome complex (e.g., ASMC) can be altered by changing the orientation of at least one nucleating polypeptide binding motif. In some embodiments, the anchor sequence comprises a nucleating polypeptide binding motif, e.g., a CTCF binding motif, and the targeting moiety introduces an alteration in at least one nucleating polypeptide binding motif, e.g., to alter binding affinity for the nucleating polypeptide.

[0107] Expression control sequences In some embodiments, the target gene (e.g., FXN) is associated with and / or operably linked to one or more expression control sequences. In one embodiment, the genome complex (e.g., ASMC) colonizes two or more genome sequences that contain one or more expression control sequences. Those skilled in the art are familiar with the various positive (e.g., promoters or enhancers) or negative (e.g., repressors or silencers) expression control sequences associated with genes. Typically, when a cognate regulatory protein binds to such an expression control sequence, transcription from the associated gene is altered (e.g., increased in the case of a positive expression control sequence; decreased in the case of a negative expression control sequence).

[0108] Promoter sequence In some embodiments, the target gene (e.g., FXN) is bound to and / or operably linked to a promoter. In one embodiment, a genomic complex (e.g., ASMC) colonizes two or more genomic sequences, wherein the two or more genomic sequences comprise a promoter. Those skilled in the art will recognize that a promoter is typically a sequence that initiates transcription of an associated gene. A promoter is typically near the 5' end of a gene, not far from the transcription start site.

[0109] As those skilled in the art will recognize, transcription of protein-coding genes in eukaryotic cells is typically initiated by the binding of general transcription factors (e.g., TFIID, TFIIE, TFIIH, etc.) and Mediator to a core promoter sequence (which targets RNA polymerase II to the transcription start site) as a transcription pre-initiation complex, and in many cases remains bound to the core promoter sequence after RNA polymerase escape and elongation of the primary transcript has been initiated.

[0110] In many embodiments, a promoter includes sequence elements such as TATA, Inr, DPE, or BRE, although one of skill in the art will appreciate that such sequences are not necessarily required to define a promoter. In some embodiments, the targeting moiety targets, e.g., binds to, a target sequence within a promoter operably linked to a target gene (the target gene is FXN). In some embodiments, FXN is located on human chromosome 9. In some embodiments, the transcription start site (TSS) is the transcription start entry of the hg19 annotation of the human genome (GRCh37), which is located at UCSC. Retrieved from the Table Browser (Karolchik D, Hinrichs AS, Furey TS, Roskin KM, Sugnet CW, Haussler D, Kent WJ. The UCSC Table Browser data retrieval tool. Nucleic Acids Res. 2004 Jan 1;32 (Database issue):D493-6). In some embodiments, the TSS is at chromosomal location 71650667 (e.g., in the Genome Reference Consortium Human Build 37 (GRCh37)). In some embodiments, an expression control sequence, e.g., a promoter, operably linked to FXN comprises sequences encompassing about 1000 bases on either side of the TSS. In some embodiments, the targeting moiety is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, Binds to a target sequence comprising a sequence position that is 0, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 bases upstream (and optionally 500, 450, 400, 350, 300, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 bases or less upstream from the TSS).In some embodiments, the targeting moiety is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, Binds to a target sequence comprising a sequence position that is 0, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 bases downstream (and optionally 500, 450, 400, 350, 300, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 bases or less downstream from the TSS).

[0111] In some embodiments, the targeting moiety binds to a target sequence, wherein the position closest to the TSS is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1 0, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 bases upstream (and optionally 500, 450, 400, 350, 300, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 bases or less upstream from the TSS). In some embodiments, the targeting moiety binds to a target sequence, wherein the position closest to the TSS is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 9 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 bases downstream (and optionally 500, 450, 400, 350, 300, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 bases or less downstream from the TSS).

[0112] In some embodiments, the targeting moiety targets, e.g., binds, a target sequence, where the position closest to the TSS is 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 from the TSS. , 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104 , 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 67, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198 8, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229 ,230,231,232,233,234,235,236,237,238,239,240,241,242,243,244,245,246,247,248,249,250,251,252,253,254,255,256,257,258,259,260,261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321 , 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 , 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 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, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, or 500 bases upstream or downstream.The targeting moiety targets, e.g., binds to, a target sequence, where the closest position to the TSS is about 150 (e.g., 150) bases upstream. In some embodiments, the targeting moiety targets, e.g., binds to, a target sequence, where the closest position to the TSS is about 50 (e.g., 50) bases downstream.

[0113] In some embodiments, the targeting moiety binds to an exemplary target sequence selected from Table 3 (e.g., listed in the upstream and downstream end columns of Table 3). In some embodiments, the targeting moiety comprises a nucleic acid sequence, e.g., an sgRNA, that is complementary or partially complementary (e.g., all but position 1, 2, 3, 4, 5, 6, 7, or 8) to a target sequence (e.g., a target sequence in Table 3). Exemplary guide sequences (e.g., used in the sgRNA of the targeting moiety) for binding to exemplary target sequences are also listed in Table 3.

[0114] [Table 3]

[0115] [Table 4]

[0116] [Table 5]

[0117] [Table 6]

[0118] Effector part The modulating agent comprises one or more effector moieties that, when localized to an appropriate site within the nucleus of a cell (e.g., by a targeting moiety), can alter (e.g., increase) expression of a target gene (e.g., FXN). In some embodiments, the effector moiety contributes to or enhances the binding of the modulating agent (e.g., targeting moiety) to a genome sequence element. In some embodiments, the effector moiety has functionality independent of targeting moiety binding. For example, the effector moiety can target, e.g., bind to, or recruit a transcription factor to, a genome sequence element or a genome complex component in proximity to the genome sequence element targeted by the targeting moiety. As yet another example, the effector moiety can comprise an enzymatic activity, e.g., a gene-modifying functionality. As yet another example, the effector moiety can be or comprise an epigenetic modification moiety.

[0119] In some embodiments, the effector moiety is or comprises a polypeptide. In some embodiments, the effector moiety is or comprises a nucleic acid. In some embodiments, the effector moiety is a chemical, for example, a chemical that modulates cytosine (C) or adenine (A) (e.g., sodium bisulfite, ammonium sulfite). In some embodiments, the effector moiety has enzymatic activity (e.g., methyltransferase, demethylase, nuclease (e.g., Cas9), or deaminase activity). The effector moiety may be or comprise one or more of a small molecule, a peptide, a nucleic acid, a nanoparticle, an aptamer, or an agent with low PK / PD.

[0120] In some embodiments, the effector moiety may comprise a peptide ligand, a full-length protein, a protein fragment, an antibody, an antibody fragment, and / or a targeting aptamer. In some embodiments, the protein may bind to a receptor such as an extracellular receptor, a neuropeptide, a hormonal peptide, a peptide drug, a toxic peptide, a viral or microbial peptide, a synthetic peptide, or an agonist or antagonist peptide.

[0121] In some embodiments, the effector moiety may comprise an antigen, an antibody, an antibody fragment (e.g., a single domain antibody, a ligand, etc.), or a receptor (e.g., glucagon-like peptide-1 (GLP-1), GLP-2 receptor 2, cholecystokinin B (CCKB), or somatostatin receptor), a peptide therapeutic (e.g., those that bind to specific cell surface receptors such as G protein-coupled receptors (GPCRs) or ion channels, synthetic or analog peptides derived from naturally occurring biologically active peptides, antimicrobial peptides, pore-forming peptides, tumor-targeting or cytotoxic peptides, etc.), or a degradative or self-destructive peptide (e.g., an apoptosis-inducing peptide signal or a photosensitizer peptide, etc.).

[0122] Peptide or protein moieties used in the effector moieties described herein can also include small antigen-binding peptides, such as antigen-binding antibodies or antibody-like fragments, e.g., single-chain antibodies, nanobodies, etc. (See, e.g., Steeland et al. 2016. Nanobodies as therapeutics: big opportunities for small antibodies. Drug Discov Today: 21(7): 1076-113.) Such small molecule antigen-binding peptides can bind, for example, to cytosolic, nuclear, or intraorganellar antigens.

[0123] In some embodiments, the effector moiety comprises a dominant-negative component (e.g., a dominant-negative moiety), such as a protein that recognizes and binds to a sequence (e.g., an anchor sequence, e.g., a CTCF binding motif) but contains an inactive (e.g., mutated) dimerization domain (e.g., a dimerization domain that is unable to form a functional anchor sequence-mediated junction), or a protein that binds to a component of a genomic complex (e.g., a transcription factor subunit) that prevents the formation of a functional transcription factor. For example, the zinc finger domain of CTCF can be altered so that it binds to a specific anchor sequence (by adding zinc fingers that recognize flanking nucleic acids), while the homodimerization domain is altered to prevent interaction between the engineered CTCF and endogenous forms of CTCF. In some embodiments, the dominant-negative component comprises a synthetic nucleation polypeptide with a selected binding affinity for the anchor sequence within the target anchor sequence-mediated junction. In some embodiments, the binding affinity may be at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% higher or lower than the binding affinity of an endogenous nucleating polypeptide (e.g., CTCF) bound to a target anchor sequence. The synthetic nucleating polypeptide may have 30-90%, 30-85%, 30-80%, 30-70%, 50-80%, or 50-90% amino acid identity to the corresponding endogenous nucleating polypeptide. The nucleating polypeptide may be modulated (e.g., interfered with) through competitive binding, e.g., by competing with the endogenous nucleating polypeptide for binding to its anchor sequence.

[0124] In some embodiments, the effector moiety comprises an antibody or fragment thereof. In some embodiments, an effector moiety that is or comprises one or more antibodies or fragments thereof is used to alter target gene (e.g., FXN) expression. In some embodiments, an effector moiety that is or comprises one or more antibodies (or fragments thereof) and dCas9 are used to alter gene expression.

[0125] In some embodiments, the antibody or fragment thereof used in the effector moiety may be monoclonal. The antibody may be a fusion, chimeric antibody, non-humanized antibody, partially or fully humanized antibody, etc. As will be appreciated by those skilled in the art, the antibody formats used may be the same or different depending on a given target.

[0126] In some embodiments, the effector moiety comprises a junction nucleation molecule, a nucleic acid encoding a junction nucleation molecule, or a combination thereof. The junction nucleation molecule may be, for example, CTCF, cohesin, USF1, YY1, TATA box-binding protein-associated factor 3 (TAF3), ZNF143-binding motif, or another polypeptide that promotes anchor sequence-mediated junction formation. The junction nucleation molecule may be an endogenous polypeptide or other protein, for example, a transcription factor, such as autoimmune regulatory factor (AIRE), another factor, such as X-inactivation specific transcript (XIST), or an engineered polypeptide engineered to recognize a specific DNA sequence of interest (e.g., having a zinc finger, leucine zipper, or bHLH domain for sequence recognition). The junction nucleation molecule may modulate DNA interactions within or around the anchor sequence-mediated junction (e.g., bound to or including a genomic sequence element targeted by the targeting moiety). For example, the junction nucleation molecule may recruit other factors to the anchor sequence that alter anchor sequence-mediated junction formation or disruption.

[0127] The junction nucleation molecule may further comprise a dimerization domain for homo- or heterodimerization. One or more junction nucleation molecules (e.g., endogenous and engineered) may interact to form an anchor sequence-mediated junction. In some embodiments, the junction nucleation molecule is engineered to further comprise a stabilization domain, e.g., a cohesin-interacting domain, to stabilize the anchor sequence-mediated junction. In some embodiments, the junction nucleation molecule is engineered to bind to a target sequence, e.g., to modulate target sequence binding affinity. In some embodiments, a junction nucleation molecule is selected or engineered to have a selected binding affinity for the anchor sequence within the anchor sequence-mediated junction.

[0128] To investigate topological interactions between topologically related domains, such as distal DNA regions or gene loci, in the absence of CTCF, cells carrying inactivating mutations in CTCF can be used with chromosome conformation capture or 3C-based techniques to identify junction nucleation molecules and their corresponding anchor sequences. Long-range DNA interactions can also be revealed. Additional analyses can include ChIA-PET analysis using baits, such as cohesin, YY1, or USF1, ZNF143-binding motifs, and MS to identify complexes associated with the bait.

[0129] In some embodiments, the effector moiety is or includes a DNA-binding domain of a protein. In some embodiments, the DNA-binding domain of the effector moiety enhances or modifies the targeting action of a modulator, but does not alone achieve complete targeting by the modulator (e.g., a targeting moiety is required to achieve targeting of the modulator). In some embodiments, the DNA-binding domain enhances the targeting of the modulator. In some embodiments, the DNA-binding domain enhances the efficacy of the modulator. DNA-binding proteins have, for example, different structural motifs that play an important role in binding to DNA, which are well known to those of skill in the art. In some embodiments, the DNA-binding domain includes a helix-turn-helix (HTH) motif, i.e., a DNA recognition motif shared in repressor proteins. Such motifs have two helices, one of which recognizes DNA with side chains that confer binding specificity (also known as the recognition helix). Such motifs are commonly used to regulate proteins involved in developmental processes. Sometimes, two or more proteins compete for the same sequence or recognize the same DNA fragment. Different proteins may differ in their affinity for the same sequence or DNA conformation due to H-bonds, salt bridges and van der Waals interactions, respectively.

[0130] In some embodiments, the DNA-binding domain comprises a helix-hairpin-helix (HhH) motif. DNA-binding proteins with the HhH structural motif can participate in non-sequence-specific DNA binding via the formation of nitrogen bonds between the protein backbone nitrogen and DNA phosphate groups.

[0131] In some embodiments, the DNA-binding domain comprises a helix-loop-helix (HLH) motif. DNA-binding domains with HLH structural motifs are transcriptional regulatory proteins, primarily involved in diverse developmental processes. HLH structural motifs are longer in terms of residues than HTH or HhH motifs. Many of these proteins interact to form homo- and heterodimers. The structural motif consists of two long helical regions and an N-terminal helix that binds to DNA, a complex that allows the proteins to dimerize.

[0132] In some embodiments, the DNA-binding domain comprises a leucine zipper. In some transcription factors, the dimer binding site containing DNA forms a leucine zipper. This motif contains two amphipathic helices, one from each subunit, that interact with each other to form a left-handed coiled-coil supersecondary structure. The leucine zipper is an interdigitation of regularly spaced leucine residues in one helix with leucines from adjacent helices. Primarily, the helices involved in the leucine zipper exhibit a heptad sequence (abcdefg), with hydrophobic residues a and d and other residues hydrophilic. The leucine zipper motif can mediate either homo- or heterodimer formation.

[0133] In some embodiments, the DNA binding domain comprises a Zn-finger domain, wherein Zn ++ The ion is coordinated by two Cys and two His residues. These transcription factors contain trimers with ββ'α stoichiometry. Zn ++The apparent effect of coordination is the stabilization of the small complex structure, rather than the hydrophobic core residues. Each Zn-finger interacts in a conformationally identical manner with a consecutive triple base-paired segment in the major groove of the double helix. The protein-DNA interaction is determined by two factors: (i) H-bond interactions between the α-helix and the DNA segment, primarily between Arg residues and guanine bases, and (ii) H-bond interactions with the DNA phosphate backbone, primarily with Arg and His. Another Zn-finger motif is a Zn-finger with six Cys residues. ++ Chelates.

[0134] In some embodiments, the DNA-binding domain also includes TATA box-binding protein (TBP), which was first identified as a component of the class II initiation factor TFIID. These binding proteins participate in transcription by all three nuclear RNA polymerases, acting as subunits in each of these. The structure of TBP reveals an 89-90 amino acid α / β structural domain. The C-terminal or core region of TBP binds with high affinity to the TATA consensus sequence (TATAa / tAa / t, SEQ ID NO: 3), which recognizes minor groove determinants and promotes DNA bending. TBP resembles a molecular saddle. The binding side aligns with the central eight strands of a ten-stranded antiparallel β-sheet. The top surface contains four α-helices and binds various components of the transcription machinery.

[0135] In some embodiments, the DNA-binding domain is or comprises a transcription factor. Transcription factors (TFs) can be regulatory proteins that contain a DNA-binding domain responsible for specific recognition of base sequences and one or more effector domains that can activate or repress transcription. TFs interact with chromatin and recruit protein complexes that serve as coactivators or corepressors.

[0136] In some embodiments, the effector moiety comprises one or more RNAs (e.g., gRNAs) and dCas9. The one or more RNAs target a genome sequence element via dCas9 and a target-specific guide RNA. As will be understood by those skilled in the art, the RNAs used for targeting can be the same or different depending on the given target.

[0137] The effector moiety may comprise an aptamer, for example an oligonucleotide aptamer or a peptide aptamer. The aptamer moiety is an oligonucleotide or a peptide aptamer.

[0138] The effector moiety may comprise an oligonucleotide aptamer, which is a single-stranded DNA or RNA (ssDNA or ssRNA) molecule capable of binding with high affinity and specificity to preselected targets, such as proteins and peptides.

[0139] Oligonucleotide aptamers are nucleic acid species that can be engineered through repeated rounds of in vitro selection or, similarly, SELEX (enrichment of exogenous sequences) to bind to a variety of molecular targets, including small molecules, proteins, nucleic acids, and even cells, tissues, and organisms. Aptamers provide distinct molecular recognition and can be generated by chemical synthesis. In addition, aptamers have desirable storage properties and induce little or no immunogenicity in therapeutic applications.

[0140] Both DNA and RNA aptamers exhibit robust binding affinities to a variety of targets. For example, DNA and RNA aptamers have been selected against lysozyme, thrombin, human immunodeficiency virus trans-acting response element (HIV TAR), hemin, interferon-γ, vascular endothelial growth factor (VEGF), prostate-specific antigen (PSA), dopamine, and the non-classical oncogene, heat shock factor (HSF1).

[0141] A diagnostic technique for aptamer-based plasma protein profiling is aptamer plasma proteomics, which will enable future multi-biomarker protein measurements that can aid in differential diagnosis of disease versus healthy conditions.

[0142] The effector moiety can include a peptide aptamer moiety. Peptide aptamers contain one or more short, variable peptides, including peptides with low molecular weights, i.e., 12-14 kDa. Peptide aptamers can be designed to specifically bind to and interfere with protein-protein interactions inside cells.

[0143] Peptide aptamers are artificial proteins selected or engineered to bind to specific genetic molecules. These proteins contain one or more complex variable sequences. They are typically isolated from combinatorial libraries and often subsequently improved by directed mutation or multiple rounds of variable region mutagenesis and selection. In vivo, peptide aptamers can bind to cellular protein targets and exert biological effects, including interfering with normal protein interactions between their target molecules and other proteins. In particular, variable peptide aptamers bound to transcription factor binding domains are screened from target proteins bound to transcription factor activators. In vivo binding of the peptide aptamer to its target via this selection strategy is detected as the expression of downstream yeast marker genes. These experiments identify the specific proteins bound by the aptamer and the protein interactions that the aptamer disrupts to result in a given phenotype. In addition, peptide aptamers derivatized with appropriate functional moieties can cause specific post-translational modifications of their target proteins or alter the subcellular localization of the target.

[0144] Peptide aptamers can also recognize targets in vitro. These peptide aptamers have proven useful in place of antibodies in biosensors and have been used to detect active isoforms of proteins from populations containing both inactive and active protein forms. Derivatives known as tadpoles (in which a peptide aptamer "head" is covalently linked to a unique double-stranded DNA "tail") allow for quantification of rare target molecules in a mixture by PCR of the DNA tail (e.g., using quantitative real-time polymerase chain reaction).

[0145] Peptide aptamer selection can be performed using various systems, but the most widely used currently is the yeast two-hybrid system. Peptide aptamers can also be selected from combinatorial peptide libraries constructed by phage display and other display technologies such as mRNA display, ribosome display, bacterial display, and yeast display. This experimental procedure is also known as biopanning. Among the peptides obtained from biopanning, mimotopes can be considered a type of peptide aptamer. Peptides panned from combinatorial peptide libraries are stored in a special database called MimoDB.

[0146] Exemplary effector moieties include, but are not limited to, the following: bicyclic peptides such as ubiquitin, ubiquitin ligase inhibitors, transcription factors, DNA and protein modifying enzymes such as topoisomerases, topoisomerase inhibitors such as topotecan, DNA methyltransferases such as those of the DNMT family (e.g., DNMT3a, DNMT3b, DNMTL), protein methyltransferases (e.g., viral lysine methyltransferase (vSET), protein-lysine N-methyltransferase (SMYD2), deaminase inhibitors, and the like. Histone methyltransferases such as enzymes (e.g., APOBEC, UG1), enhancer of zeste homolog 2 (EZH2), PRMT1, histone-lysine N-methyltransferase (Setdb1), histone methyltransferase (SET2), euchromatin histone-lysine N-methyltransferase 2 (G9a), histone-lysine N-methyltransferase (SUV39H1), and G9a), histone deacetylases (e.g., HDAC1, HDAC2, HDAC3), which play roles in DNA demethylation. These include enzymes that catalyze the oxidation of 5-methylcytosine to 5-hydroxymethylcytosine and more highly oxidized derivatives (e.g., TET family enzymes catalyze the oxidation of 5-methylcytosine to 5-hydroxymethylcytosine and more highly oxidized derivatives), protein demethylases such as KDM1A and lysine-specific histone demethylase 1 (LSD1), helicases such as DHX9, acetyltransferases, deacetylases (e.g., sirtuins 1, 2, 3, 4, 5, 6, or 7), kinases, phosphatases, DNA intercalators such as ethidium bromide, SYBR green, and proflavine, phenylalanine arginyl β-naphthyl adenosine phosphate (PAA), and phenylalanine arginyl β-naphthyl adenosine phosphate (PAA). These include efflux pump inhibitors, such as peptidomimetics like amide or quinoline derivatives, nuclear receptor activators and inhibitors, proteasome inhibitors, competitive inhibitors of enzymes (e.g., those involved in lysosomal storage diseases), protein synthesis inhibitors, nucleases (e.g., Cpf1, Cas9, zinc finger nucleases), fusions of one or more thereof (e.g., dCas9-DNMT, dCas9-APOBEC, dCas9-UG1), and specific domains from proteins (e.g., KRAB domains).

[0147] Effector moieties that affect genome complexes In some embodiments, a modulating agent comprises an effector moiety that reduces or increases the level of a genomic complex, e.g., an anchor sequence-mediated junction, that is bound to or comprises a target gene (e.g., FXN). In some embodiments, the level of a genomic complex (e.g., ASMC) comprising a target gene is reduced or increased by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% (and optionally up to 100, 90, 80, 70, 60, 50, 40, 30, or 20%) in the presence of a modulating agent comprising an effector moiety compared to the absence of the modulating agent. In some embodiments, the presence of the modulating agent alters, e.g., increases or decreases, the occupancy of a genomic complex (e.g., ASMC) at a genomic sequence element operably linked to a target gene (e.g., FXN). In some embodiments, occupancy is increased or decreased by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% (and optionally, up to 100, 90, 80, 70, 60, 50, 40, 30, or 20%) in the presence of a modulator comprising an effector moiety compared to the absence of said modulator.

[0148] In some embodiments, the modulating agent comprises an effector that disrupts an interaction between a genome sequence element and another genome sequence complex component or a transcription factor, hi some embodiments, the modulating agent comprises an effector moiety that, when present, reduces dimerization of endogenous nucleation polypeptides compared to when the effector moiety is absent.

[0149] In some embodiments, the effector moiety alters, e.g., reduces, expression of a target gene associated with a genome complex (e.g., ASMC) that includes the targeting component. In some embodiments, expression of the target gene is reduced by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% (and optionally, by up to 100, 90, 80, 70, 60, 50, 40, 30, or 20%) in the presence of a modulating agent that includes an effector moiety relative to the absence of the modulating agent.

[0150] In some embodiments, the modulating agent comprises an effector moiety that confers a steric presence (e.g., to inhibit binding of another genome complex component or transcription factor). The effector moiety may comprise a dominant negative molecule or fragment thereof (e.g., a protein that recognizes and binds to a genome complex component (e.g., a genome sequence element, e.g., an anchor sequence, (e.g., a CTCF binding motif)) but has an alteration (e.g., mutation) that prevents the formation of a functional genome complex (e.g., ASMC)), a polypeptide that interferes with the binding or function of a transcription factor (e.g., contact between a transcription factor and its target sequence to be transcribed), a nucleic acid sequence linked to a small molecule that confers steric hindrance, or any other combination of a recognition element and a steric blocking factor.

[0151] In some embodiments, the modulating agent comprises an effector moiety comprising p65 (also known as RELA), or a functional variant or fragment thereof (e.g., a portion designated by accession number NP_001138610.1). In some embodiments, the modulating agent comprises an effector moiety comprising RTA (Epstein-Barr virus BRLF1 gene product), or a functional variant or fragment thereof (e.g., a portion designated by accession number AAA66528.1).

[0152] Genetic modification part In some embodiments, a modulator comprises an effector moiety that is or comprises a gene-modifying moiety (e.g., a component of a gene editing system). In some embodiments, the gene-modifying moiety comprises one or more components of a gene editing system. Gene-modifying moieties can be used in a variety of contexts, including, but not limited to, gene editing. For example, the gene-modifying moiety can alter the sequence (e.g., introduce a mutation, e.g., a substitution, insertion, or deletion) of a target gene (e.g., FXN) or a genomic sequence element operably linked to the target gene. As another example, such a moiety can be used, for example, to localize an effector moiety to a genetic locus so that a modulator comprising the effector moiety can physically modify, genetically modify, and / or epigenetically modify a target sequence, e.g., an anchor sequence.

[0153] In some embodiments, the genetic modification moiety can target one or more nucleotides of a sequence, e.g., via a gene editing system. In some embodiments, the genetic modification moiety binds to a genomic sequence element to alter a genomic complex (e.g., ASMC), e.g., altering the topology of anchor sequence-mediated junctions comprising or associated with a target gene (e.g., FXN) and / or a genomic sequence element operably linked to the target gene.

[0154] In some embodiments, the genetic modification moiety targets one or more nucleotides of genomic DNA for substitution, addition, or deletion, e.g., via CRISPR, TALEN, dCas9, oligonucleotide pairing, recombination, transposons, etc., within or as a component of a genome complex (e.g., within an anchor sequence-mediated junction).

[0155] In some embodiments, the gene-modifying portion introduces a targeted alteration into one or more nucleosides of genomic DNA, thereby regulating transcription of a gene (e.g., FXN) in, for example, a human cell. The gene-modifying portion can introduce an alteration into a target gene (e.g., FXN), for example, into a sequence encoding an exon, intron, splice site, or 5'UTR or 3'UTR. The gene-modifying portion can introduce an alteration into a genome complex component (e.g., a promoter or enhancer) operably linked to the target gene (e.g., FXN). The gene-modifying portion can introduce an alteration into an anchor sequence that participates in ASMC, including or associated with the target gene (e.g., FXN) and / or a genome sequence element operably linked to the target gene. The alteration can include substitution, addition, or deletion of one or more nucleotides. In some embodiments, the targeted alteration alters at least one binding site of a nucleating polypeptide, for example, altering the binding affinity to an anchor sequence within an anchor sequence-mediated junction, an alternative splice site, and a binding site for untranslated RNA.

[0156] In some embodiments, the genetic modification moiety edits one component of the genome complex (e.g., one sequence within an anchor sequence-mediated junction) by at least one of the following: imparting at least one exogenous anchor sequence; altering at least one nucleating polypeptide binding motif, e.g., by altering the binding affinity for the nucleating polypeptide; altering the orientation of at least one nucleating polypeptide binding motif, such as a CTCF binding motif; and substituting, adding, or deleting at least one anchor sequence, such as a CTCF binding motif.

[0157] Exemplary gene editing systems in which components may be suitable for use in modifying portions of genes include clustered regularly interspaced short palindromic repeats (CRISPR) systems (e.g., CRISPR / Cas molecules), zinc finger nucleases (ZFNs) (e.g., Zn finger molecules), and transcription activator-like effector-based nucleases (TALENs). ZFN-, TALEN-, and CRISPR-based methods are described, for example, in Gaj et al., Trends Biotechnol. 31.7(2013):397-405; CRISPR methods of gene editing are described, for example, in Guan et al., Application of CRISPR-Cas system in gene therapy: Pre-clinical progress in animal models. DNA Repair 2016 July 30, 46:1-8; and Zheng et al., Precise gene deletion and replacement using the CRISPR / Cas9 system in human cells. BioTechniques, Vol. 57, No. 3, September 2014, pp. 115-124.

[0158] For example, in some embodiments, the genetic modification portion is site-specific and comprises a Cas nuclease (e.g., Cas9) and a site-specific guide RNA, as further described herein. In some embodiments, the genetic modification portion comprises a Cas nuclease (e.g., Cas9) and a site-specific guide RNA. In some embodiments, the Cas nuclease is enzymatically inactive, e.g., dCas9, as further described herein.

[0159] In some embodiments, the genetic modification portion can include a polypeptide (e.g., a peptide or protein portion) linked to a gRNA and a targeting nuclease, e.g., Cas9, e.g., wild-type Cas9, nickase Cas9 (e.g., Cas9 D10A), inactive Cas9 (dCas9), eSpCas9, Cpf1, C2C1, or C2C3, or a nucleic acid encoding such a nuclease. The choice of nuclease and gRNA is determined by whether the targeted mutation is a deletion, substitution, or addition of nucleotides, e.g., a deletion, substitution, or addition of nucleotides relative to the targeted sequence. Fusion of a catalytically inactive endonuclease, e.g., inactive Cas9 (dCas9, e.g., D10A; H840A), tethered with all or a portion of an effector domain(s) (e.g., epigenome editing factors, including, but not limited to, DNMT3a, DNMT3L, DNMT3b, KRAB domain, Tet1, p300, VP64, and fusions thereof) generates a chimeric protein that can be linked to a polypeptide and directed to a specific DNA site by one or more RNA sequences (e.g., DNA recognition elements, including, but not limited to, zinc finger arrays, sgRNAs, TAL arrays, peptide nucleic acids, and the like, as described herein) to modulate the activity and / or expression of one or more target nucleic acid sequences (e.g., for the purpose of methylating or demethylating DNA sequences).

[0160] As used herein, a "biologically active portion of an effector domain" is a portion that maintains (e.g., fully, partially, minimally) the function of the effector domain (e.g., a "minimal" or "core" domain). In some embodiments, fusion of all or a portion of one or more effector domains of an epigenetic modifier (e.g., a DNA methylase or enzyme playing a role in DNA demethylation, e.g., DNMT3a, DNMT3b, DNMT3L, DNMT inhibitors, combinations thereof, TET family enzymes, protein acetyltransferases or deacetylases, dCas9-DNMT3a / 3L, dCas9-DNMT3a / 3L / KRAB, dCas9 / V64) with dCas9 generates a chimeric protein, which can be linked to a polypeptide and is useful in the methods described herein. Effector moieties comprising such chimeric proteins are referred to as gene-modifying moieties (due to their use of gene editing system components, i.e., Cas9) or epigenetic-modifying moieties (due to their use of effector domains of epigenetic modifiers).

[0161] In some embodiments, the provided technology is described as comprising a gRNA that specifically targets a target gene, hi some embodiments, the target gene is an oncogene, a tumor suppressor, or a nucleotide repeat disease-associated gene.

[0162] In some embodiments, the technology provided herein includes methods of delivering one or more gene-modifying moieties (e.g., CRISPR system components) described herein to a subject, e.g., to the nucleus of a cell or tissue of a subject, by linking such moiety to a targeting moiety as part of a fusion molecule.

[0163] Epigenetic modification moiety In some embodiments, the effector moiety is or includes an epigenetic modification moiety that modulates chromatin structure or alters epigenetic markers (e.g., one or more of DNA methylation, histone methylation, histone acetylation, histone sumoylation, histone phosphorylation, and RNA-associated silencing).

[0164] Epigenetic modifying moieties useful in the disclosed methods and compositions include, for example, agents that affect DNA methylation, histone acetylation, and RNA-associated silencing. In some embodiments, the methods described herein involve sequence-specific targeting (e.g., by a modulator comprising a targeting moiety that specifically binds to a target sequence) of epigenetic enzymes (e.g., enzymes that create or remove epigenetic marks, e.g., acetylation and / or methylation). Exemplary epigenetic enzymes that can be targeted to the genome sequence elements described herein include DNA demethylases (e.g., TET family), histone methyltransferases, histone-lysine-N-methyltransferase (Setdb1), euchromatin histone-lysine-N-methyltransferase 2 (G9a), histone-lysine-N-methyltransferase (SUV39H1), enhancer of zeste homolog 2 (EZH2), viral lysine methyltransferase (vSET), histone methyltransferase (SET2), and protein-lysine N-methyltransferase (SMYD2). Examples of such epigenetic modifiers are described, for example, in de Groote et al. Nuc. Acids Res. (2012): 1-18.

[0165] In some embodiments, the epigenetic modification portion comprises histone methyltransferase activity (e.g., a protein selected from DOT1L, PRDM9, PRMT1, PRMT2, PRMT3, PRMT4, PRMT5, NSD1, NSD2, NSD3, or a functional variant or fragment of any of them). In some embodiments, the epigenetic modification portion comprises histone acetyltransferase activity (e.g., a protein selected from p300, CREB-binding protein (CBP), or a functional variant or fragment of any of them). In some embodiments, the epigenetic modification portion comprises DNA demethylase activity (e.g., a protein selected from TET1, TET2, TET3, or TDG, or a functional variant or fragment of any of them). In some embodiments, the epigenetic modification portion comprises transcription activator activity (e.g., a protein selected from VP16, VP64, VP160, VPR, or a functional variant or fragment of any of them).

[0166] In some embodiments, useful epigenetic modifying moieties described herein include constructs described in Koferle et al. Genome Medicine 7.59(2015):1-3 (e.g., Table 1), which is incorporated herein by reference. For example, in some embodiments, the expression suppressor includes or is a construct found in Table 1 of Koferle et al., e.g., a histone acetyltransferase, histone deacetylase, histone methyltransferase, DNA demethylating, or H3K4 and / or H3K9 histone demethylase (e.g., dCas9-p300, TALE-TET1) described in Table 1.

[0167] In some embodiments, the epigenetic modification portion comprises histone demethylase activity (e.g., a protein selected from KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, NO66, or a functional variant or fragment of any of them). In some embodiments, the epigenetic modification portion comprises histone deacetylase activity (e.g., a protein selected from HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9, or a functional variant or fragment of any of them). In some embodiments, the epigenetic modification portion comprises DNA methyltransferase activity (e.g., a protein selected from MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, or a functional variant or fragment of any of them). In some embodiments, the epigenetic modification portion comprises transcriptional repression activity (e.g., a protein selected from KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, or a functional variant or fragment of any of them).

[0168] Exemplary Modulating Agents In some embodiments, the modulator comprises a targeting moiety comprising a CRISPR / Cas molecule and an effector moiety having histone acetyltransferase activity, e.g., p300 or a functional fragment or variant thereof. In some embodiments, the modulator comprises a targeting moiety comprising an enzymatically inactive Cas9 molecule (e.g., dCas9) and an effector moiety having p300 or a functional fragment or variant thereof.

[0169] In some embodiments, the modulating agent is encoded by the nucleic acid sequence of SEQ ID NO: 300, or a sequence having at least 80, 85, 90, 95, 96, 97, 98, or 99% identity thereto. In some embodiments, the modulating agent is encoded by the nucleic acid sequence of SEQ ID NO: 308, or a sequence having at least 80, 85, 90, 95, 96, 97, 98, or 99% identity thereto. In some embodiments, the modulating agent comprises the amino acid sequence of SEQ ID NO: 304, or an amino acid sequence encoded by the nucleic acid sequence of either SEQ ID NO: 300 or 308, or an amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98, or 99% identity thereto. dCas9-p300 Exemplary Coding Sequence 1 [ka] [ka] [ka] dCas9-p300 exemplary coding sequence 2 [ka] [ka] [ka] [ka] [ka]

[0170] In some embodiments, the modulating agent comprises a targeting moiety comprising a CRISPR / Cas molecule and an effector moiety having transcriptional activator activity, e.g., VP64 or a functional fragment or variant thereof. In some embodiments, the modulating agent comprises a targeting moiety comprising an enzymatically inactive Cas9 molecule (e.g., dCas9) and an effector moiety having VP64 or a functional fragment or variant thereof.

[0171] In some embodiments, the modulating agent is encoded by the nucleic acid sequence of SEQ ID NO: 301, or a sequence having at least 80, 85, 90, 95, 96, 97, 98, or 99% identity thereto. In some embodiments, the modulating agent is encoded by the nucleic acid sequence of SEQ ID NO: 309, or a sequence having at least 80, 85, 90, 95, 96, 97, 98, or 99% identity thereto. In some embodiments, the modulating agent comprises the amino acid sequence of SEQ ID NO: 305, or an amino acid sequence encoded by the nucleic acid sequence of either SEQ ID NO: 301 or 309, or an amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98, or 99% identity thereto. dCas9-VPR (VP64-p65-RTA) exemplary sequence 1 [ka] [ka] [ka] dCas9-VPR (VP64-p65-RTA) exemplary sequence 2 [ka] [ka] [ka] [ka] [ka]

[0172] In some embodiments, the modulating agent comprises a targeting moiety comprising a zinc finger molecule and an effector moiety having transcriptional activator activity, e.g., VP64 or a functional fragment or variant thereof. In some embodiments, the modulating agent comprises a targeting moiety comprising a zinc finger molecule having 6, 7, 8, 9, or 10 (e.g., 9) zinc finger proteins, and an effector moiety having VP64 or a functional variant or fragment thereof.

[0173] In some embodiments, the modulating agent is encoded by the nucleic acid sequence of SEQ ID NO: 302, or a sequence having at least 80, 85, 90, 95, 96, 97, 98, or 99% identity thereto. In some embodiments, the modulating agent comprises the amino acid sequence of SEQ ID NO: 306, or an amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO: 302, or an amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98, or 99% identity thereto. ZF9-VPR [ka] [ka] [ka] [ka]

[0174] In some embodiments, the modulating agent comprises a targeting moiety comprising a TAL effector molecule and an effector moiety having transcriptional activator activity, for example, VP64 or a functional fragment or variant thereof.

[0175] In some embodiments, the modulating agent is encoded by the nucleic acid sequence of SEQ ID NO: 303, or a sequence having at least 80, 85, 90, 95, 96, 97, 98, or 99% identity thereto. In some embodiments, the modulating agent comprises the amino acid sequence of SEQ ID NO: 307, an amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO: 303, or an amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98, or 99% identity thereto. TAL-VPR [ka] [ka] [ka] [ka]

[0176] fusion molecule In some embodiments, the modulating agent may be or comprise a fusion molecule, e.g., a fusion molecule comprising two or more moieties. In some embodiments, the fusion molecule comprises one or more moieties described herein, e.g., a targeting moiety and / or an effector moiety. In some embodiments, the fusion molecule comprises one or more moieties that are covalently linked to each other. In some embodiments, one or more moieties of the fusion molecule are located on a single polypeptide chain, e.g., the polypeptide moieties of one or more moieties are located on a single polypeptide chain.

[0177] In some embodiments, for example, the fusion molecule (e.g., as part of an effector and / or targeting moiety) may comprise a dCas9-DNMT (e.g., comprising dCas9 and a DNMT, in any order, as part of the same polypeptide chain), dCas9-p300, dCas9-VP64, dCas9-VPR, dCas9-DNMT-3a-3L, dCas9-DNMT-3a-3a, dCas9-DNMT-3a-3L-3a, dCas9-DNMT-3a-3L-KRAB, dCas9-KRAB, dCas9-APOBEC, APOBEC-dCas9, dCas9-APOBEC-UGI, dCas9-UGI, UGI-dCas9-APOBEC, UGI-APOBEC-dCas9, dCas9-VP64-RelA, dCas9-VPR-RelA, d The fusion may comprise Cas9-VP64-p65, dCas9-VPR-p65, dCas9-VP64-RelA-p65, dCas9-VPR-RelA-p65, ZFM-VP64-RelA (ZFM stands for zinc finger molecule), ZFM-VPR-RelA, ZFM-VP64-p65, ZFM-VPR-p65, ZFM-VP64-RelA-p65, ZFM-VPR-RelA-p65, TEM-VP64-RelA (TEM stands for Tal effector molecule), TEM-VPR-RelA, TEM-VP64-p65, TEM-VPR-p65, TEM-VP64-RelA-p65, TEM-VPR-RelA-p65, or any variant of the protein fusions described herein, or other fusions of proteins or protein domains described herein.

[0178] Exemplary dCas9 fusion methods and compositions applicable to the methods and compositions provided by the present disclosure are known and described, for example, in Kearns et al., "Functional annotation of native enhancers with a Cas9-histone demethylase fusion." Nature Methods 12, 401-403 (2015); and McDonald et al., "Reprogrammable CRISPR / Cas9-based system for inducing site-specific DNA methylation." Biology Open 2016: doi:10.1242 / bio.019067. Using methods known in the art, dCas9 can be fused to any of the various agents and / or molecules described herein; the resulting fusion molecules can be useful in the disclosed methods.

[0179] In some embodiments, the fusion molecule may be or include a peptide oligonucleotide conjugate. Peptide oligonucleotide conjugates include chimeric molecules (e.g., peptide / nucleic acid mixmers) that include a nucleic acid portion covalently linked to a peptide portion. In some embodiments, the peptide portion may include any peptide or protein described herein. In some embodiments, the nucleic acid portion may include any nucleic acid or oligonucleotide described herein, such as DNA or RNA or modified DNA or RNA.

[0180] In some embodiments, the peptide oligonucleotide conjugate comprises a peptide antisense oligonucleotide conjugate. In some embodiments, the peptide oligonucleotide conjugate is a synthetic oligonucleotide with a chemically modified backbone. The peptide oligonucleotide conjugate can bind to both DNA and RNA targets in a sequence-specific manner to form a duplex structure. When bound to a double-stranded DNA (dsDNA) target, the peptide oligonucleotide conjugate displaces one DNA strand in the duplex by strand invasion to form a triplex structure, and the displaced DNA strand can exist as a single-stranded D-loop.

[0181] In some embodiments, peptide-oligonucleotide conjugates can be cell and / or tissue specific. In some embodiments, such conjugates can be directly conjugated to oligos, peptides, and / or proteins.

[0182] In some embodiments, the peptide-oligonucleotide conjugate comprises a membrane translocating polypeptide, eg, a membrane translocating polypeptide described elsewhere herein.

[0183] The solid-phase synthesis of several peptide-oligonucleotide conjugates is described, for example, in Williams, et al., 2010, Curr. Protoc. Nucleic Acid Chem., Chapter Unit 4.41. The synthesis and characterization of very short peptide-oligonucleotide conjugates, as well as the stepwise solid-phase synthesis of peptide-oligonucleotide conjugates on novel solid supports, are described, for example, in Bongardt, et al., Innovation Perspective. Solid Phase Synth. Comb. Libr., Collect. Pap., Int. Symp., 5th, 1999, 267-270; Antopolsky, et al. al., Helv. Chim. Acta, 1999, 82, 2130-2140.

[0184] In some embodiments, the compositions provided are pharmaceutical compositions comprising the fusion molecules described herein.

[0185] In some aspects, the present disclosure provides cells or tissues comprising the fusion molecules described herein.

[0186] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a fusion molecule described herein.

[0187] Linker In some embodiments, a modulating agent, e.g., a fusion molecule, may comprise one or more linkers. In some embodiments, a modulating agent, e.g., a fusion molecule, comprising a first moiety and a second moiety has a linker between the first moiety and the second moiety, e.g., between the targeting moiety and the effector moiety. The linker may be a chemical bond, e.g., one or more covalent or non-covalent bonds. In some embodiments, the linker is a covalent bond. In some embodiments, the linker is a non-covalent bond. In some embodiments, the linker is a peptide linker. Such linkers can be 2 to 30, 5 to 30, 10 to 30, 15 to 30, 20 to 30, 25 to 30, 2 to 25, 5 to 25, 10 to 25, 15 to 25, 20 to 25, 2 to 20, 5 to 20, 10 to 20, 15 to 20, 2 to 15, 5 to 15, 10 to 15, 2 to 10, 5 to 10, or 2 to 5 amino acids in length, or 2, 5, 10, 15, 20, 25, or 30 amino acids or more (and optionally up to 50, 40, 30, 25, 20, 15, 10, or 5 amino acids in length). In some embodiments, a linker can be used to space a first moiety and a second moiety, for example, between a targeting moiety and an effector moiety. In some embodiments, a linker can be placed between the targeting moiety and the effector moiety, for example, to confer molecular flexibility, e.g., in secondary and tertiary structure. The linker may include a flexible, rigid, and / or cleavable linker as described herein. In some embodiments, to provide flexibility, the linker comprises at least one glycine, alanine, and serine amino acid. In some embodiments, the linker is a hydrophobic linker, for example, comprising a negatively charged sulfonic acid group, a polyethylene glycol (PEG) group, or a pyrophosphate diester group. In some embodiments, the linker is cleavable to selectively release a moiety (e.g., a polypeptide) from the modulating agent, yet is sufficiently stable to prevent premature cleavage.

[0188] In some embodiments, one or more moieties of a modulator described herein are linked to one or more linkers.

[0189] As is well known to those skilled in the art, commonly used flexible linkers have sequences composed primarily of stretches of Gly and Ser residues ("GS" linkers). Flexible linkers may be useful for connecting domains that require some degree of movement or interaction and may include small nonpolar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids. Furthermore, the inclusion of Ser or Thr may maintain the stability of the linker in aqueous solution by forming hydrogen bonds with water molecules, thus reducing unfavorable interactions between the linker and the protein moiety.

[0190] Rigid linkers are useful for maintaining a fixed distance between domains and maintaining their independent functions. Rigid linkers may also be useful when spatial separation of domains is critical to preserve the stability or biological activity of one or more components in the fusion. Rigid linkers may have an α-helical structure or a Pro-rich sequence (XP)n (where X represents any amino acid, preferably Ala, Lys, or Glu).

[0191] A cleavable linker may release a free functional domain in vivo. In some embodiments, the linker may be cleaved under specific conditions, such as the presence of a reducing agent or a protease. In vivo cleavable linkers may utilize the reversibility of disulfide bonds. One example is a thrombin-sensitive sequence (e.g., PRS) between two Cys residues. In vitro thrombin treatment of CPRSC results in cleavage of the thrombin-sensitive sequence, while leaving the reversible disulfide bond intact. Such linkers are known and are described, for example, in Chen et al. 2013. Fusion Protein Linkers: Property, Design and Functionality. Adv Drug Deliv Rev. 65(10):1357-1369. In vivo cleavage of the linker during fusion may also be performed by a protease expressed in specific cells or tissues, under specific conditions, in vivo, or restricted to a specific cellular compartment. The specificity of many proteases results in slower cleavage of the linker within a restricted compartment.

[0192] Examples of linking molecules include hydrophobic linkers, such as negatively charged sulfonic acid groups; lipids, such as poly(--CH2--) hydrocarbon chains, such as polyethylene glycol (PEG) groups, unsaturated variants thereof, hydroxylated variants thereof, amidated or other N-containing variants thereof, non-carbon linkers; carbohydrate linkers; phosphodiester linkers, or other molecules capable of covalently linking two or more components of a modulating agent (e.g., two polypeptides). Non-covalent linkers are also included, such as hydrophobic lipid globules in which polypeptides are linked through hydrophobic regions of polypeptides or hydrophobic extensions of polypeptides, such as a series of residues rich in leucine, isoleucine, valine, or perhaps even alanine, phenylalanine, or even tyrosine, methionine, glycine, or other hydrophobic residues. Components of a modulating agent may also be linked using charge-based chemistry, linking a positively charged component of a modulating agent to another negatively charged component or nucleic acid.

[0193] In some embodiments, a modulating agent, e.g., a fusion molecule, has the ability, after administration (e.g., to a subject), to form a linkage, e.g., via a covalent or non-covalent bond, with another polypeptide, another moiety described herein, e.g., an effector molecule, e.g., a nucleic acid, protein, peptide, or other molecule, or another agent, e.g., an intracellular molecule. In some embodiments, one or more amino acids on the modulating agent polypeptide can be linked to a nucleic acid, e.g., by an arginine forming a pseudopair with a guanosine, or by a nucleotide tract phosphate bond or an interpolymer bond. In some embodiments, the nucleic acid is DNA, such as genomic DNA, or RNA, such as a tRNA or mRNA molecule. In some embodiments, one or more amino acids on the polypeptide can be linked to a protein or peptide.

[0194] In some embodiments, two or more entities are physically "associated" if they directly or indirectly interact to bring them into and / or remain in physical proximity to one another. In some embodiments, two or more entities that are physically associated with one another are covalently bonded to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently bonded to one another, but are non-covalently bonded by, for example, hydrogen bonding, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof.

[0195] Regulation of genome complexes In some embodiments, the modulating agent modulates (e.g., promotes or disrupts) one or more aspects of a genome complex (e.g., ASMC) associated with a target gene (e.g., FXN). In some embodiments, the modulation is or includes modulation of the topological structure of the genome complex (e.g., ASMC). In some embodiments, modulation of the topological structure of the genome complex results in altered (e.g., increased) expression of the target gene (e.g., FXN). In some embodiments, no detectable modulation of the topological structure is observed, but altered expression of the target gene (e.g., FXN) is still observed. In some embodiments, the modulation is or includes binding to a component of the genome complex (e.g., ASMC), e.g., a genome sequence element. Binding can result in sequestration of the component, thereby altering, for example, the level or occupancy of the genome complex (e.g., ASMC) at the target gene (e.g., FXN).

[0196] Those skilled in the art will understand that in certain cases, two or more complexes (e.g., ASMC) may compete with each other for a particular genomic region or a particular genomic location (e.g., the FXN gene or an expression control sequence operably linked thereto). In some embodiments, disruption of one ("first") genomic complex (e.g., ASMC) may be achieved by stabilizing one or more other genomic complexes (e.g., ASMC) that present alternative (relative to the first genomic complex) structures available for the particular genomic region or location. In some embodiments, stabilization of one ("first") genomic complex (e.g., ASMC) may be achieved by disrupting one or more other genomic complexes (e.g., ASMC) that present alternative (relative to the first genomic complex) structures available for the particular genomic region or location. Thus, in some embodiments, disruption or stabilization of a genomic complex of interest (e.g., ASMC) may be achieved by targeting one or more genomic complexes for stabilization or disruption, respectively (optionally without further providing a modulator that disrupts or stabilizes the genomic complex of interest (e.g., ASMC)).

[0197] A modulating agent may bind to its target component of a genome complex (e.g., ASMC) and alter genome complex formation (e.g., by altering the affinity of the targeting component for one or more other complex components, e.g., by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more). Alternatively, or additionally, in some embodiments, binding by a modulating agent alters the topology of genomic DNA affected by the genome complex, e.g., by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more. In some embodiments, the modulating agent alters expression of genes associated with the targeted genomic complex (e.g., ASMC) by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more. Changes in genomic complex formation, affinity of the targeting component for other complex components, and / or changes in genomic DNA topology affected by the genomic complex can be assessed, for example, by HiChIP, ChIAPET, 4C, or 3C, e.g., HiChIP.

[0198] The modulating agents described herein comprise a targeting moiety. In some embodiments, the targeting moiety binds to a component (e.g., a genome sequence element) of a target genome complex (e.g., ASMC). In some embodiments, the interaction between the targeting moiety and its targeting moiety interferes with one or more other interactions that the targeting moiety would otherwise achieve. In some embodiments, the modulating agent physically interferes with the formation and / or maintenance of a genome complex (e.g., ASMC), e.g., through the binding of the targeting moiety to a component of its target genome complex. In some embodiments, the one or more other interactions that the targeting moiety would otherwise achieve are with a polypeptide component of a genome complex (e.g., ASMC) or with the transcriptional machinery (e.g., a transcriptional activator protein or a transcriptional repressor protein).

[0199] In some embodiments, the modulating agent is complex-specific. That is, in some embodiments, the targeting moiety specifically binds to its target component, e.g., a genome sequence element, in one or more target genome complexes (e.g., within a cell), but does not bind to non-target genome complexes (e.g., within the same cell). In some embodiments, the modulating agent specifically targets a genome complex that is present only in a particular cell type and / or at a particular developmental stage or time. In some embodiments, binding of the modulating agent to a target component of a genome complex (e.g., ASMC) associated with or containing a target gene (e.g., FXN) or a genome sequence element operably linked to the target gene comprises a change (e.g., a reduction) in the frequency and / or duration of association between a polypeptide component of the genome complex and the operably linked genome sequence element.

[0200] Nucleating Polypeptides In some embodiments, the interaction between the targeting moiety and its targeting component, or the function of the effector moiety, interferes with one or more other interactions that the targeting component would otherwise achieve with a polypeptide component of a genome complex (e.g., ASMC). In some embodiments, the polypeptide component is or comprises a nucleating polypeptide. A nucleating polypeptide can promote the formation of an anchor sequence-mediated junction. Nucleating polypeptides that can be targeted by the modulating agents described herein include, for example, proteins (e.g., CTCF, USF1, YY1, TAF3, ZNF143, etc.) that specifically bind to anchor sequences or other proteins (e.g., transcription factors) whose binding to specific genome sequence elements can initiate the formation of a genome complex (e.g., ASMC) described herein. In some embodiments, a modulating agent can target one or more anchor sequences or genome sequence elements to which a nucleating polypeptide can bind within a target genome complex (e.g., ASMC). In some embodiments, a modulating agent can target (e.g., bind to) a nucleating polypeptide.

[0201] Nucleation polypeptides can be, for example, CTCF, cohesin, USF1, YY1, TATA box-binding protein-associated factor 3 (TAF3), ZNF143-binding motif, or another polypeptide that promotes anchor sequence-mediated junction formation. Nucleation polypeptides can be endogenous polypeptides or other proteins, such as transcription factors, e.g., autoimmune regulatory factor (AIRE), other factors, e.g., X-inactivation specific transcript (XIST), or engineered polypeptides engineered to recognize specific DNA sequences of interest (e.g., having zinc finger, leucine zipper, or bHLH domains for sequence recognition). Nucleation polypeptides can regulate DNA interactions within or around anchor sequence-mediated junctions. For example, nucleation polypeptides can recruit other factors to the anchor sequence, such that anchor sequence-mediated junction formation is altered (e.g., disrupted).

[0202] The nucleating polypeptide may further have a dimerization domain for homo- or heterodimerization. One or more nucleating polypeptides (e.g., endogenous and engineered) may interact to form an anchor sequence-mediated junction. In some embodiments, the modulating agent disrupts the target genome complex (e.g., ASMC) by interfering (e.g., directly or indirectly) with this interaction. In some embodiments, the nucleating polypeptide is engineered to further comprise a stabilization domain, e.g., a cohesin-interacting domain, to stabilize the anchor sequence-mediated junction. In some embodiments, the nucleating polypeptide is engineered to bind to the target sequence, e.g., to modulate target sequence binding affinity. In some embodiments, a nucleating polypeptide is selected or engineered to have a selected binding affinity for the anchor sequence within the anchor sequence-mediated junction.

[0203] To investigate topological interactions between topologically related domains, such as distant DNA regions or loci, in the absence of CTCF, cells carrying inactivating mutations in CTCF can be used to identify nucleation polypeptides and their corresponding anchor sequences using chromosome conformation capture or 3C-based methods, such as Hi-C or high-throughput sequencing. Long-range DNA interactions can also be revealed. Additional analyses can include ChIA-PET analysis using a bait, such as cohesin, YY1, or USF1, a ZNF143-binding motif, and MS to identify complexes associated with the bait.

[0204] In some embodiments, the nucleating polypeptide has a binding affinity for the anchor sequence that is higher or lower than a reference value, e.g., the binding affinity of the anchor sequence in the absence of the alteration. In some embodiments, the nucleating polypeptide is modulated to alter (e.g., disrupt) its interaction with the anchor sequence-mediated junction, e.g., its binding affinity for the anchor sequence within the anchor sequence-mediated junction.

[0205] Transcription mechanism In some embodiments, the interaction between the targeting moiety and its targeting component, or the function of the effector moiety, interferes with one or more other interactions that the targeting component would otherwise have with components of the cellular transcription machinery. Those skilled in the art are familiar with proteins that participate as part of the transcription machinery involved in the transcription of specific genes (e.g., protein-coding genes). For example, RNA polymerase (e.g., RNA polymerase II), general transcription factors such as TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH, mediators, certain elongation factors, etc.

[0206] In some embodiments, the interaction between a targeting moiety and its targeting component, or the function of an effector moiety, facilitates the interaction of the targeting component (e.g., a genomic sequence element, e.g., an expression control sequence operably linked to a target gene) and / or the target gene (e.g., FXN) with a component of the cellular transcription machinery. Those skilled in the art are familiar with proteins that participate as part of the transcription machinery involved in the transcription of specific genes (e.g., protein-coding genes), such as RNA polymerase (e.g., RNA polymerase II), general transcription factors such as TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH, mediators, specific elongation factors, etc.

[0207] transcriptional regulators In some embodiments, the modulating agent alters the interaction of a transcriptional regulatory protein with a target gene (e.g., FXN) and / or a genomic sequence element operably linked to the target gene (e.g., a target component of the targeting moiety). In some embodiments, the modulating agent promotes the interaction of a transcriptional regulatory protein with a target gene (e.g., FXN) and / or a genomic sequence element operably linked to the target gene (e.g., a target component of the targeting moiety). In some embodiments, the modulating agent interferes with (e.g., inhibits) the interaction of a transcriptional regulatory protein with a target gene (e.g., FXN) and / or a genomic sequence element operably linked to the target gene (e.g., a target component of the targeting moiety), for example, by preventing the transcriptional regulatory protein from interacting with one or more other components of a genome complex (e.g., ASMC) that includes or is associated with the target gene (or its operably linked genomic sequence element).

[0208] Those skilled in the art recognize a wide variety of transcriptional regulatory proteins, many of which are DNA-binding proteins (e.g., containing DNA-binding domains such as helix-loop-helix motifs, ETSs, forkheads, leucine zippers, Pit-Oct-Unc domains, and / or zinc fingers), many of which interact with the core transcription machinery through interactions with mediators. In some embodiments, a transcriptional regulatory protein may be or include an activator (e.g., capable of binding to an enhancer). In some embodiments, a transcriptional regulatory protein may be or include a repressor (e.g., capable of binding to a silencer).

[0209] Anchor sequence-mediated junctions (ASMCs) In some embodiments, the genome complex regulated by the modulators of the present disclosure is or comprises an anchor sequence-mediated junction (ASMC). In some embodiments, an anchor sequence-mediated junction forms when a nucleating polypeptide binds to an anchor sequence in the genome, and interactions between these proteins and, optionally, one or more other components (e.g., polypeptide components and / or non-genomic nucleic acid components) form a junction at which the anchor sequence is physically co-localized. In some embodiments, one or more genes (e.g., target genes, e.g., FXN) are bound to the anchor sequence-mediated junction. In some embodiments, the anchor sequence-mediated junction comprises one or more anchor sequences, one or more genes, and one or more expression control sequences, e.g., enhancing or silencing sequences. In some embodiments, the expression control sequences are within, partially within, or external to the anchor sequence-mediated junction.

[0210] In some embodiments, the genome complex (e.g., anchor sequence-mediated junction) comprises a first anchor sequence, a nucleic acid sequence (e.g., a gene), an expression control sequence, and a second anchor sequence. In some embodiments, the genome complex (e.g., ASMC) comprises, in order: a first anchor sequence, an expression control sequence, and a second anchor sequence; or a first anchor sequence, a nucleic acid sequence (e.g., a gene), and a second anchor sequence. In some embodiments, either the nucleic acid sequence (e.g., a gene) or the expression control sequence, or both, are located inside or outside the genome complex (e.g., ASMC). Expression control sequence. In some embodiments, the genome complex (e.g., anchor sequence-mediated junction) comprises a TATA box, a CAAT box, a GC box, or a CAP site.

[0211] In some embodiments, a genomic complex (e.g., ASMC) colocalizes (i) a gene whose expression is modulated (e.g., decreased or increased) by the formation or disruption of the genomic complex; and / or (ii) two genomic sequence elements (e.g., anchor sequences) that are external to, not part of, not contained within, or non-contiguous with one or more expression control sequences operably linked to the gene.

[0212] In some embodiments, a genomic complex (e.g., ASMC) colocalizes two genomic sequence elements: (i) a gene whose expression is modulated (e.g., decreased or increased) by the formation or disruption of the genomic complex; and / or (ii) that are within, part of, or contiguous with one or more expression control sequences operably linked to the gene.

[0213] Expression control sequence. In some embodiments, the modulating agent can regulate the transcription of a target gene associated with ASMC. For example, in some embodiments, the transcription of the target gene is activated by its inclusion in activated ASMC or its exclusion from inhibitory ASMC; in some embodiments, the modulating agent causes the target gene to be included in activated ASMC or excluded from inhibitory ASMC. In some embodiments, if the ASMC did not contain an expression control sequence before modulation, the modulating agent can cause the anchor sequence-mediated junction to contain an expression control sequence that increases the transcription of a nucleic acid sequence (e.g., a gene). In some embodiments, if the ASMC contained an expression control sequence before modulation, the modulating agent can cause the anchor sequence-mediated junction to exclude an expression control sequence that reduces the transcription of a nucleic acid sequence (e.g., a gene).

[0214] In some embodiments, transcription of the target gene is suppressed by its inclusion in inhibitory ASMCs or its exclusion from activated ASMCs. In some such embodiments, the modulating agent causes the target gene to be excluded from activated ASMCs or included in inhibitory ASMCs. In some embodiments, the anchor sequence-mediated junction comprises an expression control sequence that reduces transcription of the nucleic acid sequence (e.g., a gene). In some embodiments, the anchor sequence-mediated junction excludes an expression control sequence that increases transcription of the nucleic acid sequence (e.g., a gene).

[0215] An "activated ASMC" refers to an ASMC that is open for active gene transcription, e.g., an ASMC that includes an expression control sequence (e.g., a promoter or enhancer) that enhances transcription of an operably linked nucleic acid sequence (e.g., a gene). An "inhibitory ASMC" refers to an ASMC that is shielded from active gene transcription, e.g., an ASMC that includes an expression control sequence (e.g., a repressor sequence) that represses transcription of an operably linked nucleic acid sequence (e.g., a gene). In some embodiments, an ASMC (e.g., an activated ASMC) includes a gene and an operably linked enhancer, and the gene is actively expressed. In some embodiments, an ASMC (e.g., an activated ASMC) includes a gene and a repressor sequence located outside the ASMC, in which case the gene is actively expressed. In some embodiments, an ASMC (e.g., an inhibitory ASMC) includes a gene and an operably linked repressor sequence located within the ASMC, and the gene is not actively expressed. In some embodiments, ASMC (e.g., inhibitory ASMC) comprises a gene and an enhancer located outside the ASMC, where the gene is not actively expressed. In some embodiments, ASMC (e.g., activated ASMC) comprises a gene and an operably linked enhancer, where the repressor is located outside the ASMC, where the gene is actively expressed. In some embodiments, ASMC (e.g., inhibitory ASMC) comprises a gene and an operably linked repressor sequence, where the enhancer is located outside the ASMC, where the gene is not actively expressed.

[0216] In some embodiments, the target gene is discontinuous with one or more expression control sequences. In some embodiments in which the gene is discontinuous with its expression control sequences, the gene may be spaced from one or more expression control sequences by about 100 bp to about 500 Mb, about 500 bp to about 200 Mb, about 1 kb to about 100 Mb, about 25 kb to about 50 Mb, about 50 kb to about 1 Mb, about 100 kb to about 750 kb, about 150 kb to about 500 kb, or about 175 kb to about 500 kb. In some embodiments, the gene is located within about 100 bp, 300 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1 kb, 5 kb, 10 kb, 15 kb, 20 kb, 25 kb, 30 kb, 35 kb, 40 kb, 45 kb, 50 kb, 55 kb, 60 kb, 65 kb, 70 kb, 75 kb, 80 kb, 85 kb, 90 kb, 95 kb, 100 kb, 125 kb, 150 kb, 17 ... 00kb, 225kb, 250kb, 275kb, 300kb, 350kb, 400kb, 500kb, 600kb, 700kb, 800kb, 900kb, 1Mb, 2Mb, 3Mb, 4Mb, 5Mb, 6Mb, 7Mb, 8Mb, 9Mb, 10Mb, 15Mb, 20Mb, 25Mb, 50Mb, 75Mb, 100Mb, 200Mb, 300Mb, 400Mb, 500Mb, or any size interval therebetween.

[0217] Without wishing to be bound by theory, it is believed that in some embodiments, understanding (e.g., identifying or classifying) whether an ASMC is or corresponds to a particular type of anchor sequence-mediated junction can be helpful in determining how to modify ASMC, for example, by influencing the selection of DNA-binding or effector moieties, to regulate gene expression. For example, in some embodiments, some types of anchor sequence-mediated junctions contain one or more expression control sequences (e.g., enhancers) within the anchor sequence-mediated junction. Modulation (e.g., disruption) of a genomic complex containing ASMC and / or the presence of ASMC within the genomic complex, for example, by altering one or more anchor sequences (such alterations disrupting ASMC), can reduce transcription of target genes within the genomic complex and / or ASMC. In some embodiments, modulation (e.g., disruption) of an inhibitory ASMC or a genomic complex containing the ASMC results in increased gene expression. In some embodiments, modulation (e.g., disruption) of an activated ASMC or a genomic complex containing the ASMC results in reduced gene expression.

[0218] Compositions: Manufacturing, Formulation, Delivery, and Administration Methods The present disclosure provides, among other things, compositions comprising a modulating agent described herein and / or compositions that deliver a modulating agent to a cell, tissue, organ, and / or subject. In some embodiments, a modulating agent comprising a polypeptide (e.g., a moiety that is or contains a polypeptide) may be provided via a composition that includes the modulating agent polypeptide or polypeptide portion as a polypeptide, or via a composition that includes a nucleic acid that encodes the modulating agent or a polypeptide portion thereof, and that is linked to other sequences sufficient to achieve expression of the modulating agent or a polypeptide portion thereof in a system of interest (e.g., a particular cell, tissue, organ, etc.).

[0219] In some embodiments, the compositions provided may be pharmaceutical compositions whose active ingredient comprises or delivers a modulatory agent described herein, provided in combination with one or more pharmaceutically acceptable excipients, and optionally formulated for administration to a subject (e.g., a cell, tissue, or other site thereof).

[0220] In some aspects, the present disclosure provides a method of delivering a therapeutic agent comprising administering a composition described herein to a subject, wherein the genome complex modulating agent is a therapeutic agent and / or the delivery of the therapeutic agent targets a genome complex (e.g., ASMC) characterized by an integrity index to alter gene expression relative to gene expression in the absence of the therapeutic agent.

[0221] In some aspects, the system for pharmaceutical use includes a composition that targets a genome complex characterized by an integrity index by disrupting the genome complex. In some embodiments, the composition targets the genome complex by binding to an anchor sequence within the genome complex and altering the formation of an anchor sequence-mediated junction, wherein the composition modulates the transcription of a target gene associated with the anchor sequence-mediated junction in a human cell.

[0222] Thus, in some embodiments, the present disclosure provides compositions comprising a modulator (e.g., a disrupting agent), or a production intermediate thereof. In some specific embodiments, the present disclosure provides compositions of nucleic acids encoding a modulator (e.g., a disrupting agent) or a polypeptide portion thereof. In some such embodiments, the provided nucleic acids may be or include DNA, RNA, or any other nucleic acid moiety or entity described herein, and may be prepared by any technique described herein or otherwise available in the art (e.g., synthesis, cloning, amplification, in vitro or in vivo transcription, etc.). In some embodiments, the provided nucleic acids encoding a modulator (e.g., a disrupting agent) or a polypeptide portion thereof may be operably linked to one or more replication, integration, and / or expression signals suitable and / or sufficient to achieve integration, replication, and / or expression of the provided nucleic acid in a system of interest (e.g., a particular cell, tissue, organism, etc.).

[0223] In some embodiments, a modulating agent (e.g., an interfering agent) is or comprises a vector, e.g., a viral vector, which comprises one or more nucleic acids encoding one or more components of the modulating agent (e.g., an interfering agent) described herein.

[0224] Generate Nucleic acids as described herein or nucleic acids encoding proteins described herein may be incorporated into vectors. Vectors, such as those derived from retroviruses such as lentiviruses, are suitable tools for achieving long-term gene transfer because they allow long-term stable integration of the transgene and its propagation in daughter cells. Examples of vectors include expression vectors, replication vectors, probe generation vectors, and sequencing vectors. Expression vectors may be provided to cells in the form of viral vectors. Viral vector technology is well known in the art and is described in various virology and molecular biology manuals. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. Generally, suitable vectors have an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers.

[0225] Expression of natural or synthetic nucleic acids is typically achieved by operably linking a nucleic acid encoding a gene of interest to a promoter and incorporating the construct into an expression vector. The vector may be suitable for replication and integration in eukaryotes. Typical cloning vectors have transcription and translation terminators, initiation sequences, and promoters useful for expression of the desired nucleic acid sequence.

[0226] Additional promoter regions, such as enhancer sequences, may modulate the frequency of transcription initiation. Typically, these sequences are located within the region 30–110 bp upstream of the transcription start site, although recent studies have shown that some promoters also contain functional elements downstream of the transcription start site. Because the spacing between promoter regions is often flexible, promoter function is preserved when regions are inverted or moved relative to each other. In the thymidine kinase (tk) promoter, the spacing between promoter regions can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, each region appears to function cooperatively or independently to activate transcription.

[0227] One example of a suitable promoter is the immediate-early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong, constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operably linked to it. In some embodiments, a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences, such as, but not limited to, the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate-early promoter, Rous sarcoma virus promoter, and human gene promoters, such as, but not limited to, the actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter, may also be used.

[0228] The present disclosure should not be construed as limited to the use of any particular promoter or category of promoter (e.g., constitutive promoters). For example, in some embodiments, inducible promoters are contemplated as part of the present disclosure. In some embodiments, the use of an inducible promoter provides a molecular switch capable of activating expression of a polynucleotide sequence of interest to which it is operably linked when such expression is desired. In some embodiments, the use of an inducible promoter provides a molecular switch capable of inactivating expression when expression is not desired. Examples of inducible promoters include, but are not limited to, metallothionine promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0229] In some embodiments, the expression vector to be introduced may also contain either a selectable marker gene or a reporter gene, or both, to facilitate identification and selection of expressing cells from a population of cells sought to be transfected or infected via a viral vector. In some aspects, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection method. To enable expression in the host cell, both the selectable marker and the reporter gene may be flanked by appropriate expression control sequences. Useful selectable markers may include, for example, antibiotic resistance genes, such as neo.

[0230] In some embodiments, a reporter gene may be used to identify potentially transfected cells and / or to evaluate the functionality of expression control sequences. Generally, a reporter gene is a gene encoding a polypeptide that is absent from or expressed by the recipient source (of the reporter gene) and whose expression is manifested by some easily detectable property, such as enzymatic activity or visible fluorescence. Expression of the reporter gene is assayed at a suitable time after DNA is introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al., 2000 FEBS Letters 479:79-82). Suitable expression systems are well known and may be prepared using known techniques or obtained commercially. Generally, the construct with the minimal 5'-flanking region that exhibits the highest level of expression of the reporter gene is identified as the promoter. Such promoter regions may be linked to a reporter gene and used to assess the ability of agents to modulate promoter-driven transcription.

[0231] In some embodiments, the modulating agent comprises or is a protein and thus may be produced by a method for making a protein. As will be appreciated by those of skill in the art, methods for making proteins or polypeptides (which may be included in the modulating agents described herein) are routine in the art. For general information, see: Smales & James (Eds.), Therapeutic Proteins: Methods and Protocols (Methods in Molecular Biology), Humana Press (2005); and Crommelin, Sindelar & Meibohm (Eds.), Pharmaceutical Biotechnology: Fundamentals and Applications, Springer (2013).

[0232] Proteins or polypeptides of the disclosed compositions can be biochemically synthesized using standard solid-phase techniques. These methods include exclusive solid-phase synthesis, partial solid-phase synthesis methods, fragment condensation, and classical solution synthesis. These methods can be used when the peptide is relatively short (e.g., 10 kDa) and / or cannot be produced recombinantly (i.e., is not encoded by a nucleic acid sequence) and therefore requires alternative chemistry.

[0233] Solid phase synthesis methods are known in the art and are described in more detail in John Morrow Stewart and Janis Dillaha Young, Solid Phase Peptide Syntheses, 2nd Ed., Pierce Chemical Company, 1984; and Coin, I., et al., Nature Protocols, 2:3247-3256, 2007.

[0234] For longer peptides, recombinant methods can be used. Methods for producing recombinant therapeutic polypeptides are routine in the art. For overviews, see: Smales & James (Eds.), Therapeutic Proteins: Methods and Protocols (Methods in Molecular Biology), Humana Press (2005); and Crommelin, Sindelar & Meibohm (Eds.), Pharmaceutical Biotechnology: Fundamentals and Applications, Springer (2013).

[0235] Exemplary methods for producing therapeutic proteins or polypeptides include expression in mammalian cells, although recombinant proteins can also be produced using insect cells, yeast, bacteria, or other cells under the control of an appropriate promoter. Mammalian expression vectors can include non-transcribed elements, such as an origin of replication, a suitable promoter, and other 5'- or 3'-flanking non-transcribed sequences, and 5'- or 3'-untranslated sequences, such as necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, and termination sequences. DNA sequences from the SV40 viral genome, such as the SV40 origin, early promoter, splice, and polyadenylation sites, can also be used to obtain other genetic elements required for expression of heterologous DNA sequences. Cloning and expression vectors suitable for use with bacterial, fungal, yeast, and mammalian cell hosts are described in Green & Sambrook, Molecular Cloning: A Laboratory Manual (Fourth Edition), Cold Spring Harbor Laboratory Press (2012).

[0236] If large amounts of a protein or polypeptide are desired, see Brian Bray, Nature Reviews Drug Discovery, 2:587-593, 2003; and Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic It can be produced using techniques such as those described in The American Chemical Society, NY, Section VIII, pp 421-463.

[0237] Various mammalian cell culture systems can be used to express and produce recombinant proteins. Examples of mammalian expression systems include CHO cells, COS cells, HeLA, and BHK cell lines. The process of host cell culture for protein therapeutic production is described in Zhou and Kantardjieff (Eds.), Mammalian Cell Cultures for Biologics Manufacturing (Advances in Biochemical Engineering / Biotechnology), Springer (2014). The compositions described herein may contain a vector encoding a recombinant protein, e.g., a viral vector, e.g., a lentiviral vector. In some embodiments, the vector, e.g., a viral vector, may contain a nucleic acid encoding the recombinant protein.

[0238] For purification of protein therapeutics, see: Franks, Protein Biotechnology: Isolation, Characterization, and Stabilization, Humana Press (2013); and Cutler, Protein Purification Protocols (Methods in Molecular Biology), Humana Press (2010).

[0239] Formulation of protein therapeutics is described in Meyer (Ed.), Therapeutic Protein Drug Products: Practical Approaches to Formulation in the Laboratory, Manufacturing, and the Clinic, Woodhead Publishing Series (2012).

[0240] Proteins comprise one or more amino acids. Amino acids include any compound and / or substance that can be incorporated into a polypeptide chain, for example, via one or more peptide bonds. In some embodiments, an amino acid has the general structure: HN-C(H)(R)-COOH. In some embodiments, an amino acid is a naturally occurring amino acid. In some embodiments, an amino acid is a non-natural amino acid; in some embodiments, an amino acid is a D-amino acid; in some embodiments, an amino acid is an L-amino acid. A "standard amino acid" refers to any of the 20 standard L-amino acids commonly found in naturally occurring peptides. A "non-standard amino acid" refers to any amino acid other than the standard amino acids, whether it is synthetically prepared or obtained from a natural source. In some embodiments, an amino acid, including the carboxy- and / or amino-terminal amino acid in a polypeptide, may contain structural modifications compared to the general structures above. For example, in some embodiments, an amino acid may be modified by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and / or substitution (e.g., of an amino group, a carboxylic acid group, one or more protons, and / or a hydroxyl group) relative to the general structure. In some embodiments, such modifications may, for example, alter the circulating half-life of a polypeptide containing the modified amino acid compared to one that otherwise contains the same unmodified amino acid. In some embodiments, such modifications do not significantly alter the relevant activity of a polypeptide containing the modified amino acid compared to one that otherwise contains the same unmodified amino acid. As will be clear from the context, in some embodiments, the term "amino acid" may be used to refer to a free amino acid; in some embodiments, it may be used to refer to an amino acid residue of a polypeptide.

[0241] delivery In various embodiments, the compositions (e.g., modulators) described herein are pharmaceutical compositions. In some embodiments, the compositions (e.g., pharmaceutical compositions) described herein can be formulated for delivery to a cell and / or a subject via any route of administration. Methods of administration to a subject can include injection, infusion, inhalation, intranasal, intraocular, topical delivery, intracannular delivery, or ingestion. Injections include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intravascular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection and infusion. In some embodiments, administration includes aerosol inhalation, e.g., using nebulization. In some embodiments, administration is systemic (e.g., oral, rectal, intranasal, sublingual, buccal, or parenteral), enteral (e.g., systemic but delivered via the digestive tract), or local (e.g., topical application to the skin, intravitreal injection). In some embodiments, one or more compositions are administered systemically. In some embodiments, administration is not by injection, and the therapeutic agent is a parenteral therapeutic agent. In some specific embodiments, administration may be or include one or more of: bronchial (e.g., by intrabronchial infusion), oral, cutaneous (e.g., topical to the dermis, intradermal, intradermal, transdermal, etc.), enteral, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, intraspecific organ (e.g., intrahepatic), mucosal, intranasal, oral, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal infusion), intravaginal, intravitreal, etc. In some embodiments, administration may be a single dose. In some embodiments, administration may include intermittent (e.g., multiple doses spaced apart in time) and / or periodic (e.g., individual doses spaced apart in time) dosing. In some embodiments, administration may include continuous dosing (e.g., perfusion) over at least a selected period of time.

[0242] The pharmaceutical composition according to the present disclosure can be administered in a therapeutically effective amount. The precise therapeutically effective amount is the amount of the composition that is believed to produce the most effective results in terms of efficacy of treatment for a given subject. This amount will vary depending on various factors, including but not limited to, the characteristics of the therapeutic compound (such as activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (such as age, sex, type and stage of disease, general health, response to a given dose, and type of drug), one or more pharmaceutically acceptable carriers in the formulation, and / or the route of administration.

[0243] In some aspects, the present disclosure provides methods for delivering a therapeutic agent comprising administering a composition described herein to a subject, wherein the genome complex (e.g., ASMC) modulating agent is a therapeutic agent and / or delivery of the therapeutic agent causes a change in gene expression compared to gene expression in the absence of the therapeutic agent.

[0244] The methods provided in various embodiments herein may be used in any of several aspects delineated herein, hi some embodiments, one or more compositions are targeted to specific cells or to one or more specific tissues.

[0245] For example, in some embodiments, one or more compositions are targeted to epithelial, connective, muscle, and / or nervous tissues or cells. In some embodiments, the compositions are targeted to cells or tissues of a particular organ system, such as the cardiovascular system (heart, blood vessels); digestive system (esophagus, stomach, liver, bladder, pancreas, intestines, colon, rectum, and anus); endocrine system (hypothalamus, pituitary gland, pineal gland or gland, thyroid gland, parathyroid gland, adrenal gland); excretory system (kidneys, ureters, bladder); lymphatic system (lymph, lymph nodes, lymphatic vessels, tonsils, adenoids, thymus, spleen); integumentary system (skin, hair, nails); muscular system (e.g., skeletal muscle); nervous system (brain, spinal cord, nerves); reproductive system (ovaries, uterus, mammary glands, testes, vas deferens, seminal vesicles, prostate); respiratory system (pharynx, larynx, trachea, bronchi, lungs, diaphragm); skeletal system (bone, cartilage); and / or combinations thereof.

[0246] In some embodiments, compositions of the present disclosure cross the blood-brain barrier, placental membrane, or blood-testis barrier.

[0247] In some embodiments, the compositions provided herein are administered systemically.

[0248] In some embodiments, administration is not by injection and the therapeutic agent is a parenteral therapeutic agent.

[0249] Pharmaceutical Composition As used herein, the term "pharmaceutical composition" refers to an active agent (e.g., a disrupting agent) formulated together with one or more pharmaceutically acceptable carriers (e.g., pharmaceutically acceptable carriers known to those of skill in the art). In some embodiments, the active agent is present in a unit dose suitable for administration in a treatment regimen that, when administered to a relevant population, exhibits a statistically significant probability of achieving an intended therapeutic effect. In some embodiments, the pharmaceutical composition may be specially formulated for administration in solid or liquid form, including: oral administration, e.g., drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., buccal, sublingual, and those intended for systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, e.g., subcutaneous, intramuscular, intravenous, or epidural injection (e.g., sterile solutions or suspensions), or sustained release formulations; topical application, e.g., creams, ointments, or controlled release patches or sprays applied to the skin, lungs, or oral cavity; vaginal or rectal, e.g., as pessaries, creams, or foams; sublingual; intraocular; transdermal; or designed for the nose, lungs, and / or other mucosal surfaces.

[0250] As used herein, the term "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other impairment or complication, and commensurate with a reasonable benefit / list ratio.

[0251] As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, that is involved in carrying or transporting the subject compositions from one organ or body part to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. In some embodiments, for example, materials that can serve as pharmaceutically acceptable carriers include: sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives, for example, sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, coconut oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; ethyl alcohol; alginic acid; pyrogen-free distilled water; isotonic saline; Ringer's solution; ethyl alcohol; pH buffers; polyesters, polycarbonates, and / or polyanhydrides; and other non-toxic, compatible substances used in pharmaceutical formulations.

[0252] As used herein, the term "pharmaceutically acceptable salt" refers to a salt of a compound that is suitable for use in a pharmaceutical context, i.e., that is, a salt that, within the scope of sound medical judgment, is suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, etc., and that is commensurate with a reasonable benefit / list ratio. Pharmaceutically acceptable salts are known in the art. For example, S. M. Berge et al. describe pharmaceutically acceptable salts in J. Pharmaceutical Sciences, 66:1-19 (1977). In some embodiments, pharmaceutically acceptable salts include, but are not limited to, non-toxic acid addition salts of amino groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or organic acids such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or formed by other methods used in the art, such as ion exchange. In some embodiments, pharmaceutically acceptable salts include, but are not limited to, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydrogen phosphate, hydroxybenzoate ... Examples of suitable salts include hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, etc. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc.In some embodiments, pharmaceutically acceptable salts include, where appropriate, non-toxic ammonium, quaternary ammonium, and amine cations formed with counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, alkyls having 1 to 6 carbon atoms, sulfonates, and arylsulfonates.

[0253] In various embodiments, the present disclosure provides pharmaceutical compositions described herein with a pharmaceutically acceptable excipient. Pharmaceutically acceptable excipients generally include excipients that are safe, non-toxic, and useful for preparing desirable pharmaceutical compositions, and include excipients that are acceptable for both veterinary and human pharmaceutical use. Such excipients may be solid, liquid, semi-solid, or, in the case of aerosol compositions, gaseous.

[0254] Pharmaceutical preparations can be prepared according to conventional pharmaceutical techniques, including crushing, mixing, granulating, and optionally compressing in the case of tablets; crushing, mixing, and filling in the case of hard gelatin capsule dosage forms.When using a liquid carrier, the preparation can be in the form of a syrup, elixir, emulsion, or aqueous or non-aqueous solution or suspension.Such liquid preparations can be administered directly orally.

[0255] In some embodiments, the compositions of the disclosure have improved PK / PD, e.g., increased pharmacokinetics or pharmacodynamics, e.g., improved targeting, absorption, or transport (e.g., at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 75%, 80%, 90% or more improvement), compared to the therapeutic agent alone. In some embodiments, the compositions have reduced undesirable effects, e.g., reduced diffusion to non-target locations, off-target activity, or toxic metabolites, compared to the therapeutic agent alone (e.g., at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 75%, 80%, 90% or more reduction), compared to the therapeutic agent alone. In some embodiments, the composition increases the efficacy of the therapeutic agent and / or reduces the toxicity of the therapeutic agent (e.g., by at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 75%, 80%, 90%, or more) compared to the therapeutic agent alone.

[0256] The pharmaceutical compositions described herein can be formulated, for example, with pharmaceutical carriers and / or polymeric carriers, such as liposomes or vesicles, and delivered to a subject in need thereof (e.g., a human or non-human farm or livestock animal, e.g., cattle, dogs, cats, horses, poultry) by known methods. These methods include transfection (e.g., lipid-mediated, cationic polymers, calcium phosphate); electroporation or other membrane disruption (e.g., nucleofection); and viral delivery (e.g., lentivirus, retrovirus, adenovirus, AAV). Further delivery methods are described, for example, in Gori et al., "Delivery and Specificity of CRISPR / Cas9 Genome Editing Technologies for Human Gene Therapy." Human Gene Therapy. July 2015, 26(7):443-451. doi:10.1089 / hum.2015.074; and Zuris et al. Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat Biotechnol. 2014 Oct 30;33(1):73-80.

[0257] Liposomes are spherical vesicular structures composed of a mono- or multilamellar lipid bilayer surrounding an internal aqueous compartment and a relatively impermeable outer lipophilic phospholipid bilayer. Liposomes may be anionic, neutral, or cationic. Liposomes are biocompatible, non-toxic, and can deliver both hydrophilic and lipophilic drug molecules, protect their cargo from degradation by plasma enzymes, and transport their payload across biological membranes and the blood-brain barrier (BBB) ​​(see, e.g., Spuch and Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 for a review). pages,2011.doi:10.1155 / 2011 / 469679).

[0258] Vesicle can be made from several different types of lipid, but phospholipid is most commonly used to make liposome as drug carrier.Vesicle can comprise but is not limited to DOTMA, DOTAP, DOTIM, DDAB alone or together with cholesterol, to produce DOTMA and cholesterol, DOTAP and cholesterol, DOTIM and cholesterol, and DDAB and cholesterol.The method for preparing multilamellar vesicle lipid is known in the art (for example, see U.S. Patent No. 6,693,086, its teaching about multilamellar vesicle lipid preparation is incorporated herein by reference). When lipid membrane is mixed with aqueous solution, vesicle formation can be spontaneous, but it can also be promoted by applying force in the form of shaking by using homogenizer, sonicator or extrusion device (for example, for review, see Spuch and Navarro, Journal of Drug Delivery, vol.2011, Article ID469679, 12 pages, 2011.doi:10.1155 / 2011 / 469679).Extruded lipid can be prepared by extruding through size-reducing filter, as described in Templeton et al., Nature Biotech, 15:647-652, 1997 (the teaching of which about extruded lipid formulation is incorporated herein by reference).

[0259] The methods and compositions described herein can include pharmaceutical compositions administered in a regimen sufficient to alleviate the symptoms of a disease, disorder, and / or condition. In some aspects, the present disclosure provides methods of delivering a therapeutic agent by administering a composition described herein.

[0260] The pharmaceutical uses of the present disclosure can include compositions (e.g., modulators, e.g., interfering agents) described herein. In some embodiments, a system for pharmaceutical use includes: a protein comprising a first polypeptide domain, e.g., a Cas or modified Cas protein, and a second polypeptide domain, e.g., a polypeptide having DNA methyltransferase activity or associated with demethylation or deaminase activity, in combination with at least one guide RNA (gRNA) or antisense DNA oligonucleotide targeting an ncRNA, such as an eRNA. The system is effective to alter a genome complex, e.g., a target anchor sequence-mediated junction, characterized by an integrity index in at least one human cell.

[0261] In some aspects, a pharmaceutical composition of the present disclosure comprises a zinc finger nuclease (ZFN) that targets (e.g., cleaves) an ncRNA, such as an eRNA, or an mRNA encoding the ZFN.

[0262] In some embodiments, systems for pharmaceutical use bind to ncRNAs, e.g., eRNAs, and alter the formation of genomic complexes, e.g., anchor sequence-mediated junctions (e.g., genomic complexes characterized by an integrity index), that include the ncRNA (e.g., eRNA), wherein such compositions regulate the transcription of target genes associated with the genomic complexes, e.g., anchor sequence-mediated junctions, in human cells.

[0263] In some aspects, a system for altering expression of a target gene in a human cell includes a targeting moiety (e.g., gRNA, membrane translocating polypeptide) that binds to an ncRNA, e.g., eRNA, associated with the target gene, and an effector moiety (e.g., an enzyme, e.g., a nuclease or inactivating nuclease (e.g., Cas9, dCas9), a methylase, a demethylase, a deaminase) operably linked to the targeting moiety, wherein the system is effective to alter (e.g., reduce) expression of the target gene. The targeting moiety and the effector moiety may be different and individual moieties (e.g., comprised in different physical portions of a disrupting agent). The targeting moiety and the effector moiety may be, for example, covalently linked, e.g., by a linker. In some embodiments, the system includes a synthetic polypeptide comprising the targeting moiety and the effector moiety. In some embodiments, the system includes one or more nucleic acid vectors encoding at least one of the targeting moiety and the effector moiety.

[0264] In some aspects, the pharmaceutical composition may comprise a composition that targets a genome complex (e.g., ASMC) characterized by an integrity index by binding to an anchor sequence of the anchor sequence-mediated junction and altering the formation of the anchor sequence-mediated junction, wherein the composition regulates transcription of a target gene associated with the genome complex (e.g., ASMC) in a human cell. In some embodiments, the composition targets a genome complex characterized by an integrity index by disrupting the formation of the anchor sequence-mediated junction (e.g., reducing the affinity of the anchor sequence for a junction nucleating molecule by, e.g., at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more). In some embodiments, disrupting formation comprises altering the integrity index by modulating the affinity of the anchor sequence for the junction nucleating molecule, e.g., by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more.

[0265] In some embodiments, administration of a composition described herein improves at least one pharmacokinetic or pharmacodynamic parameter, e.g., targeting, absorption, or transport, of at least one component of the composition (e.g., a drug), compared to the other moiety alone, or reduces at least one toxicokinetic parameter, e.g., diffusion to non-target locations, off-target activity, and toxic metabolites, compared to the other moiety alone (e.g., by at least 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80% or more). In some embodiments, administration of a composition of the present disclosure increases the therapeutic range of at least one component of a modulator (e.g., by at least 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80% or more). In some embodiments, administration of a composition described herein reduces the minimum effective dose (e.g., by at least 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80% or more) compared to the other moiety alone. In some embodiments, administration of a composition described herein increases the maximum tolerated dose (e.g., by at least 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80% or more) compared to the modulator alone. In some embodiments, administration of a composition described herein increases the efficacy or reduces the toxicity of a therapeutic agent, such as administration other than injection of a parenteral therapeutic agent. In some embodiments, administration of a composition described herein increases the therapeutic range of a modulator while simultaneously reducing toxicity (e.g., by at least 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80% or more) compared to the modulator alone.

[0266] In some aspects, the present disclosure provides modulators, e.g., disruptive agents, that bind to ncRNAs, e.g., eRNAs, and alter, e.g., reduce, the formation of genome or transcription complexes, e.g., anchor sequence-mediated junctions (e.g., reduce the level of the complex by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more).

[0267] In some embodiments, the gRNA is administered in combination with a targeting nuclease, e.g., Cas9, e.g., wild-type Cas9, nickase Cas9 (e.g., Cas9 D10A), inactive Cas9 (dCas9), eSpCas9, Cpf1, C2C1, or C2C3, or a nucleic acid encoding such a nuclease. The choice of nuclease and gRNA is determined by whether the targeted mutation is a nucleotide deletion, substitution, or addition, e.g., a nucleotide deletion, substitution, or addition to an ncRNA, e.g., eRNA. For example, in some embodiments, one gRNA is administered to, e.g., cause an inactivating indel mutation in an ncRNA, e.g., eRNA; e.g., one gRNA is administered in combination with a nuclease, e.g., wtCas9.

[0268] In some aspects, the present disclosure provides compositions comprising a nucleic acid or combination of nucleic acids that, when administered to a subject in need thereof, introduce a site-specific alteration (e.g., an insertion, a deletion (e.g., a knockout), a translocation, an inversion, a single point mutation) into a target sequence of a target genome complex (e.g., ASMC) or a component of a target genome complex, e.g., ncRNA, eRNA, characterized by an integrity index, thereby modulating gene expression in the subject.

[0269] use The present disclosure further relates to uses of the modulating agents disclosed herein. Among other things, in some embodiments, such techniques provided achieve modulation, e.g., increase, of target gene (e.g., FXN) expression, e.g., allowing for control of target gene activity, delivery, and penetration, e.g., in a cell. In some embodiments, the cell is a mammalian, e.g., human, cell. In some embodiments, the cell is a somatic cell. In some embodiments, the cell is a primary cell. For example, in some embodiments, the cell is a mammalian somatic cell. In some embodiments, the mammalian somatic cell is a primary cell. In some embodiments, the mammalian somatic cell is a non-embryonic cell. In some embodiments, the cell is a muscle cell (e.g., a cardiac muscle cell, e.g., a cardiomyocyte) or a neuronal cell (e.g., a cell of the central nervous system or a cell of the spinal cord, e.g., a cell (e.g., a neuron) of the dorsal root ganglion (DRG)).

[0270] In some embodiments, such techniques provided can be used to treat FRDA or a condition associated with FRDA in a subject, e.g., a patient, in need thereof.

[0271] Regulation of gene expression The present disclosure further relates, in part, to methods of modulating, e.g., increasing, expression of a target gene (e.g., FXN), comprising providing a modulator described herein (or a nucleic acid encoding same, or a pharmaceutical composition comprising the modulator or nucleic acid) and contacting a cell, the target gene, and / or an operably linked expression control element with the modulator. In some embodiments, modulating, e.g., increasing, expression of the target gene comprises modulating transcription of the target gene compared to a reference value, e.g., transcription of the target gene in the absence of the modulator. In some embodiments, the methods of modulating, e.g., increasing, expression of the target gene are used ex vivo, e.g., on cells derived from a subject, e.g., a mammalian subject, e.g., a human subject. In some embodiments, the methods of modulating, e.g., increasing, expression of the target gene are used in vivo, e.g., on a mammalian subject, e.g., a human subject. In some embodiments, the methods of modulating, e.g., increasing, expression of the target gene are used in vivo, e.g., on a cell or cell line described herein.

[0272] The present disclosure also relates, in part, to a method of treating a condition associated with underexpression of a target gene (e.g., FXN) and / or pathologically low levels of a target gene (e.g., FXN) product (e.g., mRNA or protein) in a subject, comprising administering to the subject a modulator (or a nucleic acid encoding same, or a pharmaceutical composition comprising said modulator or nucleic acid) described herein. Conditions associated with underexpression of particular genes and / or pathologically low levels of gene products are well known to those of skill in the art. Such conditions include, but are not limited to, FRDA (associated with underexpression of FXN and / or pathologically low levels of FXN gene products), metabolic disorders, neuromuscular disorders, cancer (e.g., solid tumors), fibrosis, diabetes, urea disorders, immune disorders, inflammation, and arthritis.

[0273] The present disclosure also relates, in part, to a method of treating a condition associated with misregulated expression of a target gene in a subject, comprising administering to the subject a modulator (or a nucleic acid encoding same, or a pharmaceutical composition comprising said modulator or nucleic acid) described herein. Without wishing to be bound by theory, it is believed that the modulator is used to target (e.g., increase expression of) a gene that regulates expression of the target gene, thereby altering expression of the regulated gene. Conditions associated with misregulated expression of specific genes are well known to those of skill in the art. Such conditions include, but are not limited to, metabolic disorders, neuromuscular disorders, cancer (e.g., solid tumors), fibrosis, diabetes, urea disorders, immune disorders, inflammation, and arthritis.

[0274] The methods and compositions provided herein can treat disease conditions by stably or transiently altering (e.g., increasing) transcription of a target gene (e.g., FXN). In some embodiments, such modulation persists for at least about 1 hour to about 30 days, or at least about 2 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, or more, or any time in between. In some embodiments, such modulation lasts for 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, or 24 hours, or at least 1, 2, 3, 4, 5, 6, or 7 days, or at least 1, 2, 3, 4, or 5 weeks, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or at least 1, 2, 3, 4, or 5 years (e.g., indefinitely). Optionally, such modulation lasts for 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 year or less.

[0275] In some embodiments, the condition being treated is FRDA. FRDA is an inherited, progressive, neurodegenerative movement disorder. In some embodiments, the subject, e.g., patient, treated by the methods described herein is a subject, e.g., patient, with FRDA. In some embodiments, FRDA has a typical age of onset between 10 and 15 years of age. In some embodiments, the subject, e.g., patient, is diagnosed with FRDA before the age of 25, e.g., between 10 and 15 years of age. In some embodiments, the subject, e.g., patient, is diagnosed with FRDA between the ages of 26 and 39, e.g., the subject, e.g., patient, is diagnosed with late-onset FRDA (LOFA). In some embodiments, the subject, e.g., patient, is diagnosed with FRDA at age 40 or older, e.g., the subject, e.g., patient, is diagnosed with very late-onset FRDA (VLOFA). Early symptoms include unsteady posture due to impaired voluntary movement coordination (ataxia), frequent falls, and progressive difficulty walking. Affected individuals may develop slurred speech (dysarthria), characteristic foot deformities, and / or abnormal curvature of the spine (scoliosis). FRDA may be associated with cardiomyopathy, a disease of the heart muscle that can lead to heart failure or irregular heart rhythms (cardiac arrhythmias). Approximately one-third of patients with FRDA develop diabetes. In some embodiments, a method or composition provided herein improves (e.g., reduces the severity of) or eliminates one or more FRDA-associated symptoms selected from, but not limited to, the following: unstable posture, frequent falls, difficulty walking, ataxia, dysarthria, foot deformities, scoliosis, cardiomyopathy, cardiac arrhythmias, or diabetes.

[0276] In some embodiments, the methods provided herein may modulate, e.g., increase, expression of a target gene (e.g., FXN) by disrupting a genomic complex associated with the target gene, e.g., an anchor sequence-mediated junction. The gene associated with an anchor sequence-mediated junction may be at least partially within the junction (i.e., located sequence-wise between the first and second anchor sequences), or may be outside the junction in that it is not located sequence-wise between the first and second anchor sequences, but the gene is located on the same chromosome and in sufficient proximity to at least the first or second anchor sequence such that its expression can be modulated by controlling the topology of the anchor sequence-mediated junction. Those skilled in the art will understand that the distance in three-dimensional space between two elements (e.g., between the gene and the anchor sequence-mediated junction) may, in some embodiments, be more important than the distance in terms of base pairs. In some embodiments, the external but related gene is located within 2 Mb, 1.9 Mb, 1.8 Mb, 1.7 Mb, 1.6 Mb, 1.5 Mb, 1.4 Mb, 1.3 Mb, 1.3 Mb, 1.2 Mb, 1.1 Mb, 1 Mb, 900 kb, 800 kb, 700 kb, 500 kb, 400 kb, 300 kb, 200 kb, 100 kb, 50 kb, 20 kb, 10 kb, or 5 kb of the first or second anchor sequence.

[0277] In some embodiments, modulating expression of a gene comprises altering the accessibility of an expression control sequence to the gene. The expression control sequence can be an enhancing sequence or a silencing (or suppressing) sequence, whether inside or outside the anchor sequence-mediated junction.

[0278] Epigenetic modifications The present disclosure further relates, in part, to a method for epigenetically modifying a target gene (e.g., FXN), an expression control element operably linked to the target gene, or an anchor sequence (e.g., an anchor sequence that is proximal to the target gene or that includes or is associated with an anchor sequence-mediated junction associated with the target gene or an expression control sequence operably linked to said target gene), the method comprising the steps of providing a regulator, or a nucleic acid encoding a regulator, or a pharmaceutical composition comprising said regulator or nucleic acid; and contacting the target gene (e.g., FXN), the expression control sequence operably linked to the target gene, or a cell with the regulator, nucleic acid, or pharmaceutical composition, thereby epigenetically modifying the target gene or the expression control sequence operably linked to the target gene.

[0279] In some embodiments, the method of epigenetically modifying a target gene (e.g., FXN) or an expression control sequence operably linked to the target gene comprises increasing or decreasing DNA methylation of the target gene or the expression control sequence operably linked to the target gene. In some embodiments, the method of epigenetically modifying a target gene or a transcription control element operably linked to the target gene comprises increasing or decreasing histone methylation of histones associated with the target gene or the expression control sequence operably linked to the target gene. In some embodiments, the method of epigenetically modifying a target gene or an expression control sequence operably linked to the target gene comprises increasing or decreasing histone acetylation of histones associated with the target gene or the expression control sequence operably linked to the target gene. In some embodiments, the method of epigenetically modifying a target gene or an expression control sequence operably linked to the target gene comprises increasing or decreasing histone sumoylation of histones associated with the target gene or the expression control sequence operably linked to the target gene. In some embodiments, the method of epigenetically modifying a target gene or an expression control sequence operably linked to the target gene comprises increasing or decreasing histone phosphorylation of a histone associated with the target gene or an expression control sequence operably linked to the target gene.

[0280] In some embodiments, a method of epigenetically modifying a target gene (e.g., FXN) or an expression control sequence operably linked to a target gene may reduce the level of epigenetic modification by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% (and optionally, up to 100%) compared to the level of epigenetic modification at that site in a cell that has not been contacted with the composition or treated with the method. In some embodiments, a method of epigenetically modifying a target gene or an expression control sequence operably linked to a target gene can increase the level of epigenetic modification by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 300, 400, 500, 600, 700, 800, 900, or 1000% (and optionally up to 200, 300, 400, 500, 600, 700, 800, 900, 1000, or 2000%) relative to the level of epigenetic modification at that site in a cell that has not been contacted with the composition or treated with the method. In some embodiments, epigenetic modification of a target gene (e.g., FXN) or an expression control sequence operably linked to the target gene can modify the level of expression of the target gene, for example, as described herein.

[0281] In some embodiments, the epigenetic modifications achieved by the methods described herein persist for at least about 1 hour to about 30 days, or at least about 2 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, or more, or any time in between. In some embodiments, such modulation lasts for 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, or 24 hours, or at least 1, 2, 3, 4, 5, 6, or 7 days, or at least 1, 2, 3, 4, or 5 weeks, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or at least 1, 2, 3, 4, or 5 years (e.g., indefinitely). Optionally, such modulation lasts for 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 year or less.

[0282] In some embodiments, a modulator used in a method of epigenetically modifying a target gene (e.g., FXN) or an expression control sequence operably linked to a target gene comprises an effector moiety that is or includes an epigenetic modification moiety.

[0283] For example, the effector moiety may be or include an epigenetic modification moiety with histone acetyltransferase activity and can modify histones associated with a target gene (e.g., FXN) or an expression control sequence operably linked to the target gene so as to increase their acetylation (e.g., increase the interaction of a transcription factor with the target gene or a portion of the expression control sequence, e.g., thereby increasing transcription of the target gene).

[0284] The following examples are provided to further illustrate some embodiments of the present disclosure, but are not intended to limit the scope of the disclosure; by their illustrative nature, it will be understood that other procedures, methodologies, or techniques well known to those skilled in the art may alternatively be used. [Example]

[0285] Example 1. Increasing FXN Expression Using dCas9-p300 Modulators This example demonstrates the use of a modulator, e.g., a fusion molecule, comprising a targeting moiety with a dCas9 molecule and an effector moiety with p300 to increase FXN expression and aconitase activity in fibroblasts from a FRDA patient.

[0286] As observed by Western blot (Figure 1A) and ELISA (Figure 1B), FXN protein levels are reduced in FRDA patient-derived fibroblasts (GM04078 cells, Coriell Institute) compared with control primary fibroblasts (HDFn). Aconitase activity, which is associated with mitochondrial health, is reduced in FRDA patient-derived cells due to reduced FXN levels, as confirmed by performing an aconitase activity assay (Abcam Kit ab109712) (Figure 1C).

[0287] A modulator containing a fusion molecule of a targeting moiety with a dCas9 molecule and an effector moiety with p300 was prepared together with sgRNA, and FRDA fibroblasts were examined to determine whether the modulator could increase cellular FXN expression or aconitase activity. dCas9-p300 modulators were delivered to FRDA patient-derived fibroblasts in the form of mRNA encoding the modulators, along with a pool of sgRNA guides targeting sequences starting around the TSS (Figure 6). The region targeted by the modulators included approximately 300 bp upstream and 600 bp downstream from the TSS of the FXN gene. Three different pools of sgRNAs were used and co-delivered with the dCas9-p300 modulator-encoding mRNA using lipid nanoparticle (LNP) formulations (see Table 4). Additionally, non-targeting sgRNAs and untreated cells served as controls for these experiments.

[0288] [Table 7]

[0289] Fibroblasts (GM04078 cells) derived from FRDA patients were seeded in α-MEM 15% FBS medium in either 12- or 6-well plates. The following day, 2-6 μg of LNPs in 20-60 μl formulations were added to the cells (mixed either on the plate or in the tube before adding to the cells). After 24 hours, the medium containing LNPs was replaced with fresh medium, and samples were collected 48 and 72 hours after the initial treatment with LNPs.

[0290] RNA was isolated from four independent experiments using the RNAeasy MiniKit (Qiagen) according to the manufacturer's protocol. RNA samples were reverse transcribed to cDNA using the LunaScript RT SuperMix Kit (NEB) and analyzed by quantitative PCR (qPCR) using an FXN-specific Taqman primer / probe set assay with Taqman Fast Advanced Master Mix (Thermo Scientific). FXN expression was quantified relative to the expression of either the HPRT1 or GAPDH reference gene using the ΔΔCt method, with a non-targeting sgRNA sample used as a calibrator. Data showed that delivery of two different sgRNA pools with the dCas9-p300 modulator increased FXN gene expression by up to approximately 4.5-fold compared to the non-targeting sgRNA control (Figures 2A and 2B). The fold increase depended on the sgRNA and the concentration of LNP used. Increased FXN expression was observed 48 and 72 hours after the initial LNP treatment with one sgRNA pool (Figure 3A). The sgRNA pools that induced FXN gene expression using dCas9-p300 modulators were located up to 300 bp upstream and 150 bp downstream from the TSS of the FXN gene. In addition, these modulators also increased FXN expression in normal, unaffected fibroblasts (HDFn, ATCC) (Figures 4A and 4B).

[0291] Protein lysates were obtained using RIPA buffer supplemented with protease inhibitors. Protein was measured using a BCA assay (BioVision). Samples were run on Western blot 4-12% Bis-Tris gels (Thermo Fisher) and blotted with anti-frataxin and anti-β-actin antibodies. Results showed increased FXN protein expression in GM04078 fibroblasts treated with dCas9-p300 modulator and sgRNA against a region 150 bp downstream from the TSS of the FXN gene 72 hours after LNP delivery (Figure 3B).

[0292] Aconitase activity, which is associated with mitochondrial health, is decreased in cells derived from FRDA patients due to reduced FXN levels. We hypothesized that increased FXN expression would result in increased aconitase enzyme activity. Aconitase enzyme activity was measured using Abcam Kit ab109712 in samples collected 72 hours after LNP delivery of dCas9-p300 modulators targeted to the TSS region of the FXN gene. Increased aconitase activity was observed in cells treated with modulators targeted to the region surrounding the TSS of the FXN gene (Figure 5).

[0293] [Table 8]

[0294] Example 2. Changes in FXN expression in WT iPSCs This example demonstrates the use of a modulator, e.g., a fusion molecule, comprising a targeting moiety with a dCas9 molecule and an effector moiety with a VPR to increase FXN expression in induced pluripotent stem cell-derived cardiomyocytes.

[0295] WT iPSC-derived cardiomyocytes (iCardiomyocytes) and WT iPSC-derived glutamatergic cortical neurons (iNeurons) were treated with mRNA encoding a modulator containing a fusion molecule containing dCas9-p300 or dCas9-VPR, which was co-delivered with either a single sgRNA or a pool of three sgRNAs (Pool 1 from Example 1). The region targeted by the sgRNA is approximately 100 bp upstream from the TSS of the FXN gene. RNA was delivered using Lipofectamine MessengerMAX (ThermoFisher) according to the manufacturer's protocol. Additionally, effector-free dCas9, safe harbor (SH) sgRNA, and untreated cells served as controls for these experiments.

[0296] Cardiomyocytes (Fujifilm Cellular Dynamics International [FCDI] cat# R1017) were seeded onto 24-well plates coated with 0.1% gelatin (Stemcell Technologies) according to the manufacturer's protocol and maintained in the recommended medium. Seven days after plating, 0.5 μg of mRNA in the LNP formulation was added to the cells. The medium was changed 24 hours later, and samples were collected at various time points (24–120 hours after treatment with LNP).

[0297] iNeurons (FCDI cat#R1034) were seeded onto 24-well PDL Biocoat plates (Corning) coated with native mouse laminin (ThermoFisher). BrainPhys Biosciences supplemented with N2-A (Stemcell Technologies), iCell Neural Supplement B, and iCell Nervous System Supplement (FCDI). Cells were maintained in Stemcell Technologies medium. Seven days after plating, 0.5 μg of mRNA in the LNP formulation was added to the cells. The medium was changed 24 hours later, and samples were taken at various time points (24–96 hours after treatment with LNP).

[0298] RNA was purified from samples using the RNEasy MiniKit (Qiagen) according to the manufacturer's protocol. RNA samples were reverse transcribed to cDNA using the LunaScript RT SuperMix Kit (NEB) and then analyzed by quantitative PCR (qPCR) using an FXN-specific Taqman assay with Taqman Fast Advanced Master Mix (Thermo Scientific). Expression data were analyzed using the standard curve method. FXN expression was normalized to HPRT1 expression, and fold changes were calculated relative to the SH sgRNA-treated control at each time point. The fold change was calculated by dividing the normalized FXN mRNA amount for each sample by the mean of the SH control group.

[0299] The results in Figure 8 show that delivery of sgRNA Pool 1 with dCas9-VPR modulator-encoding RNA in i-cardiomyocytes upregulated FXN gene expression approximately threefold after 24 hours compared to dCas9-VPR and SH controls. Increased FXN expression was also observed 48 hours after LNP delivery. A single sgRNA, GD-27895, with dCas9-VPR modulator-encoding mRNA only modestly increased FXN expression in i-cardiomyocytes. A modulator containing a fusion molecule with dCas9-p300 also modestly upregulated FXN in WT i-cardiomyocytes by approximately 1.3-fold using Pool 1.

[0300] In glutamatergic i-neurons, sgRNA Pool 1 containing dCas9-VPR modulator-encoding mRNA upregulated FXN gene expression approximately 3-fold compared to SH controls after 24 hours. Increased FXN expression was similarly observed 48 hours after LNP delivery. Single sgRNA GD-27895 with dCas9-VPR modulator-encoding mRNA increased FXN expression approximately 2.5-fold compared to SH controls after 24 hours after LNP delivery. Modulators containing fusion molecules with dCas9-p300 also modestly upregulated FXN in WT glutamatergic i-neurons approximately 1.4-fold using Pool 1 sgRNA and single sgRNA GD-27895 compared to SH controls.

[0301] These results demonstrate that the modulators described herein, particularly those containing dCas9-VPR targeted approximately 100 bp upstream of the TSS, can upregulate FXN gene expression in the cell types most affected by FRDA (cardiomyocytes and neurons).

[0302] Example 3. TAL effector molecules used in modulators to increase FXN expression This example demonstrates the use of a modulator, e.g., a fusion molecule, comprising a targeting moiety with a TAL effector molecule and an effector moiety with a VPR to increase FXN expression in fibroblasts derived from a FRDA patient.

[0303] A modulator containing a fusion molecule with a TAL effector molecule fused to a VPR effector moiety was delivered in the form of mRNA to fibroblasts (GM03816) derived from a FRDA patient. The modulator containing the TAL effector molecule was designed to target approximately 100 bp upstream of the FXN gene TSS. The modulator was formulated into LNPs and then delivered to cells. Additionally, as controls for these experiments, cells treated with a modulator containing dCas9-VPR and Pool 1 sgRNA or dCas9-VPR and safe harbor (SH) sgRNA, as well as untreated cells, were used.

[0304] GM03816 cells were seeded in α-MEM medium supplemented with 15% FBS in 96- and 24-well plates. The next day, 112.5 ng and 22.5 ng of LNP (MC3) formulation per well were added to the cells in the 24- and 96-well plates, respectively. The medium was changed at 24 hours, and samples were collected at 24 and 72 hours after treatment with LNP.

[0305] RNA from samples collected 24 hours later was purified using the RNEasy MiniKit (Qiagen) according to the manufacturer's protocol. RNA samples were reverse transcribed to cDNA using the SuperMix Kit (NEB) and then analyzed by quantitative PCR (qPCR) using an FXN-specific Taqman assay with Taqman Fast Advanced Master Mix (Thermo Scientific). Expression data were analyzed using the standard curve method. FXN expression was normalized to HPRT1 expression, and fold changes were calculated relative to the SH sgRNA-treated control at each time point. Fold changes were calculated by dividing the normalized FXN mRNA amount for each sample by the mean of the SH control group.

[0306] Protein lysates from samples harvested 72 hours later were obtained using RIPA buffer supplemented with protease inhibitors (Roche), and protein concentrations were measured using a BCA Rapid Gold Kit (ThermoFisher). Samples were analyzed using an FXN ELISA kit (Abcam ab176112) according to the manufacturer's protocol.

[0307] Figures 9 and 10 show that the modulator comprising the fusion molecule with TAL-VPR increased FXN gene expression by approximately 6-fold 24 hours and FXN protein by approximately 1.5-fold 72 hours after LNP delivery.

[0308] These results revealed that modulators containing fusion molecules with TAL-based targeting moieties can deliver effector moieties to regions upstream of the TSS and that they can upregulate FXN gene expression.

[0309] Example 4. Changes in FXN expression in mice injected with dCas9-VPR This example demonstrates the use of a modulator, e.g., a fusion molecule, comprising a targeting moiety with dCas9 and an effector moiety with VPR to increase FXN expression when injected into a mouse model organism.

[0310] Wild-type C57BL / 6J mice (N = 8 mice per time point per group) were intravenously (iv) injected with 3 mg / kg of MC3 formulation containing mRNA encoding an exemplary modulator comprising a fusion molecule with dCas9-VPR, along with GD-28633 sgRNA (targeting approximately 80 bp upstream of the Fxn mouse TSS) or SH sgRNA. Additionally, a control group of mice (N = 5) injected iv with PBS was included for comparison. Liver tissue was harvested on days 3, 4, and 5 post-injection and stored in RNA-later for RNA analysis or snap-frozen for protein analysis.

[0311] RNA was purified using the RNEasy MiniKit (Qiagen) according to the manufacturer's protocol. RNA samples were reverse transcribed to cDNA using the LunaScript RT SuperMix Kit (NEB) and then analyzed by quantitative PCR (qPCR) using an Fxn-specific Taqman assay with Taqman Fast Advanced Master Mix (Thermo Scientific). Expression data were analyzed using the standard curve method. FXN expression was normalized to HPRT1 expression, and fold changes were calculated relative to the SH sgRNA-treated control at each time point. Fold changes were calculated by dividing the normalized FXN mRNA amount for each sample by the mean of the SH control group.

[0312] Protein lysates were obtained using RIPA buffer supplemented with protease inhibitors (Roche), and protein concentrations were measured using a BCA Rapid Gold Kit (ThermoFisher). Samples were analyzed using an FXN ELISA kit (Abcam ab199078) according to the manufacturer's protocol.

[0313] The results in Figure 11 showed that intravenous injection of LNPs containing the dCas9-VPR modulator with the GD-28633 sgRNA into mice increased Fxn gene expression in the liver by approximately two-fold compared to the dCas9-VPR modulator and SH control on days 3 and 4 after injection. The results in Figure 12 showed that FXN protein levels were increased by approximately 20% in the group treated with dCas9-VPR with GD-28633 compared to the SH control group on day 4 after LNP injection.

[0314] These results demonstrate that a modulator-containing LNP formulation comprising a fusion molecule harboring a dCas9-VPR with an sgRNA targeted approximately 80 bp upstream of the Fxn mouse TSS can increase Fxn gene and protein levels in surrogate tissue in vivo.

[0315] Example 5. Altered FXN expression in iPSCs derived from FRDA patients This example demonstrates the use of a modulator, e.g., a fusion molecule, comprising a targeting moiety with dCas9 and an effector moiety with either p300 or VPR to increase FXN expression in iPSCs derived from FRDA patients.

[0316] Patient (GM04078, GM03816) and wild-type (GM01717, GM03234) iPSC-derived cardiomyocytes (iCardiomyocytes) were treated with mRNA encoding a regulatory agent containing a fusion molecule containing dCas9-p300 or dCas9-VPR, which was co-delivered with a pool of three sgRNAs (Pool 1). The region targeted by the sgRNAs was approximately 100 bp upstream from the TSS of the FXN gene. RNA was delivered using Lipofectamine MessengerMAX (ThermoFisher) according to the manufacturer's protocol. Additionally, safe harbor (SH) sgRNA and untreated cells served as controls.

[0317] Patient (GM04078, GM03816) and wild-type (GM01717, GM03234) iPSC lines were derived from primary fibroblasts using the Stemcell Technologies ReproRNA OKSGM kit according to the manufacturer's protocol. Immunochemical staining for OCT4, TRA-1-60, SOX2, SSEA4, and NANOG was used to confirm iPSC identity, and STR was used to confirm the parental lines.

[0318] Patient iPSCs were differentiated into cardiomyocytes using the STEMdiff Cardiomyocyte Differentiation and Maintenance Kit (Stemcell Technologies). On day 15 of differentiation, all cells were dissociated and then incubated with the STEMdiff Cardiomyocyte Dissociation Kit. Cardiomyocytes were cryopreserved using the Stemcell Technologies EasySep Human PSC-Derived Cardiomyocyte Enrichment Kit and STEMdiff Cardiomyocyte Freezing Medium (Stemcell Technologies). Patient / WT iPSC-derived cardiomyocytes were thawed and purified using the Stemcell Technologies EasySep Human PSC-Derived Cardiomyocyte Enrichment Kit and plated onto Corning Matrigel-coated 24-well plates. The medium was replaced with fresh Cardiomyocyte Maintenance Media (Stemcell Technologies) every other day. Six days after plating, cells recovered and began beating, at which point they were treated with 0.5 μg of mRNA in the LNP formulation. After 24 hours, the medium was replaced, and samples were collected at 24, 48, and 72 hours after treatment with LNP.

[0319] RNA was purified from samples using the RNEasy MiniKit (Qiagen) according to the manufacturer's protocol. RNA samples were reverse transcribed to cDNA using the LunaScript RT SuperMix Kit (NEB) and then analyzed by quantitative PCR (qPCR) using an FXN-specific Taqman assay with Taqman Fast Advanced Master Mix (Thermo Scientific). Expression data were analyzed using the standard curve method. FXN expression was normalized to HPRT1 or GAPDH expression, and fold changes were calculated relative to the SH sgRNA-treated control at each time point. Fold changes were calculated by dividing the normalized FXN mRNA amount for each sample by the mean of the SH control group.

[0320] The results, shown in Figure 13, demonstrate a 2.5- to 3.5-fold upregulation of FXN gene expression 24 hours after treatment with dCas9-VPR modulator-encoding mRNA and Pool 1 sgRNA in all cell lines that could be assayed. In the control cell line, GM01717, expression returns to baseline by 48 hours. However, approximately 1.5-fold upregulation of FXN is sustained in the patient cell line, GM04078, after 48 hours of treatment, and in the wild-type control line, GM03234, after 48 and 72 hours. These data suggest that modulators containing fusion molecules with dCas9-VPR and Pool 1 sgRNA induce FXN upregulation, and that this upregulation may be more sustainable in some lines.

[0321] The results, shown in Figure 14, demonstrate an approximately 1.5-fold upregulation of FXN gene expression in both patient cell lines, GM04078 and GM03816, following treatment with dCas9-p300 modulator-encoding mRNA and Pool 1 sgRNA. FXN expression was upregulated approximately 1.3-fold in two wild-type lines, GM01717 and GM03234. Based on available data, modulators containing fusion molecules with p300-mediated FXN upregulation appear to remain stable after 72 hours, suggesting that they may have a more sustained effect on FXN gene expression in cardiomyocytes.

[0322] The results, shown in Figure 15, demonstrate that delivery of sgRNA pool 1 together with the dCas9-VPR modulator-encoding mRNA increases FXN protein levels in patient and wild-type cardiomyocytes by 72 hours. sgRNA pool 1 with the dCas9-p300 modulator-encoding mRNA also modestly upregulates FXN protein in patient cell lines.

[0323] These results demonstrate, inter alia, that modulators described herein (e.g., including fusion molecules) containing dCas9-VPR and targeted approximately 100 bp upstream of the TSS can upregulate FXN gene and protein expression in one of the cell types most affected by FRDA (cardiomyocytes).

Claims

[Claim 1] The invention described in this specification.

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