Compositions and methods for modulating the expression of the Forkheadbox P3 (FOXP3) gene.

Abstract

The present invention provides compositions and methods for modulating the expression of the forkheadbox P3 (FOXP3) gene. [Solution] The present invention provides agents and compositions for modulating (enhanced or reduced) the expression of the FOXP3 gene by targeting the forkhead box P3 (FOXP3) expression regulatory region, as well as methods for using them to treat FOXP3-related disorders, such as autoimmune diseases, such as IPEX syndrome. The OXP3 gene may be present in cells, such as mammalian cells, such as mammalian somatic cells, such as human or mouse somatic cells, such as naive T cells.

Description

Technical Field

[0001] Related Applications This application claims the benefit of priority of U.S. Provisional Patent Application No. 62 / 988,044, filed on March 11, 2020, the entire content of which is incorporated herein by reference.

[0002] Sequence Listing This application includes a sequence listing submitted electronically in ASCII format, which is hereby incorporated by reference in its entirety. The ASCII copy created on March 10, 2021, is named 131717-00420_SL.txt and has a size of 1,166,669 bytes.

Background Art

[0003] Background of the Invention A healthy immune system defends the body against diseases and infections. However, when the immune system is dysfunctional, it mistakenly attacks healthy cells, tissues, and organs. These attacks, which characterize autoimmune diseases or disorders, can affect any part of the body, resulting in weakened body functions and even life-threatening conditions. Some of the more common autoimmune diseases include IPEX syndrome (IPEX), type 1 diabetes, multiple sclerosis, systemic lupus erythematosus (SLE), and rheumatoid arthritis (RA).

[0004] Together, these diseases affect over 24 million people in the United States (see Progress in Autoimmune Diseases Research, https: / / www.niaid.nih.gov / sites / default / files / adccfinal.pdf). Additionally, more than 8 million people have autoantibodies, which are blood molecules that indicate a person's chance of developing an autoimmune disease. Autoimmune diseases are becoming more frequently seen.

[0005] ]]Treatment varies depending on the disease, but in most cases, one key goal is to reduce inflammation. Corticosteroids or other medications that suppress the immune response are usually prescribed.

[0006] Regulatory T cells (Tregs) are a specialized subgroup of T cells that act to suppress the immune response, thereby maintaining homeostasis and self-tolerance. Tregs have been shown to inhibit T cell proliferation and cytokine production, and can play a crucial role in preventing or treating autoimmune diseases. Forkhead box P3 (FOXP3) is a master transcription factor that controls the differentiation of naive T cells into regulatory T cells (Tregs), and forced overexpression of FOXP3 has been shown to confer the Treg phenotype to T cells. In vitro generation of Tregs has become an important area of ​​focus in ex vivo therapies targeting autoimmune disorders. However, most strategies for producing Tregs either fail to result in sustained expression of the genes leading to Tregs, or produce Tregs that do not exhibit a repressive phenotype. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Progress in Autoimmune Diseases Research, https: / / www.niaid.nih.gov / sites / default / files / adccfinal.pdf [Overview of the project] [Means for solving the problem]

[0008] Therefore, there is a need in the art for compositions and methods for treating autoimmune diseases such as IPEX syndrome.

[0009] Summary of the Invention The present invention provides agents and compositions for modulating (e.g., enhancing or reducing) the expression of the FOXP3 gene by targeting the forkhead box P3 (FOXP3) expression regulatory region. The FOXP3 gene may be present in cells, for example, mammalian cells, for example, mammalian somatic cells, for example, human or mouse somatic cells, for example, naive T cells. The present invention also provides methods of using the agents and compositions of the present invention to modulate the expression of the FOXP3 gene in subjects who would benefit from modulating the expression of the FOXP3 gene, for example, subjects suffering from or susceptible to FOXP3-related diseases, or to treat such subjects.

[0010] Therefore, in one embodiment, the present invention provides a site-specific FOXP3 disruptor comprising a site-specific FOXP3 targeting moiety that targets the forkheadbox P3 (FOXP3) expression regulatory region.

[0011] In one embodiment, the site-specific FOXP3 targeting moiety includes a polymer molecule. The polymer molecule may include a polyamide, a polynucleotide, a polynucleotide encoding a DNA-binding domain or fragment thereof, or a peptide nucleic acid (PNA) that specifically binds to the FOXP3 expression regulatory region.

[0012] In yet another embodiment, the expression regulatory region includes a region upstream of the FOXP3 transcription start site (TSS).

[0013] In one embodiment, the expression regulatory region includes one or more FOXP3-associated anchor sequences within an anchor sequence-mediated conjunction containing first and second FOXP3-associated anchor sequences.

[0014] In another embodiment, the FOXP3-associated anchor sequence includes a CCCTC-binding factor (CTCF)-binding motif.

[0015] In yet another embodiment, the FOXP3-associated anchor sequence-mediated conjunction includes one or more transcriptional regulatory elements inside the conjunction. In one embodiment, the FOXP3-associated anchor sequence-mediated conjunction includes one or more transcriptional regulatory elements outside the conjunction.

[0016] In one embodiment, the FOXP3 association anchor sequence is located within approximately 500 kb of the transcriptional regulatory element. In another embodiment, the FOXP3 association anchor sequence is located within approximately 300 kb of the transcriptional regulatory element. In yet another embodiment, the anchor sequence is located within 10 kb of the transcriptional regulatory element.

[0017] In another embodiment, the expression regulatory region includes a FOXP3-specific transcriptional regulatory element. In yet another embodiment, the transcriptional regulatory element includes a FOXP3 promoter. In yet another embodiment, the transcriptional regulatory element includes a transcriptional enhancer. In yet another embodiment, the transcriptional regulatory element includes a transcriptional repressor.

[0018] In one embodiment, the site-directed FOXP3 disruptor comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleotide identity with respect to any one of the nucleotide sequences in Table 2.

[0019] In another embodiment, the site-specific foxp3 disruptor comprises a polynucleotide encoding a DNA-binding domain or fragment of a zinc finger polypeptide (ZNF) or a transcription activator-like effector (TALE) polypeptide that specifically binds to the foxp3 expression regulatory region.

[0020] In one embodiment, the DNA-binding domain of the TALE or ZNF polypeptide contains an amino acid sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid identity with respect to any one of the amino acid sequences listed in Table 1B.

[0021] In yet another embodiment, the site-specific FOXP3 disruptor includes nucleotide modifiers, such as deoxy-nucleotides, 3'-terminal deoxythymine (dT) nucleotides, 2'-O-methyl-modified nucleotides, 2'-fluoro-modified nucleotides, 2'-deoxy-modified nucleotides, debasalized nucleotides, nucleotides containing a 5'-phosphorothioate group, nucleotides containing a 5'-methylphosphonate group, nucleotides containing a 3'-phosphorothioate group, or nucleotides containing a 3'-methylphosphonate group.

[0022] In yet another embodiment, the polymer molecule includes peptide nucleic acid (PNA).

[0023] In one embodiment, the present invention provides a vector comprising a site-specific foxp3 disruptor of various embodiments of the above-described embodiment or any other embodiment of the present invention as described in detail herein. In one embodiment, the vector is a viral expression vector.

[0024] In another aspect, the present invention provides cells. These cells provide site-specific foxp3 disruptors or vectors in various embodiments of the above-described embodiments of the present invention or any other embodiments described herein in detail.

[0025] In one embodiment, a site-specific FOXP3 disruptor is present in the composition. In another embodiment, the composition comprises a pharmaceutical composition. In yet another embodiment, the pharmaceutical composition comprises a lipid preparation. In yet another embodiment, the lipid preparation comprises one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids, or one or more PEG-modified lipids, or any combination thereof. In one embodiment, the pharmaceutical composition comprises lipid nanoparticles.

[0026] In another embodiment, the present invention provides a site-specific foxp3 disruptor. The site-specific foxp3 disruptor comprises a nucleic acid molecule encoding a fusion protein, the fusion protein comprising a site-specific foxp3 targeting moiety that targets a foxp3 expression regulatory region and an effector molecule.

[0027] In one embodiment, the site-specific FOXP3 targeting moiety comprises a polynucleotide encoding a DNA-binding domain or fragment of a zinc finger polypeptide (ZNF) or a transcription activator-like effector (TALE) polypeptide that specifically binds to the FOXP3 expression regulatory region.

[0028] In another embodiment, the effector molecule comprises a polypeptide or a nucleic acid molecule encoding a polypeptide. In yet another embodiment, the fusion protein comprises a peptide-nucleic acid fusion.

[0029] In yet another embodiment, the effector is selected from the group consisting of nucleases, physical blockers, epigenetic recruiters, and epigenetic CpG modifiers, as well as any combination thereof.

[0030] In one embodiment, the effector comprises a CRISPR-related protein (Cas) polypeptide, or a nucleic acid molecule encoding a Cas polypeptide. In another embodiment, the Cas polypeptide is an enzymatically inactive Cas polypeptide. In yet another embodiment, the site-directed FOXP3 disruptor further comprises the catalytic domain of human exonuclease 1 (hEXO1).

[0031] In another embodiment, the epigenetic recruiter includes a transcriptional enhancer or transcriptional repressor.

[0032] In one embodiment, the transfer enhancer is VPR(VP64-p65-Rta).

[0033] In one embodiment, VPR is [ka] It contains an amino acid sequence having at least approximately 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid identity with respect to the entire amino acid sequence.

[0034] In one embodiment, the transfer enhancer includes two, three, four, or five VPRs.

[0035] In one embodiment, the transfer enhancer is p300.

[0036] In one embodiment, p300 is [ka] It contains an amino acid sequence that has at least approximately 85%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with respect to the entire amino acid sequence.

[0037] In yet another embodiment, the epigenetic CpG modifier includes a DNA methylase, a DNA demethylase, a histone modifier, a histone transacetylase, or a histone deacetylase.

[0038] In one embodiment, the effector molecule comprises a zinc finger polypeptide. In another embodiment, the effector molecule comprises a transcription activator-like effector nuclease (TALEN) polypeptide.

[0039] In some embodiments, the site-directed FOXP3 disruptor further comprises a second nucleic acid molecule encoding a second fusion protein, the second fusion comprising a second site-directed FOXP3 targeting moiety targeting a second FOXP3 expression regulatory region and a second effector molecule, the second FOXP3 expression regulatory region being distinct from the FOXP3 expression regulatory region.

[0040] In one embodiment, the second effector is different from the first effector.

[0041] In one embodiment, the second effector is the same as the first effector.

[0042] In one embodiment, the fusion protein and the second fusion protein are operably linked.

[0043] In one embodiment, the fusion protein and the second fusion protein contain an amino acid sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the entire amino acid sequence of a polypeptide selected from the group consisting of dCas9-P300 and dCas9-VPR.

[0044] In one embodiment, the fusion protein is encoded by a polynucleotide containing a nucleotide sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleotide sequence identity with respect to the entire nucleotide sequence of a polynucleotide selected from the group consisting of dCas9-P300 mRNA and dCas9-VPR mRNA.

[0045] In one embodiment, the present invention provides a site-specific FOXP3 disruptor. The disruptor comprises a nucleic acid molecule encoding a fusion protein, the fusion protein comprising an amino acid sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid identity with respect to the entire amino acid sequence of a polypeptide selected from the group consisting of dCas9-P300 and dCas9-VPR.

[0046] In one embodiment, the present invention provides a site-specific foxp3 disruptor. The site-specific foxp3 disruptor is [ka] [ka] It contains a polynucleotide encoding the amino acid sequence of dCas-P300, which includes the amino acid sequence of [the specified amino acid sequence].

[0047] In another aspect, the present invention provides a site-specific foxp3 disruptor. The site-specific foxp3 disruptor is [ka] [ka] It contains a polynucleotide encoding the amino acid sequence of dCas-VPR, which includes the amino acid sequence of [the specified amino acid sequence].

[0048] In one embodiment, the present invention provides a vector. The vector comprises a nucleic acid molecule encoding a site-specific FOXP3 disruptor of various embodiments of the above embodiments or any other embodiments of the present invention described in detail herein. In one embodiment, the vector is a viral expression vector.

[0049] In another aspect, the present invention provides cells comprising site-specific foxp3 disruptors or vectors of various embodiments of the above-described embodiments or any other embodiments of the present invention as described herein.

[0050] In one embodiment, a site-specific FOXP3 disruptor is present in the composition. In another embodiment, the composition comprises a pharmaceutical composition. In yet another embodiment, the pharmaceutical composition comprises a lipid preparation. In yet another embodiment, the lipid preparation comprises one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids, or one or more PEG-modified lipids, or any combination thereof. In one embodiment, the pharmaceutical composition comprises lipid nanoparticles.

[0051] In one embodiment, the present invention provides a method for modulating the expression of forkheadbox P3 (FOXP3) in cells. The method comprises the step of contacting cells with a site-specific FOXP3 disruptor comprising a site-specific FOXP3 targeting moiety that targets a FOXP3 expression regulatory region and an effector molecule, thereby modulating the expression of FOXP3 in the cells.

[0052] In one embodiment, expression modulation is enhanced expression of FOXP3 in cells. In another embodiment, expression modulation is reduced expression of FOXP3 in cells. In yet another embodiment, the site-specific FOXP3 targeting moiety comprises a polymer molecule. In yet another embodiment, the polymer molecule comprises a polyamide. In one embodiment, the polymer molecule comprises a polynucleotide.

[0053] In another embodiment, the expression regulatory region includes a region upstream of the FOXP3 transcription start site (TSS).

[0054] In yet another embodiment, the expression regulatory region includes one or more FOXP3-associated anchor sequences within an anchor sequence-mediated conjunction containing first and second FOXP3-associated anchor sequences. In yet another embodiment, the FOXP3-associated anchor sequence includes a CCCTC-binding factor (CTCF)-binding motif.

[0055] In one embodiment, the FOXP3-associated anchor sequence-mediated conjunction includes one or more transcriptional regulatory elements inside the conjunction. In another embodiment, the FOXP3-associated anchor sequence-mediated conjunction includes one or more transcriptional regulatory elements outside the conjunction.

[0056] In another embodiment, the anchor array is located within approximately 500 kb of the transcriptional control element. In yet another embodiment, the anchor array is located within approximately 300 kb of the transcriptional control element. In yet another embodiment, the anchor array is located within 10 kb of the transcriptional control element.

[0057] In one embodiment, the expression regulatory region includes a FOXP3-specific transcription element. In another embodiment, the transcription element includes a FOXP3 promoter. In yet another embodiment, the transcription regulatory element includes a transcription enhancer. In yet another embodiment, the transcription regulatory element includes a transcription repressor.

[0058] In another embodiment, the site-directed FOXP3 disruptor comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleotide identity with respect to any of the total nucleotide sequences of the nucleotide sequences in Table 2.

[0059] In one embodiment, the site-specific foxp3 disruptor comprises a polynucleotide encoding the DNA-binding domain or a fragment of a zinc finger polypeptide (ZNF) or a transcription activator-like effector (TALE) polypeptide that specifically binds to the foxp3 expression regulatory region.

[0060] In some embodiments, the DNA-binding domain of TALE or ZNF includes an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid identity with respect to the entire amino acid sequence of an amino acid sequence selected from the amino acid sequences listed in Table 1B.

[0061] In another embodiment, the site-directed FOXP3 disruptor includes nucleotide modifications.

[0062] In yet another embodiment, the polymer molecule includes peptide nucleic acid (PNA).

[0063] In one embodiment, the effector molecule comprises a polypeptide. In another embodiment, the polypeptide constitutes a fusion protein comprising a site-specific FOXP3 targeting moiety that targets a FOXP3 expression regulatory region and the effector molecule. In yet another embodiment, the fusion protein comprises a peptide-nucleic acid fusion molecule.

[0064] In another embodiment, the effector is selected from the group consisting of nucleases, physical blockers, epigenetic recruiters, and epigenetic CpG modifiers, as well as any combination thereof. In yet another embodiment, the effector comprises a CRISPR-related protein (Cas) polypeptide, or a nucleic acid molecule encoding a Cas polypeptide. In yet another embodiment, the Cas polypeptide is an enzymatically inactive Cas polypeptide. In one embodiment, the effector further comprises the catalytic domain of human exonuclease 1 (hEXO1).

[0065] In one embodiment, the epigenetic recruiting factor includes a transcriptional enhancer or a transcriptional repressor.

[0066] In some embodiments, the transcription enhancer is a VPR.

[0067] In some embodiments, VPR is [ka] It contains an amino acid sequence having at least approximately 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid identity with respect to the entire amino acid sequence.

[0068] In some embodiments, the transcription enhancer includes two, three, four, or five VPRs.

[0069] In some embodiments, the transfer enhancer is p300.

[0070] In some embodiments, p300 is [ka] It has an amino acid sequence that has at least approximately 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid identity with respect to the entire amino acid sequence.

[0071] In another embodiment, the epigenetic CpG modifier includes a DNA methylase, a DNA demethylase, a histone modifier, a histone transacetylase, or a histone deacetylase.

[0072] In yet another embodiment, the effector molecule includes a zinc finger polypeptide.

[0073] In yet another embodiment, the effector molecule comprises a transcription activator-like effector nuclease (TALEN) polypeptide.

[0074] In one embodiment, the fusion protein comprises an enzymatically inactive Cas polypeptide and an epigenetic recruiting factor polypeptide.

[0075] In another embodiment, the fusion protein comprises an enzymatic Cas polypeptide and an epigenetic CpG modifier polypeptide.

[0076] In some embodiments, the site-directed FOXP3 disruptor further comprises a second nucleic acid molecule encoding a second fusion protein, the second fusion comprising a second site-directed FOXP3 targeting moiety targeting a second FOXP3 expression regulatory region and a second effector molecule, the second FOXP3 expression regulatory region being distinct from the FOXP3 expression regulatory region.

[0077] In one embodiment, the second effector is different from the first effector.

[0078] In one embodiment, the second effector is the same as the first effector.

[0079] In one embodiment, the fusion protein and the second fusion protein are operably linked.

[0080] In one embodiment, the fusion protein and the second fusion protein contain an amino acid sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the entire amino acid sequence of a polypeptide selected from the group consisting of dCas9-P300 and dCas9-VPR.

[0081] In one embodiment, the fusion protein is encoded by a polynucleotide containing a nucleotide sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleotide sequence identity with respect to the entire nucleotide sequence of a polynucleotide selected from the group consisting of dCas9-P300 mRNA and dCas9-VPR mRNA.

[0082] In one embodiment, the present invention provides a site-specific FOXP3 disruptor. The disruptor comprises a nucleic acid molecule encoding a fusion protein, the fusion protein comprising an amino acid sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid identity with respect to the entire amino acid sequence of a polypeptide selected from the group consisting of dCas9-P300 and dCas9-VPR.

[0083] In one embodiment, a site-specific disruptor, an effector, or both a site-specific disruptor and an effector are present within the vector. In another embodiment, the site-specific disruptor and the effector are present within the same vector. In yet another embodiment, the site-specific disruptor and the effector are present within different vectors. In yet another embodiment, the vector is a viral expression vector.

[0084] In one embodiment, a site-specific disruptor, an effector, or both a site-specific disruptor and an effector are present in the composition. In another embodiment, the site-specific disruptor and the effector are present in the same composition. In yet another embodiment, the site-specific disruptor and the effector are present in different compositions. In yet another embodiment, the composition comprises a pharmaceutical composition. In one embodiment, the pharmaceutical composition comprises a lipid formulation. In another embodiment, the lipid formulation comprises one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids, or one or more PEG-modified lipids, or any combination thereof. In yet another embodiment, the pharmaceutical composition comprises lipid nanoparticles.

[0085] In another embodiment, the cells are mammalian cells. In yet another embodiment, the mammalian cells are somatic cells. In yet another embodiment, the mammalian cells are primary cells.

[0086] In one embodiment, the contact step is performed in vitro. In another embodiment, the contact step is performed in vivo. In yet another embodiment, the contact step is performed ex vivo.

[0087] In one embodiment, the method further includes the step of administering a target to cells.

[0088] In another embodiment, the cells are located within the object.

[0089] In yet another embodiment, the subjects have a foxp3-related disease. In yet another embodiment, the foxp3-related disease is selected from the group consisting of IPEX syndrome (IPEX), type 1 diabetes, multiple sclerosis, systemic lupus erythematosus (SLE), and rheumatoid arthritis (RA).

[0090] In one embodiment, the present invention provides a method for treating a subject having a foxp3-related disorder. The method comprises administering to the subject a therapeutically effective amount of a site-specific foxp3 disruptor containing a site-specific foxp3 targeting moiety that targets a foxp3 expression regulatory region, and an effector molecule, thereby treating the subject. In one embodiment, the foxp3-related disorder is IPEX syndrome, and the site-specific foxp3 disruptor increases foxp3 expression in the subject. In another embodiment, the site-specific foxp3 disruptor and the effector molecule are administered to the subject simultaneously. In yet another embodiment, the site-specific foxp3 disruptor and the effector molecule are administered to the subject sequentially. In one embodiment, the effector molecule is administered to the subject before administration of the site-specific foxp3 disruptor. In another embodiment, the site-specific foxp3 disruptor is administered to the subject before administration of the effector molecule.

[0091] In various embodiments of the above-described aspects of the present invention or any other aspects described herein, the cells are immune cells. In one embodiment, the immune cells are naive T cells or regulatory T cells (Treg). In another embodiment,

[0092] In one embodiment, the site-directed FOXP3 disruptor of the present invention comprises a first nucleotide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleotide identity with respect to the entire nucleotide sequence of GD-28448; a second nucleotide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleotide identity with respect to the entire nucleotide sequence of GD-28449; and a third nucleotide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleotide identity with respect to the entire nucleotide sequence of GD-28450. The present invention provides, for example, the following items: (Item 1) A site-specific FOXP3 disruptor containing a site-specific FOXP3 targeting moiety that targets the forkheadbox P3 (FOXP3) expression regulatory region. (Item 2) The site-specific FOXP3 disruptor according to item 1, wherein the site-specific FOXP3 targeting portion comprises a polymer molecule. (Item 3) The aforementioned polymer molecule comprises a polyamide, and is a site-specific FOXP3 disruptor as described in item 2. (Item 4) The aforementioned polymer molecule comprises a polynucleotide, and is a site-specific FOXP3 disruptor as described in item 2. (Item 5) The site-specific foxp3 disruptor described in item 1, wherein the expression regulatory region includes a region upstream of the foxp3 transcription start site (TSS). (Item 6) The site-specific foxp3 disruptor according to item 1, wherein the expression control region comprises one or more foxp3-associated anchor sequences in an anchor sequence-mediated conjunction containing first and second foxp3-associated anchor sequences. (Item 7) The site-specific foxp3 disruptor described in item 6, wherein the anchor sequence contains a CCCTC binding factor (CTCF) binding motif. (Item 8) A site-specific foxp3 disruptor according to item 6 or 7, wherein the anchor sequence-mediated conjunction contains one or more transcriptional regulatory elements within the conjunction. (Item 9) A site-specific foxp3 disruptor according to item 6 or 7, wherein the anchor sequence-mediated conjunction includes one or more transcriptional regulatory elements outside the conjunction. (Item 10) A site-specific foxp3 disruptor according to any one of items 6 to 9, wherein the first and / or second anchor sequence is located within approximately 500 kb of the transcriptional regulatory element. (Item 11) A site-specific foxp3 disruptor according to item 10, wherein the first and / or second anchor sequence is located within approximately 300 kb of the transcriptional regulatory element. (Item 12) A site-specific foxp3 disruptor according to item 11, wherein the first and / or second anchor sequence is located within 10 kb of the transcriptional regulatory element. (Item 13) The site-specific foxp3 disruptor according to item 1, wherein the expression regulatory region includes a foxp3-specific transcriptional regulatory element. (Item 14) The site-specific FOXP3 disruptor according to item 13, wherein the transcriptional regulatory element comprises a FOXP3 promoter. (Item 15) The site-specific foxp3 disruptor according to item 13, wherein the transcriptional regulatory element includes a transcriptional enhancer. (Item 16) The site-specific foxp3 disruptor described in item 13, wherein the transcriptional regulatory element includes a transcriptional repressor. (Item 17) A site-directed foxp3 disruptor according to any one of items 1 to 16, comprising a nucleotide sequence having at least 85% nucleotide identity with respect to the total nucleotide sequence of any one of the nucleotide sequences in Table 2. (Item 18) A site-directed foxp3 disruptor as described in item 17, comprising a first nucleotide sequence having at least 85% nucleotide identity with respect to the entire nucleotide sequence of GD-28448, a second nucleotide sequence having at least 85% nucleotide identity with respect to the entire nucleotide sequence of GD-28449, and a third nucleotide sequence having at least 85% nucleotide identity with respect to the entire nucleotide sequence of GD-28450. (Item 19) The site-directed FOXP3 disruptor according to item 2, wherein the polymer molecule comprises a polynucleotide encoding a DNA-binding domain or fragment of a zinc finger polypeptide (ZNF) or a transcription activator-like effector (TALE) polypeptide that specifically binds to the FOXP3 expression regulatory region. (Item 20) A site-directed FOXP3 disruptor according to item 19, wherein the DNA-binding domain of the TALE or ZNF polypeptide contains an amino acid sequence having at least about 85% amino acid identity with respect to the total amino acid sequence of any one of the amino acid sequences listed in Table 1B. (Item 21) A site-directed foxp3 disruptor as described in any one of items 1 to 20, including nucleotide modifications. (Item 22) The polymer molecule comprises peptide nucleic acid (PNA), and is a site-specific foxp3 disruptor as described in item 2. (Item 23) A vector comprising a site-specific foxp3 disruptor as described in any one of items 1 through 22. (Item 24) A viral expression vector, as described in item 23. (Item 25) Cells containing a site-specific foxp3 disruptor as described in any one of items 1 through 22, or a vector as described in item 23 or 24. (Item 26) These are immune cells, as described in item 25. (Item 27) The cells described in item 26, wherein the immune cells are naive T cells or regulatory T cells (Treg). (Item 28) A site-specific foxp3 disruptor present in the composition, as described in any one of items 1 to 22. (Item 29) The site-specific foxp3 disruptor described in item 28, wherein the composition comprises a pharmaceutical composition. (Item 30) The aforementioned pharmaceutical composition comprises a lipid preparation, and is a site-specific foxp3 disruptor as described in item 29. (Item 31) The site-directed foxp3 disruptor described in item 30, wherein the lipid preparation comprises one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids, or one or more PEG-modified lipids, or any combination thereof. (Item 32) The aforementioned pharmaceutical composition comprises lipid nanoparticles, and is a site-specific foxp3 disruptor as described in item 30. (Item 33) A site-specific FOXP3 disruptor comprising a nucleic acid encoding a fusion protein, wherein the fusion protein comprises a site-specific FOXP3 targeting moiety that targets a FOXP3 expression regulatory region and an effector molecule. (Item 34) The site-specific FOXP3 disruptor according to item 30, wherein the site-specific FOXP3 targeting portion comprises a polynucleotide encoding a DNA-binding domain or fragment thereof of a zinc finger polypeptide (ZNF) or a transcription activator-like effector (TALE) polypeptide that specifically binds to the FOXP3 expression regulatory region. (Item 35) A site-directed FOXP3 disruptor according to item 34, wherein the DNA-binding domain of the TALE or zinc finger polypeptide contains an amino acid sequence having at least 85% amino acid identity with respect to the entire amino acid sequence of an amino acid sequence selected from the amino acid sequences listed in Table 1B. (Item 36) The site-directed foxp3 disruptor described in item 33, wherein the effector molecule comprises a nucleic acid molecule encoding a polypeptide. (Item 37) The aforementioned fusion protein comprises a peptide-nucleic acid fusion, and is a site-directed FOXP3 disruptor as described in item 33. (Item 38) A site-specific foxp3 disruptor according to any one of items 33 to 37, wherein the effector is selected from the group consisting of nucleases, physical blockers, epigenetic recruiting factors, and epigenetic CpG modifiers, as well as any combination thereof. (Item 39) The site-directed foxp3 disruptor according to item 38, wherein the effector comprises a CRISPR-related protein (Cas) polypeptide or a nucleic acid molecule encoding the Cas polypeptide. (Item 40) The site-specific foxp3 disruptor described in item 39, wherein the Cas polypeptide is an enzymatically inactive Cas polypeptide. (Item 41) A site-specific foxp3 disruptor as described in item 39, further comprising the catalytic domain of human exonuclease 1 (hEXO1). (Item 42) The site-specific foxp3 disruptor described in item 38, wherein the epigenetic mobilization factor includes a transcriptional enhancer or a transcriptional repressor. (Item 43) A site-specific foxp3 disruptor as described in item 42, wherein the transcription enhancer is VPR(VP64-p65-Rta). (Item 44) The aforementioned VPR is [ka] A site-directed FOXP3 disruptor as described in item 43, comprising an amino acid sequence having at least approximately 85% amino acid identity with respect to the entire amino acid sequence of [the specified amino acid sequence]. (Item 45) A site-specific foxp3 disruptor according to item 43 or 44, wherein the transcription enhancer comprises two, three, four, or five VPRs. (Item 46) A site-specific foxp3 disruptor as described in item 42, wherein the transcription enhancer is p300. (Item 47) The aforementioned p300 is, [ka] [ka] A site-directed foxp3 disruptor as described in item 46, comprising an amino acid sequence having at least approximately 85% identity with the entire amino acid sequence of [the specified amino acid sequence]. (Item 48) The site-directed foxp3 disruptor described in item 38, wherein the epigenetic CpG modifying factor comprises a DNA methylase, a DNA demethylase, a histone modifier, a histone transacetylase, or a histone deacetylase. (Item 49) A site-directed foxp3 disruptor according to any one of items 33 to 38, wherein the effector molecule comprises a zinc finger polypeptide. (Item 50) A site-specific foxp3 disruptor according to any one of items 33 to 38, wherein the effector molecule comprises a transcription activator-like effector nuclease (TALEN) polypeptide. (Item 51) A site-directed FOXP3 disruptor according to any one of items 33 to 50, further comprising a second nucleic acid molecule encoding a second fusion protein, wherein the second fusion comprises a second site-directed FOXP3 targeting moiety targeting a second FOXP3 expression regulatory region and a second effector molecule, wherein the second FOXP3 expression regulatory region is different from the FOXP3 expression regulatory region. (Item 52) The second effector is a site-specific foxp3 disruptor as described in item 51, which is different from the first effector. (Item 53) A site-specific foxp3 disruptor as described in item 51, wherein the second effector is the same as the first effector. (Item 54) A site-directed foxp3 disruptor according to any one of items 51 to 53, wherein the fusion protein and the second fusion protein are operably linked. (Item 55) A site-directed FOXP3 disruptor according to item 52, wherein the fusion protein and the second fusion protein contain amino acid sequences having at least about 85% amino acid sequence identity with respect to the entire amino acid sequence of a polypeptide selected from the group consisting of dCas-P300 (SEQ ID NO: 10) and dCas-VPR (SEQ ID NO: 11). (Item 56) The site-directed FOXP3 disruptor described in item 52, wherein the fusion protein is encoded by a polynucleotide containing a nucleotide sequence having at least approximately 85% amino acid sequence identity with respect to the entire nucleotide sequence of a polynucleotide selected from the group consisting of dCas-P300 mRNA (SEQ ID NO: 7) and dCas-VPR mRNA (SEQ ID NO: 8). (Item 57) A site-specific FOXP3 disruptor comprising a nucleic acid molecule encoding a fusion protein, wherein the fusion protein comprises an amino acid sequence having at least about 85% amino acid identity with the total amino acid sequence of a polypeptide selected from the group consisting of dCas-P300 (SEQ ID NO: 10) and dCas-VPR (SEQ ID NO: 11). (Item 58) [ka] [ka] A site-directed FOXP3 disruptor containing a polynucleotide encoding the amino acid sequence of dCas-P300, which includes the amino acid sequence of [specific amino acid sequence]. (Item 59) [ka] [ka] A site-specific foxp3 disruptor containing a polynucleotide encoding the amino acid sequence of dCas-VPR, which includes the amino acid sequence of [specific amino acid sequence]. (Item 60) A vector comprising a nucleic acid molecule encoding a site-specific foxp3 disruptor as described in any one of items 33 to 59. (Item 61) A viral expression vector, as described in item 60. (Item 62) Cells containing a site-specific foxp3 disruptor as described in any one of items 33 to 59, or a vector as described in item 60 or 61. (Item 63) These are immune cells, as described in item 62. (Item 64) The cells described in item 63, wherein the immune cells are naive T cells or regulatory T cells (Treg). (Item 65) A site-specific foxp3 disruptor present in a composition, as described in any one of items 33 to 59. (Item 66) The site-specific foxp3 disruptor described in item 65, wherein the composition comprises a pharmaceutical composition. (Item 67) The aforementioned pharmaceutical composition comprises a lipid preparation, and is a site-specific foxp3 disruptor as described in item 66. (Item 68) The site-directed foxp3 disruptor described in item 67, wherein the lipid preparation comprises one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids, or one or more PEG-modified lipids, or any combination thereof. (Item 69) The aforementioned pharmaceutical composition comprises lipid nanoparticles, and is a site-specific foxp3 disruptor as described in item 66. (Item 70) A method for modulating the expression of forkheadbox P3 (FOXP3) in cells, comprising the step of contacting the cells with a site-specific FOXP3 disruptor comprising a site-specific FOXP3 targeting moiety that targets a FOXP3 expression regulatory region and an effector molecule, thereby modulating the expression of FOXP3 in the cells. (Item 71) The method according to item 70, wherein the modulation of expression is enhanced expression of FOXP3 in the cells. (Item 72) The method according to item 70, wherein the modulation of expression is reduced expression of FOXP3 in the cells. (Item 73) The method according to item 70, wherein the site-specific FOXP3 targeting moiety comprises a polymer molecule. (Item 74) The method according to item 73, wherein the polymer molecule includes a polyamide. (Item 75) The method according to item 73, wherein the polymer molecule comprises a polynucleotide. (Item 76) The method according to item 70, wherein the expression regulatory region includes a region upstream of the FOXP3 transcription start site (TSS). (Item 77) The method according to item 70, wherein the expression regulatory region comprises one or more FOXP3-associated anchor sequences in an anchor sequence-mediated conjunction comprising first and second FOXP3-associated anchor sequences. (Item 78) The method according to item 77, wherein the anchor sequence includes a CCCTC binding factor (CTCF) binding motif. (Item 79) The method according to item 77, wherein the anchor sequence-intervening conjunction includes one or more transcriptional control elements within the conjunction. (Item 80) The method according to item 77, wherein the anchor sequence-intervening conjunction includes one or more transcriptional regulatory elements outside the conjunction. (Item 81) The method according to any one of items 77 to 80, wherein the anchor array is located within approximately 500 kb of the transcription control element. (Item 82) The method according to item 81, wherein the anchor array is located within approximately 300 kb of the transcription control element. (Item 83) The method according to item 82, wherein the anchor array is located within 10 kb of the transcription control element. (Item 84) The method according to item 70, wherein the expression regulatory region includes a FOXP3-specific transcriptional regulatory element. (Item 85) The method according to item 84, wherein the transcriptional regulatory element comprises a FOXP3 promoter. (Item 86) The method according to item 84, wherein the transcriptional control element includes a transcriptional enhancer. (Item 87) The method according to item 84, wherein the transcriptional control element includes a transcriptional repressor. (Item 88) The method according to any one of items 70 to 87, wherein the site-specific foxp3 disruptor comprises a nucleotide sequence having at least 85% nucleotide identity with respect to the total nucleotide sequence of any one of the nucleotide sequences in Table 2. (Item 89) The method according to item 70, wherein the site-specific foxp3 disruptor comprises a polynucleotide encoding a DNA-binding domain or fragment thereof of a zinc finger polypeptide (ZNF) or a transcription activator-like effector (TALE) polypeptide that specifically binds to the foxp3 expression regulatory region. (Item 90) The method according to item 89, wherein the DNA-binding domain of the TALE or ZNF includes an amino acid sequence having at least 85% amino acid identity with respect to the entire amino acid sequence of an amino acid sequence selected from the amino acid sequences listed in Table 1. (Item 91) The method according to any one of items 70 to 90, wherein the site-specific foxp3 disruptor includes nucleotide modifications. (Item 92) The method according to item 70, wherein the polymer molecule comprises peptide nucleic acid (PNA). (Item 93) The method according to item 70, wherein the effector molecule comprises a polypeptide. (Item 94) The method according to item 93, wherein the polypeptide constitutes a fusion protein comprising the site-specific FOXP3 targeting moiety that targets a FOXP3 expression regulatory region and the effector molecule. (Item 95) The method according to item 94, wherein the fusion protein comprises a peptide-nucleic acid fusion molecule. (Item 96) The method according to item 94, wherein the effector is selected from the group consisting of nucleases, physical blockers, epigenetic recruiting factors, and epigenetic CpG modifiers, as well as any combination thereof. (Item 97) The method according to item 96, wherein the effector comprises a CRISPR-related protein (Cas) polypeptide or a nucleic acid molecule encoding the Cas polypeptide. (Item 98) The method according to item 97, wherein the Cas polypeptide is an enzymatically inactive Cas polypeptide. (Item 99) The method according to item 97, wherein the effector further comprises the catalytically active domain of human exonuclease 1 (hEXO1). (Item 100) The method according to item 96, wherein the epigenetic recruiting factor includes a transcriptional enhancer or a transcriptional repressor. (Item 101) The method according to item 100, wherein the transcription enhancer is VPR. (Item 102) The aforementioned VPR is [ka] The method according to item 101, comprising an amino acid sequence having at least approximately 85% amino acid identity with respect to the entire amino acid sequence of [the specified amino acid sequence]. (Item 103) The method according to item 101 or 102, wherein the transcription enhancer comprises two, three, four, or five VPRs. (Item 104) The method according to item 100, wherein the transcription enhancer is p300. (Item 105) The aforementioned p300 is, [ka] [ka] The method according to item 104, having an amino acid sequence that has at least about 85% amino acid identity with respect to the entire amino acid sequence. (Item 106) The method according to item 96, wherein the epigenetic CpG modifier comprises a DNA methylase, a DNA demethylase, a histone modifier, a histone transacetylase, or a histone deacetylase. (Item 107) The method according to item 94, wherein the effector molecule comprises a zinc finger polypeptide. (Item 108) The method according to item 94, wherein the effector molecule comprises a transcription activator-like effector nuclease (TALEN) polypeptide. (Item 109) The method according to item 94, wherein the fusion protein comprises an enzymatically inactive Cas polypeptide and an epigenetic recruiting factor polypeptide. (Item 110) The method according to item 94, wherein the fusion protein comprises an enzymatic Cas polypeptide and an epigenetic CpG modifying factor polypeptide. (Item 111) The method according to any one of items 70 to 110, wherein the site-specific foxp3 disruptor comprises a second nucleic acid molecule encoding a second fusion protein, the second fusion protein comprising a second site-specific foxp3 targeting moiety targeting a second foxp3 expression regulatory region and a second effector molecule, and the second foxp3 expression regulatory region is different from the foxp3 expression regulatory region. (Item 112) The method according to item 111, wherein the second effect pedal is different from the first effect pedal. (Item 113) The method according to item 111, wherein the second effector is the same as the first effector. (Item 114) The method according to any one of items 111 to 113, wherein the fusion protein and the second fusion protein are operably linked. (Item 115) The method according to item 111, wherein the fusion protein and the second fusion protein contain amino acid sequences having at least about 85% sequence identity with respect to the entire amino acid sequence of a polypeptide selected from the group consisting of dCas-P300 (SEQ ID NO: 10) and dCas-VPR (SEQ ID NO: 11). (Item 116) The method according to any one of items 111 to 115, wherein the administration of the site-specific foxp3 disruptor and the second site-specific foxp3 disruptor has a synergistic effect in modulating foxp3 expression. (Item 117) The method according to any one of items 70 to 116, wherein the site-specific disruptor, the effector, or both the site-specific disruptor and the effector are present in the vector. (Item 118) The method according to item 117, wherein the site-specific disruptor and the effector are present in the same vector. (Item 119) The method according to item 117, wherein the site-specific disruptor and the effector are present in different vectors. (Item 120) The method according to any one of items 117 to 119, wherein the vector is a virus expression vector. (Item 121) The method according to any one of items 70 to 120, wherein the site-specific disruptor, the effector, or both the site-specific disruptor and the effector are present in the composition. (Item 122) The method according to item 121, wherein the site-specific disruptor and the effector are present in the same composition. (Item 123) The method according to item 121, wherein the site-specific disruptor and the effector are present in different compositions. (Item 124) The method according to any one of items 121 to 123, wherein the composition comprises a pharmaceutical composition. (Item 125) The method according to item 124, wherein the pharmaceutical composition comprises a lipid preparation. (Item 126) The method according to item 125, wherein the lipid preparation comprises one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids, or one or more PEG-modified lipids, or any combination thereof. (Item 127) The method according to item 124, wherein the pharmaceutical composition comprises lipid nanoparticles. (Item 128) The method according to item 70, wherein the cells are mammalian cells. (Item 129) The method according to item 128, wherein the mammalian cells are somatic cells. (Item 130) The method described in item 128, wherein the mammalian cells are primary cells. (Item 131) The method according to item 70, wherein the cells are immune cells. (Item 132) The cells described in item 131, wherein the immune cells are naive T cells or regulatory T cells (Treg). (Item 133) The method according to item 70, wherein the contact step is performed in vitro. (Item 134) The method according to item 70, wherein the contact step is performed in vivo. (Item 135) The method according to item 70, wherein the contact step is performed ex vivo. (Item 136) The method according to item 135, further comprising the step of administering the aforementioned cells. (Item 137) The method according to item 70, wherein the aforementioned cells are within the target. (Item 138) The method according to item 136 or 137, wherein the subject has a foxp3-related disease. (Item 139) The method according to item 138, wherein the FOXP3-related disease is selected from the group consisting of IPEX syndrome (IPEX), type 1 diabetes, multiple sclerosis, systemic lupus erythematosus (SLE), and rheumatoid arthritis (RA). (Item 140) A method for treating a subject with a FOXP3-related disease, wherein the subject A therapeutically effective dose of a site-specific FOXP3 disruptor containing a site-specific FOXP3 targeting moiety that targets the FOXP3 expression regulatory region, Effector molecules and A method comprising the step of administering a substance, thereby treating the subject. (Item 141) The method according to item 140, wherein the FOXP3-related disorder is IPEX syndrome, and the site-specific FOXP3 disruptor increases the expression of FOXP3 in the subject. (Item 142) The method according to item 140 or 141, wherein the site-specific foxp3 disruptor and the effector molecule are administered simultaneously to the subject. (Item 143) The method according to item 140 or 141, wherein the site-specific foxp3 disruptor and the effector molecule are administered sequentially to the subject. (Item 144) The method according to item 143, wherein the effector molecule is administered to the subject before the administration of the site-specific foxp3 disruptor. (Item 145) The method according to item 143, wherein the site-specific foxp3 disruptor is administered to the subject before the administration of the effector molecule. [Brief explanation of the drawing]

[0093] [Figure 1] Figures 1A and 1B are graphs illustrating the activation of FOXP3 expression after contacting Jurcut cells with a pool showing site-specific FOXP3 targeting regions, as well as with effector molecules including dCas, dCas9, and p300, or dCas9 and VPR.

[0094] Figure 1A shows the quantification of FOXP3 mRNA levels by qPCR 48 hours after transfection with either the dCas9+sgRNA pool (1, 2, or 3), the dCas9-p300+sgRNA pool (1, 2, or 3), or the dCas9-VPR+sgRNA pool (1, 2, or 3).

[0095] Figure 1B shows the quantitative results of FACS experiments determining the percentage of Jurcut cells that were FOXP3-positive 72 hours after transfection. All transfections were performed using Lipofectamine MessengerMax reagent (Thermofisher) according to the manufacturer's instructions.

[0096] [Figure 2] Figure 2 is a graph illustrating the activation of FOXP3 expression in Jurcut cells after contact with a pool of site-specific FOXP3 targeting regions, as well as effector molecules containing dCas9 and p300, or dCas9 and VPR. Only the combination of sgRNA pool 2 and dCas9+VPR showed significantly higher FOXP3 activation at both the mRNA and protein levels.

[0097] [Figure 3A] Figures 3A and 3B are graphs illustrating the activation of naive T cells after contact with a pool of cells showing site-specific FOXP3 targeting regions, as well as with effector molecules including dCas9, dCas9 and p300, or dCas9 and VPR. [Figure 3B] Figures 3A and 3B are graphs illustrating the activation of naive T cells after contact with a pool of cells showing site-specific FOXP3 targeting regions, as well as with effector molecules including dCas9, dCas9 and p300, or dCas9 and VPR.

[0098] Figure 3A shows the quantification of FOXP3 mRNA levels by qPCR 58 hours after transfection with either dCas9+sgRNA pool-2, dCas9-p300+sgRNA pool-2, or dCas9-VPR+sgRNA pool-2. Figure 3B shows the quantification of FACS experiments determining the percentage of naive T cells that were FOXP3 positive 72 hours after transfection. All transfections were performed using MaxCyte electroporation buffer and the ATx electroporation system according to the manufacturer's instructions. "Programmed T cells 2" and "Programmed T cells 3" are two of the electroporation settings of the instrument used to electroporate T cells with mRNA+sgRNA for delivery to cells. [Modes for carrying out the invention]

[0099] Detailed description of the invention The present invention provides agents and compositions for modulating the expression of the FOXP3 gene (e.g., enhanced or reduced expression) by targeting the forkhead box P3 (FOXP3) expression regulatory region. The FOXP3 gene may be present in cells, e.g., mammalian cells, e.g., mammalian immune cells, e.g., mammalian naive T cells, e.g., human or mouse naive T cells. The present invention also provides methods of using the agents and compositions of the present invention to modulate the expression of the FOXP3 gene in subjects who would benefit from modulating the expression of the FOXP3 gene, e.g., subjects suffering from or susceptible to autoimmune diseases, and / or to treat such subjects.

[0100] The agents of the present invention are referred to herein as site-specific foxp3 disruptors and are described in Section II below.

[0101] I. Definition Certain terms are defined first to facilitate understanding of the present invention. Furthermore, it should be noted that whenever parameter values ​​or ranges of values ​​are stated, values ​​and ranges between those stated values ​​are also intended to be part of the present invention.

[0102] The articles "a" and "an" are used herein to refer to one or more (i.e., at least one) grammatical objects of the article. For example, "an element" means one element or more than one element, such as multiple elements, such as a pool of elements, such as sgRNA.

[0103] The term "includes" is used herein to mean the phrase "includes, but not limited to," and is used synonymously with that phrase. The term "or" is used herein to mean the term "and / or," and is used synonymously with that phrase, unless the context explicitly indicates otherwise.

[0104] The term “approximately” is used herein to mean a typical tolerance in the art. For example, “approximately” may be understood as a standard deviation of about 2 from the mean. In certain embodiments, “approximately” means ±10%. In certain embodiments, “approximately” means ±5%. When “approximately” precedes a series of numbers or ranges, it is understood that “approximately” may modify each of those consecutive or ranged numbers.

[0105] The term "at least" preceding a number or range is understood to include the number adjacent to the term "at least," and all subsequent numbers or integers that could theoretically be included as is evident from the context. For example, the number of nucleotides in a nucleic acid molecule should be an integer. For example, "at least 18 nucleotides in a 21-nucleotide nucleic acid molecule" means that 18, 19, 20, or 21 nucleotides have the indicated property. When "at least" precedes a range or number, it is understood that "at least" can modify each of the consecutive or ranged numbers.

[0106] As used herein, “less than” or “less than” is understood to mean any value or integer below the value adjacent to the phrase, and any value or integer below zero that is logically reasonable in the context. If “less than” precedes a set of numbers or a range, it is understood that “less than” may modify each of the numbers in that set or range.

[0107] As used herein, the term “substantially” refers to a qualitative condition that represents the whole or nearly whole range or degree of the desired feature or characteristic. Those skilled in the art will understand that biological and chemical phenomena rarely, if not never, go to completion and / or proceed to completeness or reach or avoid absolute results. Therefore, the term “substantially” may be used herein in certain embodiments to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0108] As used herein, the terms “forkhead box P3” or “FOXP3” refer to the gene encoding a well-known member of the FOX protein family, a master transcription factor that controls the differentiation of naive T cells into regulatory T cells (Tregs). FOX proteins belong to the forkhead / winged helix family of transcription regulators and are thought to exert regulation through similar DNA-binding interactions during transcription. In regulatory T cell model systems, the FOXP3 transcription factor can occupy the promoters of genes involved in regulatory T cell function and can suppress the transcription of vital genes upon stimulation of T cell receptors. Failure to function in this gene can cause immunodysregulation, polyglandular endocrine disorders, intestinal diseases, X-linked syndromes (or IPEX), also known as X-linked autoimmune-immunodeficiency syndrome, as well as a wide range of cancers. The nucleotide and amino acid sequences of FOXP3 are publicly known and can be found, for example, in GenBank accession numbers NM_014009.4 and NM_001114377.2, the entire contents of each of these are incorporated herein by reference. The nucleotide sequence of the human X chromosome genomic region containing the endogenous promoter of FOXP3, and the FOXP3 coding sequence are also publicly known and can be found, for example, in NC_000023.11 (49250436-49264932). There are two common transcriptional variants of FOXP3 mRNA, and their sequences can be found in GenBank accession numbers NM_014009.4 and NM_001114377.2. The entire contents of each of the aforementioned GenBank accession numbers as of the filing date of this application are incorporated herein by reference.

[0109] The term “site-specific foxp3 disruptor” as used herein refers to any agent that specifically binds to a target foxp3 expression regulatory region and modulates, for example, the expression of a foxp3 gene. The site-specific foxp3 disruptor of the present invention may include a “site-specific foxp3 targeting moiety.”

[0110] As used herein, the term “site-specific FOXP3 targeting moiety” refers to a moiety that specifically binds to a FOXP3 expression regulatory region, such as a transcriptional regulatory region of the FOXP3 gene, such as a DNA region surrounding / proximal upstream of the transcription start site, a promoter, enhancer, or repressor, or to a FOXP3-associated anchor sequence, such as within a FOXP3-associated anchor sequence-mediated conjunction. Examples of “site-specific FOXP3 targeting moieties” include, but are not limited to, polyamides, nucleic acid molecules, such as RNA, DNA, or modified RNA or DNA, polypeptides, protein nucleic acid molecules, and fusion proteins.

[0111] As used herein, the terms “specific binding” or “specifically binding” refer to the ability to identify potential binding partners in the environment in which binding occurs. In some embodiments, a disruptor that interacts with one particular target in the presence of other potential disruptors, for example, preferentially, is said to “specifically bind” to the target it interacts with (i.e., a regulatory region of expression). In some embodiments, specific binding is assessed by detecting or determining the degree of association between the disruptor and its target; in some embodiments, specific binding is assessed by detecting or determining the degree of dissociation of the disruptor-target complex; in some embodiments, specific binding is assessed by detecting or determining the ability of the disruptor to compete with alternative interactions between its target and another entity; in some embodiments, specific binding is assessed by performing such detection or determination over a certain concentration range.

[0112] As used herein, the term “regulatory region” or “regulatory domain” refers to a region or domain in genomic DNA that modulates the expression of a target gene in a cell. The functionality associated with a regulatory region may directly affect the expression of a target gene, for example, by recruiting or blocking the recruitment of a transcription factor that would stimulate gene expression. The functionality associated with a regulatory region may also indirectly affect the expression of a target gene, for example, by introducing an epigenetic modification that induces a change in chromosomal topology that modulates the expression of the target gene, or by recruiting other factors that introduce an epigenetic modification. Regulatory regions may include, for example, transcriptional regulatory elements, such as DNA regions around / proximal upstream of a transcription start site, promoters, enhancers, or repressors, and anchor sequences and anchor sequence-mediated conjunctions, located upstream and / or downstream of the protein-coding sequence of a gene.

[0113] As used herein, the term “transcriptional regulatory element” refers to a nucleic acid sequence that controls the transcription of a gene. Transcriptional regulatory elements include, for example, anchor sequences, anchor sequence-mediated conjunctions, DNA regions surrounding / proximal upstream of a transcription start site, promoters, transcriptional enhancers, and transcriptional repressors.

[0114] The transcription start site (TSS) is the location at the 5' end of a gene sequence where transcription begins. The DNA region surrounding / proximal to the TSS can regulate gene expression, for example, by recruiting transcription factors. Changes in the modification status of one or more nucleotides (e.g., methylation) or one or more chromatin proteins (e.g., acetylation) in the DNA region surrounding / proximal to the TSS can regulate gene expression.

[0115] A promoter is a region of DNA recognized by RNA polymerase that initiates the transcription of a specific gene, and is generally located upstream of the 5' end of the gene's transcription start site.

[0116] A "transcriptional enhancer" increases gene transcription. A "transcriptional silencer" or "transcriptional repressor" decreases gene transcription. Enhancement and silencing sequences can be approximately 50 to 3500 base pairs long and can affect gene transcription as far as approximately 1 megabase away.

[0117] As used herein, the term “gene” refers to a sequence of nucleotides that encode a functional molecule, such as a protein. A gene contains a transcribed sequence (e.g., 3'UTR), an untranscribed sequence (e.g., promoter), a translated sequence (e.g., exon), and an untranslated sequence (e.g., intron).

[0118] As used herein, the term “target gene” means a FOXP3 gene that is targeted for modulation of expression, e.g., increase or decrease. In some embodiments, the FOXP3 target gene is a part of a targeted genomic complex (e.g., a FOXP3 gene having at least a portion of its genomic sequence as part of the target genomic complex, e.g., inside an anchor sequence-mediated conjunction), and this genomic complex is targeted by one or more site-specific disruptors described herein. In some embodiments, modulation includes activation of the expression of the target gene. In some embodiments, the FOXP3 gene is modulated by contacting the FOXP3 gene, or a transcriptional regulatory element operably linked to the FOXP3 gene, with one or more site-specific disruptors described herein. In some embodiments, the FOXP3 gene is abnormally expressed (e.g., overexpressed) in cells, e.g., in cells of a subject (e.g., a subject with a FOXP3-related disease or autoimmune disease). In some embodiments, the FOXP3 gene is abnormally expressed (e.g., underexpressed) in cells, for example, in cells of a subject (e.g., a subject with FOXP3-related disease or autoimmune disease).

[0119] The term “anchor sequence,” as used herein, refers to a nucleic acid sequence recognized by a nucleating agent that binds sufficiently to form an anchor sequence-mediated conjunction, e.g., a complex. 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 intergenetic region. In some embodiments, the anchor sequence is not located within an enhancer or a 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 within a region not associated with genomic imprinting, single-allele expression, and / or single-allele epigenetic marking. In some embodiments, the anchor sequence has one or more functions, selected from binding to an endogenous nucleating polypeptide (e.g., CTCF), interacting with a second anchor sequence to form an anchor sequence-mediated conjunction, or blocking the effects of an enhancer outside the anchor sequence-mediated conjunction. In some embodiments of the present invention, a technique is provided that allows for the specific targeting of a particular anchor sequence(s) without targeting other anchor sequences (e.g., sequences that may contain nucleating agent (e.g., CTCF) binding motifs in different contexts), and such targeted anchor sequences may be referred to as “targeted anchor sequences.” In some embodiments, the sequence and / or activity of the targeted 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 other targeted anchor sequences (e.g., in the same cell, and / or in some embodiments, on the same nucleic acid molecule, e.g., on the same chromosome) is not modulated.In some embodiments, the anchor sequence includes or is a nucleating polypeptide binding motif. In some embodiments, the anchor sequence is adjacent to the nucleating polypeptide binding motif.

[0120] The term "anchor sequence-mediated conjunction," as used herein, refers to a DNA structure, and / or complex, resulting from and / or maintained by the physical interaction or binding of at least two anchor sequences in DNA by one or more polypeptides, such as nucleating polypeptides, or one or more proteins and / or nucleic acid entities (e.g., RNA or DNA), which bind to an anchor sequence to enable spatial proximity and functional linkage between the anchor sequences.

[0121] As used herein, the term “genomic complex” is a complex formed by interactions between two geometrid sequence elements, which are spaced apart on one or more chromosomes, and which are brought together by interactions between multiple proteins and / or other components (which may include geometrid sequence elements). In some embodiments, a geometrid sequence element is an anchor sequence to which one or more protein components of the complex bind. In some embodiments, a geometrid complex may include an anchor sequence-mediated conjunction. In some embodiments, a geometrid sequence element may be a CTCF-binding motif, a promoter, and / or an enhancer, or include them. In some embodiments, a geometrid sequence element includes at least one or both of a promoter and / or a regulatory region (e.g., an enhancer). In some embodiments, complex formation is nucleated in the geometrid sequence elements and / or by the binding of protein components to one or more geometrid sequence elements. As will be understood by those skilled in the art, in some embodiments, the colocalization of genomic sites by complex formation (e.g., conjunction formation) alters the DNA topology in or near the geometrid sequence elements, including, in some embodiments, the topology between the geometrid sequence elements. In some embodiments, the genome complex comprises an anchor sequence-mediated conjunction containing one or more loops. In some embodiments, the genome complex described herein is nucleated by a nucleating polypeptide, such as CTCF and / or cohesin. In some embodiments, the genome complex described herein may comprise one or more of the following: CTCF, cohesin, non-coding RNA (e.g., eRNA), transcriptional mechanism proteins (e.g., RNA polymerase, one or more transcription factors selected from the group consisting of TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, etc.), and transcriptional regulators (e.g., mediators, P300, enhancer-binding proteins, repressor-binding proteins, histone modifiers, etc.).In some embodiments, the genome complex described herein includes one or more polypeptide components and / or one or more nucleic acid components (e.g., one or more RNA components) that, 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 constrain the stretch of genomic DNA to a topological structure (e.g., a loop) that the stretch of genomic DNA would not take if the complex were not formed.

[0122] As used herein, “effector molecule” refers to a molecule that can modulate biological activity, such as enzyme activity, gene expression, anchor sequence-mediated conjunction, or cellular signaling. Exemplary effectors are described in Section II below and, in some embodiments, include, for example, nucleases, physical blockers, epigenetic recruiters, such as transcriptional enhancers or transcriptional repressors, and epigenetic CpG modifiers, such as DNA methylases, DNA demethylases, histone modifiers, histone transacetylases, or histone deacetylases, as well as any combination thereof.

[0123] II. Site-Specific FOXP3 Disrupting Agent of the Present Invention In one aspect of the present invention, the present invention provides a site-specific FOXP3 disruptor comprising a site-specific FOXP3 targeting moiety that targets a FOXP3 expression regulatory region. In another aspect, the site-specific disruptor of the present invention comprises a site-specific FOXP3 targeting moiety that targets a FOXP3 expression regulatory region, and an effector molecule. As will be understood by those skilled in the art, such a disruptor is site-specific and therefore specifically binds to a FOXP3 expression regulatory region (e.g., one or more transcriptional regulatory elements and / or one or more target anchor sequences), for example, within a cell, but does not specifically bind to a non-target expression regulatory region (e.g., within the same cell).

[0124] FOXP3 is a master transcription factor that controls the differentiation of naive T cells into regulatory T cells (Tregs), and forced overexpression of FOXP3 has been shown to confer a Treg phenotype to T cells. The present invention features the use of an effector molecule, e.g., a chromatin remodeler, which, when fused to a DNA targeting region, can induce epigenetic changes in a specific genomic region that lead to increased transcription of a targeted gene, e.g., the FOXP3 gene. In certain embodiments, an effector molecule, p300-core or VPR, fused to the DNA targeting region, dCas9, is targeted to the FOXP3 locus using a single guide RNA (sgRNA) complementary to the DNA region surrounding / slightly upstream of the transcription start site (TSS) of the FOXP3 gene, causing changes in histone acetylation. These epigenetic changes trigger a mechanism that ultimately activates FOXP3 in naive T cells, inducing differentiation into Tregs. These Tregs can be identified based on cell surface markers such as CD127, and / or based on a repressive phenotype in which the Tregs kill effector T cells incubated in mixed culture.

[0125] In vitro generation of Tregs has been a key focus in the field of ex vivo therapy targeting autoimmune disorders. However, many strategies for producing Tregs fail to result in sustained expression of the gene leading to Tregs, nor do they produce Tregs with a repressed phenotype. The present invention features a method that directly targets FOXP3, a master regulatory transcription factor of the Treg generation and maintenance pathway, by using a targeting region (e.g., dCas9, TALE, or ZFP) to directly deliver an effector molecule (e.g., an activator) to increase the activation of the FOXP3 gene at the site of action.

[0126] The site-specific FOXP3 disruptor of the present invention includes a site-specific FOXP3 targeting moiety that targets a FOXP3 expression regulatory region. The expression regulatory region targeted by the site-specific targeting moiety may be, for example, a transcriptional regulatory element or an anchor sequence, such as an anchor sequence within an anchor-mediated conjunction.

[0127] Therefore, the site-specific FOXP3 disruptor of the present invention can modulate the expression of a gene, i.e., FOXP3, by modulating the expression of the gene from, for example, a DNA region surrounding / proximal upstream of the transcription start site, an endogenous promoter, an enhancer, or a repressor; it can alter the methylation of a regulatory region; it can alter the acetylation of a chromatin protein; it can introduce one or more mutations, e.g., nucleotide substitutions, additions, or deletions; it can alter at least one anchor sequence; it can alter at least one conjunction nucleation molecule binding site, e.g., by altering the binding affinity to a conjunction nucleation molecule; and it can alter the orientation of at least one common nucleotide sequence, such as a CTCF binding motif, e.g., by substitution, addition, or deletion in at least one anchor sequence, such as a CTCF binding motif.

[0128] In certain embodiments, the site-directed disruptors and compositions described herein modulate expression in cells by targeting an expression regulatory region comprising one or more FOXP3-specific transcriptional regulatory elements. The targetable FOXP3-specific transcriptional regulatory elements include DNA regions surrounding or proximal to a FOXP3 transcription start site, FOXP3-specific promoters, FOXP3-specific enhancers, FOXP3-specific repressors, and FOXP3-associated anchor sequences. In one embodiment, a FOXP3-specific transcriptional regulatory element, such as a DNA region surrounding or proximal to a FOXP3 transcription start site, modulates expression in immune cells.

[0129] For example, a site-directed disruptor may include a nucleic acid molecule encoding a DNA-binding domain or fragment of a transcription activator-like effector (TALE) polypeptide or zinc finger (ZNF) polypeptide that specifically targets and binds to a site-directed targeting region, such as a FOXP3 endogenous promoter region or other FOXP3 expression regulatory region, and an effector molecule, such as a transcription enhancer or transcription repressor that modulates, enhances, or suppresses the expression of a target gene from an endogenous promoter in order to modulate gene expression. In one embodiment, the disruptor is a "bisistronic nucleic acid molecule," that is, a nucleic acid molecule encoding a DNA-binding domain or fragment of a transcription activator-like effector (TALE) polypeptide or zinc finger (ZNF) polypeptide that specifically targets and binds to first and second site-specific targeting regions, such as the FOXP3 endogenous promoter region, which are FOXP3 expression regulatory regions; and an effector molecule, for example, an effector molecule containing a transcription enhancer or transcription repressor that modulates, enhances or suppresses, the expression of a target gene from the endogenous promoter in order to modulate gene expression. This fusion protein can be constructed from a single messenger RNA molecule.

[0130] In some embodiments of the present invention, the site-specific disruptor may include a guide RNA that targets a site-specific targeting moiety, for example, a nucleic acid molecule, such as a FOXP3 endogenous DNA region around or proximal to the FOXP3 transcription start site, and an effector molecule, for example, an effector molecule containing a transcription enhancer or transcription repressor that modulates, for example, enhances or suppresses the expression of a target gene from an endogenous promoter in order to modulate gene expression.

[0131] In certain embodiments of the present invention, site-specific disruptors and compositions described herein target expression regulatory regions containing one or more FOXP3-associated anchor sequences, such as within anchor sequence-mediated conjunctions, which include first and second FOXP3-associated anchor sequences, thereby modulating expression in cells, such as cells within a subject, by altering the two-dimensional chromatin structure, such as anchor sequence-mediated conjunctions, for example, by modifying anchor sequence-mediated conjunctions in DNA, such as genomic DNA.

[0132] In one embodiment, the present invention includes a site-specific foxp3 disruptor comprising a site-specific foxp3 targeting moiety that targets a foxp3 expression regulatory region containing one or more foxp3-associated anchor sequences within an anchor sequence-mediated conjunction. The disruptor binds to a specific anchor sequence-mediated conjunction, for example, specifically, and alters the topology of the anchor sequence-mediated conjunction, e.g., an anchor sequence-mediated conjunction having physical interactions of two or more DNA loci to which a conjunction nucleation molecule is bound.

[0133] The formation of anchor sequence-mediated conjunctions can cause transcriptional regulatory elements to interact with the FOXP3 gene or spatially constrain the activity of transcriptional regulatory elements. Therefore, by modifying anchor sequence-mediated conjunctions, it becomes possible to modulate FOXP3 expression without altering the coding sequence of the FOXP3 gene being modulated.

[0134] In some embodiments, the site-specific disruptors and compositions of the present invention modulate the expression of the FOXP3 gene associated with anchor sequence-mediated conjugation through physical interference between one or more anchor sequences and a conjugation nucleation molecule. For example, a DNA-binding small molecule (e.g., a minor or major groove binder), a peptide (e.g., a zinc finger, TALE, novel or modified peptide), a protein (e.g., CTCF, a modified CTCF with reduced CTCF binding and / or aggregation binding affinity), or a nucleic acid (e.g., ssDNA, modified DNA or RNA, peptide oligonucleotide conjugate, locked nucleic acid, cross-linked nucleic acid, polyamide, and / or triple-strand forming oligonucleotide) can physically prevent the conjugation nucleation molecule from interacting with one or more anchor sequences and modulate FOXP3 gene expression.

[0135] In some embodiments, the site-specific disruptors and compositions of the present invention modulate the expression of the FOXP3 gene associated with anchor sequence-mediated conjugation through modification of the anchor sequence, e.g., epigenetic modification, e.g., histone protein modification, or genome editing modification. For example, one or more anchor sequences associated with an anchor sequence-mediated conjugation containing the FOXP3 gene can be targeted for genome editing, e.g., Cas9-mediated genome editing.

[0136] In some embodiments, the site-specific disruptors and compositions of the present invention modulate the expression of the FOXP3 gene associated with anchor sequence-mediated conjugation, e.g., activate or repress transcription, e.g., induce epigenetic changes in chromatin or genome editing.

[0137] In some embodiments, the anchor sequence-mediated conjunction includes one or more anchor sequences, the FOXP3 gene, and one or more transcriptional regulatory elements, such as enhancement or silencing elements. In some embodiments, the transcriptional regulatory elements are located within, partially within, or outside of the anchor sequence-mediated conjunction.

[0138] In one embodiment, the anchor sequence-mediated conjunction includes a loop, for example, an intrachromosomal loop. In a particular embodiment, the anchor sequence-mediated conjunction has multiple loops. One or more loops may include a first anchor sequence, a nucleic acid sequence, a transcriptional regulatory element, and a second anchor sequence. In another embodiment, at least one loop includes, in order, a first anchor sequence, a transcriptional regulatory element, and a second anchor sequence, or a first anchor sequence, a nucleic acid sequence, and a second anchor sequence. In yet another embodiment, either or both of the nucleic acid sequence and the transcriptional regulatory element are located inside or outside the loop. In yet another embodiment, one or more loops include a transcriptional regulatory element.

[0139] In some embodiments, the anchor array-intervening conjunction includes a TATA box, CAAT box, GC box, or CAP site.

[0140] In some embodiments, the anchor sequence-mediated conjunction comprises multiple loops, in which case the anchor sequence-mediated conjunction includes at least one of the following in one or more of the loops: an anchor sequence, a nucleic acid sequence, and a transcriptional regulatory element.

[0141] In one embodiment, site-directed disruptors and compositions of the present invention can introduce modifications to anchor sequence-mediated conjunctions that target the modulation of nucleic acid sequence expression by a disruptor that binds to the anchor sequence. In some embodiments, the anchor sequence-mediated conjunction is modified by targeting one or more nucleotides within the anchor sequence-mediated conjunction for substitution, addition, or deletion.

[0142] In some embodiments, expression, e.g., transcription, is activated by including or excluding an activation loop. In one such embodiment, the anchor sequence-mediated conjunction includes a transcriptional regulatory sequence, e.g., a nucleic acid encoding such a FOXP3, which increases the transcription of the nucleic acid sequence. In another such embodiment, the anchor sequence-mediated conjunction does not include a transcriptional regulatory element, e.g., a nucleic acid encoding such a FOXP3, which decreases the expression, e.g., transcription of the nucleic acid sequence.

[0143] In some embodiments, expression, such as transcription, is suppressed by including or excluding a repressive loop. In one such embodiment, the anchor sequence-mediated conjunction includes a transcriptional regulatory element that reduces the expression, such as transcription, of a nucleic acid sequence, such as a nucleic acid sequence encoding FOXP3. In another such embodiment, the anchor sequence-mediated conjunction does not include a transcriptional regulatory element that increases the transcription of a nucleic acid sequence, such as a nucleic acid sequence encoding FOXP3.

[0144] Each anchor sequence-mediated conjunction comprises one or more anchor sequences, e.g., multiple. Anchor sequences can be manipulated or modified to disrupt naturally occurring loops or to form new loops (e.g., to form exogenous loops or loops not naturally occurring with exogenous or modified anchor sequences). Such modifications modulate FOXP3 gene expression by altering the two-dimensional structure of the DNA containing all or part of the FOXP3 gene, for example, by modulating the FOXP3 gene's ability to interact with transcriptional regulatory elements (e.g., enhancing and silencing / repressing sequences). 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 conjunction.

[0145] The anchor arrays may be discontinuous from one another. In embodiments involving discontinuous anchor arrays, the first anchor array may be separated from the second anchor array by approximately 500 bp to 500 Mb, approximately 750 bp to 200 Mb, approximately 1 kb to 100 Mb, approximately 25 kb to 50 Mb, approximately 50 kb to 1 Mb, approximately 100 kb to 750 kb, approximately 150 kb to 500 kb, or approximately 175 kb to 500 kb. In some embodiments, the first anchor array is approximately 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, 175 kb, 200 kb, 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 between these, separated.

[0146] In one embodiment, the anchor sequence is a common nucleotide sequence, for example, a CTCF binding motif: [ka] (where N is any nucleotide)

[0147] The CTCF binding motif can be reversed, for example, [ka] It can also be the case.

[0148] In one embodiment, the anchor sequence contains a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical to either SEQ ID NO: 1 or SEQ ID NO: 2.

[0149] In some embodiments, the anchor sequence-mediated conjunction includes at least a first anchor sequence and a second anchor sequence. The first and second anchor sequences each include a common nucleotide sequence, for example, each including a CTCF-binding motif. In some embodiments, the first and second anchor sequences include different sequences, for example, the first anchor sequence includes a CTCF-binding motif, and the second anchor sequence includes an anchor sequence other than the CTCF-binding motif. In some embodiments, each anchor sequence includes a common nucleotide sequence and one or more adjacent nucleotides on one or both sides of the common nucleotide sequence.

[0150] Two CTCF binding motifs that can form a conjunction (e.g., contiguous or discontinuous CTCF binding motifs) 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 including SEQ ID NO: 1) or 3’→5’ (right tandem, e.g., two CTCF binding motifs including SEQ ID NO: 2), or in a convergent orientation where one CTCF binding motif includes SEQ ID NO: 1 and the other includes SEQ ID NO: 2. Using CTCFBSDB 2.0: Database For CTCF binding motifs And Genome Organization (http : / / insulatordb .uthsc . edu / ), it is possible to identify CTCF binding motifs associated with a target gene, e.g., FOXP3.

[0151] In some embodiments, the anchor sequence-mediated conjunction is altered by changing the orientation of at least one common nucleotide sequence, e.g., a conjunction nucleation molecule binding site.

[0152] In some embodiments, the anchor sequence includes a conjunction nucleation molecule binding site, e.g., a CTCF binding motif, and the site-specific disruptor of the present invention introduces an alteration of at least one conjunction nucleation molecule binding site, e.g., an alteration of the binding affinity for a conjunction nucleation molecule.

[0153] In some embodiments, the anchor sequence-mediated conjunction is altered by introducing an exogenous anchor sequence. For example, the addition of a non-natural or exogenous anchor sequence to form or disrupt a naturally occurring anchor sequence-mediated conjunction by inducing the formation of a non-natural loop that alters the transcription of a nucleic acid sequence.

[0154] In some embodiments, the anchor sequence-mediated conjunction includes the FOXP3 gene and one or more other genes besides the FOXP3 gene, e.g., two, three, four, five, or __.

[0155] In some embodiments, the anchor sequence-mediated conjunction associates with one or more transcriptional regulatory elements, e.g., two, three, four, five, or more. In some embodiments, the FOXP3 gene is discontinuous with one or more transcriptional regulatory elements. In some embodiments where the FOXP3 gene is discontinuous with transcriptional regulatory elements, the gene may be separated from one or more transcriptional regulatory elements by approximately 100 bp to approximately 500 Mb, approximately 500 bp to approximately 200 Mb, approximately 1 kb to approximately 100 Mb, approximately 25 kb to approximately 50 Mb, approximately 50 kb to approximately 1 Mb, approximately 100 kb to approximately 750 kb, approximately 150 kb to approximately 500 kb, or approximately 175 kb to approximately 500 kb. In some embodiments, the gene is approximately 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, 175 kb, 200kb, 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 between these, separated.

[0156] In some embodiments, the type of anchor sequence-mediated conjunction can be useful in determining how to modulate gene expression by altering the anchor sequence-mediated conjunction, for example, in selecting site-specific targeting regions. For example, some types of anchor sequence-mediated conjunctions contain one or more transcriptional regulatory elements within the anchor sequence-mediated conjunction. Disruption of such an anchor sequence-mediated conjunction, for example by altering one or more anchor sequences, is likely to reduce the transcription of the FOXP3 gene within the anchor sequence-mediated conjunction.

[0157] In some embodiments, the expression of the FOXP3 gene is regulated, modulated, or affected by one or more transcriptional regulatory elements associated with an anchor sequence-mediated conjunction. In some embodiments, the anchor sequence-mediated conjunction includes the FOXP3 gene and one or more transcriptional regulatory elements. For example, the FOXP3 gene and one or more transcriptional regulatory sequences are at least partially located within an anchor sequence-mediated conjunction, e.g., a type 1 anchor sequence-mediated conjunction. The anchor sequence-mediated conjunction is sometimes referred to as "type 1, EP subtype". In some embodiments, the FOXP3 gene has defined expression states, e.g., in its native state, e.g., in its diseased state. For example, the FOXP3 gene may have high expression levels. The expression of the FOXP3 gene can be reduced by disrupting the anchor sequence-mediated conjunction (e.g., transcriptional reduction due to conformational changes in DNA already susceptible to transcription within the anchor sequence-mediated conjunction, e.g., transcriptional reduction due to conformational changes in DNA that create additional distance between the FOXP3 gene and the enhancement sequence). In one embodiment, both the associated FOXP3 gene and one or more transcriptional regulatory sequences, such as enhancement sequences, are located inside the anchor sequence-mediated conjunction. Disruption of the anchor sequence-mediated conjunction reduces the expression of the FOXP3 gene. In one embodiment, the FOXP3 gene associated with the anchor sequence-mediated conjunction is easily accessible to one or more transcriptional regulatory elements located at least partially inside the anchor sequence-mediated conjunction.

[0158] In some embodiments, the expression of the FOXP3 gene is regulated, modulated, or affected by one or more associated transcriptional regulatory elements, but these are difficult to access due to an anchor sequence-mediated conjunction. For example, an anchor sequence-mediated conjunction associated with the FOXP3 gene disrupts the ability of one or more transcriptional regulatory elements to regulate, modulate, or affect the expression of the FOXP3 gene. The transcriptional regulatory elements may be located away from the FOXP3 gene, for example, on the opposite side of the anchor sequence-mediated conjunction from the FOXP3 gene, at least partially, for example, inside or outside of it, so that the FOXP3 gene is difficult to access the transcriptional regulatory elements due to the proximity of the anchor sequence-mediated conjunction. In some embodiments, one or more enhancement sequences are separated from the FOXP3 gene by an anchor sequence-mediated conjunction, for example, a type 2 anchor sequence-mediated conjunction.

[0159] In some embodiments, the FOXP3 gene is difficult to access to one or more transcriptional regulatory elements due to an anchor sequence-mediated conjunction, and disruption of the anchor sequence-mediated conjunction allows the transcriptional regulatory elements to regulate, modulate, or influence the expression of the FOXP3 gene. In one embodiment, the FOXP3 gene is located both inside and outside an anchor sequence-mediated conjunction, making it difficult to access one or more transcriptional regulatory elements. Disruption of the anchor sequence-mediated conjunction increases the access of transcriptional regulatory elements to regulate, modulate, or influence the expression of the FOXP3 gene, for example, the transcriptional regulatory elements increase the expression of the FOXP3 gene. In one embodiment, the FOXP3 gene is located inside an anchor sequence-mediated conjunction, making it difficult to access one or more transcriptional regulatory elements located at least partially outside the anchor sequence-mediated conjunction. Disruption of the anchor sequence-mediated conjunction increases the expression of the FOXP3 gene. In one embodiment, the FOXP3 gene is located at least partially outside the anchor sequence-mediated conjunction, making it difficult to access one or more transcriptional regulatory elements located inside the anchor sequence-mediated conjunction. Disruption of the anchor sequence-mediated conjunction increases the expression of the FOXP3 gene.

[0160] A.FOXP3 site-specific targeting moiety The site-specific FOXP3 targeting moieties of the present invention target the FOXP3 expression regulatory region and may include polymers or polymer molecules, such as polyamides (i.e., molecules of repeating units linked by amide bonds, e.g., polypeptides), nucleotide polymers (e.g., guide RNA; nucleic acid molecules encoding TALE polypeptides or zinc finger polypeptides), peptide nucleic acids (PNAs), or amino acid polymers, such as peptides or polypeptides, e.g., fusion proteins. Preferred site-specific FOXP3 targeting moieties, compositions, and methods of use of such agents and compositions are described below and are included in their entirety in PCT Publication WO2018 / 049073, which is expressly incorporated herein by reference.

[0161] In one embodiment, the site-directed disruptor of the present invention comprises a site-directed FOXP3 targeting moiety including a nucleic acid molecule such as guide RNA (or gRNA), or a guide RNA and an effector, or a fragment thereof, or a nucleic acid molecule encoding the effector or a fragment thereof.

[0162] In another embodiment, the site-directed disruptor of the present invention comprises a site-directed FOXP3 targeting moiety comprising a nucleic acid molecule encoding a polypeptide such as a zinc finger polypeptide (ZNF) or a DNA-binding domain or fragment of a transcription activator-like effector (i.e., TALE DNA-binding domain, or TALE) polypeptide, which is engineered to specifically target a FOXP3 expression regulatory region in order to modulate the expression of the FOXP3 gene.

[0163] In another embodiment, the site-specific disruptor of the present invention comprises a site-specific FOXP3 targeting moiety comprising a polynucleotide, such as PNA, such as nucleic acid gRNA linked to an effector polypeptide, or a fragment thereof.

[0164] In another embodiment, the site-directed disruptor of the present invention comprises a site-directed FOXP3 targeting moiety including a fusion molecule such as a fusion molecule encoding a DNA-binding domain or fragment thereof of a transcription activator-like effector (TALE) polypeptide or a zinc finger (ZNF) polypeptide, and an effector.

[0165] In one embodiment, such a site-directed disruptor comprises a second fusion protein, the second fusion protein comprising a second site-directed FOXP3 targeting moiety that targets a second FOXP3 expression regulatory region, and a second effector molecule, the second FOXP3 expression regulatory region being distinct from the FOXP3 expression regulatory region.

[0166] In another embodiment, the site-directed disruptor of the present invention comprises a site-directed FOXP3 targeting moiety comprising a Cas polypeptide and a fusion molecule such as a nucleic acid molecule encoding a fusion protein containing, for example, an epigenetic recruiting factor or an epigenetic CpG modifier.

[0167] Furthermore, in another embodiment, the site-directed disruptor of the present invention comprises a site-directed FOXP3 targeting moiety comprising a Cas polypeptide and a fusion molecule such as a fusion protein containing, for example, an epigenetic recruiting factor or an epigenetic CpG modifier.

[0168] As used herein, in its broadest sense, the term “nucleic acid” refers to any compound and / or substance that is incorporated into or can be incorporated into an oligonucleotide chain. In some embodiments, a nucleic acid is a compound and / or substance that is incorporated into or can be incorporated into a polynucleotide chain by phosphodiester linkage. As will be apparent from the context, in some embodiments, “nucleic acid” refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides), and in some embodiments, “nucleic acid” refers to a polynucleotide chain comprising individual nucleic acid residues. In some embodiments, “nucleic acid” is or contains RNA, and in some embodiments, “nucleic acid” is or contains DNA. In some embodiments, “nucleic acid” is a “mixmer” comprising locked nucleic acid molecules and deoxynucleic acid molecules. In some embodiments, a nucleic acid is one or more native nucleic acid residues, or contains or consists of them. In some embodiments, a nucleic acid is one or more nucleic acid analogs, or contains or consists of them. In some embodiments, a nucleic acid analog differs from a nucleic acid in that it does not use a phosphodiester backbone. For example, in some embodiments, the nucleic acid is, contains, or consists of one or more “peptide nucleic acids,” where “peptide nucleic acids” are known in the Art, have peptide bonds rather than phosphodiester bonds in their backbone, and are considered to be 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 links rather than phosphodiester bonds. In some embodiments, the nucleic acid is, contains, or consists of one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine).In some embodiments, the nucleic acid is one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynylcytidine, C-5 propynyluridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyluridine, C5-propynylcytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof), or comprises them. In some embodiments, the nucleic acid contains one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) compared to those in natural nucleic acids. In some embodiments, the nucleic acid has a nucleotide sequence encoding a functional gene product such as RNA or protein. In some embodiments, the nucleic acid contains one or more introns. In some embodiments, the nucleic acid is prepared by one or more of the following: isolation from natural sources, enzymatic synthesis by polymerization based on complementary templates (in vivo or in vitro), reproduction in recombinant cells or systems, and chemosynthesis. In some embodiments, the nucleic acid has a residue length of 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. In some embodiments, the nucleic acid is partially or completely single-stranded, and in some embodiments, the nucleic acid is partially or completely double-stranded.In some embodiments, the nucleic acid has a nucleotide sequence comprising at least one element that encodes a polypeptide or is a complement to a polypeptide-encoding sequence. In some embodiments, the nucleic acid has enzymatic activity.

[0169] As used herein, the terms “peptide,” “polypeptide,” and “protein” refer to compounds consisting of amino acid residues covalently linked by peptide bonds or by means other than peptide bonds. A protein or peptide should contain at least two amino acids, and there is no limit imposed on the maximum number of amino acids that can constitute a protein sequence or a peptide sequence. A polypeptide includes any peptide or protein containing two or more amino acids linked to each other by peptide bonds or by means other than peptide bonds. As used herein, the terms refer to both short chains, commonly called peptides, oligopeptides, and oligomers in the art, and longer chains, of which there are many types, commonly called proteins in the art.

[0170] In certain embodiments, the polypeptide may be a chimeric or “fusion protein,” or may include one. As used herein, “chimeric protein” or “fusion protein” comprises all or part (preferably a bioactive portion) of a first protein operably linked to a heterogeneous second polypeptide (i.e., a polypeptide other than the first protein). In a fusion protein, the term “operably linked” is intended to indicate that the first protein or a segment and the heterogeneous polypeptide are fused to each other in frame. The heterogeneous polypeptide may be fused to the amino terminus or carboxyl terminus of the first protein or segment.

[0171] A polyamide is a polymer molecule having repeating units linked by amide bonds. Proteins are examples of naturally occurring polyamides. In some embodiments, polyamides include peptide nucleic acids (PNAs).

[0172] A "peptide nucleic acid" ("PNA") is a molecule having an amide-containing skeleton similar to a peptide skeleton, e.g., aminoethylglycine, in which one or more amino acid units in the PNA have a nucleic acid side chain instead of an amino acid side chain. Peptide nucleic acids (PNAs) are known to hybridize with complementary DNA and RNA with higher affinity than their oligonucleotide counterparts. This property of PNAs not only makes them stable hybrids with nucleic acid side chains, but in addition, the neutral skeleton and hydrophobic side chains result in hydrophobic units within the polypeptide. Nucleic acid side chains include, but are not limited to, purine or pyrimidine side chains, e.g., adenine, cytosine, guanine, thymine, and uracil. In one embodiment, the nucleic acid side chain includes the nucleoside analogs described herein.

[0173] In one embodiment, the site-specific FOXP3 targeting moiety of the present invention comprises a polyamide. Polyamides suitable for use in the pharmaceuticals and compositions of the present invention are known in the art.

[0174] In one embodiment, the site-specific FOXP3 targeting moiety of the present invention comprises a polynucleotide. In some embodiments, the nucleotide sequence of the polynucleotide encodes a FOXP3 gene or a FOXP3 expression product. In some embodiments, the nucleotide sequence of the polynucleotide does not include a FOXP3 coding sequence or a FOXP3 expression product. For example, in some embodiments, the site-specific FOXP3 targeting moiety of the present invention comprises a polynucleotide that hybridizes to a target expression regulatory region, such as a promoter, anchor sequence, or DNA region surrounding or proximal to the transcription start site. In some embodiments, the nucleotide sequence of the polynucleotide is a complement to the target DNA region around or proximal upstream of the transcription start site, or has a sequence that is at least 80%, at least 85%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the complement of the target sequence.

[0175] The polynucleotides of the present invention may include deoxynucleotides, ribonucleotides, modified deoxynucleotides, modified ribonucleotides (e.g., chemically modified, e.g., modified with altered skeletal linkages, sugar molecules, and / or nucleic acid bases), and artificial nucleic acids. In some embodiments, polynucleotides include, but are not limited to, genomic DNA, cDNA, peptide nucleic acid (PNA) or peptide oligonucleotide conjugates, locked nucleic acid (LNA), cross-linked nucleic acid (BNA), polyamides, triple-stranded oligonucleotides, modified DNA, antisense DNA oligonucleotides, tRNA, mPvNA, rPvNA, modified RNA, miRNA, gRNA, and siRNA or other RNA or DNA molecules.

[0176] In some embodiments, the polynucleotides of the present invention have lengths of about 2 to about 5000 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 1000 nt, about 50 to about 1000 nt, about 100 to about 1000 nt, about 1000 to about 2000 nt, about 2000 to about 3000 nt, about 3000 to about 4000 nt, about 4000 to about 5000 nt, or any range in between.

[0177] The polynucleotides of the present invention may include nucleosides, such as purines or pyrimidines, such as adenine, cytosine, guanine, thymine, and uracil. In some embodiments, the polynucleotide comprises one or more nucleoside analogs. Nucleoside analogs include, for example, 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, beta-D-galactosylkeosin, 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, beta-D-mannosylkeosin, 5'-methoxycarb Xymethyluracil, 5-Methoxyuracil, 2-Methylthio-N6-isopentenyladenine, Uracil-5-oxyacetic acid(v), Weybutoxosin, Pseudouracil, Keosin, 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, keosin, waiosin, diaminopurine, isoguanine, isocytosine, diaminopyrimidine, 2,4-difluorotoluene, isoquinoline, pyrrolo[2,3-]pyridine, and any other substances that can base pair with purine or pyrimidine side chains, but are not limited thereto.

[0178] In some embodiments, the site-specific FOXP3 targeting moiety of the present invention comprises a polynucleotide encoding a polypeptide containing a DNA-binding domain (DBD) or fragment thereof of a zinc finger polypeptide (ZNF) or a transcription activator-like effector (TALE) polypeptide, which is engineered to specifically target a FOXP3 expression regulatory region in order to modulate the expression of the FOXP3 gene.

[0179] The design and preparation of zinc finger polypeptides that specifically bind to target DNA regions of interest, such as FOXP3 expression regulatory regions, are well known in the art. For example, zinc finger (ZNF) proteins contain DNA-binding motifs that specifically bind to nucleotide triplets. Therefore, to design and prepare the site-specific FOXP3 targeting moieties of the present invention, a modular assembly process can be used, which involves combining separate zinc finger DNA-binding domains, each capable of recognizing a specific 3-base pair DNA sequence, to generate 3-finger, 4, 5, 6, 6, or 8-base finger polypeptides that recognize specific target sites in the range of 9 to 24 base pairs in length. Another preferred method may include a 2-finger module for generating a ZNF polynucleotide having up to six individual zinc fingers. For example, see Shukla VK, et al., Nature. 459 (7245) 2009: 437-41; Dreier B, et al., JBC. 280 (42) 2005: 35588-97; Dreier B, et al, JBC 276 (31) 2001: 29466-78; Bae KH, et al., Nature Biotechnology. 21 (3) 2003: 275-80.

[0180] In some embodiments, the site-specific FOXP3 targeting moiety of the present invention comprises a polynucleotide encoding a polypeptide containing a DNA-binding domain (DBD) or fragment thereof of a zinc finger, which is engineered to specifically target a FOXP3 expression regulatory region in order to modulate the expression of the FOXP3 gene. Exemplary amino acid sequences encoding zinc fingers that bind to nucleotide triplets suitable for use in the present invention are provided in Table 1A below. (e.g., Gersbach et al., Synthetic Zinc Finger Proteins: The Advent of Targeted Gene Regulation and Genome Modification) (See Technologies) Table 1A. [Table 1A-1] [Table 1A-2]

[0181] The zinc finger DNA-binding domain comprises an N-terminal region and a C-terminal region, with a "finger" between them that binds to a target DNA sequence. The N-terminal region is generally 7 amino acids long. The C-terminal region is generally 6 amino acids long. Therefore, the N-terminal region generally contains the amino acid sequence X1X2X3X4X5X6X7, where "X" can be any amino acid. In some embodiments, the N-terminal region contains the exemplary amino acid sequence LEPGEKP (SEQ ID NO: 76), where "X" can be any amino acid. The C-terminal region generally contains X 25 X 26 X 27 X 28 X 29 X 30 It includes the amino acid sequence. In certain embodiments, the C-terminal region includes the exemplary amino acid sequence of TGKKTS (SEQ ID NO: 77).

[0182] On both sides of each finger within the DNA binding domain, there are an N-terminal backbone located at the N-terminus of the finger and a C-terminal backbone located at the C-terminus of the finger. The N-terminal backbone of the finger is generally 11 amino acids long and has two conserved cysteines (C) located at positions 3 and 6. Thus, the N-terminal backbone of the finger generally has the amino acid sequence of X8X9CX 10 X 11 CX 12 X 13 X 14 X 15 X 16 where "X" can be any amino acid. The C-terminal backbone of the finger is generally 5 amino acids long and has two conserved histidines (H) located at positions 1 and 5. Thus, the C-terminal backbone of the finger generally has the amino acid sequence of HX X 17 X 18 X 19 H. "X" can be any amino acid. In some embodiments, the N-terminal backbone contains the exemplary amino acid sequence of YKCPECGKSFS (SEQ ID NO: 61), and the C-terminal backbone contains the exemplary amino acid sequence of HQRTH (SEQ ID NO: 62). The two "fingers" are linked via a linker. The linker is generally 5 amino acids in length and has the amino acid sequence of X 20 X 21 X 22 X 23 X 24 where "X" can be any amino acid. In a particular embodiment, the linker contains the exemplary amino acid sequence of TGEKP (SEQ ID NO: 63). Thus, the zinc finger of the site-specific FOXP3 site-specific disruptor has the following structure: (N-terminal backbone - finger - C-terminal backbone - linker) n and the zinc finger DNA binding domain of the site-specific FOXP3 site-specific disruptor has the following structure: [N-terminal region (N-terminal backbone - finger - C-terminal backbone - linker) n - C-terminal region]. "N" represents the number of nucleotide triplets to which the zinc finger DNA binding domain binds and thus to which the POXP3 site-specific disruptor binds.

[0183] Although the "finger" amino acid sequence of a 4-nucleotide triplet is unknown, if such a triplet is identified within the target area of ​​interest, two "linker span sequences"—linker span 1 and linker span 2—are useful to circumvent the problem. Linker span 1 is used to skip one base pair when the "finger" amino acid sequence of the triplet is unavailable. Linker span 2 is used to skip two base pairs when the "finger" amino acid sequence of the triplet is unavailable. Linker span 1 is generally 12 amino acids long. Linker span 2 is generally 16 amino acids long. Therefore, linker span 1 is generally X 31 X 32 X 33 X 34 X 35 X 36 X 37 X 38 X 39 X 40 X 41 X 42 It contains the amino acid sequence of X. Linker span 2 is generally X 43 X 44 X 45 X 46 X 47 X 48 X 49 X 50 X 51 X 52 X 53 X 54 X 55 X 56 X 57 X 58The amino acid sequence is included. In some embodiments, linker span 1 includes the amino acid sequence THPRAPIPKPFQ (SEQ ID NO: 78). In certain embodiments, linker span 2 includes the amino acid sequence TPNPHRRTDPSHKPFQ (SEQ ID NO: 79). When linker span 1 and / or linker span 2 are used, the finger-linker span 1 / span 2-finger structure includes: N-terminal skeleton-finger-C-terminal skeleton-linker span 1 / span 2-N-terminal skeleton-finger-C-terminal skeleton-linker.

[0184] Table 1B provides the amino acid sequences of exemplary zinc finger DNA-binding domains for use in the present invention and their corresponding target regions.

[0185] In some embodiments, zinc finger DNA-binding domains suitable for use in the disruptors of the present invention include amino acid sequences having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% amino acid identity with respect to any one of the total amino acid sequences of the zinc finger DNA-binding domains provided in Table 1B. Table 1B. [Table 1B-1] [Table 1B-2] [Table 1B-3] [Table 1B-4] [Table 1B-5] [Table 1B-6] Table 1B-7 Table 1B-8 Table 1B-9 Table 1B-10 Table 1B-11 Table 1B-12 Table 1B-13 Table 1B-14 Table 1B-15 Table 1B-16 Table 1B-17 Table 1B-18 Table 1B-19 Table 1B-20 Table 1B-21

Table 1B-22

[0186] Similarly, the design and preparation of such TALE polypeptides that specifically bind to target DNA regions of interest, such as FOXP3 expression regulatory regions, are well known in the art. For example, TALE DNA-binding domains contain a repeating, highly conserved 33-34 amino acid sequence in which the amino acids at positions 12 and 13 differ. These two positions, called repeating variable duos (RVDs), are highly variable and show a strong correlation with specific nucleotide recognition. This simple relationship between amino acid sequence and DNA recognition has made it possible to manipulate specific DNA-binding domains by selecting combinations of repeat segments containing appropriate RVDs. See, for example, Boch J Nature Biotechnology. 29 (2) 2011: 135-6; Boch J, et al., Science. 326 (5959) 2009: 1509-12; Moscow MJ & Bogdanove AJ Science. 326 (5959) 2009: 1501.

[0187] In some embodiments, the site-specific FOXP3 targeting moiety of the present invention, which includes a polynucleotide, includes a guide RNA (or gRNA), or a nucleic acid encoding the guide RNA. The gRNA may include, for example, a site-specific sequence of about 20 nucleotides that targets a genomic target sequence containing a FOXP3 expression regulatory element, and may include, for example, a "scaffold" sequence necessary to direct the effector to the FOXP3 expression regulatory element.

[0188] Generally, guide RNA sequences are designed to have a length between approximately 17 and 24 nucleotides (e.g., 19, 20, or 21 nucleotides) and are complementary to the target sequence. Custom gRNA generators and algorithms for use in designing effective guide RNAs are commercially available. Gene editing has also been achieved using chimeric "single guide RNA" ("sgRNA"), which are engineered (synthetic) single RNA molecules that mimic the naturally occurring crRNA-tracrRNA complex and contain both tracrRNA (for nuclease binding) and at least one crRNA (for inducing the nuclease to the sequence targeted for editing). Chemically modified sgRNAs have also been demonstrated to be effective in genome editing; see, for example, Hendel et al. (2015) Nature Biotechnol., 985-991.

[0189] In certain embodiments, the site-specific FOXP3 targeting moiety of the present invention comprises a guide RNA (or gRNA), or a nucleic acid encoding the guide RNA, and a protein or peptide. In some embodiments, the protein or peptide comprises a CRISPR-related protein (Cas) polypeptide or a fragment thereof (e.g., a Cas9 polypeptide or a fragment thereof). In one embodiment, a preferred Cas polypeptide is an enzymatically inactive Cas polypeptide, e.g., an "inactive Cas polypeptide" or "dCas" polypeptide.

[0190] Exemplary site-specific FOXP3 targeting moieties, including polynucleotides, such as gRNA, are provided in Table 2 below. In some embodiments, the polynucleotides contain nucleotide sequences that are at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical to the total nucleotide sequence of any one of the nucleotide sequences in Table 2.

[0191] Although the sequences in Table 2 are described as modified (or unmodified), it should be understood that nucleic acid molecules encompassed by the present invention, such as site-directed disruptors, may include any one of the sequences shown in Table 2, either unmodified or modified in a way different from that described in Table 2. While some of the sequences in Table 2 contain "T", it should also be understood that "T" can be replaced with "U" when used as an RNA molecule, such as a guide RNA, in the site-directed targeting portion of the present invention.

[0192] In some embodiments, the site-specific FOXP3 targeting moiety, including a polynucleotide, such as gRNA, includes a nucleotide sequence complementary to the anchor sequence. In one embodiment, the anchor sequence is a CTCF-binding motif or a consensus sequence: [ka] (where N is any nucleotide) contains the CTCF binding motif or consensus sequence in the reverse direction, for example, [ka] In some embodiments, the nucleic acid sequence includes a sequence complementary to the CTCF-binding motif or consensus sequence.

[0193] In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to the anchor sequence.

[0194] In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% complementary to the CTCF-binding motif or consensus sequence. In some embodiments, the polynucleotide is selected from the group comprising a gRNA and a sequence complementary to the anchor sequence, or a sequence that is at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% complementary to the anchor sequence.

[0195] In some embodiments, the site-specific FOXP3 targeting moiety containing polynucleotides of the present invention is an RNAi molecule. The RNAi molecule generally contains 15 to 50 base pairs (e.g., about 18 to 25 base pairs) and comprises an RNA or RNA-like structure having a nucleic acid base sequence identical (complementary) or nearly identical (substantially complementary) to the coding sequence of a target gene expressed in the cell. RNAi molecules include, but are not limited to, small interfering RNAs (siRNAs), double-stranded RNAs (dsRNAs), microRNAs (miRNAs), short hairpin RNAs (shRNAs), meloduplexes, and Dicer substrates (U.S. Patents 8,084,599, 8,349,809, and 8,513,207). In one embodiment, the present invention includes a composition for inhibiting the expression of a gene encoding a polypeptide described herein, such as a conjunction nucleation molecule.

[0196] RNAi molecules contain sequences that are substantially or completely complementary to all or a fragment of a target gene. RNAi molecules can prevent the maturation of a newly generated nuclear RNA transcript of a particular gene into mRNA for transcription by supplementing the intron-exon boundary sequence. RNAi molecules complementary to a particular gene can hybridize with the gene's mRNA and prevent its translation. Antisense molecules can be DNA, RNA, or derivatives or hybrids thereof. Examples of such derivative molecules include, but are not limited to, peptide nucleic acids (PNAs) and phosphorothioate-based molecules, such as deoxyribonucleic acid guanine (DNG) or ribonucleic acid guanine (RG).

[0197] RNAi molecules can be supplied to cells either as "ready-to-use" RNA synthesized in vitro, or as antisense genes transfected into cells that produce RNAi molecules during transcription. Hybridization with mRNA results in the degradation of the hybridized molecule by RNAse H and / or inhibition of translational complex formation. In both cases, the product of the original gene cannot be produced.

[0198] The length of the RNAi molecule hybridizing with the target transcript should be approximately 10 nucleotides, between approximately 15 and 30 nucleotides, or approximately 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotides. The degree of antisense sequence identity to the target transcript should be at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%.

[0199] RNAi molecules may also include overhangs, i.e., generally unpaired overhang nucleotides that do not directly participate in the double helix structure typically formed by the paired core sequences of the sense and antisense strands as defined herein. An RNAi molecule may independently contain 3' and / or 5' overhangs of about 1 to 5 bases from each of the sense and antisense strands. In one embodiment, both the sense and antisense strands contain 3' and 5' overhangs. In one embodiment, one or more 3' overhang nucleotides on one strand base-pair with one or more 5' overhang nucleotides on the other strand. In another embodiment, one or more 3' overhang nucleotides on one strand do not pair with one or more 5' overhang nucleotides on the other strand. The sense and antisense strands of an RNAi molecule may or may not contain the same number of nucleotide bases. The antisense and sense strands may form a double helix with only the 5' end having a blunt end, only the 3' end having a blunt end, both the 5' and 3' ends having blunt ends, or neither the 5' nor the 3' ends having blunt ends. In another embodiment, one or more nucleotides in the overhang may contain a thiophosphate, a phosphorothioate, a deoxynucleotide in reverse (3'-to-3' linked) direction, or a modified ribonucleotide or deoxynucleotide.

[0200] Small interfering RNA (siRNA) molecules contain a nucleotide sequence identical to approximately 15 to 25 consecutive nucleotides of the target mRNA. In some embodiments, the siRNA sequence begins with dinucleotide AA, contains approximately 30-70% (approximately 50-60%, 40-60%, or 45-55%) GC content, and does not have a high percentage of identity with any non-target nucleotide sequence in the mammalian genome into which it will be introduced, as determined, for example, by a standard BLAST search.

[0201] siRNA and shRNA are similar to intermediates in the processing pathway of endogenous microRNA (miRNA) genes (Bartel, Cell 116:281-297, 2004). In some embodiments, siRNA can function as miRNA, and vice versa (Zeng et al., Mol Cell 9: 1327-1333, 2002; Doench et al., Genes Dev 17:438-442, 2003). MicroRNAs, like siRNA, use RISC to downregulate target genes, but unlike siRNA, most animal miRNAs do not cleave mRNA. Instead, miRNAs reduce protein production by translational repression or poly(A) excision and mRNA degradation (Wu et al., Proc Natl Acad Sci USA 103:4034-4039, 2006). The known miRNA binding site is located within the mRNA 3'UTR, and miRNAs appear to target a site that has nearly complete complementarity to 2-8 nucleotides from the 5' end of the miRNA (Rajewsky, Nat Genet 38 Suppl: S8-13, 2006; Lim et al, Nature 433:769-773, 2005). The region is known as the seed region. Since siRNA and miRNA are interchangeable, exogenous siRNA downregulates mRNA that has seed complementarity with that siRNA (Birmingham et al., Nat Methods 3: 199-204, 2006). Multiple target sites within the 3'UTR result in stronger downregulation (Doench et al., Genes Dev). 17:438-442, 2003).

[0202] Lists of known miRNA sequences can be found in databases maintained by research organizations, including the Wellcome Trust Sanger Institute, Perm Center for Bioinformatics, Memorial Sloan Kettering Cancer Center, and European Molecule Biology Laboratory. Known effective siRNA sequences and cognitive binding sites are also well documented in the relevant literature. RNAi molecules can be readily designed and generated using techniques known in this art. In addition, computational tools exist that increase the chances of discovering effective and specific sequence motifs (Pei et al. 2006, Reynolds et al. 2004, Khvorova et al. 2003, Schwarz et al. 2003, Ui-Tei et al. 2004, Heale et al. 2005, Chalk et al. 2004, Amarzguioui et al. 2004).

[0203] RNAi molecules modulate the expression of RNA encoded by a gene. Because multiple genes may share some degree of sequence homology with one another, in some embodiments, RNAi molecules can be designed to target a class of genes with sufficient sequence homology. In some embodiments, RNAi molecules may contain sequences that are complementary to sequences shared between different gene targets or specific to a particular gene target. In some embodiments, RNAi molecules can be designed to target conserved regions of RNA sequences that are homologous among several genes, thereby targeting several genes within a gene family (e.g., different gene isoforms, splice variants, mutant genes, etc.). In some embodiments, RNAi molecules can be designed to target sequences specific to a particular RNA sequence of a single gene.

[0204] In some embodiments, the RNAi molecule targets sequences in conjunction nucleation molecules, e.g., CTCF, cohesin, USF 1, YY1, TATA box-binding protein-associated factor 3 (TAF3), ZNF 143, or other polypeptides that promote the formation of anchor sequence-mediated conjunctions, or epigenetic modifiers, e.g., enzymes involved in post-translational modification, such as DNA methylases (e.g., DNMT3a, DNMT3b, DNMTL), DNA demethylation (e.g., TET family enzymes catalyze the oxidation of 5-methylcytosine to 5-hydroxymethylcytosine and more oxidative derivatives), histone methyltransferases, histone deacetylases (e.g., HDAC1, HDAC2, HDAC3), sirtuins 1, 2, 3, 4, 5, 6, or 7 include, but are not limited to, lysine-specific histone demethylase 1 (LSD1), histone-lysine-N-methyltransferase (Setdbl), euchromatin histone-lysine-N-methyltransferase 2 (G9a), histone-lysine-N-methyltransferase (SUV39H1), zeste homolog 2 enhancer (EZH2), viral lysine methyltransferase (vSET), histone methyltransferase (SET2), protein-lysine-N-methyltransferase (SMYD2), and others. In one embodiment, the RNAi molecule targets a protein deacetylase, such as sirtuin 1, 2, 3, 4, 5, 6, or 7. In one embodiment, the present invention includes a composition comprising RNAi that targets a conjunction nucleation molecule, such as CTCF.

[0205] In some embodiments, the site-specific FOXP3 targeting moiety includes a peptide or protein moiety. In some embodiments, the site-specific disruptor includes a fusion protein. In some embodiments, the effector is a peptide or protein moiety. Examples of peptide or protein moieties include, but are not limited to, peptide ligands, antibody fragments, or targeted aptamers that bind to receptors such as extracellular receptors, neuropeptides, hormone peptides, peptide drugs, toxic peptides, viral or microbial peptides, synthetic peptides, and agonist or antagonist peptides.

[0206] Exemplary peptides or proteins include DNA-binding proteins, CRISPR component proteins, conjunction nucleation molecules, dominant-negative conjunction nucleation molecules, epigenetic modifiers, or any combination thereof. In some embodiments, peptides include nucleases, physical blockers, epigenetic recruiters, and epigenetic CpG modifiers, as well as fragments and combinations of any of the aforementioned. In some embodiments, peptides include DNA-binding domains of proteins, such as helix-turn-helix motifs, leucine zippers, Zn fingers, TATA-box binding proteins, and transcription factors.

[0207] Peptides or proteins can be linear or branched. The peptide or protein portion may have a length of approximately 5 to 200 amino acids, approximately 15 to 150 amino acids, approximately 20 to 125 amino acids, approximately 25 to 100 amino acids, approximately 20 to 70 amino acids, approximately 20 to 80 amino acids, approximately 20 to 90 amino acids, approximately 30 to 100 amino acids, approximately 30 to 60 amino acids, approximately 30 to 80 amino acids, approximately 35 to 85 amino acids, approximately 40 to 100 amino acids, or approximately 50 to 125 amino acids, or any range in between.

[0208] As described above, in some embodiments, the site-specific FOXP3 targeting portion of the present invention includes a fusion protein.

[0209] In some embodiments, the fusion protein of the present invention comprises a site-specific FOXP3 targeting moiety that targets a FOXP3 expression regulatory region, and an effector molecule. In other embodiments, the fusion protein of the present invention comprises an effector molecule. Exemplary effector molecules are described below and, in some embodiments, include, for example, nucleases, physical blockers, epigenetic recruiters, e.g., transcription enhancers or transcription repressors, and epigenetic CpG modifiers, e.g., DNA methylases, DNA demethylases, histone modifiers, histone transacetylases, or histone deacetylases, as well as any combination thereof.

[0210] For example, the site-specific targeting region may include a gRNA and an effector, such as a nuclease, such as Cas9, such as wild-type Cas9, nickase Cas9 (e.g., Cas9 D10A), inactive Cas9 (dCas9), eSpCas9, Cpfl, C2C1, or C2C3, or a nucleic acid encoding such a nuclease. The selection of the nuclease and gRNA is determined by whether the targeted mutation is a nucleotide deletion, substitution, or addition, for example, a nucleotide deletion, substitution, or addition to the target sequence. By fusing a non-catalytic endonuclease, such as inactive Cas9 (dCas9, e.g., D10A;H840A), in which all or part of (one or more) effector domains (e.g., bioactive moieties) are tethered, a chimeric protein is created that can be linked to a polypeptide for guiding the composition to a specific DNA site by one or more RNA sequences (e.g., zinc finger arrays, sgRNAs, TAL arrays, peptide nucleic acids as described herein, or DNA recognition elements) in order to modulate the activity and / or expression of one or more target nucleic acid sequences (e.g., methylate or demethylate the DNA sequences).

[0211] In one embodiment, the fusion protein of the present invention may comprise, for example, a CRISPR-related protein (Cas) polypeptide or a fragment thereof (e.g., a Cas9 polypeptide or a fragment thereof) and an effector molecule comprising an epigenetic recruiting factor or an epigenetic CpG modifier. In one embodiment, a suitable Cas polypeptide is an enzymatically inactive Cas polypeptide, such as an "inactive Cas polypeptide" or "dCas" polypeptide.

[0212] Exemplary Cas polypeptides applicable to the methods and compositions described herein are described below. Using methods known in the art, Cas polypeptides can be fused to any of the various agents and / or molecules described herein, and the resulting fusion molecules may be useful in the various methods disclosed.

[0213] In one embodiment, the present invention includes a composition comprising a protein containing a domain that acts on DNA (e.g., a nuclease domain, e.g., a Cas9 domain, e.g., a dCas9 domain; DNA methyltransferase, demethylase, deaminase), e.g., an effector, in combination with at least one guide RNA (gRNA) or antisense DNA oligonucleotide that targets the protein to a site-specific target sequence, and which is effective in altering the expression of a target gene in human cells. In some embodiments, the enzyme domain is Cas9 or dCas9. In some embodiments, the protein contains two enzyme domains, e.g., dCas9 and a methylase or demethylase domain.

[0214] In one embodiment, the present invention includes a composition comprising a protein containing a domain with a transcriptional regulatory element (e.g., a nuclease domain, e.g., a Cas9 domain, e.g., a dCas9 domain; a transcriptional enhancer; a transcriptional repressor), e.g., an effector, in combination with at least one guide RNA (gRNA) or antisense DNA oligonucleotide that targets the protein to a site-specific target sequence, and which is effective in altering the expression of a target gene in human cells. In some embodiments, the enzyme domain is Cas9 or dCas9. In some embodiments, the protein comprises two enzyme domains, e.g., dCas9 and a transcriptional enhancer or transcriptional repressor domain.

[0215] As used herein, “bioactive portion of effector domain” refers to the portion that maintains the function of an effector domain (e.g., “minimal” or “core” domain) (e.g., completely, partially, or minimally).

[0216] The chimeric proteins described herein may also include linkers, such as amino acid linkers. In some embodiments, the linker comprises two or more amino acids, e.g., one or more GS sequences. In some embodiments, the fusion of Cas9 (e.g., dCas9) with two or more effector domains (e.g., DNA methylase, or an enzyme involved in DNA demethylation, or a protein acetyltransferase or deacetylase) comprises one or more scattered linkers (e.g., GS linkers) between the domains. In some embodiments, dCas9 is fused with two to five effector domains by scattered linkers.

[0217] In some embodiments, the site-specific FOXP3 targeting moiety includes a conjunction nucleating molecule, a nucleic acid encoding the conjunction nucleating molecule, or a combination thereof. In some embodiments, the anchor sequence-mediated conjunction involves a first conjunction nucleating molecule bound to a first anchor sequence, a second conjunction nucleating molecule bound to a discontinuous second anchor sequence, and the association of the first and second conjunction nucleating molecules. In some embodiments, the conjunction nucleating molecule can disrupt the binding of an endogenous conjunction nucleating molecule to its binding site, for example, by competitive binding.

[0218] Conjunction nucleating molecules can be, for example, CTCF, cohesin, USF1, YY1, TATA box-binding protein-associated factor 3 (TAF3), ZNF143 binding motif, or other polypeptides that promote the formation of anchor sequence-mediated conjunctions. Conjunction nucleating molecules can also be endogenous polypeptides or other proteins, such as transcription factors, e.g., autoimmune modulators (AIREs), other factors, e.g., X-inactivation specific transcripts (XISTs), or engineered polypeptides that are engineered to recognize a specific DNA sequence, such as having a zinc finger, leucine zipper, or bHLH domain for sequence recognition. Conjunction nucleating molecules can modulate DNA interactions within or around anchor sequence-mediated conjunctions. For example, a conjunction nucleating molecule can recruit other factors to the anchor sequence that alter anchor sequence-mediated conjunction formation or disruption.

[0219] Conjunction nucleating molecules may also have dimerization domains for homodimerization or heterodimerization. For example, one or more endogenous and engineered conjunction nucleating molecules may interact to form an anchor sequence-mediated conjunction. In some embodiments, conjunction nucleating molecules are engineered to further include stabilization domains, e.g., aggregation interaction domains, to stabilize the anchor sequence-mediated conjunction. In some embodiments, conjunction nucleating molecules are engineered to bind to a target sequence, e.g., their target sequence binding affinity is modulated. In some embodiments, conjunction nucleating molecules are selected or engineered by selecting their binding affinity to the anchor sequence within the anchor sequence-mediated conjunction. Conjunction nucleating molecules and their corresponding anchor sequences can be identified by using cells harboring CTCF inactivating mutations and by chromosome conformation capture or 3C-based methods, e.g., Hi-C or high-throughput sequencing, to investigate topological interactions between topologically related domains, e.g., distal DNA regions or loci, in the absence of CTCF. Long-term DNA interactions can also be identified. Further analysis may include ChlA-PET analysis using baits, such as cohesin, YY1, or USF1, and ZNF143 binding motifs, and MS to identify complexes associated with the baits.

[0220] B. Effector Molecules Effector molecules for use in the compositions and methods of the present invention may include those that modulate biological activity, such as enhancing or reducing enzyme activity, gene expression, cell signaling, and cell or organ function. Preferred effector molecules of the present invention include nucleases, physical blockers, epigenetic recruiters, such as transcription enhancers or transcription repressors, and epigenetic CpG modifiers, such as DNA methylases, DNA demethylases, histone modifiers, histone transacetylases, or histone deacetylases, as well as any combination thereof.

[0221] Further effector activity may include binding regulatory proteins for modulating the activity of regulatory factors, such as transcription or translation. Effector molecules may also include activator or inhibitor (or “negative effector”) functions as described herein. In another example, an effector molecule may inhibit the binding of a substrate to a receptor and inhibit its activation; for example, naltrexone and naloxone bind to opioid receptors without activating them, blocking the receptor’s ability to bind opioids. Effector molecules may also modulate protein stability / degradation and / or transcript stability / degradation. For example, a protein may be marked for degradation on the protein surface, making it a target for degradation by the polypeptide cofactor ubiquitin. In another example, effector molecules inhibit enzyme activity by blocking the active site of the enzyme. For instance, methotrexate is a structural analog of tetrahydrofolate, a coenzyme of dihydrofolate reductase, an enzyme that inhibits nucleotide base synthesis, and binds to dihydrofolate reductase 1000 times more strongly than its native substrate.

[0222] In some embodiments, the effector molecule is a chemical substance that modulates cytosine (C) or adenine (A) (e.g., sodium bisulfite, ammonium bisulfite). In some embodiments, the effector molecule has enzymatic activity (methyltransferase, demethylase, nuclease (e.g., Cas9), deaminase). In some embodiments, the effector molecule sterically interferes with the formation of an anchor sequence-mediated conjunction or the binding of RNA polymerase to a promoter.

[0223] The effector molecule having effector activity may be any one of the following: a small molecule, a peptide, a fusion protein, a nucleic acid, a nanoparticle, an aptamer, or a drug having insufficient PK / PD as described herein.

[0224] In some embodiments, the effector molecule is an inhibitor or a "negative effector molecule." In the context of a negative effector molecule that modulates the formation of an anchor sequence-mediated conjunction, in some embodiments, the negative effector molecule is characterized by the fact that the dimerization of the endogenous nucleating polypeptide is reduced in the presence of the negative effector molecule compared to the absence of the negative effector molecule. For example, in some embodiments, the negative effector molecule is a variant of the dimerization domain of the endogenous nucleating polypeptide, or a dimerization moiety thereof, or includes such variant.

[0225] For example, in certain embodiments, the anchor sequence-mediated conjunction is modified (e.g., disrupted) by the use of a protein having a dominant-negative effector, e.g., a protein that recognizes and binds to an anchor sequence (e.g., a CTCF binding motif) but has an inactive (e.g., mutated) dimerization domain, e.g., a dimerization domain that cannot form a functional anchor sequence-mediated conjunction. For example, the zinc finger domain of CTCF can be modified to bind to a specific anchor sequence (by adding a zinc finger that recognizes an adjacent nucleic acid), while the homodimerization domain can be modified to prevent interaction between the manipulated CTCF and the endogenous form of CTCF.

[0226] In some embodiments, the effector molecule includes a synthetic conjunction nucleating molecule having a selected binding affinity to the anchor sequence within the target anchor sequence-mediated conjunction (the binding affinity may be at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% of the affinity of the endogenous conjunction nucleating molecule associated with the target anchor sequence, or higher or lower than such an affinity. The synthetic conjunction nucleating molecule may have amino acid sequence identity between 30-90%, 30-85%, 30-80%, 30-70%, 50-80%, and 50-90% with respect to the endogenous conjunction nucleating molecule). Conjunction nucleating molecules can disrupt the binding of endogenous conjunction nucleating molecules to their anchor sequences, for example, by competitive binding. In some further embodiments, conjunction nucleating molecules are manipulated to bind to novel anchor sequences within anchor sequence-mediated conjunctions.

[0227] In some embodiments, the dominant-negative effector molecule has a domain that recognizes a specific DNA sequence (e.g., an anchor sequence, a CTCF anchor sequence, adjacent to a sequence-specificity-constituting sequence) and a second domain that provides steric presence near the anchoring sequence. The second domain may include a dominant-negative conjunction nucleating molecule or a fragment thereof, a polypeptide that interferes with the recognition of the conjunction nucleating molecule sequence (e.g., a peptide / nucleic acid or PNA amino acid backbone), a nucleic acid sequence ligated to a steric-constituting small molecule, or any other combination of a DNA-recognizing element and a steric blocker.

[0228] In some embodiments, the effector molecule is an epigenetic modifier. Useful epigenetic modifiers in the methods and compositions described herein include, for example, agents that affect DNA methylation / demethylation, histone acetylation / deacetylation, and RNA-related silencing. In some embodiments, the effector sequence-specifically targets an epigenetic enzyme (e.g., an enzyme that causes or removes an epigenetic mark, e.g., acetylation and / or methylation). An exemplary epigenetic effector can target an expression regulatory region, such as a transcriptional regulatory element or anchor sequence, with a site-specific disruptor containing a site-specific targeting moiety.

[0229] In some embodiments, the effector molecule comprises one or more components of a gene editing system. These gene editing system components can be used in a variety of situations, including but not limited to gene editing. For example, such components can be used to target drugs that physically, genetically, and / or epigenetically modify the FOXP3 sequence.

[0230] Examples of gene editing systems include clustered regulatory interspaced short palindromic repeat (CRISPR) systems, zinc finger nucleases (ZFNs), and activator-like effector-based nucleases (TALENs). Methods based on ZFNs, TALENs, and CRISPR are described, for example, in Gaj et al. Trends Biotechnol. 31.7(2013):397-405, and the CRISPR method for gene editing is described, for example, in Guan et al, Application of CRISPR-Cas system in gene therapy: Pre-clinical progress in animal model. DNA Repair 2016 July 30 [Epub ahead of print]; 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.

[0231] The CRISPR system is an adaptive defense system originally discovered in bacteria and archaea. The CRISPR system uses RNA-inducible nucleases called CRISPR-associated or "Cas" endonucleases (e.g., Cas9 or Cpfl) to cleave foreign DNA. In a typical CRISPR / Cas system, the endonuclease is directed to a target nucleotide sequence (e.g., a site in the genome to be sequence-edited) by a sequence-specific, non-coding "guide RNA" that targets a single-stranded 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). One class II CRISPR system includes a type II Cas endonuclease, e.g., Cas9, CRISPR RNA ("crRNA"), and transactivating crRNA ("tracrRNA"). The crRNA contains a "guide RNA," which is typically an approximately 20-nucleotide RNA sequence corresponding to the target DNA sequence. crRNA also contains a region that binds to tracrRNA to form a partially double-stranded structure, which is cleaved by RNase III, resulting in a crRNA / tracrRNA hybrid. The crRNA / tracrRNA hybrid then instructs Cas9 endonuclease to recognize and cleave target DNA. The target DNA sequence should generally be adjacent to a "protospacer facilitation motif ("PAM")" that is specific to a given Cas endonuclease, but PAM sequences appear throughout a given genome. CRISPR endonucleases identified from various prokaryotic species require specific PAM sequences, and examples of PAM sequences include 5'-NGG (Streptococcus pyogenes), 5'-NNAGAA (Streptococcus thermophilus CRISPR1), 5'-NGGNG (Streptococcus thermophilus CRISPR3), and 5'-NNNGATT (Neisseria meningiditis).Some endonucleases, such as Cas9 endonuclease, associate with G-rich PAM sites, e.g., 5'-NGG, and perform blunt-end cleavage of target DNA at a position 3 nucleotides upstream (5' side) from the PAM site. Another class II CRISPR system includes smaller V-type endonucleases than Cas9, Cpfl, examples of which include AsCpfl (from Acidaminococcus sp.) and LbCpfl (from Lachnospiraceae sp.). Cpf1-associated CRISPR arrays process to mature crRNA without requiring tracrRNA. In other words, the Cpfl system requires only the Cpfl nuclease and crRNA to cleave the target DNA sequence. Cpfl endonuclease associates with T-rich PAM sites, e.g., 5'-TTN. Cpfl can also recognize the 5'-CTA PAM motif. Cpfl cleaves target DNA by introducing offset or staggered double-strand breaks at the 5' overhang of 4 or 5 nucleotides, for example, cleaving the target DNA with a 5-nucleotide offset or staggered cut located 18 nucleotides downstream (3' side) from the PAM site on the coding strand and 23 nucleotides downstream from the PAM site on the complementary strand. The resulting 5-nucleotide overhang from such offset cleavage allows for more precise genome editing by homologous recombination DNA insertion than insertion with blunt-end cut DNA. See, for example, Zetsche et al. (2015) Cell, 163:759-771.

[0232] Various CRISPR-related (Cas) genes or proteins can be used in this invention, and the selection of the Cas protein will depend on the specific conditions of the method.

[0233] Specific examples of Cas proteins include the Class II system, which includes Casl, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Casl0, Cpfl, C2C1, or C2C3. In some embodiments, the Cas protein, e.g., the Cas9 protein, may be from any of various prokaryotic species. In some embodiments, a specific Cas protein, e.g., a specific Cas9 protein, is selected to recognize a specific protospacer-adjacent motif (PAM) sequence. In some embodiments, the site-directed targeting moiety includes a sequence-targeting polypeptide, e.g., an enzyme, e.g., Cas9. In certain embodiments, the Cas protein, e.g., the Cas9 protein, may be obtained from bacteria or archaea or synthesized using known methods. In certain embodiments, the Cas protein may be from Gram-positive or Gram-negative bacteria. In certain embodiments, the Cas protein may be from Streptococcus (e.g., S. pyogenes, S. thermophilus), Crptoococcus, Corynebacterium, Haemophilus, Eubacterium, Pasteurella, Prevotella, Veillonella, or Marinobacter. In some embodiments, nucleic acids encoding two or more different Cas proteins, or two or more Cas proteins, may be introduced into cells, zygotes, embryos, or animals to enable, for example, the recognition and modification of sites containing the same, similar, or different PAM motifs.In some embodiments, the Cas protein is modified to inactivate nucleases, for example, to nuclease-deficient Cas9; to recruit transcription activators or repressors, such as the cosubunit of E. coli Pol, VP64, the activation domain of p65, KRAB, or SID4X; and to induce epigenetic alterations, such as recruiting histone acetyltransferases, histone methyltransferases and demethylases, DNA methyltransferases, and enzymes involved in DNA demethylation (e.g., TET family enzymes catalyze the oxidation of 5-methylcytosine to 5-hydroxymethylcytosine and more oxidative derivatives).

[0234] For gene editing purposes, CRISPR arrays can be designed to contain one or more guide RNA sequences corresponding to a desired target DNA sequence. See, for example, Cong et al. (2013) Science, 339:819-823; Ran et al. (2013) Nature Protocols, 8:2281-2308. Creotides are required for Cas9 to perform DNA cleavage, and for Cpfl, at least approximately 16 nucleotides of the gRNA sequence are required to achieve detectable DNA cleavage.

[0235] Wild-type Cas9 induces double-strand breaks (DSBs) at specific DNA sequences targeted by gRNAs, but several functionally modified CRISPR endonucleases are available. For example, the "nickase" version of Cas9 induces only single-strand breaks, and non-catalytic Cas9 ("dCas9") interferes with transcription through steric hindrance rather than cleaving target DNA. dCas9 can be further fused with heterologous effectors (CRISPRi) to suppress the expression of target genes or heterologous effectors (CRISPRa) to activate them. For example, Cas9 can be fused with a transcription silencer (e.g., a KRAB domain) or a transcription activator (e.g., a dCas9-VP64 fusion). Non-catalytical Cas9 (dCas9) fused with a Fokl nuclease ("dCas9-FokI") can be used to induce DSBs at target sequences homologous to two gRNAs. For example, refer to the numerous CRISPR / Cas9 plasmids disclosed and publicly available in the Addgene repository (Addgene, 75 Sidney St., Suite 550A, Cambridge, MA 02139; addgene.org / crispr). “Double nickase” Cas9, which introduces two separate double-strand breaks, each directed by a separate guide RNA, achieves more precise genome editing, as described by Ran et al. (2013) Cell, 154: 1380-1389.

[0236] CRISPR technology for editing eukaryotic genes is disclosed in U.S. Patent Applications Publications 2016 / 0138008A1 and 2015 / 0344912A1, and in U.S. Patents 8,697,359, 8,771,945, 8,945,839, 8,999,641, 8,993,233, 8,895,308, 8,865,406, 8,889,418, 8,871,445, 8,889,356, 8,932,814, 8,795,965, and 8,906,616. The Cpfl endonuclease, along with its corresponding guide RNA and PAM site, is disclosed in U.S. Patent Application Publication No. 2016 / 0208243A1.

[0237] In some embodiments, the effector comprises one or more CRISPR-based components described above in this specification.

[0238] In some embodiments, effectors suitable for use in the agents, compositions, and methods of the present invention include, for example, nucleases, physical blockers, epigenetic recruiters such as transcription enhancers or transcription repressors, and epigenetic CpG modifiers such as DNA methylases, DNA demethylases, histone modifiers, histone transacetylases, or histone deacetylases, as well as any combination thereof.

[0239] Preferred effectors include polypeptides or their variants. The term “variant,” as used herein, refers to a polypeptide obtained by incorporating one or more amino acid insertions, substitutions, or deletions into a precursor polypeptide (e.g., the “parent” polypeptide). In certain embodiments, the variant polypeptide has at least about 85% amino acid sequence identity with respect to the entire amino acid sequence of the parent polypeptide, e.g., about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% amino acid sequence identity.

[0240] As used herein, the term “sequence identity” refers to the comparison between a pair of nucleic acids or amino acid molecules, i.e., the relationship between two amino acid sequences or two nucleotide sequences. Generally, sequences are aligned to obtain the highest-order match. Methods for determining sequence identity are publicly known and can be determined by commercially available computer programs capable of calculating the identity percentage between two or more sequences. A representative example of such a computer program is CLUSTAL.

[0241] Exemplary effectors include ubiquitin, bicyclic peptides as ubiquitin ligase inhibitors, transcription factors, DNA and protein modifying enzymes such as topoisomerases, topoisomerase inhibitors such as topotecans, DNA methyltransferases such as the DNMT family (e.g., DNMT3a, DNMT3b, DNMTL), protein methyltransferases (e.g., viral lysine methyltransferase (vSET), protein lysine N-methyltransferase (SMYD2), deaminases (e.g., FOXP3)EC, UG1), histone methyltransferases, e.g., zeste homolog 2 enhancer (EZH2), PRMT1, histone-lysine-N-methyltransferase (Setdbl), histone methyltransferase (SET2), euchromatin histone-lysine-N-methyltransferase 2 (G9a), histone-lysine-N-methyltransferase (SUV39H1, and G9a), histone deacetylases (e.g., HDACl, HDAC2, HDAC3), enzymes involved in DNA demethylation (e.g., TET family enzymes catalyze the oxidation of 5-methylcytosine to 5-hydroxymethylcytosine and more oxidative derivatives), protein demethylases, e.g., KDMIA and lysine-specific histone demethylase 1 (LSD1), helicases, e.g., DHX9, acetyltransferases, deacetylases (e.g., sirtuin 1, 2, 3, 4, 5, 6 or 7) kinases, phosphatases, DNA insertion agents, e.g., ethidium bromide, cybergreen and proflavin, efflux pump inhibitors, e.g., peptide mimetic agents such as phenylalanine arginyl naphthylamide or quinoline derivatives, nuclear receptor activators and inhibitors, proteasome inhibitors, competitive inhibitors against enzymes such as those involved in lysosomal storage disorders, zinc finger proteins, TALENs, specific domains from proteins, e.g., KRAB domain, VP64 domain, p300 domain (e.g., p300 core domain), MeCP2 domain, MQ1 domain, DNMT3a-3L domain, TET1 domain and / or TET2 domain, protein synthesis inhibitors, nucleases (e.g., Cpfl, Cas9, zinc finger nucleases), one or more fusions thereof (e.g., dCas9-DNMT, dCas9-FOXP3) Examples include EC, dCas9-UGl, dCas9-VP64, dCas9-p300 core, dCas9-KRAB, dCas9-KRAB-MeCP2, dCas9-MQ1, dCas9-DNMT3a-3L, dCas9-TET1 / TET2, and dCas9-MC / MN.

[0242] In some embodiments, the nuclease suitable for use in the drugs, compositions, and methods of the present invention comprises a Cas9 polypeptide or an enzymatically active moiety thereof. In one embodiment, the Cas9 polypeptide or its enzymatically active moiety further comprises the catalytic domain of human exonuclease 1 (hEXO1), e.g., 5'-to-3' exonuclease activity and / or RNase H activity. In other embodiments, the suitable nuclease comprises a transcription activator-like effector nuclease (TALEN). In yet another embodiment, the suitable nuclease comprises a zinc finger protein.

[0243] The term TALEN, as used herein, is broad and includes monomeric TALENs capable of cleaving double-stranded DNA without the assistance of another TALEN. The term TALEN is also used to refer to one or both members of a pair of TALENs that are manipulated to work together to cleave DNA at the same site. The working TALENs are sometimes referred to as left TALEN and right TALEN, in reference to the palmatity of the DNA. See USSN12 / 965,590, USSN13 / 426,991 (US8,450,471), USSN13 / 427,040 (US8,440,431), USSN13 / 427,137 (US8,440,432), and USSN13 / 738,381, all of which are incorporated herein by reference.

[0244] TAL effector (TALE) is a protein secreted by the bacterium Xanthomonas. Its DNA-binding domain contains a 33-34 amino acid sequence that is highly conserved except for the 12th and 13th amino acids. These two positions are highly variable (repeat variable duo (RVD)) and show a strong correlation with specific nucleotide recognition. This simple relationship between the amino acid sequence and DNA recognition has made it possible to manipulate the specific DNA-binding domain by selecting a combination of repeat segments containing the appropriate RVD.

[0245] Using the nonspecific DNA cleavage domain from the terminal of the FokI endonuclease, hybrid nucleases active in yeast assays can be constructed. These reagents are also active in plant and animal cells. Early TALEN studies used the wild-type FokI cleavage domain, but some subsequent TALEN studies also used FokI cleavage domain variants with mutations designed to improve cleavage specificity and activity. The FokI domain functions as a dimer, requiring two constructs with specific DNA-binding domains for a site in the target genome, oriented and spaced appropriately. Both the number of amino acid residues between the TALE DNA-binding domain and the FokI cleavage domain, and the number of bases between the two individual TALE binding sites, are parameters for achieving high activity levels. The number of amino acid residues between the TALE DNA-binding domain and the FokI cleavage domain can be modified by introducing spacers (clearly distinct from spacer sequences) between multiple TAL effector repeat sequences and the FokI endonuclease domain. The spacer sequence can consist of 12 to 30 nucleotides, for example, 12 to 15, 12 to 20, 20 to 25, or 15 to 30 nucleotides.

[0246] The relationship between the amino acid sequence of the TALE-binding domain and DNA recognition allows for the consideration of designable proteins. In this case, artificial gene synthesis presents many problems due to the improper annealing of the repetitive sequences found in the TALE-binding domain. One solution to this problem is to use publicly available software programs (DNAWorks) to calculate oligonucleotides suitable for assembly in a two-step PCR: assembly of oligonucleotides followed by whole-gene amplification. Several modular assembly schemes for generating manipulated TALE constructs have also been reported. Both methods provide systematic approaches for manipulating DNA-binding domains, conceptually similar to modular assembly methods for generating zinc finger DNA recognition domains.

[0247] Once assembled, TALEN genes are inserted into plasmids, which are then used to transfect target cells. In these target cells, the gene product is expressed, enters the nucleus, and approaches the genome. Using TALENs, the genome can be edited by inducing double-strand breaks (DSBs), which the cell responds to with repair mechanisms. In this manner, TALENs can be used to correct genomic mutations that cause diseases, for example.

[0248] As used herein, “zinc finger polypeptide” or “zinc finger protein” is a protein that binds sequence-specifically to DNA, RNA, and / or proteins via a metal-stabilizing domain known as a zinc finger. A zinc finger protein is a nuclease having a DNA cleavage domain and a DNA-binding zinc finger domain. Zinc finger polypeptides can be constructed by fusing the nonspecific DNA cleavage domain of an endonuclease with a site-specific DNA-binding zinc finger domain. Such nucleases are powerful tools for gene editing and can be assembled to induce site-specific double-strand breaks (DSBs) in genomic DNA. ZFNs enable specific gene disruption during DNA repair, such that the targeted gene can be disrupted by mutagenic non-homologous end joining (NHEJ) or modified by homologous recombination (HR) when a closely related DNA template is supplied.

[0249] Zinc finger nucleases are chimeric enzymes created by fusing the nonspecific DNA cleavage domain of the endonuclease FokI with a site-specific DNA-binding zinc finger domain. Due to the flexible nature of zinc finger proteins (ZFPs), ZFNs can be assembled to induce site-specific double-strand breaks (DSBs) in genomic DNA. ZFNs enable specific gene disruption during DNA repair, where the targeted gene can be disrupted by mutagenic non-homologous end joining (NHEJ) or modified by homologous recombination (HR) when a closely related DNA template is supplied.

[0250] In some embodiments, the physical blockers suitable for use in the agents, compositions, and methods of the present invention include gRNA, antisense DNA, or triple-stranded oligonucleotides (which may target expression regulatory units), steric blocks, transcriptional regulatory elements, or anchoring sequences. The gRNA includes sequences that recognize specific DNA sequences and further function as steric blockers by interfering, for example, with conjunction nucleation molecular sequences. In some embodiments, the gRNA is compounded with one or more peptides that function as steric entities, such as S-adenosylmethionine (SAM). In other embodiments, the physical blocker includes an enzymatically inactive Cas9 polypeptide or a fragment thereof (e.g., dCas9).

[0251] In one embodiment, the epigenetic recruiter activates or enhances the transcription of a target gene. In some embodiments, the epigenetic recruiter suitable for use in the drugs, compositions, and methods of the present invention comprises a VP64 domain or a p300 core domain.

[0252] In one embodiment, the epigenetic recruiter silences or represses the transcription of a target gene. In some embodiments, the epigenetic recruiter suitable for use in the drugs, compositions, and methods of the present invention comprises a KRAB domain or a MeCP2 domain.

[0253] In one embodiment, suitable epigenetic recruiting factors for use in the drugs, compositions, and methods of the present invention include dCas9-VP64 fusions, dCas9-p300 core fusions, dCas9-KRAB fusions, or dCas9-KRAB-MeCP2 fusions.

[0254] As used herein, "VP64" is a transcription activator consisting of four tandem copies of VP16 (herpes simplex virus protein 16, amino acids 437-447*: DALDDFDLDML (SEQ ID NO: 95)) linked by a glycine-serine (GS) linker. In one embodiment, VP64 further includes the transcription factors p65 and Rta at its C-terminus. VP64 containing p65 and Rta is sometimes referred to as "VPR" or "VP64-p65-Rta". VP64-p65-Rta, or VPR, was created by adding the transcription factors p65 and Rta to the C-terminus of Vp64. Thus, all three transcription factors can target the same gene. The use of all three transcription factors, not just Vp64, can result in increased expression of the targeted gene. The GenBank accession number for VP64 is ADD60007.1, the GenBank accession number for p65 is NP_001138610.1, and the GenBank accession number for Rta is AAA66528.1.

[0255] An example amino acid sequence of VPR is as follows: [ka]

[0256] As used herein, “p300 core domain” refers to the catalytic core of the human acetyltransferase p300. The GenBank accession number for the protein containing p300 is NP_001420.2.

[0257] An example amino acid sequence of p300 is as follows: [ka]

[0258] As used herein, "KRAB" refers to the Kruppel-associated box (KRAB) transcriptional repression domain present in the human zinc finger protein-based transcription factor (KRAB zinc finger protein).

[0259] As used herein, "MeCp2" refers to a methyl CpG-binding protein 2 that, for example, inhibits transcription by binding to a promoter containing methylated DNA.

[0260] In one embodiment, the epigenetic CpG modifier methylates DNA, inactivating or repressing transcription. In some embodiments, the epigenetic CpG modifier suitable for use in the drugs, compositions, and methods of the present invention comprises an MQ1 domain or a DNMT3a-3L domain.

[0261] In one embodiment, the epigenetic CpG modifier demethylates DNA and activates or stimulates transcription. In some embodiments, the epigenetic recruiting factors suitable for use in the agents, compositions, and methods of the present invention include a TET1 or TET2 domain.

[0262] As used herein, "MQ1" refers to prokaryotic DNA methyltransferase.

[0263] As used herein, "DNMT3a-3L" refers to a fusion of the DNA methyltransferase Dnmt3a and Dnmt3L, which is non-catalyzed but directly interacts with the catalytic domain of Dnmt3a.

[0264] As used herein, “TET1” refers to “10-11 translocation methylcytosine dioxygenase 1,” a member of the TET enzyme family, encoded by the TET1 gene. TET1 is a dioxygenase that catalyzes the conversion of the modified DNA base 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) by oxidation of 5-mC in an iron and alpha-ketoglutarate-dependent manner, which is the first step in active DNA demethylation in mammals. Methylation of the C5 position of cytosine bases is an epigenetic modification of the mammalian genome that plays a crucial role in transcriptional regulation. In addition to its role in DNA demethylation, it plays a more common role in chromatin regulation. It preferentially binds to CpG-rich sequences in the promoters of both transcriptional activating genes and Polycomb repressing genes. It is involved in the recruitment of O-GlcNAc transferase OGT to the CpG-rich transcription start site of activating genes, thereby promoting histone H2B GlcN acylation by OGT. Exemplary TET1 nucleotide and amino acid sequences can be found in GenBank accessions NM_030625.3 and NP_085128.2.

[0265] As used herein, “TET2” refers to “10-11 translocation 2 (TET2),” a member of the TET enzyme family encoded by the TET1 gene. Similar to TET1, TET2 is a dioxygenase that catalyzes the conversion of the modified genomic base 5-methylcitrin (5mC) to 5-hydroxymethylcytosine (5hmC), playing a crucial role in active DNA demethylation. TET2 prefers 5-hydroxymethylcytosine in the CpG motif. TET2 also mediates the subsequent conversion of 5hmC to 5-formylcytosine (5fC), and the conversion of 5fC to 5-carboxylcytosine (5caC). The conversion of 5mC to 5hmC, 5fC, and 5caC likely constitutes the first step in cytosine demethylation. Methylation of cytosine bases at the C5 position is an epigenetic modification of the mammalian genome that plays a vital role in transcriptional regulation. In addition to its role in DNA demethylation, it is also involved in the recruitment of the O-GlcNAc transferase OGT to the CpG-rich transcription start site of active genes, thereby promoting histone H2B GlcN acylation by OGT. Exemplary nucleotide and amino acid sequences can be found in GenBank accessions NM_001127208.2 and NP_001120680.1.

[0266] In some embodiments, the epigenetic mobilizers suitable for use in the drugs, compositions, and methods of the present invention include the MQ1 domain, DNMT3a-3L, TET1, or TET2 domain. In one embodiment, the epigenetic mobilizers suitable for use in the drugs, compositions, and methods of the present invention include the dCas9-MQ1 fusion, the dCas9-DNMT3a-3L fusion, the dCas9-TET1 fusion, or the -dCas9-TET2 fusion.

[0267] III. Delivery of the site-specific foxp3 disruptor of the present invention and compositions containing the site-specific foxp3 disruptor of the present invention The delivery of the disruptor of the present invention to cells in a subject, such as a human subject (e.g., a subject requiring it, e.g., a subject with autoimmune diseases such as FOXP3-related disorders, e.g., IPEX syndrome), can be accomplished in several different ways. For example, delivery can be carried out by contacting cells with the disruptor of the present invention in vitro, ex vivo, or in vivo. In vivo delivery can be carried out directly by administering a composition containing the disruptor, e.g., a lipid composition, to the subject. Alternatively, in vivo delivery can be carried out indirectly by administering one or more vectors that encode the disruptor and direct its expression in the cells of the subject. These alternatives are discussed further below. In vitro introduction into cells includes methods known in the art, e.g., electroporation and lipofection. Further methods are described below herein and / or are known in the art.

[0268] In some embodiments, the disruptor comprises a nucleic acid molecule encoding a fusion protein, the fusion protein comprising a site-specific FOXP3 targeting moiety, for example, a polynucleotide encoding the DNA-binding domain or a fragment of a transcription activator-like effector (TALE) polypeptide or zinc finger (ZNF) polypeptide, which specifically targets and binds to a FOXP3 expression regulatory region, and an effector molecule, for example, a VPR.

[0269] In other embodiments, the disruptor comprises a guide RNA and mRNA encoding an effector molecule. The ratio of guide RNA to mRNA may be approximately 100:1 to approximately 1:100 (weight:weight).

[0270] In general, any delivery method (in vitro, ex vivo, or in vivo) of the site-specific foxp3 disruptor of the present invention can be adapted for use with the disruptor of the present invention (for example, Akhtar S. and Julian RL., (1992) Trends Cell. Biol. 2(5):139-144 and WO94 / 02, which are incorporated herein by reference in their entirety). (See 595). In the case of in vivo delivery, factors to be considered for delivering the site-specific foxp3 disruptor of the present invention include, for example, the biological stability of the disruptor, prevention of nonspecific effects, and accumulation of the disruptor in the target tissue. Nonspecific effects of the disruptor can be minimized by local administration, for example, by direct injection or implantation into tissue, or by local administration of a composition containing the disruptor. Local administration to the treatment site maximizes the local concentration of the disruptor; limits exposure of systemic tissues to the disruptor that could otherwise be harmed or degraded by the disruptor; and allows for the administration of a lower total dose of the disruptor.

[0271] When site-specific foxp3 disruptors are administered systemically for the treatment of diseases such as foxp3-related disorders, the disruptor, for example, can be modified to include a site-specific targeting moiety containing a nucleic acid molecule, or it can be delivered using a drug delivery system. Both methods act to prevent rapid in vivo degradation of the site-specific targeting moiety containing the nucleic acid molecule by endo- and exonucleases. Modification of the disruptor or drug carrier containing the site-specific targeting moiety containing the nucleic acid molecule also enables targeting of the disruptor to target tissues and avoidance of undesirable off-target effects. For example, the disruptor of the present invention can be modified to enhance cellular uptake and prevent degradation by chemical conjugation to a lipophilic group such as cholesterol.

[0272] Alternatively, the disruptor of the present invention can be delivered using a drug delivery system, such as nanoparticles, dendrimers, polymers, liposomes, or cationic delivery systems. Positively charged cationic delivery systems facilitate the binding of the disruptor (e.g., load molecules) and also enhance interactions at the load cell membrane, enabling efficient uptake of the disruptor by cells. A cationic lipid, dendrimer, or polymer can be bound to the disruptor, or the disruptor can be contained in vesicles or micelles (e.g., Kim SH. et al., (2008)). It can be induced to form vesicles or micelles (see Journal of Controlled Release 129(2):107-116). The formation of vesicles or micelles further prevents the degradation of the disruptor when administered systemically. Methods for preparing and administering cationic complexes are well within the capabilities of those skilled in the art (e.g., Sorensen, DR., et al. (2003) J. Mol. Biol 327:761-766; Verma, UN. et al., (2003) Clin. Cancer Res. 9:1291-1300; Arnold, AS et al. (2007) J. See Hypertens. 25:197-205). Some non-limiting examples of drug delivery systems useful for systemic delivery of the disruptor of the present invention include DOTAP (Sorensen, DR., et al (2003)). See above; Verma, UN. et al., (2003), see above), oligofectamine, "solid nucleic acid lipid particles" (Zimmermann, TS. et al., (2006) Nature 441:111-114), cardio Pin (Chien, PY. et al., (2005) Cancer Gene Ther. 12:321-328; Pal, A. et al., (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine (Bonnet ME. et al., (2008) Pharm. Res. Aug 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp(RGD) Examples include butylated amines (Liu, S. (2006) Mol. Pharm. 3:472-487) and polyamidoamines (Tomalia, DA. et al., (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H. et al., (1999) Pharm. Res. 16:1799-1804). In some embodiments, a disruptor (e.g., gRNA or mRNA) forms a complex with cyclodextrin for systemic administration. Methods for administration and pharmaceutical compositions containing cyclodextrin can be found in U.S. Patent No. 7,427,605, the entire contents of which patent document are incorporated herein by reference.

[0273] The disruptors of the present invention can be incorporated into pharmaceutical compositions suitable for administration. Such compositions generally comprise one or more types of disruptors and a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersions, coatings, antimicrobial and antifungal agents, isotonic and absorption retarders, etc., that are suitable for drug administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agent is incompatible with the active compound, its use in a composition is intended. Co-active compounds can also be incorporated into the composition.

[0274] The pharmaceutical compositions of the present invention can be administered in several ways, depending on whether topical or systemic treatment is desired and the area to be treated. Administration may be topical (including ocular, vaginal, rectal, nasal, and percutaneous), oral, or parenteral. Parenteral administration includes intravenous infusion, subcutaneous, intraperitoneal or intramuscular injection, or subarachnoid or intraventricular administration.

[0275] The route and site of administration may be selected to enhance the delivery or targeting of the disruptor to a specific location, including the site-specific targeting portion. For example, intravenous injection can be used to target liver cells. Lung cells can be targeted by administering the disruptor in aerosol form. Jerusalem cells can be targeted by rectal administration.

[0276] Formulations for local administration include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional drug carriers, aqueous, powder, or oily bases, and thickeners may be necessary or desirable. Coated condoms and gloves may also be useful.

[0277] Compositions for oral administration include powders or granules, suspensions or solutions in water, syrups, elixirs, or non-aqueous media, tablets, capsules, licks, or lozenges. For tablets, possible carriers include salts of lactose, sodium citrate, and phosphoric acid. Various disintegrants, such as starch, and lubricants, such as magnesium stearate, sodium lauryl sulfate, and talc, are commonly used for tablets. For oral administration in capsule form, useful diluents are lactose and high molecular weight polyethylene glycol. When an aqueous suspension is required for oral use, the nucleic acid composition can be combined with emulsifiers and suspending agents. Certain sweeteners or flavorings may be added if desired.

[0278] Examples of compositions for intravenous administration include sterile aqueous solutions, which may also contain buffers, diluents, and other suitable additives.

[0279] Examples of formulations for parenteral administration include sterile aqueous solutions, which may also contain buffers, diluents, and other suitable additives. For intravenous use, the total concentration of the solute can be controlled to make the preparation isotonic.

[0280] In one embodiment, the administration of the disruptor composition of the present invention is parenteral, for example, intravenous (e.g., as a bolus or as a diffusible injector), intradermal, intraperitoneal, intramuscular, subarachnoid, intraventricular, intracranial, subcutaneous, transmucosal, buccal, sublingual, endoscopic, rectal, oral, vaginal, local, pulmonary, intranasal, urethral, ​​or ocular. Administration may be provided by the subject or by another person, for example, a healthcare provider. The composition may be provided in measured doses or in dispensers that deliver measured doses. The selected mode of delivery will be discussed in more detail below.

[0281] In certain embodiments, the disruptor of the present invention is a polynucleotide, such as mRNA, which is formulated with lipid nanoparticles (LNPs).

[0282] A. Composition comprising the site-specific foxp3 disruptor of the present invention The site-directed foxp3 disruptor of the present invention can be formulated into compositions such as pharmaceutical compositions with one or more excipients for the following purposes: (1) increasing stability, (2) increasing cell transfection, (3) enabling sustained or delayed release (e.g., from a depot formulation), (4) altering in vivo distribution (e.g., targeting the disruptor to a specific tissue or cell type), (5) increasing in vivo translation of the encoded protein, and / or (6) altering the in vivo release profile of the encoded protein. In addition to conventional excipients, such as any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surfactants, isotonic agents, thickeners or emulsifiers, and preservatives, excipients for use in the compositions of the present invention may, without limitation, include lipidoids; liposomes; lipid nanoparticles; polymers; lipoplexes; core-shell nanoparticles; peptides; proteins; nucleic acid molecules, modified nucleic acid molecules, or cells transfected with RNA (e.g., for transplantation into a subject); hyaluronidases; nanoparticle mimics; and combinations thereof. Accordingly, the pharmaceutical compositions of the present invention may contain one or more excipients, each in amounts that increase the stability of the disruptor, increase cell transfection by the disruptor, increase the expression of modified nucleic acids or mRNA-encoded proteins, and / or alter the release profile of the disruptor. Furthermore, the disruptors of the present invention can be formulated using self-assembling nucleic acid nanoparticles (see, for example, U.S. Patent Application Publication 2016 / 0038612A1, which is incorporated herein by reference in its entirety).

[0283] i. Lipidoids The synthesis of lipidoids is widely described, and formulations containing these compounds are particularly suitable for the delivery of the disruptors of the present invention, for example, disruptors containing site-specific FOXP3 targeting moieties, including nucleic acid molecules, modified nucleic acid molecules, or mRNA (see Mahon et al., Bioconjug Chem. 2010 21:1448-1454; Schroeder et al., J Intern Med. 2010 267:9-21; Akinc et al., Nat Biotechnol. 2008 26:561-569; Love et al., Proc Natl Acad Sci USA. 2010 107: 1864-1869; Siegwart et al., Proc Natl Acad Sci USA. 2011 108:12996-3001). All of the contents are incorporated herein.

[0284] For example, lipidoids have been used to effectively deliver double-stranded small interfering RNA molecules, single-stranded nucleic acid molecules, modified nucleic acid molecules, or modified mRNA. (See, for example, U.S. Patent Application Publication No. 2016 / 0038612A1). Complexes, micelles, liposomes, or particles containing these lipidoids can be prepared, resulting in effective delivery, for example, by localized and / or systemic administration, after administration of the lipidoid formulation, as determined by the production of the encoded protein of a site-specific FOXP3 targeting moiety containing the nucleic acid molecule. These lipidoid complexes can be administered by a variety of means, including but not limited to intravenous, intramuscular, intradermal, intraperitoneal, or subcutaneous routes.

[0285] For example, the in vivo delivery of site-specific FOXP3 targeting moieties containing nucleic acid molecules may be affected by many parameters, such as formulation composition, particle PEGylation properties, degree of loading, polynucleotide-to-lipid ratio, and biophysical parameters, including, but not limited to, particle size (the whole is incorporated herein by reference, Akinc et al.). (Mol Ther. 2009 17:872-879). As an example, small changes in the anchor chain length of poly(ethylene glycol) (PEG) lipids can have a significant effect on in vivo efficacy. Formulations containing different lipidoids, including but not limited to penta[3-(1-laurylaminopropiony I)]-triethylenetetramine hydrochloride (TETA-5LAP; also known as 98NI2-5, see Murugaiah et al., Analytical Biochemistry, 401:61 (2010), the contents of which this reference is incorporated herein by reference in their entirety), C12-200 (including derivatives and variants), and MDl, can be used.

[0286] In one embodiment, a disruptor containing a site-specific FOXP3 targeting moiety, for example, a nucleic acid molecule, is formulated with a lipidoid for systemic intravenous administration to target cells in the liver. For example, a final optimized intravenous formulation containing a disruptor containing a site-specific FOXP3 targeting moiety, with a lipid molar composition of 42% 98NI2-5, 48% cholesterol, and 10% PEG-lipid, a final weight ratio of total lipids to nucleic acid molecules of approximately 7.5:1, a C14 alkyl chain length on the PEG-lipid, and an average particle size of approximately 50-60 nm, can result in a hepatic distribution of more than 90% of the formulation (Akinc et al., Mol Please refer to Ther. 2009 17:872-879, as the entire content of this reference is referenced. (More incorporated herein by reference). In another example, an intravenous formulation using a C12-200 lipidoid having a molar ratio of 50 / 10 / 38.5 / 1.5 for C12-200 / disteroylphosphatidylcholine / cholesterol / PEG-DMG, a weight ratio of total lipids to nucleic acid molecules of 7:1, and an average particle size of 80 nm (see, for example, PCT publication number WO2010 / 129709, which is entirely incorporated herein by reference) can be used to deliver a disruptor containing a site-specific FOXP3 targeting moiety containing nucleic acid molecules to hepatocytes (Love et al., Proc Natl Academia). See Sci USA. 2010 107:1864-1869 (the contents of this reference are incorporated herein by reference in their entirety). In another embodiment, an MD1 lipidoid-containing formulation can be used to effectively deliver a disruptor containing a site-specific FOXP3 targeting moiety, which includes a nucleic acid molecule, to hepatocytes in vivo. The characteristics of optimized lipidoid formulations for intramuscular or subcutaneous pathways can vary considerably depending on the target cell type and the formulation's ability to diffuse into the bloodstream through the extracellular matrix. Due to the size of the endothelial pores, particle sizes less than 150 nm may be desirable for effective hepatocyte delivery (see Akinc et al., Mol Ther. 2009 17:872-879). Please refer to the reference (the entire contents of which are incorporated herein by reference), but the use of lipidoid formulation nucleic acid molecules for delivering formulations to other cell types, including but not limited to endothelial cells, myeloid cells, and muscle cells, may not be subject to similar size limitations. The use of lipidoid formulations for in vivo delivery of siRNA to non-hepatocyte cells, such as myeloid cells and endothelium, has been reported (Akinc et al., Nat Biotechnol. 200826:561-569;Leuschner et al., Nat Biotechnol. 2011 See 29: 1005-101 0; Cho et al. Adv. Funct. Mater. 2009 19:3112-3118; 8th International Judah Folkman Conference, Cambridge, Mass. Oct. 8-9, 2010 (the contents of each of these references are incorporated herein by reference in their entirety). For delivery to myeloid cells such as monocytes, lipidoid formulations may have similar molar ratios of components. By using different ratios of lipidoids with other components, including but not limited to distearoylphosphatidylcholine, cholesterol, and PEG-DMG, formulations can be optimized for delivery to different cell types, including but not limited to hepatocytes, myeloid cells, and muscle cells. For example, possible molar ratios of components include, but are not limited to, 50% CI2-200, 10% distearoylphosphatidylcholine, 38.5% cholesterol, and 1.5% PEG-DMG (see Leuschner et al., Nat Biotechnol 2011 29: 1005-101 0, the contents of which are incorporated herein by reference in their entirety). The use of lipidoid formulations for localized delivery to cells (e.g., adipocytes and muscle cells, but not limited to these) by subcutaneous, intradermal, or intramuscular delivery may not require all of the formulation components desired for systemic delivery, and may, for example, only include the lipidoid and a disruptor containing a site-specific FOXP3 targeting moiety, such as a nucleic acid molecule, as described herein.

[0287] The efficacy of a formulation can be improved by using combinations of different lipidoids to increase cell transfection and / or increase the translation of the encoded proteins contained therein (see Whitehead et al., Mol. Ther. 2011, 19:1688-1694, the entire content of this reference is referenced in this specification). (It will be incorporated into the book.)

[0288] In one embodiment, the lipidoid can be prepared by the conjugate addition of an alkylamine (alklamines) to an acrylate. In a non-limiting example, the lipidoid can be prepared by the method described in PCT Patent Publication No. WO2014 / 028487, the contents of which are incorporated herein by reference in their entirety. In one embodiment, the lipidoid may include a compound having formula (I), formula (II), formula (III), formula (IV), or formula (V) as described in PCT Patent Publication No. WO2014 / 028487, the contents of which are incorporated herein by reference in their entirety. In one embodiment, the lipidoid may be biodegradable.

[0289] ii. Liposomes, lipoplexes, and lipid nanoparticles The disruptors of the present invention can be formulated using one or more liposomes, lipoplexes, or lipid nanoparticles. In one embodiment, the pharmaceutical composition of the present invention comprises liposomes. Liposomes are artificially prepared vesicles composed primarily of lipid bilayers and can be used as delivery vehicles for administering nutrients and pharmaceutical formulations. Liposomes can be of various sizes, for example, multilayer vesicles (MLVs) which may have a diameter of several hundred nanometers and contain a series of concentric bilayers separated by narrow aqueous compartments, small single-cell vesicles (SUVs) which may have a diameter of less than 50 nm, and large monolayer vesicles (LUVs) which may have a diameter between 50 and 500 nm. The liposome design may include, but are not limited to, opsonins or ligands to improve the adhesion of liposomes to unhealthy tissues or to activate events such as endocytosis. Liposomes may contain low or high pH to improve the delivery of pharmaceutical formulations. Liposome formation may depend on physicochemical characteristics, such as, but not limited to, the pharmaceutical formulation and liposomal components to be encapsulated, the properties of the medium in which the lipid vesicles are dispersed, the effective concentration of the encapsulated material and its potential toxicity, any further processes required during the utilization and / or delivery of the vesicles, the size of the vesicles optimized for their intended use, polydispersibility and shelf life, and the possibility of batch-to-batch reproducibility and mass production of safe and efficient liposomal products.

[0290] As a non-limiting example, liposomes such as synthetic membrane vesicles can be prepared by methods, apparatus and devices described in U.S. Patent Publication Nos. 2013 / 0177638, 2013 / 0177637, 2013 / 0177636, 201 / 30177635, 2013 / 0177634, 2013 / 0177633, 2013 / 0183375, 2013 / 0183373, 2013 / 0183372 and 2016 / 0038612, and PCT Patent Publication No. WO2008 / 042973, the contents of each of these patent documents incorporated herein by reference in their entirety.

[0291] In one embodiment, the pharmaceutical composition described herein, without limitation, contains 1,2-dioleyloxy-N,N-dimethylaminopropane (ami-nopropane) (DODMA) lipo Liposomes may include those formed from DiLa2 liposomes from Marina Biotech (Bothell, Wash.), 1,2-dilinoleyloxy-3-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), and MC3 (the whole of which is incorporated herein by reference, US20100324120), and liposomes capable of delivering small molecule drugs such as DOXIL® from Janssen Biotech, Inc. (Horsham, Pa.). In one embodiment, the pharmaceutical compositions described herein may, without limitation, include liposomes such as those formed from the synthesis of stabilized plasmid-lipid particles (SPLPs) or stabilized nucleic acid-lipid particles (SNALPs), which have been previously described and shown to be suitable for oligonucleotide delivery in vitro and in vivo (Wheeler et al. Gene Therapy. 1999 6:271-281; ​​Zhang et al. Gene Therapy. 1999 6:1438-1447; Jeffs et al. Pharm Res. 2005 22:362-372; Morrissey et al., Nat Biotechnol. 2005 2:1002-1007). See also Zimmermann et al., Nature. 2006 441:111-114; Heyes et al. J Contr Rel. 2005 107:276-287; Semple et al. Nature Biotech. 2010 28:172-176; Judge et al. J Clin Invest. 2009 119:661-673; deFougerolles Hum Gene Ther. 2008 19:125-132; U.S. Patent Publications 2013 / 0122104, 2013 / 0303587, and 2016 / 0038612, the contents of each of these references being incorporated herein by reference). The initial manufacturing method by Wheeler et al. was a surfactant dialysis method, which was later improved by Jeffs et al. and is called a spontaneous vesicle formation method. The liposome formulation of the present invention may consist of three to four lipid components in addition to a disruptor containing a site-specific FOXP3 targeting moiety. As an example, the liposome of the present invention may contain, but is not limited to, 55% cholesterol, 20% distearoylphosphatidylcholine (DSPC), 10% PEG-SDSG, and 15% 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), as described by Jeffs et al. As another example, the liposomal formulations of the present invention may contain, but are not limited to, 48% cholesterol, 20% DSPC, 2% PEG-c-DMA, and 30% cationic lipids, as described by Heyes et al., the cationic lipids may be 1,2-distearoyl-N,N-dimethylaminopropane (DSDMA), DODMA, DLin-DMA, or 1,2-dilinolenyloxy-3-dimethylaminopropane (DLenDMA). In some embodiments, the liposomal formulations may contain about 25.0% to about 40.0% cholesterol, about 30.0% to about 45.0% cholesterol, about 35.0% to about 50.0% cholesterol, and / or about 48.5% to about 60% cholesterol.In another embodiment, the formulation of the present invention may contain a percentage of cholesterol selected from the group consisting of 28.5%, 31.5%, 33.5%, 36.5%, 37.0%, 38.5%, 39.0%, and 43.5%. In some embodiments, the liposomal formulation of the present invention may contain about 5.0% to about 10.0% DSPC and / or about 7.0% to about 15.0% DSPC.

[0292] In one embodiment, the pharmaceutical composition may comprise liposomes that can be formed to deliver the disruptor of the present invention. A disruptor comprising a site-specific FOXP3 targeting moiety containing __ can be encapsulated by liposomes and / or housed in an aqueous core, which can then be encapsulated by liposomes (see, for example, PCT Patent Publications WO2012 / 031046, WO2012 / 031043, WO2012 / 030901 and WO2012 / 006378 and U.S. Patent Publications 2013 / 0189351, 2013 / 0195969 and 201 / 30202684, the contents of each of these patents being incorporated herein by reference in their entirety).

[0293] In another embodiment, the liposomes for use in the present invention can be formulated for targeted delivery. In a non-limiting example, the liposomes can be formulated for targeted delivery to the liver. Such liposomes include, but are not limited to, those described in U.S. Patent Publication 2013 / 0195967, the entirety of which is incorporated herein by reference.

[0294] In one embodiment, a formulation containing liposomes and a disruptor can be administered intramuscularly, intradermally, or intravenously.

[0295] In another embodiment, the lipid formulation of the present invention may comprise at least one cationic lipid, a transfection-enhancing lipid, and at least one lipid containing a hydrophilic head group linked to the lipid moiety (International Publication No. WO2011076807 and U.S. Patent Application Publication No. 20110200582; the entire contents of each of these patent documents are incorporated herein by reference). In another embodiment, the lipid formulation of the present invention is a lipid vesicle that may have crosslinks between functionalized lipid bilayers (see U.S. Patent Application Publication No. 2012 / 0177724, the entire contents of which are incorporated herein by reference).

[0296] In one embodiment, the formulation containing the disruptor is a lipid nanoparticle (LNP) which may contain at least one lipid. The lipid may be selected from, but is not limited to, DLin-DMA, DLin-K-DMA, 98NI2-5, CI2-200, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, PLGA, PEG, PEG-DMG, PEGylated lipids, and amino alcohol lipids. In another embodiment, the lipid may be a cationic lipid, for example, but is not limited to, DLin-DMA, DLin-D-DMA, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, and amino alcohol lipids. The amino alcohol cationic lipid may be a lipid described in U.S. Patent Application Publication No. 2013 / 0150625 and / or may be prepared by the method described therein.

[0297] In one embodiment, the cationic lipid is PCT publication numbers WO2012 / 040184, WO2011 / 153120, WO2011 / 149733, WO2011 / 090965, WO2011 / 043913, WO2011 / 022460, WO2012 / 061259, WO2012 / 054365, WO2012 / 044638, WO2010 / 080724, WO2010 / 21865, WO2008 / 103276, WO2013 / 086373 and WO2013 / 086354, U.S. Patent No. 7,893,302, and No. 7,404. Cationic lipids may be selected from, but are not limited to, those described in U.S. Patent Publications No. 969, 8,283,333, 8,466,122 and 8,569,256, and U.S. Patent Application Publications No. 2010 / 0036115, 2012 / 0202871, 2013 / 0064894, 2013 / 0129785, 2013 / 0150625, 2013 / 0178541, 2013 / 0225836 and 2014 / 0039032, and the contents of each of these patent documents are incorporated herein by reference in their entirety. In another embodiment, the cationic lipid may be selected from, but is not limited to, formula A as described in PCT publication numbers WO2012 / 040184, WO0111 / 53120, WO2011 / 149733, WO2011 / 090965, WO2011 / 043913, WO2011 / 022460, WO2012 / 061259, WO2012 / 054365, WO2012 / 044638 and WO2013 / 116126, or U.S. Patent Application Publications 2013 / 0178541 and 2013 / 0225836, and the contents of each of these patent documents are incorporated herein by reference in their entirety.In yet another embodiment, the cationic lipid may be selected from, but is not limited to, formulas CLI-CLXXIX of PCT Publication No. WO2008 / 103276, formula CLICLXXIX of U.S. Patent No. 7,893,302, formula CLICLXXXXII of U.S. Patent No. 7,404,969, and formulas I-VI of U.S. Patent Application Publication No. 2010 / 0036115 and formula I of U.S. Patent Application Publication No. 2013 / 0123338, each of these patent documents is incorporated herein by reference in whole.

[0298] In one embodiment, cationic lipids are used as known in the art and / or PCT publication numbers WO2012 / 040184, WO2011 / 153120, WO2011 / 149733, WO2011 / 090965, WO2011 / 043913, WO2011 / 022460, WO2012 / 061259, and WO2012 / 054. They can be synthesized in the manner described in WO2012 / 044638, WO2010 / 080724, WO2010 / 21865, WO2013 / 126803, WO2013 / 086373, and WO2013 / 086354, the contents of each of these patent documents being incorporated herein by reference in their entirety.

[0299] In one embodiment, the lipids that may be used in and / or for delivery of the disruptor formulations described herein may be cleavable lipids. As a non-limiting example, cleavable lipids and / or pharmaceutical compositions comprising cleavable lipids include those described in PCT Patent Publication No. WO2012 / 170889, the contents of which this patent document is incorporated herein by reference in whole. As another non-limiting example, cleavable lipids may be HGT400l, HGT4002, HGT4003, HGT4004 and / or HGT4005, as described in PCT Patent Publication No. WO2012 / 170889, the contents of which this patent document is incorporated herein by reference in whole.

[0300] In one embodiment, polymers that may be used in and / or for delivery of the disruptor formulations described herein include poly(ethylene) glycol (PEG), polyethyleneimine (PEI), dithiobis(succinimidylpropionate) (DSP), dimethyl 1-3,3'-dithiobispropionimidate (DTBP), poly(ethyleneimine) biscarbamate (PEIC), poly(L-lysine) (PLL), histidine-modified PLL, poly(N-vinylpyrrolidone) (PVP), and poly(propionide). Poly(PPI), poly(amideamine) (PAMAM), poly(amideethyleneimine) (SS-PAEI), triethylenetetramine (TETA), poly(β-aminoester), poly(4-hydroxy-L-proline (proine) ester) (PHP), poly(allylamine), poly(α-[4-aminobutyl]-L-glycolic acid (PAGA), poly(D,L-lactic-coglycolic acid) (Poly(D,L-lactic-coglycolic acid) Examples include, but are not limited to, poly(N-ethyl-4-vinylpyridinium bromide), poly(phosphazenes) (PPZ), poly(phosphoesters) (PPE), poly(phosphoamides) (PPA), poly(N-2-hydroxypropyl methacrylamide) (pHPMA), poly(2-(dimethylamino)ethyl methacrylate) (pDMAEMA), poly(2-aminoethylpropylene phosphate) (PPE_EA), chitosan, galactosylated chitosan, N-dodecylated chitosan, histones, collagen, and dextran-spermine. In one embodiment, the polymer may be an inert polymer, for example, PEG, but not limited to these. In one embodiment, the polymer may be a cationic polymer, for example, PLL, TETA, poly(allylamine), poly(N-ethyl-4-vinylpyridinium bromide), pHPMA, and pDMAEMA, but not limited to these. In one embodiment, the polymer may be a biodegradable PEI, for example, DSP, DTBP, and PEIC, but not limited to these. In one embodiment, the polymer may be a biodegradable polymer, for example, histidine, modified PLL SSPAEI, poly(β-aminoester), PHP, PAGA, PLGA, PPZ, PPE, PPA, and PPE-EA, but not limited to these.

[0301] In one embodiment, the LNP formulation of the present invention can be prepared according to the method described in PCT publication numbers WO2011 / 127255 or WO2008 / 103276, the contents of each of these patent documents being incorporated herein by reference in their entirety. As a non-limiting example, a disruptor containing a site-specific FOXP3 targeting moiety can be encapsulated in an LNP formulation as described in PCT publication numbers WO2011 / 127255 and / or WO2008 / 103276, the contents of each of these patent documents being incorporated herein by reference in their entirety. As another non-limiting example, a disruptor containing a site-specific FOXP3 targeting moiety as described herein can be formulated with nanoparticles delivered by parenteral routes as described in U.S. Patent Application Publication No. 2012 / 0207845 and PCT publication number WO2014 / 008334, the contents of each of these patent documents being incorporated herein by reference in their entirety.

[0302] In one embodiment, the LNP formulation described herein can be administered intramuscularly. The LNP formulation may include cationic lipids described herein, for example, but not limited to, DLin-DMA, DLin-KC2-DMA, DLin-MC3-DMA, DODMA, and C12-200.

[0303] In one embodiment, an LNP formulation described herein, comprising a disruptor described herein, can be administered intradermally. The LNP formulation may include cationic lipids described herein, for example, but not limited to, DLin-DMA, DLin-KC2-DMA, DLin-MC3-DMA, DODMA, and C12-200.

[0304] Nanoparticle formulations may include conjugates, such as phosphate conjugates, polymer conjugates, and conjugates that enhance the delivery of nanoparticles, as described in U.S. Patent Application Publication US20160038612A1.

[0305] In one embodiment, the lipid nanoparticle formulation includes DLin-MC3-DMA as described in U.S. Patent Application Publication US20100324120.

[0306] In one embodiment, lipid nanoparticles are specified in U.S. Patent No. US10723692B2, U.S. Patent Application Publication No. US20200172472A1, U.S. Patent Application Publication No. US20200163878A1, U.S. Patent Application Publication No. US20200046838A1, U.S. Patent Application Publication No. US20190359556A1, U.S. Patent Application Publication No. US20190314524A1, U.S. Patent Application Publication No. US20190274968A1, U.S. Patent Application Publication No. US20190022247A1, U.S. Patent Application Publication No. US20180303925A1, U.S. Patent Application Publication No. US20180185516A1, U.S. Patent Application Publication No. US20160317676A1, and International Patent Publication No. This includes lipid compounds, or pharmaceutically acceptable salts thereof, tautomers or stereoisomers thereof, or lipid nanoparticle formulations, as described in patent documents (Acuitas) WO20200146805A1, WO2020081938A1, WO2019089828A1, WO2019036030A1, WO2019036028A1, WO2019036008A1, WO2018200943A1, WO2018191719A1, WO2018107026A1, and WO2018081480A1, and these patent documents (Acuitas) The contents of each publication of Therapeutics, Inc. are incorporated herein by reference in their entirety.

[0307] In one embodiment, the lipid nanoparticles include aminolipids, or pharmaceutically acceptable salts thereof, tautomers or stereoisomers thereof, or lipid nanoparticle formulations, as described by Tekmira Pharmaceuticals Corp. in US9139554B2, US9051567B2, US8883203B2, and U.S. Patent Application Publication US20110117125A1, the contents of each of these patent documents are incorporated herein by reference in their entirety. In one particular example, the compound described in US9139554B2 is DLin-kC2-DMA.

[0308] In one embodiment, the lipid nanoparticles include aminolipids, or pharmaceutically acceptable salts thereof, tautomers or stereoisomers thereof, or lipid nanoparticle formulations, as described by Arbutus Biopharma Corp. in US10561732B2, US9938236B2, US9687550B2, U.S. Patent Application Publication No. US20190240354A1, US20170027658A1, WO2020097493A1, WO2020097520A1, WO2020097540A1, and WO2020097548A1, the contents of each of these patent documents are incorporated herein by reference in their entirety.

[0309] Lipid nanoparticles can be manipulated to alter the surface properties of the particles, allowing them to penetrate mucosal barriers. Mucus is found in mucosal tissues, including, but not limited to, the oral cavity (e.g., cheek and esophageal membranes, as well as tonsil tissue), eyes, gastrointestinal tract (e.g., stomach, small intestine, large intestine, colon, rectum), nasal cavity, respiratory system (e.g., nose, pharynx, trachea, and bronchial membranes), and genital organs (e.g., vagina, cervix, and urethral membranes). Nanoparticles larger than 10-200 nm, preferred for their ability to provide higher drug encapsulation efficiency and sustained delivery of diverse drugs, have been considered too large to rapidly diffuse through mucosal barriers. Because mucus is continuously secreted, excreted, discarded, or digested and recycled, the majority of captured particles can be removed from mucosal tissue within seconds or hours. Large polymer nanoparticles (200 nm to 500 nm in diameter) densely coated with low molecular weight polyethylene glycol (PEG) diffused through mucous membranes at only one-quarter to one-sixth the rate of the same particles diffusing into water (Lai et al. PNAS 2007 104(5): 1482-487; Lai et al. et al. Adv Drug Deliv Rev. 200961(2): 158-171; the contents of each of these references are incorporated herein by reference in their entirety. Nanoparticle transport can be determined using transmittance and / or fluorescence microscopy techniques, including but not limited to photobleaching-free fluorescence recovery (FRAP) and high-resolution multiparticle tracking (MPT). As a non-limiting example, compositions that can penetrate mucosal barriers can be prepared as described in U.S. Patent No. 8,241,670 or International Patent Publication No. WO2013110028, the contents of each of these patents are incorporated herein by reference in their entirety.

[0310] In one embodiment, the disruptor comprising the site-specific FOXP3 targeting moiety described herein is formulated as a lipoplex, without limitation, including: ATUPLEX®, DACC, DBTC, and other siRNA lipoplex technologies from Silence Therapeutics (London, United Kingdom), STEMFECFM from STEMGENT® (Cambridge, Mass.), and targeted and untargeted delivery of nucleic acids based on polyethyleneimine (PEI) or protamine (Aleku et al. Cancer Res. 2008 68:9788-9798; Strumberg et al. Int J Clin Pharmacol Ther 2012 50:76-78; Santel et al., Gene Ther 2006 13:1222-1234; Santel et al., Gene Ther 2006 13:1360-1370; Gutbier et al., PulmPharmacol. Ther. 201023:334-344;Kaufmann et al. Microvasc Res 2010 80:286-293;Weide et al. J Immunother. 2009 32:498- 507;Weide et al. J Immunother. 2008 31:180-188;Pascolo Expert Opin. Biol. Ther. 4:1285-1294;Fotin-Mleczek et al., 2011 J. Immunother. 34: 1-15;Song et al., Nature Biotechnol. 2005,23:709-717;Peer et al., Proc NatlAcad Sci USA. 2007 6;104:4095-4100;deFougerolles Hum Gene Ther. 2008 19: 125-132; all of these references are incorporated herein by reference in their entirety.

[0311] In one embodiment, such formulations may also be constructed or modified compositions such that they are passively or actively directed in vivo to different cell types, including but not limited to hepatocytes, immune cells, tumor cells, endothelial cells, antigen-presenting cells, and leukocytes (Akinc et al. Mol Ther. 2010 18:1357-1364; Song et al., Nat Biotechnol. 2005 23:709-717; Judge et al., J Clinlnvest. 2009 119:661-673; Kaufmann et al., Microvasc Res 2010 80:286- 293;Santel et al., Gene Ther 200613:1222-1234;Santel et al., Gene Ther 2006 13: 1360-1370;Gutbier et al., Pulm Pharmacol. Ther. 2010 23:334-344;Basha et al., Mol. Ther. 2011 19:2186-2200;Fenske and Cullis, Expert Opin Drug Deliv. 2008 5:25-44; Peer et al., Science. 2008 319:627-630; Peer and Lieberman, Gene Ther. 2011 18: 1127-1133; all of these references are incorporated herein by reference in their entirety). An example of passive targeting of formulations to hepatocytes includes lipid nanoparticle formulations based on DLin-DMA, DLin-KC2-DMA, and DLin-MC3-DMA, which have been shown to bind to apolipoprotein E and promote their binding to and uptake into hepatocytes in vivo (Akinc et al. Mol Ther. 2010 18: 1357-1364; the contents of this reference are incorporated herein by reference in their entirety). Formulations can also be selectively targeted by the expression of different ligands on their surfaces, such as folate, transferrin, N-acetylgalactosamine (GaINAc), and antibody-targeted approaches, as exemplified by these methods (Kolhatkar et al., Curr Drug Discov Technol. 2011 8: 197-206; Musacchio and Torchilin, Front Biosci. 201116: 1388-1412; Yu et al., Mol Membr BioI. 2010 27:286-298; Patil et al., Crit Rev Ther Drug Carrier Syst. 2008 25: 1-61; Benoit et al., Biomacromolecules. 2011 12:2708-2714; Zhao et al., Expert Opin Drug Deliv. 2008). 5:309-319;Akinc et al., Mol Ther. 2010 18:1357-1364;Srinivasan et al., Methods Mol BioI. 2012 820: 105-116;Ben-Arie et al., Methods Mol Biol. 2012 757:497-507;Peer 2010 J Control Release. 20:63-68;Peer et al., Proc Natl Acad Sci USA. 2007 104:4095-4100;Kim et al., Methods Mol BioI. 2011 721:339-353;Subramanya et al., Mol Ther. 2010 18:2028-2037;Song et al., Nat Biotechnol. 2005 23:709-717;Peer et al., Science. 2008 319:627-630; Peer and Lieberman, Gene Ther. 2011 18:1127-1133; The entire contents of these references are incorporated herein by reference. (to be able to).

[0312] In one embodiment, the disruptor containing the site-specific FOXP3 targeting moiety of the present invention can be formulated as solid lipid nanoparticles. The solid lipid nanoparticles (SLNs) may be spherical with an average diameter between 10 and 1000 nm. The SLNs possess a solid lipid core matrix that can solubilize lipophilic molecules and can be stabilized with surfactants and / or emulsifiers. In further embodiments, the lipid nanoparticles may be self-assembled lipid-polymer nanoparticles (see Zhang et al., ACS Nano, 2008, 2 (8), pp 1696-1702, the whole of which is incorporated herein by reference). As a non-limiting example, the SLNs may be those described in PCT Patent Publication No. WO2013 / 105101, the contents of which the whole of which is incorporated herein by reference. As another non-limiting example, SLN can be produced by the method or process described in PCT Patent Publication No. WO2013 / 105101, the contents of which are incorporated herein by reference in their entirety.

[0313] Liposomes, lipoplexes, or lipid nanoparticles can be used to improve the efficacy of disruptors containing site-specific FOXP3 targeting moieties, such as nucleic acid molecules, in directing protein production. These formulations may be capable of increasing cell transfection by nucleic acid molecules and / or increasing the translation of encoded proteins (e.g., effectors of the present invention). One such example is the use of lipid encapsulation to enable effective systemic delivery of polyplex plasmid DNA (Heyes et al., Mol Ther. 2007 15:713-720; the entire content of this reference is referenced). The liposomes, lipoplexes, or lipid nanoparticles of the present invention can also increase the stability of disruptors containing site-specific FOXP3 targeting moieties, for example, nucleic acid molecules. The liposomes, lipoplexes, or lipid nanoparticles are described in U.S. Patent Application Publication No. 2016 / 0038612, the contents of which are incorporated herein by reference in their entirety.

[0314] In one embodiment, a disruptor comprising a site-specific FOXP3-targeting moiety containing __ can be formulated for controlled release and / or targeted delivery. As used herein, “controlled release” refers to a pharmaceutical composition or compound release profile that follows a specific release pattern that results in a therapeutic outcome. In one embodiment, a disruptor comprising a site-specific FOXP3-targeting moiety as described herein can be encapsulated in a delivery agent described herein and / or known in the art for controlled release and / or targeted delivery. As used herein, the term “encapsulate” means to wrap, surround, or contain. If it relates to the formulation of a compound of the present invention, encapsulation can be substantial, complete, or partial. The term “substantially encapsulated” means that at least 50%, and more than 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.9%, or 99.999% of the pharmaceutical composition or disruptor of the present invention may be encapsulated, surrounded, or contained within a delivery agent. “Partially encapsulated” means that less than 10%, 10, 20, 30, 40, 50, or less of the pharmaceutical composition or disruptor of the present invention may be encapsulated, surrounded, or contained within a delivery agent. Advantageously, encapsulation can be determined by measuring the escaping or activity of the pharmaceutical composition or compound of the present invention using fluorescence and / or electron microscopy. For example, at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, or more than 99.99% of the pharmaceutical composition or disruptor of the present invention is encapsulated in the delivery agent.

[0315] In one embodiment, a disruptor containing a site-specific FOXP3 targeting moiety, including the __ described herein, can be encapsulated in therapeutic nanoparticles. The therapeutic nanoparticles are described herein and in the art, for example, but are not limited to those described herein, but are PCT publication numbers WO2010 / 005740, WO2010 / 030763, WO2010 / 005721, WO2010 / 005723, WO2012 / 054923, and U.S. Patent Application Publication No. 2201 / 102 No. 62491, No. 2010 / 0104645, No. 2010 / 0087337, No. 2010 / 0068285, No. 2011 / 0274759, Same No. 2010 / 0068286, No. 2012 / 0288541, No. 2013 / 0123351, No. 2013 / 0230567, No. 2013 / 0 It can be formulated by methods known in Patent No. 236500, No. 2013 / 0302433, No. 2013 / 0302432, No. 1013 / 0280339 and No. 2013 / 0251757, and in U.S. Patent No. 8,206,747, No. 8,293,276, No. 8,318,208, No. 8,318,211, No. 8,623,417, No. 8,617,608, No. 8,613,954, No. 8,613,951, No. 8,609,142, No. 8,603,534 and No. 8,563,041, and the contents of each of these patent documents are incorporated herein by reference in their entirety. In another embodiment, therapeutic polymer nanoparticles can be prepared by the method described in U.S. Patent Application Publication 2012 / 0140790, which is incorporated herein by reference in its entirety. In non-limiting examples, therapeutic nanoparticles may comprise about 4 to about 25 weight percent of a disruptor and about 10 to about 99 weight percent of a diblock poly(lactic acid)-poly(ethylene) glycol copolymer containing poly(lactic acid) as described in U.S. Patent Application Publication 2013 / 0236500, which is incorporated herein by reference in its entirety.As another non-limiting example, the nanoparticles may comprise about 0.2 to about 35 weight percent of a disruptor and about 10 to about 99 weight percent of a diblock poly(lactic acid)-poly(ethylene) glycol copolymer, as described in U.S. Patent Application Publications 2013 / 0280339 and 2010251757 and U.S. Patent No. 8,652,528, the contents of each of these patents are incorporated herein by reference in their entirety.

[0316] In one embodiment, a disruptor formulated with therapeutic nanoparticles can be administered intramuscularly, intradermally, or intravenously.

[0317] In one embodiment, the disruptor can be delivered by therapeutic nanoparticles having a high glass transition temperature, for example, but not limited to, the nanoparticles described in U.S. Patent Application Publications 2014 / 0030351 and 2011 / 0294717, the entire contents of each of these patent documents incorporated herein by reference.

[0318] In one embodiment, therapeutic nanoparticles can be formulated for sustained release. As used herein, “sustained release” refers to a pharmaceutical composition or compound that releases at a certain rate over a specific period of time. This period may include, but is not limited to, hours, days, weeks, months, and years. Non-limiting examples include the polymers and disruptors of the present invention (see PCT Publication No. WO2010075072 and U.S. Patent Applications Publications 2010 / 0216804, 2011 / 0217377, 2012 / 0201859, 2013 / 0243848, and 2013 / 0243827, each of which is incorporated herein by reference in its entirety).

[0319] In one embodiment, the disruptor of the present invention can be encapsulated in a synthetic nanocarrier, linked to a synthetic nanocarrier, and / or associated with a synthetic nanocarrier. The synthetic nanocarrier is PCT publication number WO2010 / 005740, WO2010 / 030763, WO2012 / 13501, WO2012 / 149252, WO2012149255, WO2012149259, WO2012149265, WO2012149268, WO2012149282, WO2012149301, WO2012149393, WO2012149405, WO201214 This includes, but is not limited to, the contents of each of these patent documents, which are incorporated herein by reference in their entirety. Synthetic nanocarriers can be formulated using methods known in the art and / or described herein. As a non-limiting example, synthetic nanocarriers can be formulated by the methods described in PCT publication numbers WO2010005740, WO2010030763 and WO201213501, and U.S. Patent applications published 20110262491, 20100104645, 20100087337 and 20120244222, each of which is incorporated herein by reference in its entirety. In another embodiment, synthetic nanocarrier formulations can be freeze-dried by the methods described in PCT publication number WO2011072218 and U.S. Patent No. 8,211,473, each of which is incorporated herein by reference in its entirety. In yet another embodiment, formulations of the present invention, including but not limited to synthetic nanocarriers, can be freeze-dried or restored by the method described in U.S. Patent Application Publication No. 20130230568, the contents of which are incorporated herein by reference in their entirety.

[0320] In one embodiment, a synthetic nanocarrier containing a disruptor can be administered intramuscularly, intradermally, or intravenously.

[0321] In some embodiments, the disruptor can be formulated for delivery using smaller LNPs. Such particles range from less than 0.1 μm to a maximum of 1000 μm, for example, but are not limited to, less than 0.1 μm, less than 1.0 μm, less than 5 μm, less than 10 μm, less than 15 μm, less than 20 μm, less than 25 μm, less than 30 μm, less than 35 μm, less than 40 μm, less than 50 μm, less than 55 μm, less than 60 μm, less than 65 μm, less than 70 μm, less than 75 μm, less than 80 μm, less than 85 μm, less than 90 μm, less than 95 μm, less than 100 μm, less than 125 μm, less than 150 μm, less than 175 μm, less than 200 μm, less than 225 μm, less than 250 μm. May include diameters less than 275 μm, less than 300 μm, less than 325 μm, less than 350 μm, less than 375 μm, less than 400 μm, less than 425 μm, less than 450 μm, less than 475 μm, less than 500 μm, less than 525 μm, less than 550 μm, less than 575 μm, less than 600 μm, less than 625 μm, less than 650 μm, less than 675 μm, less than 700 μm, less than 725 μm, less than 750 μm, less than 775 μm, less than 800 μm, less than 825 μm, less than 850 μm, less than 875 μm, less than 900 μm, less than 925 μm, less than 950 μm, and less than 975 μm.

[0322] In another embodiment, approximately 1nm to 100nm, approximately 1nm to 10nm, approximately 1nm to 20nm, approximately 1nm to 30nm, approximately 1nm to 40nm, approximately 1nm to 50nm, approximately 1nm to 60nm, approximately 1nm to 70nm, approximately 1nm to 80nm, approximately 1nm to 90nm, approximately 5nm to 100nm, approximately 5nm to 10nm, approximately 5nm to 20nm, approximately 5nm to 30nm, approximately 5nm to 40nm, approximately 5nm to 50nm, approximately 5nm to 60nm, approximately 5nm to 70nm, approximately 5nm to 80nm, approximately 5nm to 90nm, approximately 10nm to 50nm, approximately 20nm to 50nm, approximately 30nm Smaller LNPs, which may include diameters of approximately 50 nm, 40-50 nm, 20-60 nm, 30-60 nm, 40-60 nm, 20-70 nm, 30-70 nm, 40-70 nm, 50-70 nm, 60-70 nm, 20-80 nm, 30-80 nm, 40-80 nm, 50-80 nm, 60-80 nm, 20-90 nm, 30-90 nm, 40-90 nm, 50-90 nm, 60-90 nm, and / or 70-90 nm, can be used to formulate disruptors for delivery.

[0323] In one embodiment, the disruptor can be formulated with smaller LNPs and administered intramuscularly, intradermally, or intravenously.

[0324] In some embodiments, the disruptor can be formulated for delivery using drug-encapsulated microspheres described in PCT Patent Publication No. WO2013063468 or U.S. Patent No. 8,440,614, each of which is incorporated herein by reference in its entirety. In other embodiments, amino acids, peptides, polypeptides, and lipids (APPL) are useful for the delivery of the disruptor of the present invention to cells (see PCT Patent Publication No. WO2013063468, which is incorporated herein by reference in its entirety).

[0325] In one embodiment, the lipid nanoparticles may be limit-size lipid nanoparticles described in PCT Patent Publication No. WO2013059922, which is incorporated herein by reference in whole. The limit-size lipid nanoparticles may comprise a lipid bilayer surrounding an aqueous or hydrophobic core, the lipid bilayer of which may comprise phospholipids, for example, but not limited to, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, cerebroside, C8-C20 fatty acid diacylphosphatidylcholine, and 1-palmitoyl-2-oleylphosphatidylcholine (POPC). In some embodiments, the limit-size lipid nanoparticles may include polyethylene glycol-lipids, for example, but not limited to, DLPEPEG, DMPE-PEG, DPPC-PEG, and DSPE-PEG.

[0326] In one embodiment, the disruptor of the present invention can be delivered, localized, and / or concentrated at a specific location using the delivery method described in PCT Patent Publication No. WO2013063530, the contents of which are incorporated herein by reference in their entirety. In a non-limiting example, empty polymer particles can be administered to a target before, simultaneously with, or after the delivery of the disruptor to the target. The empty polymer particles change volume upon contact with the target and remain, embed, immobilize, or capture at a specific location in the target.

[0327] In one embodiment, the disruptor can be formulated in an active substance release system (see, for example, U.S. Patent Application Publication No. 20130102545, which is incorporated herein by reference in whole). The active substance release system may include 1) at least one nanoparticle bound to an oligonucleotide inhibitor chain hybridized with a catalytically active nucleic acid, and 2) a compound bound to at least one substrate molecule bound to a therapeutically active substance (e.g., the disruptor of the present invention), the therapeutically active substance being released by cleavage of the substrate molecule by the catalytically active nucleic acid.

[0328] In one embodiment, the nanoparticles of the present invention may be water-soluble nanoparticles, for example, but not limited to those described in PCT Publication No. WO2013090601, the contents of which are incorporated herein by reference in their entirety. The nanoparticles may be inorganic nanoparticles having dense zwitterionic ligands to exhibit good water solubility. The nanoparticles may also have a small hydrodynamic diameter (HD), stability to time, pH and salinity, and low nonspecific protein binding levels.

[0329] In one embodiment, the nanoparticles of the present invention are stealth nanoparticles or target-specific stealth nanoparticles, for example, but not limited to those described in U.S. Patent Publication Nos. 20130172406 (Bind), US20130251817 (Bind), US2013251816 (Bind), and US20130251766 (Bind), the contents of each of these patent documents being incorporated herein by reference in their entirety. The stealth nanoparticles may comprise diblock copolymers and chemotherapeutic agents. These stealth nanoparticles can be prepared by the methods described in U.S. Patent Publication Nos. 20130172406, US20130251817, US2013251816, and US20130251766, the contents of each of these patent documents being incorporated herein by reference in their entirety. As a non-limiting example, stealth nanoparticles, such as those described in U.S. Patent Application Publications 20130172406, 20130251817, 2013251816, and 20130251766, can target cancer cells, and the contents of each of these patents are incorporated herein by reference in their entirety.

[0330] In one embodiment, stealth nanoparticles containing the disruptor of the present invention can be administered intramuscularly, intradermally, or intravenously.

[0331] In one embodiment, the disruptor of the present invention can be formulated and / or delivered in lipid nanoparticles containing a plurality of cationic lipids, for example, but not limited to, those described in U.S. Patent Application Publication No. 20130017223, the contents of which are incorporated herein by reference in their entirety. In an unlimiting example, the LNP formulation may contain a first cationic lipid and a second cationic lipid. In another unlimiting example, the LNP formulation may contain DLin-MC2-DMA and DLinMC4-DMA. In yet another unlimiting example, the LNP formulation may contain DLin-MC3-DMA and CI2-200. In one embodiment, the LNP formulation containing a plurality of cationic lipids (for example, but not limited to, those described in U.S. Patent Application Publication No. US20130017223; the contents of which are incorporated herein by reference in their entirety) can be administered intramuscularly, intradermally, or intravenously.

[0332] In one embodiment, the disruptor described herein can be formulated and / or delivered in lipid nanoparticles comprising the cationic lipid DLin-MC3-DMA and the neutral lipid DOPE. The lipid nanoparticles may also comprise PEG-based lipids and cholesterol or antioxidants. These lipid nanoparticle formulations comprising DLin-MC3-DMA, DOPE, and the disruptor can be administered intramuscularly, intradermally, or intravenously.

[0333] In one embodiment, lipid nanoparticles comprising DLin-MC3-DMA and DOPE may include, but are not limited to, PEG lipids such as pentaerythritol PEG ester tetrasuccinimidyl and pentaerythritol PEG ether tetrathiol, PEGc-DOMG, PEG-DMG (1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol), PEG-DSG (1,2-distearoyl-sn-glycerol, methoxypolyethylene glycol), PEG-DPG (1,2-dipalmitoyl-sn-glycerol, methoxypolyethylene glycol), PEG-DSA (PEG coupled with 1,2-distearoyloxypropyl-3-amine), PEG-DMA (PEG coupled with 1,2-dimyristyloxypropyl-3-amine), PEG-c-DNA, PEG-c-DMA, PEG-S-DSG, PEG-c-DMA, PEG-DPG, PEG-DMG 2000, and others described herein and / or known in the art.

[0334] In one embodiment, lipid nanoparticles containing DLin-MC3-DMA and DOPE may have lipid molar ratios of 0.5% to about 3.0%, about 1.0% to about 3.5%, about 1.5% to about 4.0%, about 2.0% to about 4.5%, about 2.5% to about 5.0%, and / or about 3.0% to about 6.0% of PEG lipids.

[0335] In one embodiment, lipid nanoparticles containing DLin-MC3-DMA and DOPE may contain 25.0% to about 50.0% cholesterol, about 30.0% to about 45.0% cholesterol, about 35.0% to about 50.0% cholesterol, and / or about 48.5% to about 60% cholesterol. In one embodiment, the formulation may contain a percentage of cholesterol selected from the group consisting of 28.5%, 31.5%, 33.5%, 36.5%, 37.0%, 38.5%, 39.0%, 43.5%, and 48.5%.

[0336] In one embodiment, lipid nanoparticles containing DLin-MC3-DMA and DOPE may contain 25.0% to about 50.0% antioxidants, about 30.0% to about 45.0% antioxidants, about 35.0% to about 50.0% antioxidants, and / or about 48.5% to about 60% antioxidants. In one embodiment, the formulation may contain an antioxidant in a percentage selected from the group consisting of 28.5%, 31.5%, 33.5%, 36.5%, 37.0%, 38.5%, 39.0%, 43.5%, and 48.5%.

[0337] The disruptors of the present invention can be formulated using natural and / or synthetic polymers. Non-limiting examples of polymers that may be used for delivery include, but are not limited to, DYNAMIC POLYCONJUGATE® (Arrowhead Research Corp., Pasadena, Calif.) formulations from MIRUS® Bio (Madison, Wis.) and Roche Madison (Madison, Wis.), PHASERX® polymer formulations, and, for example, SMARTT POLYMER TECHNOLOGY® (Seattle, Wash.), DMRIIDOPE, poloxamer, VAXFECTIN® adjuvant from Vical (San Diego, Calif.), chitosan, cyclodextrins, dendrimers and poly(lactic acid-co-glycolic acid) (PLGA) polymers from Calando Pharmaceuticals (Pasadena, Calif.), and RONDEL® (RNAi / Oligonucleotide Nanoparticle Delivery) polymers (Arrowhead Research). Examples include, but are not limited to, PHASERX® (Seattle, Wash.), a pH-responsive coblock polymer (PASADENA Corporation, Calif.), and PHASERX® (Seattle, Wash.).

[0338] Polymer formulations can enable sustained or delayed release of disruptors (e.g., after intramuscular, intradermal, or subcutaneous injection). As a result of the altered release profile of the disruptor, for example, the encoded protein may be translated over a longer period. Polymer formulations can also be used to increase the stability of disruptors. For example, biodegradable polymers have been previously used to protect nucleic acids other than modified mRNA from degradation and have been shown to result in sustained release of the payload in vivo (Rozema et al., Proc Natl Acad Sci USA. 2007 104:12982-12887; Sullivan et al., Expert Opin Drug Deliv.). 2010 7:1433-1446;Convertine et al., Biomacromolecules. 2010 Oct. 1;Chu et al., Acc Chern Res. 2012 Jan. 13;Manganiello et al., Biomaterials. 2012 33:2301-2309;Benoit et a!., Biomacromolecules. 2011 12:2708-2714;Singha et al., Nucleic Acid Ther. 2011 2: 133- 147;deFougerolles Hum Gene Ther. 2008 19:125-132;Schaffert and Wagner, Gene Ther. 2008 16:1131-1138;Chaturvedi et al., Expert Opin Drug Deliv. 2011 8: 1455–1468; Davis, Mol Pharm. 2009 6:659–668; Davis, Nature 201 0464: 1067–1070; Each of these references is incorporated herein by reference in its entirety. .

[0339] In one embodiment, the pharmaceutical composition may be a sustained-release formulation. In a further embodiment, the sustained-release formulation may be for subcutaneous delivery. The sustained-release formulation is not limited to these, but may include PLGA microspheres, ethylene vinyl acetate (EVAc), poloxamer, GELSITE® (Nanotherapeutics, Inc., Alachua, Fla.), HYLENEX® (Halozyme Therapeutics, San This may include surgical sealants, such as fibrinogen polymers (Ethic on Inc., Cornelia, Ga.), TISSELL® (Baxter International, Inc., Deerfield, Ill.), PEG-based sealants, and COSEAL® (Baxter International, Inc., Deerfield, Ill.).

[0340] B. Site-specific FOXP3 disruptor of the present invention encoded in a vector For example, a disruptor containing a site-specific FOXP3 targeting moiety, including nucleic acid molecules, can be expressed from a transcription unit inserted into a DNA or RNA vector (e.g., Couture, See A, et al., TIG. (1996), 12:5-10; WO00 / 22113, WO00 / 22114, and US6,054,299). In some embodiments, expression is sustained (for several months or longer) depending on the specific construct used and the target tissue or cell type. These transgenes can be introduced as linear constructs, circular plasmids, or viral vectors, which may be integrated or non-integrated vectors. Transgenes can also be constructed to allow inheritance as extrachromosomal plasmids (Gassmann, et al., (1995) Proc. Natl. Acad. Sci. USA 92:1292). Different components of a disruptor, e.g., gRNA and effector, can be located on separate expression vectors that can be simultaneously introduced into target cells (e.g., by transfection or infection). Alternatively, each individual component can be transcribed by promoters both located on the same expression plasmid.

[0341] The delivery of the disruptor expression vector may be systemic, for example, by intravenous or intramuscular administration to target cells explanted from the patient, subsequent reintroduction into the patient, or by any other means that enable introduction into the desired target cells.

[0342] In certain embodiments, nucleic acids encoding nucleic acids described herein, or proteins described herein, such as effectors, are incorporated into a vector, such as a viral vector.

[0343] Individual strands (one or more) of a disruptor containing a site-specific FOXP3 targeting moiety, including a nucleic acid molecule, can be transcribed from a promoter within an expression vector. If two separate strands are to be expressed, for example, to generate dsRNA, two separate expression vectors can be simultaneously introduced into target cells (e.g., by transfection or infection). Alternatively, each individual strand of the nucleic acid molecule can be transcribed by promoters both located on the same expression plasmid. In one embodiment, the nucleic acid molecule is expressed as a reverse repeat polynucleotide joined by a linker polynucleotide sequence, and therefore the nucleic acid molecule has a stem-and-loop structure.

[0344] Expression vectors are generally DNA plasmids or viral vectors. Recombinant constructs for the expression of the disruptors described herein can be produced using expression vectors compatible with eukaryotic cells, preferably those compatible with vertebrate cells.

[0345] Constructs for the recombinant expression of disruptors generally require regulatory elements, such as promoters and enhancers, to ensure the expression of the disruptor in target cells.

[0346] The expression of natural or synthetic nucleic acids is generally achieved by operably ligating the nucleic acid encoding the target nucleic acid to a regulatory region such as a promoter, and by incorporating the construct into an expression vector. Vectors may be suitable for replication and integration in eukaryotes.

[0347] Regulatory regions, such as promoters, suitable for operably ligating to nucleic acid molecules can be operably ligated to regulatory regions, such as promoters, which may originate from any species. Any type of promoter can be operably ligated to a nucleic acid sequence. Examples of promoters include, without limitation, tissue-specific promoters, constitutive promoters, and promoters that are responsive or unresponsive to specific stimuli (e.g., inductive promoters). Further promoter elements, such as enhancement sequences, regulate the frequency of transcription initiation. Generally, these are located 30–110 bp upstream of the initiation site, although some promoters have recently been shown to also contain functional elements downstream of the initiation site. The spacing between promoter elements is often flexible, and therefore promoter function is preserved even if the elements are oriented in opposite directions relative to each other or are moved. In the thymidine kinase (TK) promoter, the spacing between promoter elements can be increased to 50 bp before activity begins to decline. Depending on the promoter, individual elements can function cooperatively or independently to activate transcription.

[0348] A suitable promoter is the very early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a potent constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operably ligated to it. Another suitable promoter is elongation growth factor-la (EF-la). However, other constitutive promoter sequences can also be used, including, but not limited to, the Simian virus 40 (SV40) early promoter, mouse mammary cancer virus (MMTV), human immunodeficiency virus (HIV) long-term repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus very early promoter, Roussarcoma virus promoter, and human gene promoters, including, but not limited to, actin promoters, myosin promoters, hemoglobin promoters, and creatine kinase promoters.

[0349] Furthermore, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also intended as part of the present invention. The use of an inducible promoter provides a molecular switch that can turn on the expression of the polynucleotide sequence to which it is operably ligated when such expression is desired, and turn off when expression is not desired. An example of an inducible promoter is metallothionein promoter. Examples include, but are not limited to, the ter, glucocorticoid promoter, progesterone promoter, and tetracycline promoter.

[0350] Further regulatory regions that may be useful in nucleic acid constructs include, but are not limited to, transcriptional and translational terminators, start sequences, polyadenylation sequences, translational regulatory sequences (e.g., internal ribosome entry segments, IRESs), enhancers, inducible elements, or introns. While such regulatory regions may not always be necessary, they can increase expression by influencing transcription, mRNA stability, translation efficiency, etc. Such regulatory regions may be included in nucleic acid constructs as needed to achieve optimal nucleic acid expression in cells. However, sufficient expression can sometimes be achieved without such additional elements.

[0351] The introduced expression vector may contain either or both a selectable marker gene or a reporter gene to facilitate the identification and selection of cells expressing the marker from a population of cells intended to be transfected or infected with the viral vector. In other embodiments, the selectable marker may be supported on a separate DNA fragment and used in a cotransfection procedure. Both the selectable marker and the reporter gene can be flanked by appropriate transcriptional regulatory sequences to enable expression in host cells. Useful selectable markers include, for example, antibiotic resistance genes, such as neo. Non-limiting examples of selectable markers include puromycin, ganciclovir, adenosine deaminase (ADA), aminoglycoside phosphotransferase (neo, G418, APH), dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase, thymidine kinase (TK), and xanthine-guanine phosphoribosyltransferase (XGPRT). Such markers are useful for selecting stable transformants under culture. Other selectable markers include fluorescent polypeptides, such as green fluorescent protein or yellow fluorescent protein.

[0352] Signal peptides can also be included and used to orient the encoded polypeptide to a specific cellular location (e.g., the cell surface).

[0353] Reporter genes can be used to identify potentially transfected cells and to evaluate the functionality of transcriptional regulatory sequences. Generally, a reporter gene is a gene that is not present in the recipient source and is not expressed by the recipient source, and whose expression is elucidated by some easily detectable characteristic, such as enzymatic activity, which encodes a polypeptide. Reporter gene expression is assayed at a suitable time after the DNA has been introduced into recipient cells. Suitable reporter genes include those encoding luciferase, beta-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 can be prepared using known techniques, Alternatively, they can be purchased. Generally, a construct having a minimum 5' adjacent region that exhibits the highest expression level of the reporter gene is identified as a promoter. Such a promoter region can be ligated to the reporter gene and used to evaluate a drug's ability to modulate promoter-driven transcription.

[0354] Other aspects of vectors and structures to be considered are known in the art.

[0355] In some embodiments, the vector, for example, a viral vector, includes a disruptor that comprises a site-specific FOXP3 targeting moiety containing a nucleic acid molecule.

[0356] Viral vector systems that may be used with the methods and compositions described herein include, but are not limited to, (a) adenovirus vectors (e.g., Ad5 / F35 vector), (b) retroviral vectors (including, but not limited to, lentiviral vectors with or without integration ability), Moloney's mouse leukemia virus, etc., (c) adeno-associated virus vectors, (d) herpes simplex virus vectors, (e) SV40 vector, (f) polyomavirus vectors, (g) papillomavirus vectors, (h) picornavirus vectors, (i) poxvirus vectors, e.g., orthopox, e.g., vaccinia virus vector, or tripox, e.g., canary pox or fowlpox, and (j) helper-dependent or gutless adenoviruses. Replication-deficient viruses may also be advantageous. Different vectors may or may not be incorporated into the cell genome. The construct may, if necessary, include a viral sequence for transfection. Alternatively, the constructs can be incorporated into episomal replication vectors, such as EPV and EBV vectors. See, for example, U.S. Patents 6,534,261, 6,607,882, 6,824,978, 6,933,113, 6,979,539, 7,013,219 and 7,163,824, the entire contents of each of these patents being incorporated herein by reference.

[0357] Vectors, including those derived from retroviruses such as lentiviruses, are suitable tools for achieving long-term gene transfer because they enable the long-term, stable integration of transgenes and their transmission in daughter cells. Examples of vectors include expression vectors, replication vectors, probe-generating vectors, and sequencing vectors. Expression vectors can be supplied to cells in the form of viral vectors. Viral vector technology is well known in this art and is described in various virology and molecular biology manuals.

[0358] In some embodiments, a viral vector suitable for use in the present invention is an adeno-associated virus vector, such as a recombinant adeno-associated virus vector.

[0359] Recombinant adeno-associated virus vectors (rAAV) are gene delivery systems based on defective, non-pathogenic parvovirus adeno-associated virus type 2. All vectors are derived from plasmids that retain only the AAV 145bp reverse terminal repeat adjacent to the transgene expression cassette. Efficient gene transfer and stable transgene delivery, resulting from integration into the genome of transduced cells, are key features of this vector system. (Wagner et al., Lancet 351:9117 1702-3 (1998), Kearns et al., Gene Ther. 9:748-55 (1996)) AAV serotypes, including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9, can be used in accordance with the present invention.

[0360] Replication-deficient recombinant adenovirus vectors (Ad) can be produced at high titers and readily infect several different cell types. Most adenovirus vectors are engineered so that the transgene replaces the Ad E1a, E1b, and / or E3 genes, and then the replication-deficient vector is transmitted in human 293 cells that trans-supply the deleted gene function. Ad vectors can transduce in vivo into several types of tissues, including differentiated non-dividing cells found in the liver, kidney, and muscle. Conventional Ad vectors have high transport capacity. One example of Ad vector use in clinical trials includes polynucleotide therapy for anti-tumor immunization via intramuscular injection (Sterman et al., Hum. Gene Ther. 7:1083-9 (1998)). Further examples of adenovirus vector use for gene transduction in clinical trials include Rosenecker et al. al., Infection 24:1 5-10 (1996); Sterman et al., Hum. Gene Ther. 9:7 1083-1089 (1998); Welsh et al., Hum. Gene Ther. 2:205-18 (1995); Alvarez et al., Hum. Gene Ther. 5:597-613 (1997); Topf et al., Gene Ther. 5:507-513 (1998); Sterman et al., Hum. Gene Ther. 7:1083-1089 (1998).

[0361] Packaging cells are used to form viral particles capable of infecting host cells. Examples of such cells include 293 cells for packaging adenoviruses, and ψ2 or PA317 cells for packaging retroviruses. Viral vectors used in gene therapy are typically produced by producer cell systems that package nucleic acid vectors into viral particles. The vectors generally contain the minimum viral sequences required for packaging and subsequent integration into the host (if applicable), with other viral sequences replaced by expression cassettes encoding proteins that will be expressed. Missing viral functions are supplied trans by the packaging cell system. For example, AAV vectors used in gene therapy typically contain only the reverse terminal repeat (ITR) sequences from the AAV genome required for packaging and integration into the host genome. The viral DNA is packaged into a cell system containing helper plasmids that encode other AAV genes, namely rep and cap, but lack the ITR sequences. The cell system is also infected with adenovirus as a helper. Helper viruses promote the replication of AAV vectors and the expression of AAV genes from helper plasmids. Because helper plasmids lack an ITR sequence, they are not packaged in significant quantities. For example, heat treatment, which makes adenoviruses more susceptible than AAV, can reduce contamination by adenoviruses.

[0362] IV. Method of the present invention A. Modulation of FOXP3 expression in cells The present invention also provides methods for using the agents and compositions described herein to modulate the expression of forkheadbox P3 (FOXP3) in cells. The method comprises the step of contacting cells, for example, naive T cells, with a site-specific FOXP3 disruptor containing a site-specific FOXP3 targeting moiety that targets a FOXP3 expression regulatory region, and an effector molecule, thereby modulating the expression of FOXP3 in the cells. The site-specific disruptor, the effector, or both the site-specific disruptor and the effector may be present in a composition, for example, the above composition. In some embodiments, the site-specific disruptor and the effector are present in the same composition. In other embodiments, the site-specific disruptor and the effector are present in different compositions. In some embodiments, the method of the present invention comprises the step of contacting cells with two site-specific FOXP3 disruptors (first and second agents). The two site-specific foxp3 disruptors may be present in the same composition, e.g., a pharmaceutical composition, e.g., a pharmaceutical composition containing LNP, or in separate compositions, e.g., a pharmaceutical composition, e.g., a pharmaceutical composition containing LNP. Cells may be brought into contact with the first site-specific foxp3 disruptor at a first time point and with the second site-specific foxp3 disruptor at a second time point, or cells may be brought into contact with both agents simultaneously.

[0363] FOXP3 expression may be enhanced or reduced, for example, compared to cells not in contact with a site-specific FOXP3 disruptor. Modulation of gene expression can be assessed by any method known in the art. For example, expression modulation can be determined by determining the mRNA expression level of a gene, for example, in cells, multiple cells, and / or tissue samples, using methods common to those skilled in the art, such as Northern blotting or qRT-PCR; or by determining the protein level of a gene, using methods common to those skilled in the art, such as Western blotting or immunological techniques.

[0364] The term "reduced" in the context of FOXP3 gene expression or FOXP3 protein production in a subject, or in relation to disease markers or symptoms, is used in that context. This refers to a statistically significant decrease of such a level. The decrease may be, for example, at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the detection level in the detection method, or less. In certain embodiments, the expression of the target is normalized, i.e., reduced to a level that would be considered within the normal range for an individual without such impairment. As used herein, “lower” in the subject does not require a decrease in expression in all cells or tissues of the subject, and may refer to a decrease in gene expression or protein production in the cells of the subject. For example, as used herein, a decrease in the subject may include a decrease in gene expression or protein production in the liver of the subject.

[0365] The term "reduced" means normalizing the symptoms of a disease or condition, that is, It may also be used in relation to reducing the difference between the level in a subject with an autoimmune disease or FOXP3-related disease and the level in a normal subject who does not have either an autoimmune disease or FOXP3-related disease, toward or to the level of the normal subject. As used herein, if the disease is associated with an increase in the value of a symptom, "normal" is considered to be the upper limit of normal. If the disease is associated with a decrease in the value of a symptom, "normal" is considered to be the lower limit of normal.

[0366] In the context of levels of FOXP3 gene expression or FOXP3 protein production in a subject, or of disease markers or symptoms, the term “improved” refers to a statistically significant increase in such levels. An increase may be, for example, at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more of the detection level in a detection method. In certain embodiments, the expression of the target increases toward or to a level that is normalized, i.e., within the normal range for individuals without such impairment. “Higher” in a subject, as used herein, may refer to an increase in gene expression or protein production in the cells of the subject, and does not require an increase in expression in all cells or tissues of the subject. For example, as used herein, an increase in a subject may include an increase in gene expression or protein production in the liver of the subject.

[0367] The term “improved” may also be used in relation to normalizing the symptoms of a disease or condition, i.e., raising the difference between the level in a subject with a FOXP3-related disease or autoimmune disease and the level in a normal subject without either FOXP3-related disease or autoimmune disease toward or to the level of the normal subject. As used herein, “normal” is considered the upper limit of normal if the disease is related to an increase in the value of the symptoms. “Normal” is considered the lower limit of normal if the disease is related to a decrease in the value of the symptoms.

[0368] In some embodiments, the cells suitable for use in the method of the present invention are mammalian cells. In some embodiments, the cells are somatic cells. In some embodiments, the cells are primary cells. For example, in some embodiments, the cells are mammalian somatic cells. In some embodiments, the mammalian somatic cells are primary cells. In some embodiments, the mammalian somatic cells are non-embryonic cells.

[0369] B. In vitro generation of immune cells The contact step may be performed in vitro, in vivo (i.e., the cells may be within the subject), or ex vivo. In some embodiments, the cell contact step is performed ex vivo, and the method further includes the step of removing cells (e.g., mammalian cells) from the subject before the contact step. In some embodiments, the method further includes the step of administering cells (e.g., mammalian cells) to the subject after the contact step.

[0370] In one aspect of the present invention, the present invention provides a method for generating immune cells, such as Treg cells, comprising the site-specific FOXP3 disruptor of the present invention. The FOXP3 disruptor can modulate, for example, increase the expression of the FOXP3 gene for a period of time sufficient to direct immune cells into a differentiation pathway or alter their activation status, for example, to induce naive T cells to differentiate into Treg cells or to activate Treg cells.

[0371] Methods for manipulating immune cells In one embodiment, the present invention provides a method for manipulating cells, such as immune cells or subpopulations thereof (e.g., Treg or naive T cells). In this context, the term “manipulation” includes, for example, activation, division, differentiation, proliferation, expansion, reprogramming, anergy, quiescence, senescence, apoptosis, or death of target cells.

[0372] Various cells, including fresh samples derived from the target, primary cultured cells, immortalized cells, cell lines, and hybridomas, such as immune cells, can be manipulated. The cells to be manipulated may also include stem cells, such as embryonic stem cells, induced pluripotent stem cells, and mobilized peripheral blood stem cells. The manipulated cells can be used for various immunotherapeutic applications as well as research.

[0373] In certain embodiments of the present invention, cells can be manipulated ex vivo by culturing a sample containing immune cells, such as a sample obtained from a subject that would benefit from the modulation of FOXP3 expression, with the FOXP3 disruptor of the present invention.

[0374] In certain embodiments, the immune cells to be manipulated may be naive T cells isolated from umbilical cord blood or peripheral blood. Naive T cells can be manipulated (e.g., differentiated and / or activated) by contacting the cells with the FOXP3 disruptor of the present invention. In some embodiments, naive T cells can be further differentiated into antigen-specific Tregs by contacting them with an antigen or antigen-presenting cells. In some embodiments, the immune cells to be manipulated may be Treg cells. Tregs can be manipulated (e.g., activated) by contacting the cells with the FOXP3 disruptor of the present invention.

[0375] Methods for isolating the aforementioned T cells from samples such as those derived from the target are well known in the art and are described below.

[0376] As used herein, the terms “regulatory T cells,” “Treg cells,” or “Treg,” also known as “suppressor T cells,” refer to a population of T cells that modulate the immune system, maintain tolerance to autoantigens, and prevent autoimmune diseases. Tregs are immunosuppressive and generally suppress or downregulate the induction and proliferation of effector T cells. Tregs express the biomarkers CD4, FOXP3, and CD25 and are thought to originate from the same lineage as naive CD4 cells.

[0377] As used herein, the term “naive T cells” refers to a population of T cells differentiated in the bone marrow that successfully undergoes positive or negative central selection processes in the thymus. These include naive helper T cells (CD4 + ) and cytotoxic T cells (CD8 +) exists. Naive T cells are thought to be mature and, unlike activated or memory T cells, have never encountered their alloantigen in the periphery.

[0378] Expansion of the T cell population In related embodiments, the present invention further relates to a method for expanding a specific immune cell, such as a naive T cell or Treg, from an immune cell population, for example, a Treg or naive T cell contained in a sample containing B cells, dendritic cells, macrophages, plasma cells, etc. In another embodiment, the present invention also relates to a method for expanding a specific population of T cells, for example, a differentiated / activated Treg.

[0379] In one embodiment, immune cells, such as Tregs, are expanded (e.g., proliferated or differentiated) by culturing a sample containing immune cells with the FOXP3 disruptor of the present invention in ex vivo. In one embodiment, T cell expansion in ex vivo may be performed by first isolating Tregs or naive T cells from a sample, and then stimulating the T cells so that the Tregs are activated and / or expanded by contacting the T cells with the FOXP3 disruptor of the present invention.

[0380] In one embodiment of the present invention, the T cells are primary T cells obtained from a subject. T cells can be obtained from several sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymic tissue, tissue from an infection site, spleen tissue, and tumors. In certain embodiments of the present invention, any number of primary T cells and / or T cell lines available in the art can be used.

[0381] Studies of whole blood counts clearly demonstrate the very small number of T cells in whole blood. For example, according to a product catalog (document number 23629, version 2.1.0) published by Stem Cell Technologies, Vancouver, BC, CANADA, the white blood cell population in whole blood is approximately 0.1-0.2% (due to red blood cell dominance), of which T cells constitute approximately 7-24% of the total white blood cell population. Of these T cells, CD4 + T cells make up about 4-20% of the total white blood cell population (which means less than 0.04% of the total cell population in whole blood), and CD8 + T cells constitute approximately 2–11% of the total leukocyte population (which is less than 0.022% of the total cell population in whole blood). Therefore, in certain embodiments of the present invention, the method of the present invention can be used in combination with other techniques known in the art for enriching immune cells, such as naive T cells or Tregs. The enrichment step can be performed before contacting the sample with the FOXP3 disruptor of the present invention. In another embodiment, the enrichment step can be performed after contacting the sample with the FOXP3 disruptor of the present invention.

[0382] In one embodiment, a population of Tregs can be enriched using Ficol separation. In one embodiment, cells from the circulating blood of an individual are obtained by apheresis or leukocyte apheresis. The apheresis product generally contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. Cells collected by apheresis can be washed to remove the plasma fraction and to place the cells into a suitable buffer or medium for subsequent processing steps. The cells are then washed with phosphate-buffered saline (PBS). Alternatively, the washing solution may be calcium-deficient, magnesium-deficient, or lack many but not all divalent cations. A semi-automatic "flow-through" centrifuge can also be used according to the manufacturer's instructions. After washing, the cells can be resuspended in various biocompatible buffers, such as Ca-free, Mg-free PBS. Alternatively, undesirable components of the apheresis sample can be removed, and the cells can be resuspended directly in a culture medium.

[0383] In another embodiment, peripheral or whole blood T cells can be enriched by lysing red blood cells and depleting monocytes, for example, by centrifugation using a PERCOLL® gradient. A specific subpopulation of T cells, for example, CD28 + CD4 + CD8 + CD45RA + and CD45RO + T cells can be further isolated using positive or negative selection techniques.

[0384] In accordance with the present invention, various sorting techniques can be used as needed. For example, an enlarged or manipulated T cell population can be further sorted using a combination of antibodies against cell-specific surface markers. A preferred method is cell sorting and / or selection by magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies against cell surface markers present on the selected cells. For example, to enrich Tregs, CD4 +CD25 + CD62L hi GITR + , and FoxP3 + It is sometimes desirable to select regulatory T cells that normally express [specific gene / symptom].

[0385] To isolate a desired population of cells, the cell concentration and scaffold surface can be varied. In certain embodiments, it may be desirable to significantly reduce the volume in which the FOXP3 disruptor and T cells are mixed together (i.e., increase the cell concentration) in order to ensure that contact between cells and the FOXP3 disruptor is maximized. For example, in one embodiment, a concentration of 2 billion cells / ml is used. In another embodiment, a concentration of 1 billion cells / ml is used. In further embodiments, concentrations higher than 100 million cells / ml are used. In further embodiments, cell concentrations of 10 million, 15 million, 20 million, 25 million, 30 million, 35 million, 40 million, 45 million, or 50 million cells / ml are used. In yet another embodiment, cell concentrations ranging from 75 million, 80 million, 85 million, 90 million, 95 million, or 100 million cells / ml are used. In yet another embodiment, concentrations of 125 million or 150 million cells / ml may be used. Using higher concentrations can result in increased cell yield, cell activation, and cell expansion. Furthermore, the use of high cell concentrations allows for more efficient capture of cells that may weakly express the target antigen of interest.

[0386] In one embodiment, the present invention may include procedures known in the art for sample preparation. For example, T cells can be frozen after a washing step and thawed before use. Freezing and subsequent thawing result in a more homogeneous product by removing granulocytes and, to some extent, monocytes from the cell population. After a washing step to remove plasma and platelets, the cells can be suspended in a freezing solution. Many freezing solutions and parameters are known in the art and may be useful in this regard, but one method involves using PBS containing 20% ​​DMSO and 8% human serum albumin, or other suitable cell freezing media containing, for example, HESPAN and PLASMALYTE A, and then the cells are frozen to -80°C at a rate of 1° per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other controlled freezing methods, as well as immediate, uncontrolled freezing at -20°C or in liquid nitrogen, may be used.

[0387] The collection of blood samples or leukocyte apheresis products from subjects during the period until the enlarged cells described herein may be required is also contemplated in connection with the present invention. Therefore, the cell source to be enlarged can be collected at any point in time as needed, and desired cells, such as T cells, can be isolated and frozen for later use in T cell therapy for any number of diseases or conditions that would benefit from T cell therapy, such as those described herein. In one embodiment, the blood sample or leukocyte apheresis product is collected from a generally healthy subject. In a particular embodiment, the blood sample or leukocyte apheresis product is collected from a generally healthy subject at risk of developing the disease but who has not yet developed the disease, and the cells of interest are isolated and frozen for later use. In a particular embodiment, T cells can be enlarged, frozen, and used at a later point in time. In a particular embodiment, the sample is collected from a patient immediately after diagnosis of a particular disease described herein but before any treatment.

[0388] In related embodiments, the present invention is CD4 + / FOXP3 + or CD4+ / FOXP3 - The present invention relates to a method for obtaining a polyclonal population of cells. The method is a step of contacting a biological sample of the present invention with a FOXP3 disruptor, thereby activating and, if necessary, expanding a population of T cells present in the sample; and a step of the T cells in the sample with CD4 + Steps to bring T cells into contact with reagents for cell detection; FOXP3 + Steps include further contact with reagents for cell detection; and detected CD4 + / FOXP3 + or CD4 + / FOXP3 - The step includes isolating a subpopulation of T cells from the sample. In these embodiments, CD4 + and / or FOXP3 + The reagent for detecting and / or isolating T cells is preferably CD4 + and an antibody or its antigen-binding fragment that specifically binds to the FOXP3 marker.

[0389] In yet another embodiment, the present invention relates to a method for obtaining a population of naive T cells. Methods for isolating naive T cells are known in the art, for example, STEMCELL Technologies' EasySep® Human Naive CD4 + Use commercially available kits such as T cell isolation kits.

[0390] In certain embodiments, differentiated / activated immune cells can be further expanded. For example, activated Treg cells can be further expanded by culturing them in the presence of a specific cytokine, such as IL-2.

[0391] Accordingly, in another embodiment, the present invention provides immune cells comprising a FOXP3 disruptor. In some embodiments, the FOXP3 disruptor may be present in immune cells for a period of time sufficient to induce immune cells, such as naive T cells, to differentiate into Tregs or to activate Tregs. In certain embodiments, the immune cells may contain one or more genetic modifications that modulate, for example, activate, the expression of the FOXP3 gene. Such genetic modifications may be present in the cells after the FOXP3 disruptor has been eliminated from the cells or remains in the cells at very low levels. Thus, the expression of the FOXP3 gene may remain activated even after the FOXP3 disruptor has ceased to function.

[0392] In such circumstances, gene modification can be introduced using site-specific FOXP3 disruptors. Gene modification involves the addition, deletion, or substitution of one or more nucleotides to a target sequence, e.g., the DNA region surrounding / proximal upstream of the TSS of the FOXP3 gene. Gene modification may be an epigenetic modification (e.g., methylation / demethylation) of one or more nucleotides in the target sequence, or an epigenetic modification (e.g., acetylation / deacetylation) of one or more chromatin proteins in the target sequence, e.g., the DNA region surrounding / proximal upstream of the TSS of the FOXP3 gene.

[0393] C. In vivo method of the present invention The in vivo method of the present invention may include the step of administering the drug, composition, or cells of the present invention to a subject.

[0394] In one embodiment, immune cells, such as naive T cells or Tregs, are manipulated (e.g., activated) in vivo by providing the FOXP3 disruptor of the present invention to the immune cells, such as naive T cells or Tregs, so that they come into contact with the disruptor. To facilitate contact, the FOXP3 disruptor can be administered to the target, for example, subcutaneously or intravenously.

[0395] The term “subject” as used herein refers to an organism, e.g., a mammal (e.g., human, non-human mammal, non-human primate, primate, laboratory animal, mouse, rat, hamster, gerbil, cat, or dog). In some embodiments, a human subject is an adult, adolescent, or child subject. In some embodiments, a subject has a disease or condition. In some embodiments, a subject suffers from a disease, disorder, or condition, e.g., a disease, disorder, or condition that can be treated as provided herein. In some embodiments, a subject is susceptible to a disease, disorder, or condition, and in some embodiments, a susceptible subject is predisposed to developing a disease, disorder, or condition and / or exhibits an increased risk of developing a disease, disorder, or condition (compared to the average risk observed in a reference subject or population). In some embodiments, a subject exhibits one or more symptoms of a disease, disorder, or condition. In some embodiments, a subject does not exhibit a specific symptom (e.g., clinical findings of a disease) or feature of a disease, disorder, or condition. In some embodiments, a subject does not exhibit any symptom or feature of a disease, disorder, or condition. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual who is and / or has been administered a diagnosis and / or treatment.

[0396] Subjects who will benefit from the method of the present invention include subjects with autoimmune diseases, subjects at risk of autoimmune diseases, subjects with "FOXP3-related disease," or subjects at risk of "FOXP3-related disease."

[0397] Accordingly, the present invention further provides methods for treating subjects that require such treatment. A treatment method of the present invention includes the step of administering a therapeutically effective dose of the agent, composition, or cells of the present invention to a subject, for example, a subject who would benefit from the modulation of FOXP3 expression, for example, a subject having an autoimmune disease or a FOXP3-related disease. In some embodiments, the method of the present invention includes a subject to whom two site-specific FOX3P disruptors (first and second agents) may be administered. The two site-specific FOX3P disruptors may be present in the same composition, for example, a pharmaceutical composition, for example, a pharmaceutical composition containing LNP, or in separate compositions, for example, a pharmaceutical composition, for example, a pharmaceutical composition containing LNP. The subject may be administered the first site-specific FOX3P disruptor at a first time point and the second site-specific FOX3P disruptor at a second time point, or both agents may be administered to the subject simultaneously.

[0398] In addition, the present invention provides a method for preventing at least one symptom in subjects who would benefit from modulation of FOXP3 expression, for example, subjects with autoimmune diseases or FOXP3-related diseases, by administering the agent, composition, or cells of the present invention to the subjects in a prophylactic effective dose.

[0399] When used herein, “therapeutic dose” is intended to include the amount of a drug, composition, or cells that, when administered to a patient to treat a subject having an autoimmune disease or a FOXP3-related disease, is sufficient to treat the disease (for example, by reducing, improving, or maintaining one or more symptoms of the existing disease or disease or associated comorbidities). “Therapeutic dose” may vary depending on the drug, composition, or cells, the method of administration, the disease and its severity, and the patient being treated, including medical history, age, weight, family history, genetic structure, stage of the pathological process mediated by FOXP3 gene expression, type of prior or concomitant treatment, if any, and other personal characteristics.

[0400] When used herein, “Prophylactic effective dose” is intended to include an amount of drug, composition, or cells sufficient to prevent or delay, for a clinically significant period, the onset or progression of the disease or one or more symptoms of the disease, when administered to a subject who has not yet experienced or shown symptoms of the disease but may be predisposed to the disease. The “Prophylactic effective dose” may vary depending on the drug or composition, the method of administration, the degree of risk of the disease, and the patient being treated, including medical history, age, weight, family history, genetic makeup, type of prior or concomitant treatment, if any, and other personal characteristics.

[0401] As used herein, “prevention” or “prevention” when used in relation to a disease, disorder, or condition that would benefit from the expression of the FOXP3 gene or the activation of the production of the FOXP3 protein means a reduction in the likelihood that the subject would develop symptoms associated with such disease, disorder, or condition, such as signs or symptoms of Treg or FOXP3 gene dysfunction.

[0402] The “therapeutic dose” or “preventive dose” also includes the amount of a drug, composition, or cells that produces any desired local or systemic effect in a reasonable cost-benefit ratio appropriate to any treatment. The drugs and compositions or cells used in the methods of the present invention can be administered in an amount sufficient to produce a reasonable cost-benefit ratio appropriate to such treatment. In some embodiments, the therapeutic dose or preventive dose is administered as a single dose, and in some embodiments, multiple unit doses are required to deliver the therapeutic or preventive dose.

[0403] As used herein, the phrase “symptoms are reduced” may be used when one or more symptoms of a particular disease, disorder, or condition are reduced in magnitude (e.g., intensity, severity, etc.) and / or frequency. In some embodiments, a delay in the onset of a particular symptom is considered a form of reduction in the frequency of that symptom.

[0404] When the subject being treated is a mammal such as a human, the composition or cells may be administered by any means known in the art, including but not limited to oral, intraperitoneal, or parenteral routes, including intracranial (e.g., intravenous, intraparenchymal, and subarachnoid), intravenous, intramuscular, subcutaneous, transdermal, respiratory (aerosol), nasal, rectal, and local (including buccal and sublingual) administration. In certain embodiments, the composition is administered by intravenous infusion or injection. In certain embodiments, the composition is administered by subcutaneous injection.

[0405] As used herein, the term “FOXP3-related disease” includes diseases, disorders, or conditions that would benefit from modulation, e.g., increased, of FOXP3 gene expression, replication, or protein activity, such as autoimmune diseases or diseases associated with Treg dysfunction. Non-exclusive examples of FOXP3-related diseases include autoimmune diseases such as IPEX syndrome (IPEX), type 1 diabetes, multiple sclerosis, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), esophageal achalasia, Addison's disease, adult-onset Still's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, antiphospholipid syndrome, autoimmune angioedema, autoimmune autonomic neuropathy, autoimmune encephalomyelitis, autoimmune hepatitis, and autoimmune inner ear disease (AI). ED), autoimmune myocarditis, autoimmune oophoritis, autoimmune orchitis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune urticaria, axonal and neuronal neuropathy (AMAN), Baro's disease, Behçet's disease, benign mucosal pemphigoid, bullous pemphigoid, Castleman disease (CD), celiac disease, Chagas disease, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic relapsing polymyelitis (CRMO), Churg-Strauss syndrome (CSS) or eosinophilic granulomatosis (EGPA), scarring pemphigoid, Cogan syndrome, cold agglutinin disease, congenital heart block, coxsackie myocarditis, CREST syndrome, Crohn's disease, herpetiform dermatitis, dermatomyositis, Devic's disease (neuromyelitis optica), lupus discoid, Dressler syndrome, endometriosis, eosinophilic esophagitis (EoE), eosinophilic fasciitis, erythema nodosum, essential mixed cryoglobulinemia, Evans syndrome, fibromyalgia, fibrotic alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, Goodpasture syndrome, granulomatosis with polyangiitis, Grave Sz. scourge, Guillain-Barré syndrome, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schönlein purpura (HSP), herpes zoster of pregnancy or bullous pemphigoid of pregnancy (PG), hidradenitis suppurativa (HS) (reverse acne), hypogammaglobulinemia, IgA nephropathy, IgG4-related sclerosing disease, immune thrombocytopenic purpura (ITP), inclusion body myositis (IBM), interstitial cystitis (IC), juvenile arthritis, juvenile diabetes mellitus (type 1 diabetes mellitus), juvenile myositis (JM), Kawasaki disease, Lambert-Eaton syndrome, leukocytosis-destructive vasculitis, lichen planus,Lichen sclerosing, woody conjunctivitis, linear IgA disease (LAD), lupus, chronic Lyme disease, Meniere's disease, microscopic polyangiitis (MPA), mixed connective tissue disease (MCTD), Mohren's ulcer, Mucha-Habermann disease, multifocal motor neuropathy (MMN) or MMNCB, myasthenia gravis, myositis, narcolepsy, neonatal lupus, neuromyelitis optica, neutropenia, ocular scarring pemphigoid, optic neuritis, recurrent lupus Rheumatism (PR), PANDAS, paraneoplastic cerebellar degeneration (PCD), paroxysmal nocturnal hemoglobinuria (PNH), Parry-Romberg syndrome, peripheral uveitis, Personage-Turner syndrome, pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia (PA), POEMS syndrome, polyarteritis nodosa, polymyositis type I, II, III, polymyalgia rheumatica, polymyositis Post-myocardial infarction syndrome, post-pericardiotomy syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, progesterone-induced dermatitis, psoriasis, psoriatic arthritis, pure red cell fistula (PRCA), pyoderma gangrenosum, Raynaud's phenomenon, reactive arthritis, reflex sympathetic dystrophy, relapsing polychondritis, restless legs syndrome (RLS), retroperitoneal fibrosis, rheumatic fever, sarcoidosis, Schmidt syndrome, scleritis, scleroderma, Sjögren's syndrome These include group uritis, sperm and testicular autoimmunity, generalized rigidus syndrome (SPS), subacute bacterial endocarditis (SBE), Suzak syndrome, sympathetic ophthalmitis (SO), Takayasu's arteritis, temporal arteritis / giant cell arteritis, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome (THS), transverse myelitis, ulcerative colitis (UC), undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vitiligo, and Vogt-Koyanagi-Harada disease.

[0406] In one embodiment, the FOXP3-related disease is selected from the group consisting of IPEX syndrome (IPEX), type 1 diabetes mellitus, multiple sclerosis, systemic lupus erythematosus (SLE), and rheumatoid arthritis (RA).

[0407] Further details regarding the signs and symptoms of various diseases or conditions are provided herein and are well known in the art (see, for example, ghr.nlm.nih.gov).

[0408] Administration of the agent, composition, or cells of the present invention by the method of the present invention may result in a reduction of the severity, signs, symptoms, or markers of foxp3-related disease or disorder in patients with foxp3-related disease or disorder. In this context, “reduction” means a statistically significant decrease in such levels. The reduction (absolute reduction, or reduction in the difference between the elevated level and the normal level in the subject) may be, for example, at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the detection level of the assay used, or less.

[0409] Administration of a drug, composition, or cells by the method of the present invention can stably or transiently modulate the expression of a target gene, or stably or transiently increase the amount or activation level of Tregs. In some embodiments, the modulation of expression lasts 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 longer, or any time in between. In some other embodiments, the modulation of expression lasts for about 30 minutes to about 7 days, or about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, 4 days, 5 days, 6 days, 7 days, or any time in between. In certain embodiments, the amount of Treg can increase by at least about 5% to about 10 times, or at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, or more, or any multiplier in between. In certain embodiments, the percentage of activated Tregs, such as Tregs characterized by increased expression of FOXP3, can increase by at least about 5% to about 10 times, or by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times, or more, or any multiplier in between.In some embodiments, FOXP3 expression in or in a Treg population may increase by at least about 5% to about 10 times, or by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times, or more, or any multiplier in between.

[0410] A drug, composition, or cell may be administered to a subject as a single dose, or instead, multiple doses may be administered over a period of time. For example, two, three, four, five, or more doses may be administered to a subject during a single treatment or over a period of time. In some embodiments, six, eight, ten, twelve, fifteen, or twenty, or more doses may be administered to a subject as a treatment regimen during a single treatment or over a period of time.

[0411] In some embodiments, administration may be given as needed, for example, as long as symptoms associated with the disease, disorder, or condition persist. In some embodiments, repeated administration may be directed over the remainder of the subject's life. The duration of treatment may vary, for example, to one day, two days, three days, one week, two weeks, one month, two months, three months, six months, one year, or longer.

[0412] The effectiveness of disease treatment or prevention can be assessed by measuring, for example, disease progression, disease remission, symptom severity, pain relief, quality of life, dosage required to sustain the treatment effect, levels of disease markers, or any other measurable parameters appropriate to a given disease being treated or targeted for prevention. Monitoring the effectiveness of treatment or prevention by measuring any one of such parameters, or any combination of parameters, is well within the capabilities of those skilled in the art. As discussed herein, the specific parameters to be measured will depend on the autoimmune disease or FOXP3-related disease to which the subject is suffering.

[0413] By comparing the initial reading with subsequent readings, a physician can obtain an indicator of whether the treatment is effective. Monitoring the effectiveness of a treatment or prevention by measuring any one of such parameters, or any combination of parameters, is well within the capabilities of a person skilled in the art. In relation to the administration of a drug or composition, "effective against" autoimmune disease or FOXP3-related disorder indicates that administration in a clinically appropriate manner results in a beneficial effect for at least a statistically significant proportion of patients, e.g., improvement of symptoms, cure, reduction of disease, extension of lifespan, improvement of quality of life, or other effects generally perceived as positive by physicians familiar with the treatment of FOXP3-related disorder.

[0414] The therapeutic or preventive effect is evident when there is a statistically significant improvement in one or more parameters of the disease status, or when there is no worsening or onset of symptoms that would otherwise be expected. For example, a favorable change of at least 10%, preferably at least 20%, 30%, 40%, 50%, or higher, in the measurable parameters of the disease may indicate an effective treatment. The efficacy of a given drug or composition can also be determined using an experimental animal model of a given disease known in the art. When using an experimental animal model, the efficacy of the treatment is demonstrated when a statistically significant reduction in markers or symptom reduction is observed.

[0415] Alternatively, efficacy may be measured by a reduction in disease severity, as determined by those skilled in the field of diagnostic technology based on clinically accepted disease severity assessment scales. For example, any desirable change resulting in a reduction in disease severity as measured using an appropriate scale also represents an appropriate treatment using the agents or compositions described herein.

[0416] As used herein, the terms “to treat” or “treatment” refer to a beneficial or desired outcome, including but not limited to the alleviation or improvement of one or more signs or symptoms associated with an autoimmune disease, or the reduction of FOXP3 gene expression or FOXP3 protein production. “Treatment” may also mean an extension of survival compared to the survival expected in the absence of treatment.

[0417] D. Combination Methods The present invention further provides a combination method for activating Treg cells. In certain embodiments, the in vitro or ex vivo differentiation or activation of Tregs by FOXP3 activation can be combined with stimulation by TGFβ, for example, by including TGFβ in the growth medium used when culturing cells in vitro or ex vivo, as described herein. TGFβ is a key growth factor in T cell differentiation and is known to induce FOXP3 activation.

[0418] The present invention will now be illustrated by the following embodiments. However, the use of these and other examples found throughout this specification is for illustrative purposes only and does not limit in any way the scope and meaning of the present invention or any of the illustrated embodiments. The present invention is not limited to any particular preferred embodiment described herein. Many modified and altered forms of the present invention may be apparent to those skilled in the art and can be made without departing from the spirit and scope of the invention. All references, patents and published patent applications referenced throughout this application, including the figures, are incorporated herein by reference. [Examples]

[0419] (Example 1) FOXP3 activation in Jarcut cells This embodiment describes the activation of FOXP3 expression in Jurcut cells, measured by increases in FOXP3 mRNA and protein levels, using a site-specific FOXP3 disruptor comprising a site-specific FOXP3 targeting moiety, i.e., sgRNA, that targets the FOXP3 expression regulatory region, and an effector containing dCas9, dCas9 and p300, or dCas9 and VPR.

[0420] To understand the effect of the activator of the present invention on FOXP3 gene expression, Jurcat cells, a human leukemia T cell line, were transfected with either dCas9-encoding mRNA, dCas9-p300-encoding mRNA, or dCas9-VPR-encoding mRNA, along with sgRNAs targeting different regions around and upstream of the transcription start site (TSS), using Lipofectamine Messeger Max as recommended by the manufacturer. Three guide RNA pools were used, and one pool was found to activate FOXP3 mRNA possessing both p300 (9x) and VPR (100x) (Figure 1A). The sgRNA pool was found to induce FOXP3 protein production in combination with the VPR protein. After treatment with the combination of sgRNA (pool 2) and dCas9-VPR-encoding mRNA, 8% of cells were assayed by FACS and became FOXP3-positive (Figure 1B).

[0421] When using a guide pool or individual guides, an activation response via VPR can be achieved. While we do not wish to be constrained by theory, the activation observed in this experiment is thought to be a result of the recruitment of multiple effectors / activators to the target site, or rather, the activation mechanism (Figure 2). The guide pools used in Examples 1 and 2 are summarized in Table 2.

[0422] (Example 2) FOXP3 activation in naive T cells This embodiment describes the activation of FOXP3 expression in naive T cells, measured by increases in FOXP3 mRNA and protein levels, using a site-specific FOXP3 disruptor comprising a site-specific FOXP3 targeting moiety that targets the FOXP3 expression regulatory region, i.e., sgRNA, and an effector containing dCas9, dCas9 and p300, or dCas9 and VPR.

[0423] To compare the effects of dCas9 alone versus dCas9-p300 or dCas9-VPR activator fusions on activation in naive T cells, electroporation was performed using a MaxCyte ATx with the manufacturer's recommended electroporation settings, transfecting with the same mRNA and three sgRNA guide combinations (Pool 2) optimized in the Jarcut experiment detailed above. Similar to the Jarcut cells, VPR was found to elicit the greatest response to FOXP3 mRNA expression (up to 600-fold compared to dCas9 alone), as determined by qPCR, and assay by FACS analysis revealed that 10-14% of cells were FOXP3-activated. + It was determined to be a cell (Figure 3). Table 2: Site-specific FOXP3 targeting regions - The first 20 nucleotides in each region below constitute the targeting region. [Table 2-1] [Table 2-2] Table 3: Site-specific FOXP3 targeting regions - Nucleotides of FOXP3 guides used in Examples 1 and 2 Sequences and complementary target sequences within the genome [Table 3] Note: *: All single guides were 100 nucleotides long. The 20-mer targeting region shown is the portion of the SpCas9 PAM single guide RNA that has the following 80-nt sequence for dCas9 binding. tta: [ka] #: The pool numbers are the same as those in Figures 1A and 1B. **Pool 2 strongly induced an upregulatory response in Jurcut cells, and this pool was successfully used to activate FOXP3 in naive T cells. Table 4: Site-specific FOXP3 targeting moieties [Table 4-1] [Table 4-2] Table 5. Abbreviations for nucleotide monomers used in nucleic acid sequence representation. It should be understood that when these monomers are present in oligonucleotides, they are linked to each other by 5'-3'-phosphodiester bonds. [Table 5]

Claims

[Claim 1] The invention described in the specification.

Images