WO2017181026A1

WO2017181026A1 - Selective modulation of foxp3 expression

Info

Publication number: WO2017181026A1
Application number: PCT/US2017/027655
Authority: WO
Inventors: Bethany FORTNAM; Caroline WOO
Original assignee: Translate Bio Ma, Inc.
Priority date: 2016-04-15
Filing date: 2017-04-14
Publication date: 2017-10-19

Abstract

Aspects of the disclosure provide compositions, kits, and methods for selective modulation of FOXP3 expression. Single stranded oligonucleotides for activating or enhancing expression of FOXP3 are also provided herein.

Description

SELECTIVE MODULATION OF FOXP3 EXPRESSION

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. US 62/323,510, filed on April 15, 2016 and U.S. Provisional Application No. US 62/374,168, filed on August 12, 2016, the contents of each of which are incorporated herein by reference in its entirety.

FIELD OF THE DISCLOSURE

The disclosure relates to compositions (e.g., oligonucleotide-based compositions) and methods for modulating FOXP3 expression. Methods of using FOXP3 expression- modulating compositions for treating diseases or disorders are also described herein.

BACKGROUND OF THE DISCLOSURE FOXP3 (forkhead box P3), a member of the FOX protein family, is a master regulator transcription factor that drives the differentiation and activity of immune suppressive regulatory T cells (Tregs). Tregs are Foxp3+CD4+CD25+ T lymphocytes which have immune suppressive activity and can establish a toleragenic response. It has been shown previously that administration of Foxp3+ Treg cells leads to marked reductions in

inflammatory/autoimmune disease severity in animal models of type 1 diabetes, multiple sclerosis, asthma, inflammatory bowel disease, and thyroiditis. Expression of FOXP3 decreases effector T cell proliferation and activity. Additionally, Foxp3+ T cells can control a Thl response, Thl7 response, suppress antibody production, CD8+ cytotoxic T cell activity and antigen presentation.

Alterations in the number or function of Tregs, such as those that express Foxp3, are associated with several disease states. For example, patients with autoimmune diseases such as systemic lupus erythematosus (SLE) have been found to have defective regulatory function of Tregs. The FOXP3 gene has also been shown to be mutated in patients with IPEX

(Immunodysregulation, Polyendocrinopathy, and Enteropathy, X-linked) syndrome. IPEX syndrome is characterized by the development of multiple autoimmune disorders, such as enteropathy, dermatitis, and Type 1 diabetes, in affected patients. Accordingly, there is a need to develop compositions and methods for selective modulation of FOXP3 expression, which can be used to treat FOXP3-associated diseases or disorders.

SUMMARY OF THE DISCLOSURE

Aspects of the disclosure relate to methods and compositions that are useful for upregulating expression of FOXP3 in cells. In some aspects, single stranded oligonucleotides are provided that target a portion of a PRC2-associated region of an FOXP3 gene (e.g., as set forth in SEQ ID NO: 3) and thereby cause upregulation of the gene. In other aspects, compositions (e.g., single stranded oligonucleotides) are provided that downregulate expression of CCDC22 in cells and thereby upregulate expression of FOXP3 in cells.

Aspects of the disclosure provide methods and compositions that are useful for upregulating FOXP3 for the treatment and/or prevention of diseases or disorders associated with aberrant immune cell (e.g., T cell) activation, e.g., autoimmune or inflammatory diseases or disorders.

Other aspects of the disclosure relate to methods and compositions that are useful for upregulating FOXP3 in cells of the immune system such as T cells or other lymphocytes. In some embodiments, methods are provided for increasing FOXP3 expression in lymphocytes ex vivo for administration to a subject. Because FOXP3 is a transcription factor that drives T cell differentiation and activity of T regulatory cells (Tregs), such embodiments are useful, for example, for generating Tregs ex vivo from isolated T cell or lymphocyte populations obtained from a subject. Such Tregs can be delivered to a subject {e.g., allogenically or autologously) to promote immune suppressive activity and/or a toleragenic response.

Accordingly, methods provided herein may be useful in some embodiments for treating autoimmune conditions or transplant rejection or graft versus host disease or other related conditions for which suppression of an immune response is desired. For example, in some embodiments, the Tregs are useful for suppressing T cell-mediated immunity and self- reactive T cells that have escaped negative selection. In some embodiments, Tregs produced by upregulating FOXP3 expression can be further modified by engineering them to express a chimeric antigen receptor (CAR). For example, in some embodiments, lymphocytes {e.g., Tregs) can be engineered to express CARs that target antigens that cause inflammatory or autoimmune responses, such as self-antigens.

According to some aspects of the disclosure, single stranded oligonucleotides are provided that have a region of complementarity that is complementary with (e.g., at least 8 consecutive nucleotides of) a PRC2-associated region of a FOXP3 gene, e.g., a PRC2- associated region located within a sequence corresponding to nucleotide 511 to nucleotide 920 of SEQ ID NO: 3. In some embodiments, the oligonucleotide has at least one of the following features: a) a sequence that is 5'X-Y-Z, in which X is any nucleotide and in which X is at the 5' end of the oligonucleotide, Y is a nucleotide sequence of 6 nucleotides in length that is not a human seed sequence of a microRNA, and Z is a nucleotide sequence of 1 to 23 nucleotides in length; b) a sequence that does not comprise three or more consecutive guanosine nucleotides; c) a sequence that does not bind to an off-target sequence that is located beyond 50 kilobases upstream of a 5 '-end of a FOXP3 gene or beyond 50 kilobases downstream of a 3 '-end of the FOXP3 gene; d) a sequence comprising the region of complementarity that is complementary to the at least 8 consecutive nucleotides of the first sequence, wherein the at least 8 consecutive nucleotides encode a portion of an RNA that forms a secondary structure comprising at least two single stranded loops; e) a sequence that has greater than 60% G-C content; and f) a sequence that is not complementary with (e.g., at least 8 consecutive nucleotides) of a sequence corresponding to nucleotide 1 to nucleotide 510 of SEQ ID NO: 3. In some embodiments, the single stranded oligonucleotide has at least two of features a), b), c), d), e), and f), each independently selected. In some embodiments, the single stranded oligonucleotide has at least three of features a), b), c), d), e), and f), each independently selected. In some embodiments, the single stranded oligonucleotide has at least four of features a), b), c), d), e), and f), each independently selected. In some embodiments, the single stranded oligonucleotide has each of features a), b), c), d), e), and f).

In some embodiments, the single stranded oligonucleotide is 8 to 30 nucleotides in length. In some embodiments, the single stranded oligonucleotide is up to 50 nucleotides in length.

In some embodiments, at least one nucleotide of the oligonucleotide is a nucleotide analogue. In some embodiments, the at least one nucleotide analogue results in an increase in Tm of the oligonucleotide in a range of 1 to 5 °C compared with an oligonucleotide that does not have the at least one nucleotide analogue.

In some embodiments, at least one nucleotide of the oligonucleotide comprises a 2' O-methyl. In some embodiments, each nucleotide of the oligonucleotide comprises a 2' O- methyl. In some embodiments, the oligonucleotide comprises at least one ribonucleotide, at least one deoxyribonucleotide, or at least one bridged nucleotide. In some embodiments, the bridged nucleotide is a LNA nucleotide, a cEt nucleotide or a ENA modified nucleotide. In some embodiments, each nucleotide of the oligonucleotide is a LNA nucleotide.

In some embodiments, the nucleotides of the oligonucleotide comprise alternating deoxyribonucleotides and 2'-fluoro-deoxyribonucleotides. In some embodiments, the nucleotides of the oligonucleotide comprise alternating deoxyribonucleotides and 2'-0- methyl nucleotides. In some embodiments, the nucleotides of the oligonucleotide comprise alternating deoxyribonucleotides and ENA nucleotide analogues. In some embodiments, the nucleotides of the oligonucleotide comprise alternating deoxyribonucleotides and LNA nucleotides. In some embodiments, the 5' nucleotide of the oligonucleotide is a

deoxyribonucleotide. In some embodiments, the nucleotides of the oligonucleotide comprise alternating LNA nucleotides and 2'-0-methyl nucleotides. In some embodiments, the 5' nucleotide of the oligonucleotide is a LNA nucleotide. In some embodiments, the nucleotides of the oligonucleotide comprise deoxyribonucleotides flanked by at least one LNA nucleotide on each of the 5' and 3' ends of the deoxyribonucleotides.

In some embodiments, the single stranded oligonucleotide comprises modified internucleotide linkages (e.g., phosphorothioate internucleotide linkages or other linkages) between at least two, at least three, at least four, at least five or more nucleotides. In some embodiments, the single stranded oligonucleotide comprises modified internucleotide linkages (e.g., phosphorothioate internucleotide linkages or other linkages) between all nucleotides.

In some embodiments, the nucleotide at the 3' position of the oligonucleotide has a 3' hydroxyl group. In some embodiments, the nucleotide at the 3' position of the

oligonucleotide has a 3' thiophosphate. In some embodiments, the single stranded oligonucleotide has a biotin moiety or other moiety conjugated to its 5' or 3' nucleotide. In some embodiments, the single stranded oligonucleotide has cholesterol, Vitamin A, folate, sigma receptor ligands, aptamers, peptides, such as CPP, hydrophobic molecules, such as lipids, ASGPR or dynamic polyconjugates and variants thereof at its 5' or 3' end.

According to some aspects of the disclosure, single stranded oligonucleotides are provided that have a region of complementarity that is complementary with (e.g., at least 8 consecutive nucleotides of) a PRC2-associated region of a FOXP3 gene, e.g., a PRC2- associated region located within a sequence as set forth in SEQ ID NO: 3, wherein nucleotides of the oligonucleotide comprise alternating LNA nucleotides and 2'-0-methyl nucleotides. In some embodiments, such oligonucleotides further comprise phosphorothioate internucleotide linkages between all nucleotides. In some embodiments, the oligonucleotide has at least one of the following features: a) a sequence that does not comprise three or more consecutive guanosine nucleotides; b) a sequence that does not bind to an off-target sequence that is located beyond 50 kilobases upstream of a 5 '-end of a FOXP3 gene or beyond 50 kilobases downstream of a 3 '-end of the FOXP3 gene; c) a sequence comprising the region of complementarity that is complementary to the at least 8 consecutive nucleotides of the first sequence, wherein the at least 8 consecutive nucleotides encode a portion of an RNA that forms a secondary structure comprising at least two single stranded loops; d) a sequence that has greater than 60% G-C content; and e) a sequence that is not complementary with (e.g., at least 8 consecutive nucleotides) of a sequence corresponding to nucleotide 1 to nucleotide 510 of SEQ ID NO: 3. In some embodiments, the single stranded oligonucleotide has at least two of features a), b), c), d), and e), each independently selected. In some embodiments, the single stranded oligonucleotide has at least three of features a), b), c), d), and e), each independently selected. In some embodiments, the single stranded oligonucleotide has at least four of features a), b), c), d), and e), each independently selected. In some

embodiments, the single stranded oligonucleotide has each of features a), b), c), d), and e). In some embodiments, the single stranded oligonucleotide comprises a sequence selected from SEQ ID Nos. 4 to 13.

In some embodiments, the single stranded oligonucleotide is 8 to 30 nucleotides in length. In some embodiments, the single stranded oligonucleotide is up to 50 nucleotides in length. In some embodiments, the single stranded oligonucleotide is 15 nucleotides in length.

In some embodiments, at least one nucleotide of the oligonucleotide is a nucleotide analogue. In some embodiments, the at least one nucleotide analogue results in an increase in Tm of the oligonucleotide in a range of 1 to 5 °C compared with an oligonucleotide that does not have the at least one nucleotide analogue. In some embodiments, the 5' nucleotide of the oligonucleotide is a LNA nucleotide.

According to some aspects of the disclosure, methods of increasing expression of FOXP3 in a cell (e.g., a T cell) that comprise delivering to the cell an inhibitor of CCDC22 in an amount effective for increasing expression of FOXP3 in the cell are provided herein. In some embodiments, delivery of the inhibitor of CCDC22 into the cell results in (i) a level of expression of CCDC22 that is at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 90% or more) lower than a level of expression of CCDC22 in a control cell that does not comprise the inhibitor of CCDC22; and (ii) a level of expression of FOXP3 that is at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 90% or more) higher than a level of expression of FOXP3 in the control cell.

In some embodiments, the inhibitor of CCDC22 is an oligonucleotide having a region of complementarity that is complementary with (e.g., at least 8 consecutive nucleotides of) a portion of a CCDCC22 mRNA. In some embodiments, the inhibitor of CCDC22 is an oligonucleotide having a region of complementarity that is complementary with (e.g., at least 8 consecutive nucleotides of) exon 1 of the CCDCC22 mRNA. In some embodiments, the inhibitor of CCDC22 is an oligonucleotide having a region of complementarity that is complementary with (e.g., at least 8 consecutive nucleotides of) exon 2 of the CCDCC22 mRNA. In some embodiments, the oligonucleotide is in a form of a gapmer.

In some aspects, a method of increasing FOXP3 expression in a T cell ex vivo for administration to a subject is provided, the method comprising: (a) providing a population of T cells comprising a FOXP3 gene; (b) contacting the T cells ex vivo with a single stranded oligonucleotide as described herein or an inhibitor of CCDC22 as described herein; and (c) administering the contacted T cells to the subject.

In some embodiments, the population of T cells comprises activated T cells. In some embodiments, the activated T cells are produced by contacting CD4-positive T cells with an activating agent. In some embodiments, the activating agent is an anti-CD3 and/or anti- CD28 antibody. In some embodiments, the activated T cells express CD69 or IL-2RA.

In some embodiments, contacting the T cells with the single stranded oligonucleotide or inhibitor of CCDC22 increases the number of CD4+CD25+FOXP3+ T cells in the population.

In some embodiments, the method further comprises transfecting the T cells with an expression construct encoding a chimeric antigen receptor (CAR) before, after or

simultaneously with step (b). In some embodiments, the method further comprises transfecting the T cells with an expression construct encoding a chimeric antigen receptor (CAR) after step (b) and before step (c). In some embodiments, the CAR is specific for a self-antigen or an antigen that causes an inflammatory response. In some embodiments, the subject has an autoimmune or inflammatory disease or disorder. In some embodiments, the method further comprises (d) transplanting a cell, tissue or organ into the subject. In some embodiments, the method alleviates or prevents development of graft-versus-host disease in the subject. In some embodiments, the cell, tissue or organ is allogeneic to the subject.

In some embodiments, delivery of the single stranded oligonucleotide or inhibitor of

CCDC22 into the T cells results in a level of expression of FOXP3 in the T cells that is at least 50% greater than a level of expression of FOXP3 in a control cell that does not comprise the single stranded oligonucleotide or inhibitor of CCDC22. In some embodiments, delivery of the single stranded oligonucleotide or inhibitor of CCDC22 into the T cells results in an increased level of CTLA4, GITR, and/or IL-10 expression in the T cells compared to an appropriate control cell that does not comprise the single stranded oligonucleotide or inhibitor of CCDC22. In some embodiments, delivery of the single stranded oligonucleotide or inhibitor of CCDC22 into the T cells results in a level of expression of CTLA4, GITR, and/or IL-10 in the T cells that is at least 30% greater than a level of expression of CTLA4, GITR, and/or IL-10 in a control cell that does not comprise the single stranded oligonucleotide or inhibitor of CCDC22. In some embodiments, delivery of the inhibitor of CCDC22 into the T cells results in (i) a level of expression of CCDC22 that is at least 50% lower than a level of expression of CCDC22 in a control cell that does not comprise the inhibitor of CCDC22; and (ii) a level of expression of FOXP3 that is at least 50% higher than a level of expression of FOXP3 in the control cell.

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a graph illustrating that FOXP3 mRNA is upregulated when CCDC22 mRNA is knocked down with gapmers targeting an exon of CCDC22. The RQ (relative quantification) values for FOXP3 and CDC22 on the Y-axis are calculated relative to the untreated control. The left bar in each pair shows CCDC22 RQ and the right bar in each pair shows FOXP3 RQ.

FIG. 2 shows the design of single stranded oligonucleotides (e.g., mixmers) for modulation of FOXP3 expression, which target a PRC2-associated region that includes a sequence corresponding to the coding region for a portion of the 3' UTR of CCDC22 and a further sequence that is antisense to the coding region for the 3 'UTR of FOXP3. FIG. 3 is a graph showing effects of single stranded oligonucleotides on modulation of FOXP3 mRNA expression in donor T cells after seven days (Donor 48) and 4 days (Donor 49). The RQ (relative quantification) value for FOXP3 on the Y-axis is calculated relative to the untreated control. In each grouping of bars, the left bar shows 20 micromolar, the middle bar shows 10 micromolar, and the right bar shows 3 micromolar.

FIG. 4 is a graph showing the effect of an example single stranded oligonucleotide on modulation of FOXP3 mRNA expression in different donor T cells over a period of time. The RQ (relative quantification) value for FOXP3 on the Y-axis is calculated relative to the untreated control. In each grouping of bars, the left bar shows 20 micromolar and the right bar shows 10 micromolar.

FIGs. 5A-5B are graphs showing the effects of two different example single stranded oligonucleotides (Oligo-2 in FIG. 5A and Oligo-3 in FIG. 5B) on modulation of FOXP3 mRNA expression in different donor T cells over a period of time. In each grouping of bars, the left bar shows 20 micromolar and the right bar shows 10 micromolar.

FIGs. 6A-6B are graphs showing the effects of two different example single stranded oligonucleotides (Oligo-7 in FIG. 6A and Oligo-8 in FIG. 6B) on modulation of FOXP3 mRNA expression in different donor T cells over a period of time.

FIGs. 7 A -7B show the fold change in FOXP3 mRNA expression in donor T cells after four (FIG. 7A) and seven (FIG. 7B) days of treatment with example single stranded oligonucleotides as described herein. The fold changes were assessed by droplet digital PCR (left bar in each pair) and TaqMan (right bar in each pair).

FIG. 8 shows a genome browser view (e.g., using Integrative Genomics Viewer (IGV)) of the RIPseq against the EZH2 subunit of PRC2.

FIG. 9A shows FOXP3 protein expression as measured by flow cytometry in activated CD4+ T cells from a donor treated with an example single stranded oligonucleotide.

FIG. 9B shows the fold change of FOXP3+ cells (TF+ cells) after treatment of activated CD4+ T cells from a donor with EZH2 gapmer (positive control), an example single stranded oligonucleotide, a negative control oligonucleotide or untreated cells.

BRIEF DESCRIPTION OF CERTAIN TABLES

Table 1: Oligonucleotide sequences of FOXP3 mixmers that were evaluated as shown in the Examples. Nucleotide modifications are also included, where bX represents an LNA nucleotide, mX is a 2'-0-methyl nucleotide. An s at the end of a nucleotide code indicates that the nucleotide had a 3' phosphorothioate linkage.

Table 2: Hexamers that are not seed sequences of human miRNAs.

Table 3: A listing of oligonucleotide modifications that can be used in the single stranded oligonucleotides described herein.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE DISCLOSURE

Aspects of the disclosure provided herein relate to compositions (e.g.,

oligonucleotide-based compositions) and methods that are useful for upregulating FOXP3 expression. Some aspects relate to compositions (e.g., oligonucleotide-based compositions) and methods that target a PRC2-associated region of an FOXP3 gene (e.g., located within a sequence as set forth in SEQ ID NO: 3) and thereby cause upregulation of the gene. Some aspects relate to compositions (e.g., single stranded oligonucleotides) and methods that downregulate expression of CCDC22 in cells and thereby upregulate expression of FOXP3 in cells.

Some aspects of the disclosure provided herein relate to the discovery of polycomb repressive complex 2 (PRC2)-interacting RNAs. Polycomb repressive complex 2 (PRC2) is a histone methyltransferase and a known epigenetic regulator involved in silencing of genomic regions through methylation of histone H3. Among other functions, PRC2 interacts with long noncoding RNAs (IncRNAs), such as Rep A, Xist, and Tsix, to catalyze

trimethylation of histone H3-lysine27. PRC2 contains four subunits, Eed, Suzl2, RbAp48, and Ezh2.

Some aspects of the disclosure relate to the recognition that single stranded oligonucleotides that bind to PRC2-associated regions of RNAs (e.g., IncRNAs) that are expressed from within a genomic region that encompasses or that is in functional proximity to the FOXP3 gene can induce or enhance expression of FOXP3. In some embodiments, this upregulation is believed to result from inhibition of PRC2 mediated repression of FOXP3. FOXP3 is a master regulator transcription factor that drives T cell differentiation and activity of T regulatory cells (Tregs). Tregs have immune suppressive activity and can help to promote a toleragenic response. Tregs have been shown to be helpful in shutting down T cell-mediated immunity toward the end of an immune reaction and in suppressing self- reactive T cells that have escaped the process of negative selection in the thymus. Activated T cells are important for immunoprotection of a host from pathogens and tumor cells.

However, inappropriately activated or self-reactive T cells may have deleterious effects, e.g., by causing uncontrolled immune responses or a self-targeting autoimmune response. It is contemplated herein that upregulation of FOXP3 may be used to drive T cell differentiation and/or activity toward a T regulatory state. This may be useful, e.g., to drive activated T cells to differentiate into Tregs or to suppress activated T cell activity. Accordingly, aspects of the disclosure relate to compositions and methods for upregulating FOXP3.

In some embodiments, the disclosure provides methods of increasing FOXP3 expression in a cell (e.g., a T cell or population of T cells) ex vivo for administration to a subject, e.g., by administering an oligonucleotide described herein or by administering an inhibitor of CCDC22 as described herein. The cell (e.g., a T cell or population of T cells) may be derived from the subject, such as from a peripheral blood mononuclear cell (PBMC) sample from the subject, or may be from another source such as a donor or a cell line. In some embodiments, the method comprises providing a cell comprising a FOXP3 gene (e.g., a T cell comprising a FOXP3 gene, such as a human T cell comprising a human FOXP3 gene); contacting the cell with an oligonucleotide described herein or an inhibitor of CCDC22 as described herein ex vivo; and administering the contacted cell to a subject in need thereof (e.g., a human subject having an autoimmune or inflammatory disease or disorder or a human subject who will receive a transplant). The contacted cell may be administered to the subject in any appropriate way known in the art or described herein, e.g., by intravenous injection or by catheter.

In some embodiments, where the cell is a T cell or population of T cells, the method further comprises activating the T cell prior to contacting the cell with the oligonucleotide. The T cell or population thereof may be activated using any method known in the art or described herein. For example, the T cell or population thereof may be contacted with an activating agent such as an anti-CD3 and/or anti-CD28 antibody, which may optionally be coupled to a solid substrate, such as a bead. In some embodiments, activated T cells express CD69 and/or IL-2RA. In some embodiments, where the cell is a population of T cells, contacting the T cells with the oligonucleotide increases the number of CD4⁺CD25⁺FOXP3⁺ T cells in the population of T cells (e.g., compared to a control population of T cells that is not contacted with the oligonucleotide). In some embodiments, the concentration of oligonucleotide or inhibitor of CCDC22 delivered to the cell is 0.5 μΜ to 10 μΜ, 1 μΜ to 20 μΜ, or 0.01 μΜ to 50 μΜ. In some embodiments, the concentration of oligonucleotide or inhibitor of CCDC22 delivered to the cell is up to 1 μΜ, up to 5 μΜ, up to 10 μΜ, up to 20 μΜ, up to 50 μΜ, or up to 100 μΜ.

In some embodiments of methods provided herein, if the subject is a subject who will receive a transplant, the method further comprises transplanting a cell, tissue or organ into the subject. The cell, tissue or organ may be transplanted before, after or simultaneously with administration of a cell (e.g., T cell or population of T cells) that has contacted with an oligonucleotide or inhibitor of CCDC22 provided herein that increases FOXP3 expression. In some embodiments, methods provided herein alleviate or prevent development of an adverse response to the transplant, such as graft-versus-host disease, in the subject. The cell, tissue or organ to be transplanted may be autologous, allogeneic, or xenogeneic to the subject. Exemplary cells, tissue and organs for transplantation into a subject include stem cells, bone marrow, liver, kidney, skin, cornea, heart, lung, intestine, pancreas, islet cells, tendon, and ligament.

In some embodiments of methods provided herein, the cell is a T cell (such as a population of T cells) and the methods further comprise transfecting the T cell with an expression construct encoding a chimeric antigen receptor (CAR). CARs have been utilized to engineer T cells to target selected antigens. For example, CARs have been utilized to engineer T cells (e.g., Tregs) to target antigens that cause inflammatory or autoimmune responses, such as self-antigens (see, e.g., Fransson et al. CAR/FoxP3 -engineered T regulatory cells target the CNS and suppress EAE upon intranasal delivery. Journal of Neuroinflammation (2012) 9: 112 and Dotti. The Other Face of Chimeric Antigen Receptors. Molecular Therapy (2014) 22(5): 899-900). Accordingly, in some embodiments, the CAR is specific for a self-antigen or an antigen that causes an inflammatory response in the subject. In some embodiments, CARs comprise an extracellular antigen-binding domain (e.g., a single chain variable fragment (scFv) from an antibody), a transmembrane domain (e.g., a transmembrane domain of any one of the following: alpha, beta or zeta chain of the T- cell receptor, CD28, CD3 epsilon, CD3 zeta, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD27, CD33, CD37, CD64, CD80, CD86, CD134, CD137,and CD154) and an intracellular domain. In some embodiments, CARs may further comprise a hinge region (such as a human IgGl, IgG4, or IgD hinge region or a CD8 hinge region). In some embodiments, CARs may comprise an intracellular domain comprising one or more signaling or co-stimulatory domains (e.g., one or more signaling domains of the CD3ζ chain, 4-1BB and CD28 and/or one or more co-stimulatory domains of 4-1BB (CD137), CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, and HVEM). Any appropriate CARs and/or methods of making CARs may be used (see, e.g., PCT publication numbers WO2014184744A1, WO2014184143A1,

WO2014059173A2 and WO2015179801A1 and Fransson et al. CAR/FoxP3 -engineered T regulatory cells target the CNS and suppress EAE upon intranasal delivery. Journal of Neuroinflammation (2012) 9: 112, the contents of each of which relating to CARs are incorporated herein by reference). In some embodiments, a CAR comprises (a) a scFv specific for a self-antigen or an antigen that causes an inflammatory response in the subject, (b) an Ig hinge region, (c) a CD3ζ chain transmembrane domain, (d) a CD3ζ chain signaling domain and/or (e) a CD28 signaling domain. In some embodiments, the CAR comprises (a) a scFv specific for a self-antigen or an antigen that causes an inflammatory response in the subject, (b) an Ig hinge region, (c) a CD3ζ chain transmembrane domain, and (d) a CD3ζ chain signaling domain.

In some embodiments, transfection of the T cell with the CAR expression construct occurs before the T cell is contacted with the oligonucleotide or inhibitor of CCDC22. In some embodiments, transfection occurs after the T cell is contacted with the oligonucleotide inhibitor of CCDC22. In some embodiments, the T cell is activated prior to transfection, e.g., by contacting with an activating agent such as an anti-CD3 and/or anti-CD28 antibody optionally immobilized on a solid substrate. In some embodiments, the T cell is activated after transfection, e.g., by contacting with an activating agent such as an anti-CD3 and/or anti-CD28 antibody. In some embodiments, transfection is achieved by viral infection (e.g., lentiviral infection) of the T cell with the expression construct encoding the CAR. The expression construct may comprise the coding sequence of the CAR optionally along with one or more regulatory sequences that drive expression of the coding sequence, e.g., a promoter and/or enhancer sequence. In some embodiments, the expression construct is a lentiviral construct comprising 5' and 3' long terminal repeats (LTRs). Lentiviruses for use in transfecting T cells can be produced using any method known in the art or described herein. For example, 293FT cells may be co-transfected with lentiviral helper plasmids and a lentiviral construct comprising the coding sequence of the CAR optionally with regulatory sequences. Virus supernatants can be isolated from the 293T cells and then concentrated, e.g., by ultracentrifugation. The T cells for use in developing a CAR T cells may be obtained using any method known in the art or described herein (see, e.g., PCT publication numbers WO2014184744A1, WO2014184143A1, WO2014059173A2 and WO2015179801A1 and Fransson et al.

CAR/FoxP3 -engineered T regulatory cells target the CNS and suppress EAE upon intranasal delivery. Journal of Neuroinflammation (2012) 9: 112). For example, T cells can be obtained from a number of sources, including peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, cord blood, thymus tissue and spleen tissue from the subject or a donor. PBMCs can be obtained, e.g., by Ficoll™ separation from blood. Alternatively, the T cells may be obtained from a T cell line. A specific subpopulation of T cells, such as CD4⁺ T cells, can be further isolated by positive or negative selection techniques, such as by fluorescent activated cell sorting or magnetic cell sorting.

PRC2-associated regions

As used herein, the term "PRC2-associated region" refers to a region of a nucleic acid that comprises or encodes a sequence of nucleotides that interact directly or indirectly with a component of PRC2. A PRC2-associated region may be present in a RNA (e.g., a long non- coding RNA (IncRNA)) that interacts with a PRC2. A PRC2-associated region may be present in a DNA that encodes an RNA that interacts with PRC2. In some cases, the PRC2- associated region is equivalently referred to as a PRC2-interacting region.

In some embodiments, a PRC2-associated region is a region of an RNA that crosslinks to a component of PRC2 in response to in situ ultraviolet irradiation of a cell that expresses the RNA, or a region of genomic DNA that encodes that RNA region. In some embodiments, a PRC2-associated region is a region of an RNA that immunoprecipitates with an antibody that targets a component of PRC2, or a region of genomic DNA that encodes that RNA region. In some embodiments, a PRC2- associated region is a region of an RNA that immunoprecipitates with an antibody that binds specifically to SUZ12, EED, EZH2 or RBBP4 (which as noted above are components of PRC2), or a region of genomic DNA that encodes that RNA region.

In some embodiments, a PRC2-associated region is a region of an RNA that is protected from nucleases (e.g., RNases) in an RNA-immunoprecipitation assay that employs an antibody that targets a component of PRC2, or a region of genomic DNA that encodes that protected RNA region. In some embodiments, a PRC2-associated region is a region of an RNA that is protected from nucleases (e.g., RNases) in an RNA-immunoprecipitation assay that employs an antibody that targets SUZ12, EED, EZH2 or RBBP4, or a region of genomic DNA that encodes that protected RNA region.

In some embodiments, a PRC2-associated region is a region of an RNA within which occur a relatively high frequency of sequence reads in a sequencing reaction of products of an RNA-immunoprecipitation assay that employs an antibody that targets a component of PRC2, or a region of genomic DNA that encodes that RNA region. In some embodiments, a PRC2- associated region is a region of an RNA within which occur a relatively high frequency of sequence reads in a sequencing reaction of products of an RNA-immunoprecipitation assay that employs an antibody that binds specifically to SUZ12, EED, EZH2 or RBBP4, or a region of genomic DNA that encodes that protected RNA region. In such embodiments, the PRC2-associated region may be referred to as a "peak."

In some embodiments, a PRC2-associated region comprises a sequence of 40 to 60 nucleotides that interact with PRC2 complex. In some embodiments, a PRC2-associated region comprises a sequence of 40 to 60 nucleotides that encode an RNA that interacts with PRC2. In some embodiments, a PRC2-associated region comprises a sequence of up to 5kb in length that comprises a sequence (e.g., of 40 to 60 nucleotides) that interacts with PRC2. In some embodiments, a PRC2-associated region comprises a sequence of up to 5kb in length within which an RNA is encoded that has a sequence (e.g., of 40 to 60 nucleotides) that is known to interact with PRC2. In some embodiments, a PRC2-associated region comprises a sequence of about 4kb in length that comprise a sequence (e.g., of 40 to 60 nucleotides) that interacts with PRC2. In some embodiments, a PRC2-associated region comprises a sequence of about 4kb in length within which an RNA is encoded that includes a sequence (e.g., of 40 to 60 nucleotides) that is known to interact with PRC2. In some embodiments, a PRC2- associated region is located within a sequence as set forth in SEQ ID NO: 3. In some embodiments, a PRC2-associated region is located within a sequence corresponding to nucleotide 511 to nucleotide 920 of SEQ ID NO: 3.

In some embodiments, single stranded oligonucleotides are provided that specifically bind to, or are complementary to, a PRC2-associated region in a genomic region that encompasses or that is in proximity to the FOXP3 gene. In some embodiments, single stranded oligonucleotides are provided that specifically bind to, or are complementary to, a PRC2-associated region located within a sequence as set forth in SEQ ID NO: 3. In some embodiments, single stranded oligonucleotides are provided that specifically bind to, or are complementary to, a PRC2-associated region located within a sequence corresponding to nucleotide 511 to nucleotide 920 of SEQ ID NO: 3. In some embodiments, single stranded oligonucleotides are provided that specifically bind to, or are complementary to, a PRC2- associated region that is located within a sequence as set forth in SEQ ID NO: 3 or a sequence corresponding to nucleotide 511 to nucleotide 920 of SEQ ID NO: 3, combined with up to 2kb, up to 5kb, or up to lOkb of flanking sequences from a corresponding genomic region to which these SEQ IDs map (e.g., in a human genome). Examples of such single stranded oligonucleotides have a sequence as set forth in any one of SEQ ID Nos: 4-13 (as set forth in Table 1).

Without being bound by a theory, these oligonucleotides are able to interfere with the binding of and function of PRC2, by preventing recruitment of PRC2 to a specific

chromosomal locus. For example, a single administration of single stranded oligonucleotides designed to specifically bind a PRC2-associated region IncRNA can stably displace not only the IncRNA, but also the PRC2 that binds to the IncRNA, from binding chromatin. After displacement, the full complement of PRC2 is not recovered for up to 24 hours. Further, IncRNA can recruit PRC2 in a cis fashion, repressing gene expression at or near the specific chromosomal locus from which the IncRNA was transcribed.

Some aspects of the disclosure provided herein relate to the discovery of

downregulation of CCDC22 expression or silencing CCDC22 gene resulting in increased expression of FOXP3 in cells. CCDC22 (coiled-coil domain-containing protein 22) gene encodes a protein containing a coiled-coil domain. It is previously reported that the encoded protein functions in the regulation of NF-kB (nuclear factor kappa-light-chain-enhancer of activated B cells) by interacting with COMMD (copper metabolism Murrl domain- containing) proteins. CCDC22 is broadly expressed in human tissues and CCDC22 ortholog is also previously reported to be detected in other mammalian tissues such as mouse tissues. Starokadomskyy et al., (2013) The Journal of Clinical Investigation, 123(5): 2244-2256. The mouse orthologous protein is previously shown to bind copines, which are calcium- dependent, membrane-binding proteins that may function in calcium signaling. The human CCDC22 gene is also previously reported to be associated with syndromic X-linked intellectual disability. Some aspects of the disclosure relate to the recognition that single stranded oligonucleotides that target an exon of CCDC22 protein-coding mRNA (e.g., exon 1 or exon 2 of CCDC22 protein-coding mRNA) can induce or enhance expression of FOXP3. In some embodiments, this upregulation is believed to result from downregulation of CCDC22 expression. As shown in FIG. 2, CCDC22 gene is one of the genes that is located in close proximity to FOXP3 within a genomic region of FOXP3.

Methods of modulating gene expression are provided, in some embodiments, that may be carried out in vitro, ex vivo, or in vivo. It is understood that any reference to uses of compounds, molecules, or agents throughout the description contemplates use of the compound, molecule, or agent in preparation of a pharmaceutical composition or medicament for use in the treatment of condition or a disease (e.g., a disease or disorder associated with aberrant immune cell activation such as an autoimmune or inflammatory disease or disorder) associated with decreased levels or activity of FOXP3. Thus, as one nonlimiting example, one aspect of the disclosure includes use of such single stranded oligonucleotides in the preparation of a medicament for use in the treatment of disease, wherein the treatment involves upregulating expression of FOXP3.

In further aspects of the disclosure, methods are provided for selecting a candidate oligonucleotide for activating expression of FOXP3. The methods generally involve selecting as a candidate oligonucleotide, a single stranded oligonucleotide comprising a nucleotide sequence that is complementary to a PRC2-associated region (e.g., located within a sequence as set forth SEQ ID NO: 3 or a sequence corresponding to nucleotide 511 to nucleotide 920 of SEQ ID NO:3). In some embodiments, sets of oligonucleotides may be selected that are enriched (e.g., compared with a random selection of oligonucleotides) in oligonucleotides that activate expression of FOXP3.

Single Stranded Oligonucleotides for Modulating Expression ofFOXP3

In one aspect of the disclosure, single stranded oligonucleotides complementary to the PRC2-associated regions are provided for modulating expression of FOXP3 in a cell. In some embodiments, expression of FOXP3 is upregulated or increased. In some

embodiments, single stranded oligonucleotides complementary to these PRC2-associated regions inhibit the interaction of PRC2 with long RNA transcripts such that gene expression is upregulated or increased. In some embodiments, single stranded oligonucleotides complementary to these PRC2-associated regions inhibit the interaction of PRC2 with long RNA transcripts, resulting in reduced methylation of histone H3 and reduced gene inactivation, such that gene expression is upregulated or increased. In some embodiments, this interaction may be disrupted or inhibited due to a change in the structure of the long RNA that prevents or reduces binding to PRC2. The oligonucleotide may be selected using any of the methods disclosed herein for selecting a candidate oligonucleotide for activating expression of FOXP3.

The PRC2-associated region of a FOXP3 gene may map to a position in a

chromosome between 50 kilobases upstream of a 5 '-end of the FOXP3 gene and 50 kilobases downstream of a 3 '-end of the FOXP3 gene. For example, the PRC2 associated region of a FOXP3 gene may have a sequence that maps to a position in chromosome X of a human genome within the coordinates chrX:49,057,795-49, 164,962, based on the February 2009 UCSC genome assembly (GRCh37/hgl9). The PRC2-associated region may map to a position in a chromosome between 25 kilobases upstream of a 5 '-end of the FOXP3 gene and 25 kilobases downstream of a 3 '-end of the FOXP3 gene. The PRC2-associated region may map to a position in a chromosome between 12 kilobases upstream of a 5'-end of the FOXP3 gene and 12 kilobases downstream of a 3'-end of the FOXP3 gene. The PRC2-associated region may map to a position in a chromosome between 5 kilobases upstream of a 5 '-end of the FOXP3 gene and 5 kilobases downstream of a 3 '-end of the FOXP3 gene.

The genomic position of the selected PRC2-associated region relative to the FOXP3 gene may vary. For example, the PRC2-associated region may be upstream of the 5' end of the FOXP3 gene. The PRC2-associated region may be downstream of the 3' end of the FOXP3 gene. For example, the CCDC22 gene is located downstream in close proximity to the 3' end of the FOXP3 gene. Thus, in some embodiments, the PRC2-associated region may be within the 3'-UTR portion of the CCDC22 gene. The PRC2-associated region may be within an intron of the FOXP3 gene. The PRC2-associated region may be within an exon of the FOXP3 gene. The PRC2-associated region may traverse an intron-exon junction, a 5'- UTR-exon junction or a 3 '-UTR-exon junction of the FOXP3 gene. The PRC2-associated region may be within the 3'-UTR of the FOXP3 gene. The PRC2-associated region may be located on the sense (plus) strand of the FOXP3 gene. The PRC2-associated region may be located on the antisense (minus) strand of the FOXP3 gene. For example, the PRC2- associated region may be located on an antisense (minus) strand of the FOXP3 gene downstream of the 3' end of the CCDC22 gene. In other words, the PRC2- associated region may be located downstream of the 3' end of a sense (plus) strand of the CCDC22 gene, which is antisense to a 3' end portion of the FOXP3 gene.

The single stranded oligonucleotide may comprise a region of complementarity that is complementary with a PRC2-associated region located within a nucleotide sequence set forth in SEQ ID NO: 3 or a nucleotide sequence corresponding to nucleotide 511 to nucleotide 920 of SEQ ID NO:3. The region of complementarity of the single stranded oligonucleotide may be complementary with at least 6, e.g., at least 7, at least 8, at least 9, at least 10, at least 15 or more consecutive nucleotides of the PRC2-associated region.

The single stranded oligonucleotide may have a sequence that is not complementary to at least 6, e.g., at least 7, at least 8, at least 9, at least 10, at least 15 or more consecutive nucleotides of a nucleotide sequence corresponding to nucleotide 1 to nucleotide 510 of SEQ ID NO: 3.

The single stranded oligonucleotide may comprise a sequence having the formula X- Y-Z, in which X is any nucleotide, Y is a nucleotide sequence of 6 nucleotides in length that is not a human seed sequence of a microRNA, and Z is a nucleotide sequence of varying length. In some embodiments X is the 5' nucleotide of the oligonucleotide. In some embodiments, when X is anchored at the 5' end of the oligonucleotide, the oligonucleotide does not have any nucleotides or nucleotide analogs linked 5' to X. In some embodiments, other compounds such as peptides or sterols may be linked at the 5' end in this embodiment as long as they are not nucleotides or nucleotide analogs. In some embodiments, the single stranded oligonucleotide has a sequence 5'X-Y-Z and is 8-50 nucleotides in length.

Oligonucleotides that have these sequence characteristics are predicted to avoid the miRNA pathway. Therefore, in some embodiments, oligonucleotides having these sequence characteristics are unlikely to have an unintended consequence of functioning in a cell as a miRNA molecule. The Y sequence may be a nucleotide sequence of 6 nucleotides in length set forth in Table 2.

The single stranded oligonucleotide may have a sequence that does not contain guanosine nucleotide stretches (e.g., 3 or more, 4 or more, 5 or more, 6 or more consecutive guanosine nucleotides). In some embodiments, oligonucleotides having guanosine nucleotide stretches have increased non-specific binding and/or off-target effects, compared with oligonucleotides that do not have guanosine nucleotide stretches. The single stranded oligonucleotide may have a sequence that has less than a threshold level of sequence identity with every sequence of nucleotides, of equivalent length, that map to a genomic position encompassing or in proximity to an off-target gene. For example, an oligonucleotide may be designed to ensure that it does not have a sequence that maps to genomic positions encompassing or in proximity with all known genes (e.g., all known protein coding genes) other than FOXP3. In a similar embodiment, an

oligonucleotide may be designed to ensure that it does not have a sequence that maps to any other known PRC2-associated region, particularly PRC2-associated regions that are functionally related to any other known gene (e.g., any other known protein coding gene). In either case, the oligonucleotide is expected to have a reduced likelihood of having off-target effects, e.g., modulating activity and/or expression level of a gene that is not FOXP3. The threshold level of sequence identity may be 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity.

In some embodiments, the single stranded oligonucleotide may have a sequence that has less than a threshold level of sequence identity with every sequence of nucleotides, of equivalent length, that map to a genomic position in a chromosome outside a sequence window, the boundaries of which are defined by 50 kilobases upstream of a 5 '-end of the FOXP3 gene and 50 kilobases downstream of a 3 '-end of the FOXP3 gene, respectively. The threshold level of sequence identity may be 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity. The oligonucleotide is expected to have a reduced likelihood of having off-target effects, e.g., modulating activity and/or expression level of a gene that is not FOXP3.

The single stranded oligonucleotide may have a sequence that is complementary to a PRC2-associated region that encodes an RNA that forms a secondary structure comprising at least two single stranded loops. In has been discovered that, in some embodiments, oligonucleotides that are complementary to a PRC2-associated region that encodes an RNA that forms a secondary structure comprising one or more single stranded loops (e.g., at least two single stranded loops) have a greater likelihood of being active (e.g., of being capable of activating or enhancing expression of a target gene) than a randomly selected

oligonucleotide. In some cases, the secondary structure may comprise a double stranded stem between the at least two single stranded loops. Accordingly, the region of

complementarity between the oligonucleotide and the PRC2-associated region may be at a location of the PRC2 associated region that encodes at least a portion of at least one of the loops. In some cases, the region of complementarity between the oligonucleotide and the PRC2-associated region may be at a location of the PRC2-associated region that encodes at least a portion of at least two of the loops. In some cases, the region of complementarity between the oligonucleotide and the PRC2-associated region may be at a location of the

PRC2 associated region that encodes at least a portion of the double stranded stem. In some embodiments, a PRC2-associated region (e.g., of an IncRNA) is identified (e.g., using RIP- Seq methodology or information derived therefrom [see, e.g., Zhao et al. Genome-wide identification of Polycomb-associated RNAs by RIP-seq. Mol Cell. 2010 December 22; 40(6): 939-953]). In some embodiments, the predicted secondary structure RNA (e.g., IncRNA) containing the PRC2-associated region is determined using RNA secondary structure prediction algorithms, e.g., RNAfold, mfold. In some embodiments,

oligonucleotides are designed to target a region of the RNA that forms a secondary structure comprising one or more single stranded loop (e.g., at least two single stranded loops) structures which may comprise a double stranded stem between the at least two single stranded loops.

The single stranded oligonucleotide may have a sequence that is has greater than 30% G-C content, greater than 40% G-C content, greater than 50% G-C content, greater than 60% G-C content, greater than 70% G-C content, or greater than 80% G-C content. The single stranded oligonucleotide may have a sequence that has up to 100% G-C content, up to 95% G-C content, up to 90% G-C content, or up to 80% G-C content. In some embodiments in which the oligonucleotide is 8 to 10 nucleotides in length, all but 1, 2, 3, 4, or 5 of the nucleotides of the complementary sequence of the PRC2-associated region are cytosine or guanosine nucleotides. In some embodiments, the sequence of the PRC2- associated region to which the single stranded oligonucleotide is complementary comprises no more than 3 nucleotides selected from adenine and uracil.

The single stranded oligonucleotide may be complementary to a chromosome of more than one species. For example, the oligonucleotide that is complementary to a chromosome of one species may be also complementary to a chromosome of a different species (e.g., a mouse, rat, rabbit, goat, monkey, etc.) at a position that encompasses or that is in proximity to that species' homolog of FOXP3. The single stranded oligonucleotide may be complementary to a human genomic region encompassing or in proximity to the FOXP3 gene and also be complementary to a mouse genomic region encompassing or in proximity to the mouse homolog of FOXP3. For example, the single stranded oligonucleotide may be

complementary to a region located within a sequence as set forth in SEQ ID NO: 3, which is a human genomic region encompassing or in proximity to the FOXP3 gene, and also be complementary to a sequence (e.g., correspoinding to a PRC2-associated region), which is a mouse genomic region encompassing or in proximity to the mouse homolog of the FOXP3 gene. Oligonucleotides having these characteristics may be tested in vivo or in vitro for efficacy in multiple species (e.g., human and mouse). This approach also facilitates development of clinical candidates for treating human disease by selecting a species in which an appropriate animal exists for the disease.

In some embodiments, the region of complementarity of the single stranded oligonucleotide is complementary with at least 8 to 15, 8 to 30, 8 to 40, or 10 to 50, or 5 to 50, or 5 to 40 bases, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 consecutive nucleotides of a PRC2-associated region. In some embodiments, the region of complementarity is complementary with at least 8 consecutive nucleotides of a PRC2-associated region located within a nucleotide sequence set forth in SEQ ID NO: 3 or a nucleotide sequence corresponding to nucleotide 511 to nucleotide 920 of SEQ ID NO:3. In some embodiments the sequence of the single stranded oligonucleotide is based on an RNA sequence that binds to PRC2, or a portion thereof, said portion having a length of from 5 to 40 contiguous base pairs, or about 8 to 40 bases, or about 5 to 15, or about 5 to 30, or about 5 to 40 bases, or about 5 to 50 bases.

Complementary, as the term is used in the art, refers to the capacity for precise pairing between two nucleotides. For example, if a nucleotide at a certain position of an

oligonucleotide is capable of hydrogen bonding with a nucleotide at the same position of

PRC2-associated region, then the single stranded nucleotide and PRC2-associated region are considered to be complementary to each other at that position. The single stranded nucleotide and PRC2-associated region are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides that can hydrogen bond with each other through their bases. Thus, "complementary" is a term which is used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the single stranded nucleotide and PRC2-associated region. For example, if a base at one position of a single stranded nucleotide is capable of hydrogen bonding with a base at the corresponding position of a PRC2-associated region, then the bases are considered to be complementary to each other at that position. 100% complementarity is not required.

The single stranded oligonucleotide may be at least 80% complementary to

(optionally one of at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementary to) the consecutive nucleotides of a PRC2-associated region. In some embodiments, the single stranded oligonucleotide may be at least 80% complementary to (optionally one of at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementary to) the consecutive nucleotides of a PRC2-associated region located within a nucleotide sequence set forth in SEQ ID NO: 3 or a nucleotide sequence

corresponding to nucleotide 511 to nucleotide 920 of SEQ ID NO: 3. In some embodiments the single stranded oligonucleotide may contain 1 , 2 or 3 base mismatches compared to the portion of the consecutive nucleotides of a PRC2-associated region. In some

embodimentsthe single stranded oligonucleotide may have up to 3 mismatches over 15 bases, or up to 2 mismatches over 10 bases.

It is understood in the art that a complementary nucleotide sequence need not be 100% complementary to that of its target to be specifically hybridizable. In some

embodiments, a complementary nucleic acid sequence for purposes of the present disclosure is specifically hybridizable when binding of the sequence to the target molecule (e.g.,

IncRNA) interferes with the normal function of the target (e.g., IncRNA) to cause a loss of activity (e.g., inhibiting PRC2-associated repression with consequent up-regulation of gene expression) and there is a sufficient degree of complementarity to avoid non-specific binding of the sequence to non-target sequences under conditions in which avoidance of non-specific binding is desired, e.g., under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed under suitable conditions of stringency.

In some embodiments, the single stranded oligonucleotide is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or more nucleotides in length. In a preferred embodiment, the oligonucleotide is 8 to 30 nucleotides in length. In some embodiments, the PRC2-associated region occurs on the same DNA strand as a gene sequence (sense). In some embodiments, the PRC2-associated region occurs on the opposite DNA strand as a gene sequence (anti-sense). Oligonucleotides complementary to a PRC2-associated region can bind either sense or anti-sense sequences. Base pairings may include both canonical Watson-Crick base pairing and non-Watson-Crick base pairing (e.g., Wobble base pairing and Hoogsteen base pairing). It is understood that for complementary base pairings, adenosine-type bases (A) are complementary to thymidine-type bases (T) or uracil-type bases (U), that cytosine-type bases (C) are complementary to guanosine-type bases (G), and that universal bases such as 3-nitropyrrole or 5-nitroindole can hybridize to and are considered complementary to any A, C, U, or T. Inosine (I) has also been considered in the art to be a universal base and is considered complementary to any A, C, U or T.

In some embodiments, any one or more thymidine (T) nucleotides (or modified nucleotide thereof) or uridine (U) nucleotides (or a modified nucleotide thereof) in a sequence provided herein, including a sequence provided in the sequence listing, may be replaced with any other nucleotide suitable for base pairing (e.g., via a Watson-Crick base pair) with an adenosine nucleotide. In some embodiments, any one or more thymidine (T) nucleotides (or modified nucleotide thereof) or uridine (U) nucleotides (or a modified nucleotide thereof) in a sequence provided herein, including a sequence provided in the sequence listing, may be suitably replaced with a different pyrimidine nucleotide or vice versa. In some embodiments, any one or more thymidine (T) nucleotides (or modified nucleotide thereof) in a sequence provided herein, including a sequence provided in the sequence listing, may be suitably replaced with a uridine (U) nucleotide (or a modified nucleotide thereof) or vice versa.

In some embodiments, GC content of the single stranded oligonucleotide is preferably between about 30-60 %. Contiguous runs of three or more Gs or Cs may not be preferable in some embodiments. Accordingly, in some embodiments, the oligonucleotide does not comprise a stretch of three or more guanosine nucleotides.

In some embodiments, the single stranded oligonucleotide specifically binds to, or is complementary to an RNA that is encoded in a genome (e.g., a human genome) as a single contiguous transcript (e.g., a non-spliced RNA). In some embodiments, the single stranded oligonucleotide specifically binds to, or is complementary to an RNA that is encoded in a genome (e.g., a human genome), in which the distance in the genome between the 5 'end of the coding region of the RNA and the 3' end of the coding region of the RNA is less than 1 kb, less than 2 kb, less than 3 kb, less than 4 kb, less than 5 kb, less than 7 kb, less than 8 kb, less than 9 kb, less than 10 kb, or less than 20 kb.

It is to be understood that any oligonucleotide provided herein can be excluded.

In some embodiments, it has been found that single stranded oligonucleotides disclosed herein may increase expression of mRNA corresponding to a FOXP3 gene by at least about 50% (i.e., 150% of normal or 1.5 fold), or by about 2 fold to about 5 fold. In some embodiments, expression may be increased by at least about 15 fold, 20 fold, 30 fold, 40 fold, 50 fold or 100 fold, or any range between any of the foregoing numbers. It has also been found that increased mRNA expression has been shown to correlate to increased protein expression.

In some embodiments, single stranded oligonucleotides disclosed herein may increase expression of mRNA or protein corresponding to CTLA4, GITR, and/or IL-10 by at least about 30% (i.e. 130% of normal or 1.3 fold), or by about 1.5 fold, or by about 2 fold to about 5 fold. In some embodiments, expression may be increased by at least about 5 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold or 100 fold, or any range between any of the foregoing numbers. For example, mRNA or protein corresponding to CTLA4, GITR, and/or IL-10 may be increased by an amount in a range of 1.3 fold to 2 fold, 1.3 fold to 5 fold, 1.3 fold to 10 fold, 1.3 fold to 20 fold, 1.3 fold to 50 fold, 1.3 fold to 100 fold, 2 fold to 5 fold, 2 fold to 10 fold, 2 fold to 20 fold, 2 fold to 10 fold. 2 fold to 20 fold, 2 fold to 50 fold, or 2 fold to 100 fold. Exemplary human mRNA and protein sequence identifiers for CTLA4, GITR, and IL-10 are provided below. These sequence identifiers can be used to identify exemplary mRNA and protein sequences for CTLA4, GITR, and IL-10 by using the NCBI Gene search as of the filing of the instant application.

CTLA4: NM 001037631.2, NM_005214.4, NP 001032720.1, NP_005205.2

GITR (also called TNFRSF18): NM_004195.2, NM_148901.1, NM_148902.1, NP 004186.1, NP_683699.1, NP_683700.1

IL-10: NM_000572.2, NP_000563.1

In some embodiments, single stranded oligonucleotides disclosed herein may increase the number of CD4+CD25+FOXP3+ T cells by at least about 30% (i.e. 130% of normal or 1.3 fold), or by about 1.5 fold, or by about 2 fold to about 5 fold. In some embodiments, the number may be increased by at least about 5 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold or 100 fold, or any range between any of the foregoing numbers. For example, numbers of CD4+CD25+FOXP3+ T cells may be increased in a population of T cells by an amount in a range of 1.3 fold to 2 fold, 1.3 fold to 5 fold, 1.3 fold to 10 fold, 1.3 fold to 20 fold, 1.3 fold to 50 fold, 1.3 fold to 100 fold, 2 fold to 5 fold, 2 fold to 10 fold, 2 fold to 20 fold, 2 fold to 10 fold. 2 fold to 20 fold, 2 fold to 50 fold, or 2 fold to 100 fold.

In some or any of the embodiments of the oligonucleotides described herein, or processes for designing or synthesizing them, the oligonucleotides will upregulate gene expression and may specifically bind or specifically hybridize or be complementary to the PRC2 binding RNA that is transcribed from the same strand as a protein coding reference gene. The oligonucleotide may bind to a region of the PRC2 binding RNA that originates within or overlaps an intron, exon, intron exon junction, 5' UTR, 3' UTR, a translation initiation region, or a translation termination region of a protein coding sense strand of a reference gene (refGene).

In some or any of the embodiments of oligonucleotides described herein, or processes for designing or synthesizing them, the oligonucleotides will upregulate gene expression and may specifically bind or specifically hybridize or be complementary to a PRC2 binding RNA that transcribed from the opposite strand (the antisense strand) of a protein coding reference gene. The oligonucleotide may bind to a region of the PRC2 binding RNA that originates within or overlaps an intron, exon, intron exon junction, 5' UTR, 3' UTR, a translation initiation region, or a translation termination region of a protein coding antisense strand of a reference gene

The oligonucleotides described herein may be modified, e.g., comprise a modified sugar moiety, a modified internucleoside linkage, a modified nucleotide and/or combinations thereof. In addition, the oligonucleotides can exhibit one or more of the following properties: do not induce substantial cleavage or degradation of the target RNA; do not cause

substantially complete cleavage or degradation of the target RNA; do not activate the RNAse H pathway; do not activate RISC; do not recruit any Argonaute family protein; are not cleaved by Dicer; do not mediate alternative splicing; are not immune stimulatory; are nuclease resistant; have improved cell uptake compared to unmodified oligonucleotides; are not toxic to cells or mammals; may have improved endosomal exit; do interfere with interaction of IncRNA with PRC2, preferably the Ezh2 subunit but optionally the Suzl2, Eed, RbAp46/48 subunits or accessory factors such as Jarid2; do decrease histone H3 lysine27 methylation and/or do upregulate gene expression.

Oligonucleotides that are designed to interact with RNA to modulate gene expression are a distinct subset of base sequences from those that are designed to bind a DNA target (e.g., are complementary to the underlying genomic DNA sequence from which the RNA is transcribed).

Any of the oligonucleotides disclosed herein may be linked to one or more other oligonucleotides disclosed herein by a linker, e.g., a cleavable linker. Method for Selecting Candidate Oligonucleotides for Activating Expression ofFOXP3

Methods are provided herein for selecting a candidate oligonucleotide for activating or enhancing expression of FOXP3. The target selection methods may generally involve steps for selecting single stranded oligonucleotides having any of the structural and functional characteristics disclosed herein. Typically, the methods involve one or more steps aimed at identifying oligonucleotides that target a PRC2-associated region that is functionally related to FOXP3, for example a PRC2-associated region of a IncRNA that regulates expression of FOXP3 by facilitating (e.g., in a cis-regulatory manner) the recruitment of PRC2 to the FOXP3 gene. Such oligonucleotides are expected to be candidates for activating expression of FOXP3 because of their ability to hybridize with the PRC2-associated region of a nucleic acid (e.g., a IncRNA). In some embodiments, this hybridization event is understood to disrupt interaction of PRC2 with the nucleic acid (e.g., a IncRNA) and as a result disrupt recruitment of PRC2 and its associated co-repressors (e.g., chromatin remodeling factors) to the FOXP3 gene locus.

Methods of selecting a candidate oligonucleotide may involve selecting a PRC2- associated region that maps to a chromosomal position encompassing or in proximity to the FOXP3 gene. The PRC2-associated region may map to the strand of a chromosome comprising the sense strand of the FOXP3 gene, in which case the candidate oligonucleotide is complementary to the sense strand of the FOXP3 gene (i.e., is antisense to the FOXP3 gene). Alternatively, the PRC2-associated region may map to the strand of the chromosome comprising the antisense strand of the FOXP3 gene, in which case the oligonucleotide is complementary to the antisense strand (the template strand) of the FOXP3 gene (i.e., is sense to the FOXP3 gene). In some embodiments, the PRC2-associated region may map to a chromosomal region having a sequence as set forth in SEQ ID NO: 3, or a sequence corresponding to nucleotide 511 to nucleotide 920 of SEQ ID NO: 3.

Methods for selecting a set of candidate oligonucleotides that is enriched in oligonucleotides that activate expression of FOXP3 may involve selecting one or more PRC2-associated regions that map to a chromosomal position that encompasses or that is in proximity to the FOXP3 gene and selecting a set of oligonucleotides, in which each oligonucleotide in the set comprises a nucleotide sequence that is complementary with the one or more PRC2-associated regions. In some embodiments, the PRC2- associated region may map to a chromosomal region having a sequence as set forth in SEQ ID NO: 3, or a sequence corresponding to nucleotide 511 to nucleotide 920 of SEQ ID NO: 3. As used herein, the phrase, "a set of oligonucleotides that is enriched in oligonucleotides that activate expression of refers to a set of oligonucleotides that has a greater number of

oligonucleotides that activate expression of a target gene (e.g., FOXP3) compared with a random selection of oligonucleotides of the same physicochemical properties (e.g., the same GC content, Tm, length etc.) as the enriched set.

Where the design and/or synthesis of a single stranded oligonucleotide involves design and/or synthesis of a sequence that is complementary to a nucleic acid or PRC2- associated region described by such sequence information, the skilled person is readily able to determine the complementary sequence, e.g., through understanding of Watson Crick base pairing rules which form part of the common general knowledge in the field.

In some embodiments design and/or synthesis of a single stranded oligonucleotide involves manufacture of an oligonucleotide from starting materials by techniques known to those of skill in the art, where the synthesis may be based on a sequence of a PRC2- associated region, or portion thereof.

Methods of design and/or synthesis of a single stranded oligonucleotide may involve one or more of the steps of:

Identifying and/or selecting PRC2-associated region;

Designing a nucleic acid sequence having a desired degree of sequence identity or complementarity to a PRC2-associated region or a portion thereof;

Synthesizing a single stranded oligonucleotide to the designed sequence;

Purifying the synthesized single stranded oligonucleotide; and Optionally mixing the synthesized single stranded oligonucleotide with at least one pharmaceutically acceptable diluent, carrier or excipient to form a pharmaceutical

composition or medicament.

Single stranded oligonucleotides so designed and/or synthesized may be useful in method of modulating gene expression as described herein.

Preferably, single stranded oligonucleotides as described herein are synthesized chemically. Oligonucleotides used to practice the methods of modulating FOXP3 expression can be synthesized in vitro by well-known chemical synthesis techniques.

Oligonucleotides described herein can be stabilized against nucleolytic degradation such as by the incorporation of a modification, e.g., a nucleotide modification. For example, nucleic acid sequences described herein can include a phosphorothioate at least the first, second, or third internucleotide linkage at the 5' or 3' end of the nucleotide sequence. As another example, the nucleic acid sequence can include a 2'-modified nucleotide, e.g., a - deoxy, 2'-deoxy-2'-fluoro, 2'-0-methyl, 2'-0-methoxyethyl (2'-0-MOE), 2'-0-aminopropyl (2'-0-AP), 2'-0-dimethylaminoethyl (2'-0-DMAOE), 2'-0-dimethylaminopropyl (2'-0- DMAP), 2'-0-dimethylaminoethyloxyethyl (2'-0-DMAEOE), or 2'-0-N-methylacetamido (2'-0— NMA). As another example, the nucleic acid sequence can include at least one 2'-0- methyl-modified nucleotide, and in some embodiments, all of the nucleotides include a 2'-0- methyl modification. In some embodiments, the nucleic acids are "locked," i.e., comprise nucleic acid analogues in which the ribose ring is "locked" by a methylene bridge connecting the 2'-0 atom and the 4'-C atom.

It is understood that any of the modified chemistries or formats of single stranded oligonucleotides described herein can be combined with each other, and that one, two, three, four, five, or more different types of modifications can be included within the same molecule.

In some embodiments, the method may further comprise the steps of amplifying the synthesized single stranded oligonucleotide, and/or purifying the single stranded

oligonucleotide (or amplified single stranded oligonucleotide), and/or sequencing the single stranded oligonucleotide so obtained.

As such, the process of preparing a single stranded oligonucleotide may be a process that is for use in the manufacture of a pharmaceutical composition or medicament for use in the treatment of disease, optionally wherein the treatment involves modulating expression of a gene associated with a PRC2-associated region. In the methods described above a PRC2-associated region may be, or have been, identified, or obtained, by a method that involves identifying RNA that binds to PRC2.

Such methods may involve the following steps: providing a sample containing nuclear ribonucleic acids, contacting the sample with an agent that binds specifically to PRC2 or a subunit thereof, allowing complexes to form between the agent and protein in the sample, partitioning the complexes, synthesizing nucleic acid that is complementary to nucleic acid present in the complexes.

Where the single stranded oligonucleotide is based on a PRC2-associated region, or a portion of such a sequence, it may be based on information about that sequence, e.g., sequence information available in written or electronic form, which may include sequence information contained in publicly available scientific publications or sequence databases.

Nucleotide Analogues and oligonucleotides comprising at least one nucleotide analogue

In some embodiments, the oligonucleotide may comprise at least one ribonucleotide, at least one deoxyribonucleotide, and/or at least one bridged nucleotide. In some

embodiments, the oligonucleotide may comprise a bridged nucleotide, such as a locked nucleic acid (LNA) nucleotide, a constrained ethyl (cEt) nucleotide, or an ethylene bridged nucleic acid (ENA) nucleotide. Examples of such nucleotides are disclosed herein and known in the art. In some embodiments, the oligonucleotide comprises a nucleotide analog disclosed in one of the following United States Patent or Patent Application Publications: US 7,399,845, US 7,741,457, US 8,022,193, US 7,569,686, US 7,335,765, US 7,314,923, US 7,335,765, and US 7,816,333, US 20110009471, the entire contents of each of which are incorporated herein by reference for all purposes. The oligonucleotide may have one or more 2' O-methyl nucleotides. The oligonucleotide may consist entirely of 2' O-methyl nucleotides.

Often the single stranded oligonucleotide has one or more nucleotide analogues. For example, the single stranded oligonucleotide may have at least one nucleotide analogue that results in an increase in Tm of the oligonucleotide in a range of 1°C, 2 °C, 3°C, 4 °C, or 5°C compared with an oligonucleotide that does not have the at least one nucleotide analogue. The single stranded oligonucleotide may have a plurality of nucleotide analogues that results in a total increase in Tm of the oligonucleotide in a range of 2 °C, 3 °C, 4 °C, 5 °C, 6 °C, 7 °C, 8 °C, 9 °C, 10 °C, 15 °C, 20 °C, 25 °C, 30 °C, 35 °C, 40 °C, 45 °C or more compared with an oligonucleotide that does not have the nucleotide analogue.

The oligonucleotide may be of up to 50 nucleotides in length in which 2 to 10, 2 to 15, 2 to 16, 2 to 17, 2 to 18, 2 to 19, 2 to 20, 2 to 25, 2 to 30, 2 to 40, 2 to 45, or more nucleotides of the oligonucleotide are nucleotide analogues. The oligonucleotide may be of 8 to 30 nucleotides in length in which 2 to 10, 2 to 15, 2 to 16, 2 to 17, 2 to 18, 2 to 19, 2 to 20, 2 to 25, 2 to 30 nucleotides of the oligonucleotide are nucleotide analogues.

The oligonucleotide may be of 8 to 15 nucleotides in length in which 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 2 to 11, 2 to 12, 2 to 13, 2 to 14 nucleotides of the oligonucleotide are nucleotide analogues. Optionally, the oligonucleotides may have every nucleotide except 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides modified.

The oligonucleotide may consist entirely of bridged nucleotides (e.g., LNA nucleotides, cEt nucleotides, ENA nucleotides). The oligonucleotide may comprise alternating deoxyribonucleotides and 2'-fluoro-deoxyribonucleotides. The oligonucleotide may comprise alternating deoxyribonucleotides and 2'-0-methyl nucleotides. The

oligonucleotide may comprise alternating deoxyribonucleotides and ENA nucleotide analogues. The oligonucleotide may comprise alternating deoxyribonucleotides and LNA nucleotides. The oligonucleotide may comprise alternating LNA nucleotides and 2'-0- methyl nucleotides. The oligonucleotide may have a 5' nucleotide that is a bridged nucleotide (e.g., a LNA nucleotide, cEt nucleotide, ENA nucleotide). The oligonucleotide may have a 5' nucleotide that is a deoxyribonucleotide.

The oligonucleotide may comprise deoxyribonucleotides flanked by at least one bridged nucleotide (e.g., a LNA nucleotide, cEt nucleotide, ENA nucleotide) on each of the 5' and 3' ends of the deoxyribonucleotides. The oligonucleotide may comprise

deoxyribonucleotides flanked by 1, 2, 3, 4, 5, 6, 7, 8 or more bridged nucleotides (e.g., LNA nucleotides, cEt nucleotides, ENA nucleotides) on each of the 5' and 3' ends of the deoxyribonucleotides. The 3' position of the oligonucleotide may have a 3' hydroxyl group. The 3' position of the oligonucleotide may have a 3' thiophosphate.

The oligonucleotide may be conjugated with a label. For example, the

oligonucleotide may be conjugated with a biotin moiety, cholesterol, Vitamin A, folate, sigma receptor ligands, aptamers, peptides, such as CPP, hydrophobic molecules, such as lipids, ASGPR or dynamic polyconjugates and variants thereof at its 5' or 3' end. Preferably the single stranded oligonucleotide comprises one or more modifications comprising: a modified sugar moiety, and/or a modified internucleoside linkage, and/or a modified nucleotide and/or combinations thereof. It is not necessary for all positions in a given oligonucleotide to be uniformly modified, and in fact more than one of the

modifications described herein may be incorporated in a single oligonucleotide or even at within a single nucleoside within an oligonucleotide.

In some embodiments, the single stranded oligonucleotides are chimeric

oligonucleotides that contain two or more chemically distinct regions, each made up of at least one nucleotide. These oligonucleotides typically contain at least one region of modified nucleotides that confers one or more beneficial properties (such as, for example, increased nuclease resistance, increased uptake into cells, increased binding affinity for the target) and a region that is a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. Chimeric single stranded oligonucleotides may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or

oligonucleotide mimetics as described above. Such compounds have also been referred to in the art as hybrids or gapmers. Representative United States patents that teach the preparation of such hybrid structures comprise, but are not limited to, US patent nos. 5,013,830;

5,149,797; 5, 220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, each of which is herein incorporated by reference.

In some embodiments, the single stranded oligonucleotide comprises at least one nucleotide modified at the 2' position of the sugar, most preferably a 2'-0-alkyl, 2'-0-alkyl-0- alkyl or 2'-fluoro-modified nucleotide. In other preferred embodiments, RNA modifications include 2'-fluoro, 2'-amino and 2' O-methyl modifications on the ribose of pyrimidines, abasic residues or an inverted base at the 3' end of the RNA. Such modifications are routinely incorporated into oligonucleotides and these oligonucleotides have been shown to have a higher Tm (i.e., higher target binding affinity) than 2'-deoxyoligonucleotides against a given target.

A number of nucleotide and nucleoside modifications have been shown to make the oligonucleotide into which they are incorporated more resistant to nuclease digestion than the native oligodeoxynucleotide; these modified oligos survive intact for a longer time than unmodified oligonucleotides. Specific examples of modified oligonucleotides include those comprising modified backbones, for example, phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages. Most preferred are oligonucleotides with

phosphorothioate backbones and those with heteroatom backbones, particularly CH2 -NH-O- CH2, CH,~N(CH3)~0~CH2 (known as a methylene(methylimino) or MMI backbone, CH2 - -O-N (CH3)-CH2, CH2 -N (CH3)-N (CH3)-CH2 and O-N (CH3)- CH2 -CH2 backbones, wherein the native phosphodiester backbone is represented as O- P— O- CH,); amide backbones (see De Mesmaeker et al. Ace. Chem. Res. 1995, 28:366-374); morpholino backbone structures (see Summerton and Weller, U.S. Pat. No. 5,034,506); peptide nucleic acid (PNA) backbone (wherein the phosphodiester backbone of the oligonucleotide is replaced with a polyamide backbone, the nucleotides being bound directly or indirectly to the aza nitrogen atoms of the polyamide backbone, see Nielsen et al., Science 1991, 254, 1497). Phosphorus-containing linkages include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates comprising 3'alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates comprising 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3 -5' to 5'-3' or 2 -5' to 5'-2'; see US patent nos. 3,687,808; 4,469,863;

4,476,301; 5,023,243; 5, 177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455, 233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563, 253; 5,571,799; 5,587,361; and 5,625,050.

Morpholino-based oligomeric compounds are described in Dwaine A. Braasch and David R. Corey, Biochemistry, 2002, 41(14), 4503-4510); Genesis, volume 30, issue 3, 2001; Heasman, J., Dev. Biol., 2002, 243, 209-214; Nasevicius et al., Nat. Genet., 2000, 26, 216- 220; Lacerra et al., Proc. Natl. Acad. Sci., 2000, 97, 9591-9596; and U.S. Pat. No. 5,034,506, issued Jul. 23, 1991. In some embodiments, the morpholino-based oligomeric compound is a phosphorodiamidate morpholino oligomer (PMO) (e.g., as described in Iverson, Curr. Opin. Mol. Ther., 3:235-238, 2001; and Wang et al., J. Gene Med., 12:354-364, 2010; the disclosures of which are incorporated herein by reference in their entireties).

Cyclohexenyl nucleic acid oligonucleotide mimetics are described in Wang et al., J. Am. Chem. Soc, 2000, 122, 8595-8602. Modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These comprise those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts; see US patent nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264, 562; 5, 264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596, 086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623, 070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, each of which is herein incorporated by reference.

Modified oligonucleotides are also known that include oligonucleotides that are based on or constructed from arabinonucleotide or modified arabinonucleotide residues.

Arabinonucleosides are stereoisomers of ribonucleosides, differing only in the configuration at the 2'-position of the sugar ring. In some embodiments, a 2'-arabino modification is 2'-F arabino. In some embodiments, the modified oligonucleotide is 2'-fluoro-D-arabinonucleic acid (FANA) (as described in, for example, Lon et al., Biochem., 41:3457-3467, 2002 and Min et al., Bioorg. Med. Chem. Lett., 12:2651-2654, 2002; the disclosures of which are incorporated herein by reference in their entireties). Similar modifications can also be made at other positions on the sugar, particularly the 3' position of the sugar on a 3' terminal nucleoside or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide.

PCT Publication No. WO 99/67378 discloses arabinonucleic acids (ANA) oligomers and their analogues for improved sequence specific inhibition of gene expression via association to complementary messenger RNA.

Other preferred modifications include ethylene-bridged nucleic acids (ENAs) (e.g., International Patent Publication No. WO 2005/042777, Morita et al., Nucleic Acid Res., Suppl 1:241-242, 2001; Surono et al., Hum. Gene Ther., 15:749-757, 2004; Koizumi, Curr. Opin. Mol. Ther., 8: 144-149, 2006 and Horie et al., Nucleic Acids Symp. Ser (Oxf), 49: 171- 172, 2005; the disclosures of which are incorporated herein by reference in their entireties). Preferred ENAs include, but are not limited to, 2'-0,4'-C-ethylene-bridged nucleic acids.

Examples of LNAs are described in WO/2008/043753 and include compounds of the following general formula.

where X and Y are independently selected among the groups -0-,

-S-, -N(H)-, N(R)-, -CH2- or -CH- (if part of a double bond),

-CH2-0-, -CH2-S-, -CH2-N(H)-, -CH2-N(R)-, -CH2-CH2- or -CH2-CH- (if part of a double bond),

-CH=CH-, where R is selected from hydrogen and Cl-4-alkyl; Z and Z* are independently selected among an intemucleoside linkage, a terminal group or a protecting group; B constitutes a natural or non-natural nucleotide base moiety; and the asymmetric groups may be found in either orientation.

Preferably, the LNA used in the oligonucleotides described herein comprises at least one LNA unit according any of the formulas

wherein Y is -0-, -S-, -NH-, or N(RH); Z and Z* are independently selected among an intemucleoside linkage, a terminal group or a protecting group; B constitutes a natural or non-natural nucleotide base moiety, and RH is selected from hydrogen and Cl-4-alkyl.

In some embodiments, the Locked Nucleic Acid (LNA) used in the oligonucleotides described herein comprises at least one Locked Nucleic Acid (LNA) unit according any of the formulas shown in Scheme 2 of PCT/DK2006/000512.

In some embodiments, the LNA used in the oligomer comprises intemucleoside linkages selected from -0-P(O)2-O-, -0-P(0,S)-0-, -0-P(S)2-O-, -S-P(0)2-0-, -S-P(0,S)-0-, -S-P(S)2-0-, -0-P(O)2-S-, -0-P(0,S)-S-, -S-P(0)2-S-, -0-PO(RH)-0-, 0-PO(OCH3)-0-, -O- PO(NRH)-0-, -0-PO(OCH2CH2S-R)-O-, -0-PO(BH3)-0-, -0-PO(NHRH)-0-, -0-P(0)2- NRH-, -NRH-P(0)2-0-, -NRH-CO-0-, where RH is selected from hydrogen and Cl-4-alkyl.

Specifically preferred LNA units are shown in scheme 2:

Scheme 2

The term "thio-LNA" comprises a locked nucleotide in which at least one of X or Y in the general formula above is selected from S or -CH2-S-. Thio-LNA can be in both beta-D and alpha-L-configuration.

The term "amino-LNA" comprises a locked nucleotide in which at least one of X or Y in the general formula above is selected from -N(H)-, N(R)-, CH2-N(H)-, and -CH2-N(R)- where R is selected from hydrogen and Cl-4-alkyl. Amino-LNA can be in both beta-D and alpha-L-configuration.

The term "oxy-LNA" comprises a locked nucleotide in which at least one of X or Y in the general formula above represents -O- or -CH2-0-. Oxy-LNA can be in both beta-D and alpha-L-configuration. The term "ena-LNA" comprises a locked nucleotide in which Y in the general formula above is -CH2-0- (where the oxygen atom of -CH2-0- is attached to the 2'-position relative to the base B).

LNAs are described in additional detail herein.

One or more substituted sugar moieties can also be included, e.g., one of the following at the 2' position: OH, SH, SCH3, F, OCN, OCH3 OCH3, OCH3 0(CH2)n CH3, 0(CH2)n NH2 or 0(CH2)n CH3 where n is from 1 to about 10; CI to CIO lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyl; CI; Br; CN; CF3 ; OCF3; 0-, S-, or N-alkyl; 0-, S-, or N-alkenyl; SOCH3; S02 CH3; ON02; N02; N3; NH2; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleaving group; a reporter group; an intercalator; a group for improving the pharmacokinetic properties of an oligonucleotide; or a group for improving the pharmacodynamic properties of an oligonucleotide and other substituents having similar properties. A preferred modification includes 2'-methoxyethoxy [2'-0-CH2CH20CH3, also known as 2'-0-(2-methoxyethyl)] (Martin et al, Helv. Chim. Acta, 1995, 78, 486). Other preferred modifications include 2'- methoxy (2'-0-CH3), 2'-propoxy (2'-OCH2 CH2CH3) and 2'-fluoro (2'-F). Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide and the 5' position of 5' terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyls in place of the pentofuranosyl group.

Single stranded oligonucleotides can also include, additionally or alternatively, nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include adenine (A), guanine (G), thymine (T), cytosine (C) and uracil (U). Modified nucleobases include nucleobases found only infrequently or transiently in natural nucleic acids, e.g., hypoxanthine, 6-methyladenine, 5-Me pyrimidines, particularly 5-methylcytosine (also referred to as 5-methyl-2'

deoxycytosine and often referred to in the art as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC and gentobiosyl HMC, isocytosine, pseudoisocytosine, as well as synthetic nucleobases, e.g., 2-aminoadenine, 2- (methylamino)adenine, 2-(imidazolylalkyl)adenine, 2- (aminoalklyamino)adenine or other heterosubstituted alkyladenines, 2-thiouracil, 2- thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 5-propynyluracil, 8-azaguanine, 7- deazaguanine, N6 (6-aminohexyl)adenine, 6-aminopurine, 2-aminopurine, 2-chloro-6- aminopurine and 2,6-diaminopurine or other diaminopurines. See, e.g., Kornberg, "DNA Replication," W. H. Freeman & Co., San Francisco, 1980, pp75-77; and Gebeyehu, G., et al. Nucl. Acids Res., 15:4513 (1987)). A "universal" base known in the art, e.g., inosine, can also be included. 5-Me-C substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C. (Sanghvi, in Crooke, and Lebleu, eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and may be used as base substitutions.

It is not necessary for all positions in a given oligonucleotide to be uniformly modified, and in fact more than one of the modifications described herein may be

incorporated in a single oligonucleotide or even at within a single nucleoside within an oligonucleotide.

In some embodiments, both a sugar and an internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar- backbone of an oligonucleotide is replaced with an amide containing backbone, for example, an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, US patent nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al, Science, 1991, 254, 1497-1500.

Single stranded oligonucleotides can also include one or more nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein,

"unmodified" or "natural" nucleobases comprise the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified

nucleobases comprise other synthetic and natural nucleobases such as 5-methylcytosine (5- me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudo-uracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8- thioalkyl, 8-hydroxyl and other 8- substituted adenines and guanines, 5-halo particularly 5- bromo, 5-triiluoromethyl and other 5-substituted uracils and cytosines, 7-methylquanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3- deazaguanine and 3-deazaadenine.

Further, nucleobases comprise those disclosed in United States Patent No. 3,687,808, those disclosed in "The Concise Encyclopedia of Polymer Science And Engineering", pages 858-859, Kroschwitz, ed. John Wiley & Sons, 1990; those disclosed by Englisch et al., Angewandle Chemie, International Edition, 1991, 30, page 613, and those disclosed by Sanghvi, Chapter 15, Antisense Research and Applications," pages 289- 302, Crooke, and Lebleu, eds., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligonucleotides as described herein. These include 5- substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, comprising 2-aminopropyladenine, 5-propynyluracil and 5- propynylcytosine. 5- methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2<0>C (Sanghvi, et al., eds, "Antisense Research and Applications," CRC Press, Boca Raton, 1993, pp. 276-278) and are presently preferred base substitutions, even more particularly when combined with 2'-0-methoxyethyl sugar modifications. Modified nucleobases are described in US patent nos. 3,687,808, as well as 4,845,205; 5,130,302; 5,134,066; 5,175, 273; 5, 367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,596,091; 5,614,617; 5,750,692, and 5,681,941, each of which is herein incorporated by reference.

In some embodiments, the single stranded oligonucleotides are chemically linked to one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide. For example, one or more single stranded oligonucleotides, of the same or different types, can be conjugated to each other; or single stranded

oligonucleotides can be conjugated to targeting moieties with enhanced specificity for a cell type or tissue type. Such moieties include, but are not limited to, lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S- tritylthiol (Manoharan et al, Ann. N. Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49- 54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2- di-O-hexadecyl- rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Mancharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-t oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937). See also US patent nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552, 538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486, 603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762, 779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082, 830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5, 245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391, 723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5, 565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599, 928 and 5,688,941, each of which is herein incorporated by reference.

These moieties or conjugates can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Conjugate groups include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugate groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, in the context of this disclosure, include groups that improve uptake, enhance resistance to degradation, and/or strengthen sequence-specific hybridization with the target nucleic acid. Groups that enhance the pharmacokinetic properties, in the context of this disclosure, include groups that improve uptake, distribution, metabolism or excretion of the compounds, molecules, or agents described herein. Representative conjugate groups are disclosed in International Patent Application No. PCT/US92/09196, filed Oct. 23, 1992, and U.S. Pat. No. 6,287,860, which are incorporated herein by reference. Conjugate moieties include, but are not limited to, lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-5-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac- glycerol or triethylammonium 1,2- di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxy cholesterol moiety. See, e.g., U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941.

In some embodiments, single stranded oligonucleotide modification include modification of the 5' or 3' end of the oligonucleotide. In some embodiments, the 3' end of the oligonucleotide comprises a hydroxyl group or a thiophosphate. It should be appreciated that additional molecules (e.g. a biotin moiety or a fluorophor) can be conjugated to the 5' or 3' end of the single stranded oligonucleotide. In some embodiments, the single stranded oligonucleotide comprises a biotin moiety conjugated to the 5' nucleotide.

In some embodiments, the single stranded oligonucleotide comprises locked nucleic acids (LNA), ENA modified nucleotides, 2'-0-methyl nucleotides, or 2'-fluoro- deoxyribonucleotides. In some embodiments, the single stranded oligonucleotide comprises alternating deoxyribonucleotides and 2'-fluoro-deoxyribonucleotides. In some embodiments, the single stranded oligonucleotide comprises alternating deoxyribonucleotides and 2'-0- methyl nucleotides. In some embodiments, the single stranded oligonucleotide comprises alternating deoxyribonucleotides and ENA modified nucleotides. In some embodiments, the single stranded oligonucleotide comprises alternating deoxyribonucleotides and locked nucleic acid nucleotides. In some embodiments, the single stranded oligonucleotide comprises alternating locked nucleic acid nucleotides and 2'-0-methyl nucleotides.

In some embodiments, the 5' nucleotide of the oligonucleotide is a

deoxyribonucleotide. In some embodiments, the 5' nucleotide of the oligonucleotide is a locked nucleic acid nucleotide. In some embodiments, the nucleotides of the oligonucleotide comprise deoxyribonucleotides flanked by at least one locked nucleic acid nucleotide on each of the 5' and 3' ends of the deoxyribonucleotides. In some embodiments, the nucleotide at the 3' position of the oligonucleotide has a 3' hydroxyl group or a 3' thiophosphate.

In some embodiments, the single stranded oligonucleotide comprises

phosphorothioate internucleotide linkages. In some embodiments, the single stranded oligonucleotide comprises phosphorothioate internucleotide linkages between at least two nucleotides. In some embodiments, the single stranded oligonucleotide comprises phosphorothioate internucleotide linkages between all nucleotides.

It should be appreciated that the single stranded oligonucleotide can have any combination of modifications as described herein.

The oligonucleotide may comprise a nucleotide sequence having one or more of the following modification patterns.

(a) (X)Xxxxxx, (X)xXxxxx, (X)xxXxxx, (X)xxxXxx, (X)xxxxXx and (X)xxxxxX,

(b) (X)XXxxxx, (X)XxXxxx, (X)XxxXxx, (X)XxxxXx, (X)XxxxxX, (X)xXXxxx, (X)xXxXxx, (X)xXxxXx, (X)xXxxxX, (X)xxXXxx, (X)xxXxXx, (X)xxXxxX, (X)xxxXXx, (X)xxxXxX and (X)xxxxXX,

(c) (X)XXXxxx, (X)xXXXxx, (X)xxXXXx, (X)xxxXXX, (X)XXxXxx, (X)XXxxXx, (X)XXxxxX, (X)xXXxXx, (X)xXXxxX, (X)xxXXxX, (X)XxXXxx, (X)XxxXXx

(X)XxxxXX, (X)xXxXXx, (X)xXxxXX, (X)xxXxXX, (X)xXxXxX and (X)XxXxXx,

(d) (X)xxXXX, (X)xXxXXX, (X)xXXxXX, (X)xXXXxX, (X)xXXXXx,

(X)XxxXXXX, (X)XxXxXX, (X)XxXXxX, (X)XxXXx, (X)XXxxXX, (X)XXxXxX, (X)XXxXXx, (X)XXXxxX, (X)XXXxXx, and (X)XXXXxx,

(e) (X)xXXXXX, (X)XxXXXX, (X)XXxXXX, (X)XXXxXX, (X)XXXXxX and (X)XXXXXx, and

(f) XXXXXX, XxXXXXX, XXxXXXX, XXXxXXX, XXXXxXX, XXXXXxX and XXXXXXx, in which "X" denotes a nucleotide analogue, (X) denotes an optional nucleotide analogue, and "x" denotes a DNA or RNA nucleotide unit. Each of the above listed patterns may appear one or more times within an oligonucleotide, alone or in combination with any of the other disclosed modification patterns.

Aspects of the disclosure relate to methods for inducing FOXP3 expression, activating T cells, and/or treating a condition or disease (e.g., a disease or disorder associated with aberrant immune cell activation such as an autoimmune or inflammatory disease or disorder) associated with decreased levels of FOXP3 that involve inhibiting expression or activity of EZH1 and/or EZH2 or another component of PRC2, e.g., Suzl2, EED1 or RbAp48. For example, expression of EZH1 and/or EZH2 may inhibited through the using any of oligonucleotides (e.g., single stranded oligonucleotides) disclosed herein. In some embodiments, expression or activity may be inhibited through the use of a mixmer, gapmer, siRNA, miRNA or other oligonucleotide that inhibits expression of a FOXP3 mRNA.

Exemplary human mRNA and protein sequence identifiers for EZH1, EZH2, Suzl2, EED1 and RbAp48 are provided below. These sequence identifiers can be used to identify exemplary mRNA and protein sequences by using the NCBI Gene search as of the filing of the instant application.

EZH1: NM 001991.3, NP_001982.2

EZH2: NM 001203247.1, NM OO 1203248.1, NM 001203249.1, NM_004456.4, NP_004447.2, NM_152998.2, NP_001190177.1, NP_001190176.1, NP_001190178.1, NP_694543.1

Suzl2: NM_015355.2, NP_056170.2

EED1: NM_003797.3, NM_152991.2, NP_003788.2, NP_694536.1

RbAp48: NM_001135255.1, NM_001135256.1, NM_005610.2, NP 001128727.1, NP_001128728.1, NP_005601.1.

Accordingly, in some embodiments, an oligonucleotide described herein may be a mixmer or comprise a mixmer sequence pattern. The term 'mixmer' refers to

oligonucleotides which comprise both naturally and non-naturally occurring nucleotides or comprise two different types of non-naturally occurring nucleotides. Mixmers are generally known in the art to have a higher binding affinity than unmodified oligonucleotides and may be used to specifically bind a target molecule, e.g., to block a binding site on the target molecule. Generally, mixmers do not recruit an RNAse to the target molecule and thus do not promote cleavage of the target molecule. Accordingly, in some embodiments, an oligonucleotide provided herein may be cleavage promoting (e.g., an siRNA or gapmer) or not cleavage promoting (e.g., a mixmer, siRNA, single stranded RNA or double stranded RNA).

In some embodiments, the mixmer comprises or consists of a repeating pattern of nucleotide analogues and naturally occurring nucleotides, or one type of nucleotide analogue and a second type of nucleotide analogue. However, it is to be understood that the mixmer need not comprise a repeating pattern and may instead comprise any arrangement of nucleotide analogues and naturally occurring nucleotides or any arrangement of one type of nucleotide analogue and a second type of nucleotide analogue. The repeating pattern, may, for instance be every second or every third nucleotide is a nucleotide analogue, such as LNA, and the remaining nucleotides are naturally occurring nucleotides, such as DNA, or are a 2' substituted nucleotide analogue such as 2'MOE or 2' fluoro analogues, or any other nucleotide analogues described herein. It is recognised that the repeating pattern of nucleotide analogues, such as LNA units, may be combined with nucleotide analogues at fixed positions— e.g. at the 5 Or 3' termini.

In some embodiments, the mixmer does not comprise a region of more than 5, more than 4, more than 3, or more than 2 consecutive naturally occurring nucleotides, such as DNA nucleotides. In some embodiments, the mixmer comprises at least a region consisting of at least two consecutive nucleotide analogues, such as at least two consecutive LNAs. In some embodiments, the mixmer comprises at least a region consisting of at least three consecutive nucleotide analogue units, such as at least three consecutive LNAs.

In some embodiments, the mixmer does not comprise a region of more than 7, more than 6, more than 5, more than 4, more than 3, or more than 2 consecutive nucleotide analogues, such as LNAs. It is to be understood that the LNA units may be replaced with other nucleotide analogues, such as those referred to herein.

In some embodiments, the mixmer comprises at least one nucleotide analogue in one or more of six consecutive nucleotides. The substitution pattern for the nucleotides may be selected from the group consisting of Xxxxxx, xXxxxx, xxXxxx, xxxXxx, xxxxXx and xxxxxX, wherein "X" denotes a nucleotide analogue, such as an LNA, and "x" denotes a naturally occurring nucleotide, such as DNA or RNA.

In some embodiments, the mixmer comprises at least two nucleotide analogues in one or more of six consecutive nucleotides. The substitution pattern for the nucleotides may be selected from the group consisting of XXxxxx, XxXxxx, XxxXxx, XxxxXx, XxxxxX, xXXxxx, xXxXxx, xXxxXx, xXxxxX, xxXXxx, xxXxXx, xxXxxX, xxxXXx, xxxXxX and xxxxXX, wherein "X" denotes a nucleotide analogue, such as an LNA, and "x" denotes a naturally occuring nucleotide, such as DNA or RNA. In some embodiments, the substitution pattern for the nucleotides may be selected from the group consisting of XxXxxx, XxxXxx, XxxxXx, XxxxxX, xXxXxx, xXxxXx, xXxxxX, xxXxXx, xxXxxX and xxxXxX. In some embodiments, the substitution pattern is selected from the group consisting of xXxXxx, xXxxXx, xXxxxX, xxXxXx, xxXxxX and xxxXxX. In some embodiments, the substitution pattern is selected from the group consisting of xXxXxx, xXxxXx and xxXxXx. In some embodiments, the substitution pattern for the nucleotides is xXxXxx.

In some embodiments, the mixmer comprises at least three nucleotide analogues in one or more of six consecutive nucleotides. The substitution pattern for the nucleotides may be selected from the group consisting of XXXxxx, xXXXxx, xxXXXx, xxxXXX, XXxXxx, XXxxXx, XXxxxX, xXXxXx, xXXxxX, xxXXxX, XxXXxx, XxxXXx, XxxxXX, xXxXXx, xXxxXX, xxXxXX, xXxXxX and XxXxXx, wherein "X" denotes a nucleotide analogue, such as an LNA, and "x" denotes a naturally occuring nucleotide, such as DNA or RNA. In some embodiments, the substitution pattern for the nucleotides is selected from the group consisting of XXxXxx, XXxxXx, XXxxxX, xXXxXx, xXXxxX, xxXXxX, XxXXxx, XxxXXx, XxxxXX, xXxXXx, xXxxXX, xxXxXX, xXxXxX and XxXxXx. In some embodiments, the substitution pattern for the nucleotides is selected from the group consisting of xXXxXx, xXXxxX, xxXXxX, xXxXXx, xXxxXX, xxXxXX and xXxXxX. n some embodiments, the substitution pattern for the nucleotides is xXxXxX or XxXxXx. In some embodiments, the substitution pattern for the nucleotides is xXxXxX.

In some embodiments, the mixmer comprises at least four nucleotide analogues in one or more of six consecutive nucleotides. The substitution pattern for the nucleotides may be selected from the group consisting of xXXXX, xXxXXX, xXXxXX, xXXXxX, xXXXXx, XxxXXX, XxXxXX, XxXXxX, XxXXXx, XXxxXX, XXxXxX, XXxXXx, XXXxxX,

XXXxXx and XXXXxx, wherein "X" denotes a nucleotide analogue, such as an LNA, and "x" denotes a naturally occuring nucleotide, such as DNA or RNA.

In some embodiments, the mixmer comprises at least five nucleotide analogues in one or more of six consecutive nucleotides. The substitution pattern for the nucleotides may be selected from the group consisting of xXXXXX, XxXXXX, XXxXXX, XXXxXX,

In some embodiments, the mixmer contains a modified nucleotide, e.g., an LNA, at the 5' end. In some embodiments, the mixmer contains a modified nucleotide, e.g., an LNA, at the first two positions, counting from the 5' end.

In some embodiments, the mixmer is incapable of recruiting RNAseH.

Oligonucleotides that are incapable of recruiting RNAseH are well known in the literature, in example see WO2007/112754, WO2007/112753, or PCT/DK2008/000344. Mixmers may be designed to comprise a mixture of affinity enhancing nucleotide analogues, such as in non- limiting example LNA nucleotides and 2'-0-methyl nucleotides. In some embodiments, the mixmer comprises modified internucleoside linkages (e.g., phosphorothioate internucleoside linkages or other linkages) between at least two, at least three, at least four, at least five or more nucleotides.

In some embodiments, the oligonucletoide is a mixmer comprising alternating LNA nucleotides and 2'-0-methyl nucleotides. In some embodiments, the oligonucleotide can further comprise phosphorothioate internucleotide linkages between some or all nucleotides. In some embodiments, such oligonucleotides can have a sequence set forth as one of the following:

GCTCGGTAGTCCTCC (SEQ ID NO: 4);

GCGGAGGAAGTAGCT (SEQ ID NO: 5);

GGTTGCGGTCAGTGG (SEQ ID NO: 6);

CCCACAGTACCGTCC (SEQ ID NO: 7);

CCCTGATCCATGCCT (SEQ ID NO: 8);

ATACCCCGTGTCTCC (SEQ ID NO: 9);

CTTGAGTCCCGTGCA (SEQ ID NO: 10);

AGCAGCGTCAGTACC (SEQ ID NO: 11);

ACGCACCCACAGCCA (SEQ ID NO: 12); and

AGCCAAACAGAGCCT (SEQ ID NO: 13).

A mixmer may be produced using any method known in the art or described herein. Representative U.S. patents, U.S. patent publications, and PCT publications that teach the preparation of mixmers include U.S. patent publication Nos. US20060128646,

US20090209748, US20090298916, US20110077288, and US20120322851, and U.S. patent No. 7687617.

In some embodiments, the oligonucleotide is a gapmer. In some embodiments, a gapmer oligonucleotide has the formula 5'-X-Y-Z-3', with X and Z as flanking regions around a gap region Y. In some embodiments, the Y region is a contiguous stretch of nucleotides, e.g., a region of at least 6 DNA nucleotides, which are capable of recruiting an RNAse, such as RNAseH. Without wishing to be bound by theory, it is thought that the gapmer binds to the target nucleic acid, at which point an RNAse is recruited and can then cleave the target nucleic acid. In some embodiments, the Y region is flanked both 5' and 3' by regions X and Z comprising high-affinity modified nucleotides, e.g., 1 - 6 modified nucleotides. Exemplary modified oligonucleotides include, but are not limited to, 2' MOE or 2'OMe or Locked Nucleic Acid bases (LNA). The flanks X and Z may have a of length 1 - 20 nucleotides, preferably 1-8 nucleotides and even more preferred 1 - 5 nucleotides. The flanks X and Z may be of similar length or of dissimilar lengths. The gap-segment Y may be a nucleotide sequence of length 5 - 20 nucleotides, preferably 6-12 nucleotides and even more preferred 6 - 10 nucleotides. In some aspects, the gap region of the gapmer oligonucleotides described herein may contain modified nucleotides known to be acceptable for efficient RNase H action in addition to DNA nucleotides, such as C4'-substituted nucleotides, acyclic nucleotides, and arabino-configured nucleotides. In some embodiments, the gap region comprises one or more unmodified internucleosides. In some embodiments, one or both flanking regions each independently comprise one or more phosphorothioate internucleoside linkages (e.g., phosphorothioate internucleoside linkages or other linkages) between at least two, at least three, at least four, at least five or more nucleotides. In some embodiments, the gap region and two flanking regions each independently comprise modified internucleoside linkages (e.g., phosphorothioate internucleoside linkages or other linkages) between at least two, at least three, at least four, at least five or more nucleotides.

A gapmer may be produced using any method known in the art or described herein. Representative U.S. patents, U.S. patent publications, and PCT publications that teach the preparation of gapmers include, but are not limited to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; 5,700,922; 5,898,031; 7,432,250; and 7,683,036; U.S. patent publication Nos. US20090286969, US20100197762, and US20110112170; and PCT publication Nos.

WO2008049085 and WO2009090182, each of which is herein incorporated by reference in its entirety.

In some embodiments, oligonucleotides provided herein may be in the form of small interfering RNAs (siRNA), also known as short interfering RNA or silencing RNA. SiRNA, is a class of double-stranded RNA molecules, typically about 18-23 or 20-25 base pairs in length that target nucleic acids (e.g., mRNAs) for degradation via the RNA interference

(RNAi) pathway in cells. Specificity of siRNA molecules may be determined by the binding of the antisense strand of the molecule to its target RNA. Effective siRNA molecules are generally less than 30 to 35 base pairs in length to prevent the triggering of non-specific RNA interference pathways in the cell via the interferon response, although longer siRNA can also be effective. The siRNA molecule can be double stranded (i.e. a dsRNA molecule comprising an antisense strand and a complementary sense strand) or single- stranded (i.e. a ssRNA molecule comprising just an antisense strand). The siRNA molecules can comprise a duplex, asymmetric duplex, hairpin or asymmetric hairpin secondary structure, having self- complementary sense and antisense strands.

Double-stranded siRNA may comprise RNA strands that are the same length or different lengths. Double- stranded siRNA molecules can also be assembled from a single oligonucleotide in a stem-loop structure, wherein self-complementary sense and antisense regions of the siRNA molecule are linked by means of a nucleic acid based or non-nucleic acid-based linker(s), as well as circular single-stranded RNA having two or more loop structures and a stem comprising self-complementary sense and antisense strands, wherein the circular RNA can be processed either in vivo or in vitro to generate an active siRNA molecule capable of mediating RNAi. Small hairpin RNA (shRNA) molecules thus are also contemplated herein. These molecules comprise a specific antisense sequence in addition to the reverse complement (sense) sequence, typically separated by a spacer or loop sequence. Cleavage of the spacer or loop provides a single- stranded RNA molecule and its reverse complement, such that they may anneal to form a dsRNA molecule (optionally with additional processing steps that may result in addition or removal of one, two, three or more nucleotides from the 3' end and/or the 5' end of either or both strands). A spacer can be of a sufficient length to permit the antisense and sense sequences to anneal and form a double- stranded structure (or stem) prior to cleavage of the spacer (and, optionally, subsequent processing steps that may result in addition or removal of one, two, three, four, or more nucleotides from the 3' end and/or the 5' end of either or both strands). A spacer sequence is may be an unrelated nucleotide sequence that is situated between two complementary nucleotide sequence regions which, when annealed into a double- stranded nucleic acid, comprise a shRNA.

The overall length of the siRNA molecules can vary from about 14 to about

200nucleotides, e.g., about 14-100, 14-50, 14-30 or 18-23 nucleotides, depending on the type of siRNA molecule being designed. Generally, between about 14 and about 50 of these nucleotides are complementary to the RNA target sequence, i.e. constitute the specific antisense sequence of the siRNA molecule. For example, when the siRNA is a double- or single-stranded siRNA, the length can vary from about 14 to about 50 nucleotides, whereas when the siRNA is a shRNA or circular molecule, the length can vary from about 40 nucleotides to about 200 nucleotides.

An siRNA molecule may comprise a 3' overhang at one end of the molecule, the other end may be blunt-ended or have also an overhang (5' or 3')· When the siRNA molecule comprises an overhang at both ends of the molecule, the length of the overhangs may be the same or different. In one embodiment, the siRNA molecule as described herein comprises 3' overhangs of about 1 to about 3 nucleotides on both ends of the molecule.

In some embodiments, an oligonucleotide may be a microRNA (miRNA).

MicroRNAs (referred to as "miRNAs") are small non-coding RNAs, belonging to a class of regulatory molecules found in plants and animals that control gene expression by binding to complementary sites on a target RNA transcript. miRNAs are generated from large RNA precursors (termed pri-miRNAs) that are processed in the nucleus into approximately 70 nucleotide pre-miRNAs, which fold into imperfect stem-loop structures (Lee, Y., et al., Nature (2003) 425(6956):415-9). The pre-miRNAs undergo an additional processing step within the cytoplasm where mature miRNAs of 18-25 nucleotides in length are excised from one side of the pre-miRNA hairpin by an RNase III enzyme, Dicer (Hutvagner, G., et al., Science (2001) 12: 12 and Grishok, A., et al., Cell (2001) 106(l):23-34).

As used herein, miRNAs including pri-miRNA, pre-miRNA, mature miRNA or fragments of variants thereof that retain the biological activity of mature miRNA. In one embodiment, the size range of the miRNA can be from 21 nucleotides to 170 nucleotides, although miRNAs of up to 2000 nucleotides can be utilized. In a preferred embodiment, the size range of the miRNA is from 70 to 170 nucleotides in length. In another preferred embodiment, mature miRNAs of from 21 to 25 nucleotides in length can be used.

In some embodiments, the miRNA may be a miR-30 precursor. As used herein, an "miR-30 precursor", also called an miR-30 hairpin, is a precursor of the human microRNA miR-30, as it is understood in the literature (e.g., Zeng and Cullen, 2003; Zeng and Cullen, 2005; Zeng et al., 2005; United States Patent Application Publication No. US 2004/005341), where the precursor could be modified from the wild-type miR-30 precursor in any manner described or implied by that literature, while retaining the ability to be processed into an miRNA. In some embodiments, a miR-30 precursor is at least 80 nucleotides long and comprises a stem-loop structure. In some embodiments, the miR-30 precursor further comprises a first miRNA sequence of 20- 22 nucleotides on the stem of the stem-loop structure complementary to a portion of a first target sequence (e.g., a sequence within a euchromatic region of a target gene disclosed herein).

A miRNA may be isolated from a variety of sources or may be synthesized according to methods well known in the art (see, e.g., Current Protocols in Molecular Biology, Wiley Online Library; US Patent Number 8354384; and Wahid et al. MicroRNAs: synthesis, mechanism, function, and recent clinical trials. Biochim Biophys Acta. 2010; 1803(11): 1231- 43). In some embodiments, a miRNA is expressed from a vector as known in the art or described herein. In some embodiments, the vector may include a sequence encoding a mature miRNA. In some embodiments, the vector may include a sequence encoding a pre- miRNA such that the pre-miRNA is expressed and processed in a cell into a mature miRNA. In some embodiments, the vector may include a sequence encoding a pri-miRNA. In this embodiment, the primary transcript is first processed to produce the stem-loop precursor miRNA molecule. The stem-loop precursor is then processed to produce the mature microRNA.

In some embodiments, an oligonucleotide described herein comprises a synthetic cap, e.g., to increase efficiency of translation, RNA half-life and/or function within cells.

Synthetic caps are known in the art. Exemplary synthetic caps include, but are not limited to, N7-Methyl-Guanosine-5'-Triphosphate-5'-Guanosine, Guanosine-5'-Triphosphate-5'-

Guanosine, N7-Methyl-3'-0-Methyl-Guanosine-5'-Triphosphate-5'-Guanosine (see, e.g., products available from TrilinkBiotech), and N7-benzylated dinucleoside tetraphosphate analogs (see, e.g., Grudzien et al. Novel cap analogs for in vitro synthesis of mRNAs with high translational efficiency. RNA. 2004 Sep; 10(9): 1479-1487).

Methods for Modulating Gene Expression

In one aspect, the disclosure relates to methods for modulating gene expression in a cell (e.g., a cell for which FOXP3 levels are reduced) for research purposes (e.g., to study the function of the gene in the cell). In another aspect, the disclosure relates to methods for modulating gene expression in a cell (e.g., a cell for which FOXP3 levels are reduced) for gene or epigenetic therapy. The cells can be in vitro, ex vivo, or in vivo (e.g., in a subject who has a disease or condition resulting from reduced expression or activity of FOXP3). In some embodiments, methods for modulating gene expression in a cell comprise delivering a single stranded oligonucleotide as described herein. In some embodiments, delivery of the single stranded oligonucleotide to the cell results in a level of expression of gene that is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or more greater than a level of expression of gene in a control cell to which the single stranded oligonucleotide has not been delivered. In certain embodiments, delivery of the single stranded oligonucleotide to the cell results in a level of expression of gene that is at least 50% greater than a level of expression of gene in a control cell to which the single stranded oligonucleotide has not been delivered.

In another aspect of the disclosure, methods comprise administering to a subject (e.g. a human) a composition comprising a single stranded oligonucleotide as described herein to increase protein levels in the subject. In some embodiments, the increase in protein levels is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, or more, higher than the amount of a protein in the subject before administering.

As another example, to increase expression of FOXP3 in a cell, the methods include introducing into the cell a single stranded oligonucleotide that is sufficiently complementary to a PRC2-associated region (e.g., of a long non-coding RNA) that maps to a genomic position encompassing or in proximity to the FOXP3 gene. The PRC2-associated region may map to a genomic region having a sequence as set forth in SEQ ID NO: 3, or a sequence corresponding to nucleotide 511 to nucleotide 920 of SEQ ID NO: 3.

In another aspect, provided herein relate to methods for modulating expression of FOXP3 in cells by modulating expression of CCDC22 or activity of the CCDC22 gene in cells. For example, as shown in Example 1, downregulation of CCDC22 expression or silencing CCDC22 gene resulted in increased expression of FOXP3 in cells. Accordingly, in some embodiments, methods for upregulating or increasing expression of FOXP3 in cells comprising delivering to cells an inhibitor of CCDC22 (also referred to herein as "CCDC22 inhibitor"). In some embodiments, delivery of the CCDC22 inhibitor to cells results in a level of expression of the CCDC22 gene that is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or more lower than a level of expression of the CCDC22 gene in a control cell to which the CCDC22 inhibitor has not been delivered. In certain embodiments, delivery of the CCDC22 inhibitor to cells results in a level of expression of the CCDC22 gene that is at least 50% lower than a level of expression of the CCDC22 gene in a control cell to which the CCDC22 inhibitor has not been delivered.

In another aspect, methods for upregulating or increasing expression of FOXP3 in a subject in need thereof (e.g., a subject with decreased expression of FOXP3) comprising administering to the subject (e.g., a human) a composition comprising an CCDC22 inhibitor to decrease CCDC22 expression or activity in the subject, thereby causing an increase in expression of FOXP3. In some embodiments, the decrease in CCDC22 expression or activity is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more, lower than the CCDC22 expression or activity in the subject before administering. In some

embodiments, the increase in expression of FOXP3 is at least 5%, 10%, 20%, 30%, 40%,

50%, 60%, 70%, 80%, 90%, 100%, 200%, or more, higher than the FOXP3 expression in the subject before administering. In some embodiments, RQ (relative quantification) is used as a read-out of FOXP3 or CCDC22 expression, such as mRNA levels. In some embodiments, RQ is a calculated as follows: Target dCT= Target Ct - Housekeeper Ct; ddCT= Target dCT - Negative control dCT ; RQ= Log2 - ddCT .

A CCDC22 inhibitor or an inhibitor of CCDC22 is an agent that reduces or inhibits expression or activity of CCDC22 in a cell, for example, either by decreasing transcription or translation of CCDC22-encoding nucleic acid, or by inhibiting or blocking CCDC22 protein activity, or both. In some embodiments, a CCDC22 inhibitor is an agent that directly or indirectly upregulates or increases the CCDC22-mediated expression of FOXP3 in cells. As an example, a CCDC22 inhibitor can be an oligonucleotide that reduces or inhibits expression or activity of CCDC22 in a cell. The oligonucleotide may comprise a region of

complementarity that is complementary with at least 6 or more (e.g., at least 7, at least 8, at least 9, at least 10, at least 15, at least 20 or more) consecutive nucleotides of a CCDC22 mRNA. In some embodiments, the region of complementarity can be complementary with at least 6 or more (e.g., at least 7, at least 8, at least 9, at least 10, at least 15, at least 20 or more) consecutive nucleotides of exon 1 of a CCDC22 mRNA. In some embodiments, the region of complementarity can be complementary with at least 6 or more (e.g., at least 7, at least 8, at least 9, at least 10, at least 15, at least 20 or more) consecutive nucleotides of exon 2 of a CCDC22 mRNA. The CCDC22 inhibitor oligonucleotide can be in a form of a gapmer, small interfering RNA (siRNA), or microRNA (miRNA). In some embodiments, the CCDC22 inhibitor oligonucleotide can be in a form of a gapmer as described herein.

In another aspect of the disclosure provides methods of treating a condition (e.g., a disease or disorder associated with aberrant immune cell activation such as an autoimmune disease or disorder) associated with decreased levels of expression of FOXP3 in a subject, the method comprising administering a single stranded oligonucleotide as described herein or an CCDC22 inhibitor as described herein.

A subject can include a non-human mammal, e.g. mouse, rat, guinea pig, rabbit, cat, dog, goat, cow, or horse. In preferred embodiments, a subject is a human. Single stranded oligonucleotides have been employed as therapeutic moieties in the treatment of disease states in animals, including humans. Single stranded oligonucleotides can be useful therapeutic modalities that can be configured to be useful in treatment regimens for the treatment of cells, tissues and animals, especially humans.

For therapeutics, an animal, preferably a human, suspected of having a disease or disorder associated with aberrant immune cell activation such as an autoimmune disease or disorder is treated for the disease or disorder by administering single stranded oligonucleotide as described herein. For example, in one non-limiting embodiment, the methods comprise the step of administering to an animal in need of treatment, a therapeutically effective amount of a single stranded oligonucleotide as described herein.

Examples of autoimmune diseases and disorders that may be treated according to the methods disclosed herein include, but are not limited to, Acute Disseminated

Encephalomyelitis (ADEM), Acute necrotizing hemorrhagic leukoencephalitis, Addison's disease, Agammaglobulinemia, Alopecia areata, Amyloidosis, Ankylosing spondylitis, Anti- GBM/Anti-TBM nephritis, Antiphospholipid syndrome (APS), Autoimmune angioedema, Autoimmune aplastic anemia, Autoimmune dysautonomia, Autoimmune hepatitis,

Autoimmune hyperlipidemia, Autoimmune immunodeficiency, Autoimmune inner ear disease (AIED), Autoimmune myocarditis, Autoimmune oophoritis, Autoimmune

pancreatitis, Autoimmune retinopathy, Autoimmune thrombocytopenic purpura (ATP), Autoimmune thyroid disease, Autoimmune urticaria, Axonal & neuronal neuropathies, Balo disease, Behcet's disease, Bullous pemphigoid, Cardiomyopathy, Castleman disease, Celiac disease, Chagas disease, Chronic inflammatory demyelinating polyneuropathy (CIDP), Chronic recurrent multifocal ostomyelitis (CRMO), Churg-Strauss syndrome, Cicatricial pemphigoid/benign mucosal pemphigoid, inflammatory bowel disease (e.g., Crohn's disease or Ulcerative colitis), Cogans syndrome, Cold agglutinin disease, Congenital heart block, Coxsackie myocarditis, CREST disease, Essential mixed cryoglobulinemia, Demyelinating neuropathies, Dermatitis herpetiformis, Dermatomyositis, Devic's disease (neuromyelitis optica), Discoid lupus, Dressier' s syndrome, Endometriosis, Eosinophilic esophagitis, Eosinophilic fasciitis, Erythema nodosum, Experimental allergic encephalomyelitis, Evans syndrome, Fibrosing alveolitis, Giant cell arteritis (temporal arteritis), Giant cell myocarditis, Glomerulonephritis, Goodpasture's syndrome, Granulomatosis with Polyangiitis (GPA) (formerly called Wegener's Granulomatosis), Graves' disease, Guillain-Barre syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, Hemolytic anemia, Henoch-Schonlein purpura, Herpes gestationis, Hypogammaglobulinemia, Idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, IgG4-related sclerosing disease, Immunoregulatory lipoproteins, Inclusion body myositis, Interstitial cystitis, IPEX (Immunodysregulation,

Polyendocrinopathy, and Enteropathy, X-linked) syndrome, Juvenile arthritis, Juvenile diabetes (Type 1 diabetes), Juvenile myositis, Kawasaki syndrome, Lambert-Eaton syndrome, Leukocytoclastic vasculitis, Lichen planus, Lichen sclerosus, Ligneous conjunctivitis, Linear IgA disease (LAD), systemic lupus erythematosus (SLE), chronic Lyme disease, Meniere's disease, Microscopic polyangiitis, Mixed connective tissue disease (MCTD), Mooren' s ulcer, Mucha-Habermann disease, Multiple sclerosis, Myasthenia gravis, Myositis, Narcolepsy, Neuromyelitis optica (Devic's), Neutropenia , Ocular cicatricial pemphigoid, Optic neuritis, Palindromic rheumatism, PANDAS (Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus), Paraneoplastic cerebellar degeneration, Paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome, Parsonnage-Turner syndrome, Pars planitis (peripheral uveitis), Pemphigus, Peripheral neuropathy, Perivenous encephalomyelitis, Pernicious anemia, POEMS syndrome,

Polyarteritis nodosa, Type I, II, & III autoimmune polyglandular syndromes, Polymyalgia rheumatica, Polymyositis, Postmyocardial infarction syndrome, Postpericardiotomy syndrome, Progesterone dermatitis, Primary biliary cirrhosis, Primary sclerosing cholangitis, Psoriasis, Psoriatic arthritis, Idiopathic pulmonary fibrosis, Pyoderma gangrenosum, Pure red cell aplasia, Raynauds phenomenon, Reactive Arthritis, Reflex sympathetic dystrophy, Reiter's syndrome, Relapsing polychondritis, Restless legs syndrome, Retroperitoneal fibrosis, Rheumatic fever, Rheumatoid arthritis, Sarcoidosis, Schmidt syndrome, Scleritis, Scleroderma, Sjogren's syndrome, Sperm & testicular autoimmunity, Stiff person syndrome, Subacute bacterial endocarditis (SBE), Susac's syndrome, Sympathetic ophthalmia,

Takayasu' s arteritis, Temporal arteritis/Giant cell arteritis, Thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome, Transverse myelitis, Type 1 diabetes, Undifferentiated connective tissue disease (UCTD), Uveitis, Vasculitis, Vesiculobullous dermatosis, Vitiligo, and

Wegener's granulomatosis (also called Granulomatosis with Polyangiitis (GPA)). In some embodiments, the autoimmune disease or disorder is inflammatory bowel disease (e.g., Crohn's disease or Ulcerative colitis), IPEX syndrome, Multiple sclerosis, Psoriasis,

Rheumatoid arthritis, SLE or Type 1 diabetes.

Examples of inflammatory diseases or disorders that may be treated according to the methods disclosed herein include, but are not limited to, Acne Vulgaris, Appendicitis, Arthritis, Asthma, Atherosclerosis, Allergies (Type 1 Hypersensitivity), Bursitis, Colitis, Chronic Prostatitis, Cystitis, Dermatitis, Glomerulonephritis, Inflammatory Bowel Disease, Inflammatory Myopathy (e.g., Polymyositis, Dermatomyositis, or Inclusion-body Myositis), Inflammatory Lung Disease, Interstitial Cystitis, Meningitis, Pelvic Inflammatory Disease, Phlebitis, Psoriasis, Reperfusion Injury, Rheumatoid Arthritis, Sarcoidosis, Tendonitis, Tonsilitis, Transplant Rejection, and Vasculitis. In some embodiments, the inflammatory disease or disorder is asthma.

Formulation, Delivery, And Dosing

The oligonucleotides described herein can be formulated for administration to a subject for treating a condition (e.g., a disease or disorder associated with aberrant immune cell activation such as an autoimmune or inflammatory disease or disorder) associated with decreased levels of FOXP3. It should be understood that the formulations, compositions and methods can be practiced with any of the oligonucleotides disclosed herein.

The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient (e.g., an oligonucleotide or compound as described herein) which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration, e.g., intradermal or inhalation. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect, e.g. tumor regression.

Pharmaceutical formulations as described herein can be prepared according to any method known to the art for the manufacture of pharmaceuticals. Such formulations can contain sweetening agents, flavoring agents, coloring agents and preserving agents. A formulation can be admixtured with nontoxic pharmaceutically acceptable excipients which are suitable for manufacture. Formulations may comprise one or more diluents, emulsifiers, preservatives, buffers, excipients, etc. and may be provided in such forms as liquids, powders, emulsions, lyophilized powders, sprays, creams, lotions, controlled release formulations, tablets, pills, gels, on patches, in implants, etc.

A formulated single stranded oligonucleotide composition can assume a variety of states. In some examples, the composition is at least partially crystalline, uniformly crystalline, and/or anhydrous (e.g., less than 80, 50, 30, 20, or 10% water). In another example, the single stranded oligonucleotide is in an aqueous phase, e.g., in a solution that includes water. The aqueous phase or the crystalline compositions can, e.g., be incorporated into a delivery vehicle, e.g., a liposome (particularly for the aqueous phase) or a particle (e.g., a microparticle as can be appropriate for a crystalline composition). Generally, the single stranded oligonucleotide composition is formulated in a manner that is compatible with the intended method of administration.

In some embodiments, the composition is prepared by at least one of the following methods: spray drying, lyophilization, vacuum drying, evaporation, fluid bed drying, or a combination of these techniques; or sonication with a lipid, freeze-drying, condensation and other self-assembly.

A single stranded oligonucleotide preparation can be formulated or administered (together or separately) in combination with another agent, e.g., another therapeutic agent or an agent that stabilizes a single stranded oligonucleotide, e.g., a protein that complexes with single stranded oligonucleotide. Still other agents include chelators, e.g., EDTA (e.g., to remove divalent cations such as Mg2+), salts, RNAse inhibitors (e.g., a broad specificity RNAse inhibitor such as RNAsin) and so forth.

In one embodiment, the single stranded oligonucleotide preparation includes another single stranded oligonucleotide, e.g., a second single stranded oligonucleotide that modulates expression of a second gene or a second single stranded oligonucleotide that modulates expression of the first gene. Still other preparation can include at least 3, 5, ten, twenty, fifty, or a hundred or more different single stranded oligonucleotide species. Such single stranded oligonucleotides can mediate gene expression with respect to a similar number of different genes. In one embodiment, the single stranded oligonucleotide preparation includes at least a second therapeutic agent (e.g., an agent other than an oligonucleotide).

Route of Delivery

A composition that includes a single stranded oligonucleotide can be delivered to a subject by a variety of routes. Exemplary routes include: intravenous, intradermal, topical, rectal, parenteral, anal, intravaginal, intranasal, pulmonary, ocular, subcutaneous, intramuscular, intraperitoneal, and intra- articular (e.g., injection into a joint for, e.g., rheumatoid arthritis) administration. The term "therapeutically effective amount" is the amount of oligonucleotide present in the composition that is needed to provide the desired level of FOXP3 expression in the subject to be treated to give the anticipated physiological response. The term "physiologically effective amount" is that amount delivered to a subject to give the desired palliative or curative effect. The term "pharmaceutically acceptable carrier" means that the carrier can be administered to a subject with no significant adverse

toxicological effects to the subject.

The single stranded oligonucleotide molecules as described herein can be

incorporated into pharmaceutical compositions suitable for administration. Such

compositions typically include one or more species of single stranded oligonucleotide and a pharmaceutically acceptable carrier. As used herein the language "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

The pharmaceutical compositions as described herein may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic, vaginal, rectal, intranasal, transdermal), oral or parenteral. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, or intrathecal or

intraventricular administration.

The route and site of administration may be chosen to enhance targeting. For example, to target muscle cells, intramuscular injection into the muscles of interest would be a logical choice. Lung cells might be targeted by administering the single stranded oligonucleotide in aerosol form. The vascular endothelial cells could be targeted by coating a balloon catheter with the single stranded oligonucleotide and mechanically introducing the oligonucleotide.

In some embodiments, a T cell or population of T cells may be obtained from a subject, e.g., a human subject, and contacted with a single-stranded oligonucleotide as described herein. In some embodiments, the T cell or population of T cells contacted with a single-stranded oligonucleotide as described herein are readminstered to the subject. In some embodiments, the T cell or population of T cells contacted with a single- stranded

oligonucleotide as described herein are cultured for a time period (e.g., 1 hour, 2 hours, 3 hours, 4 hours, or more; 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days or more) before being readministered to the subject.

Topical administration refers to the delivery to a subject by contacting the formulation directly to a surface of the subject. The most common form of topical delivery is to the skin, but a composition disclosed herein can also be directly applied to other surfaces of the body, e.g., to the eye, a mucous membrane, to surfaces of a body cavity or to an internal surface. As mentioned above, the most common topical delivery is to the skin. The term encompasses several routes of administration including, but not limited to, topical and transdermal. These modes of administration typically include penetration of the skin's permeability barrier and efficient delivery to the target tissue or stratum. Topical administration can be used as a means to penetrate the epidermis and dermis and ultimately achieve systemic delivery of the composition. Topical administration can also be used as a means to selectively deliver oligonucleotides to the epidermis or dermis of a subject, or to specific strata thereof, or to an underlying tissue.

Formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful.

Transdermal delivery is a valuable route for the administration of lipid soluble therapeutics. The dermis is more permeable than the epidermis and therefore absorption is much more rapid through abraded, burned or denuded skin. Inflammation and other physiologic conditions that increase blood flow to the skin also enhance transdermal adsorption. Absorption via this route may be enhanced by the use of an oily vehicle

(inunction) or through the use of one or more penetration enhancers. Other effective ways to deliver a composition disclosed herein via the transdermal route include hydration of the skin and the use of controlled release topical patches. The transdermal route provides a potentially effective means to deliver a composition disclosed herein for systemic and/or local therapy. In addition, iontophoresis (transfer of ionic solutes through biological membranes under the influence of an electric field), phonophoresis or sonophoresis (use of ultrasound to enhance the absorption of various therapeutic agents across biological membranes, notably the skin and the cornea), and optimization of vehicle characteristics relative to dose position and retention at the site of administration may be useful methods for enhancing the transport of topically applied compositions across skin and mucosal sites.

Both the oral and nasal membranes offer advantages over other routes of

administration. For example, oligonucleotides administered through these membranes may have a rapid onset of action, provide therapeutic plasma levels, avoid first pass effect of hepatic metabolism, and avoid exposure of the oligonucleotides to the hostile gastrointestinal (GI) environment. Additional advantages include easy access to the membrane sites so that the oligonucleotide can be applied, localized and removed easily.

In oral delivery, compositions can be targeted to a surface of the oral cavity, e.g., to sublingual mucosa which includes the membrane of ventral surface of the tongue and the floor of the mouth or the buccal mucosa which constitutes the lining of the cheek. The sublingual mucosa is relatively permeable thus giving rapid absorption and acceptable bioavailability of many agents. Further, the sublingual mucosa is convenient, acceptable and easily accessible.

A pharmaceutical composition of single stranded oligonucleotide may also be administered to the buccal cavity of a human being by spraying into the cavity, without inhalation, from a metered dose spray dispenser, a mixed micellar pharmaceutical formulation as described above and a propellant. In one embodiment, the dispenser is first shaken prior to spraying the pharmaceutical formulation and propellant into the buccal cavity.

Compositions for oral administration include powders or granules, suspensions or solutions in water, syrups, slurries, emulsions, elixirs or non-aqueous media, tablets, capsules, lozenges, or troches. In the case of tablets, carriers that can be used include lactose, sodium citrate and salts of phosphoric acid. Various disintegrants such as starch, and lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc, are commonly used in tablets. For oral administration in capsule form, useful diluents are lactose and high molecular weight polyethylene glycols. When aqueous suspensions are required for oral use, the nucleic acid compositions can be combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavoring agents can be added.

Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, intrathecal or intraventricular administration. In some embodiments, parental administration involves administration directly to the site of disease (e.g. injection into a tumor).

Formulations for parenteral administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives. Intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir. For intravenous use, the total concentration of solutes should be controlled to render the preparation isotonic.

Any of the single stranded oligonucleotides described herein can be administered to ocular tissue. For example, the compositions can be applied to the surface of the eye or nearby tissue, e.g., the inside of the eyelid. For ocular administration, ointments or droppable liquids may be delivered by ocular delivery systems known to the art such as applicators or eye droppers. Such compositions can include mucomimetics such as hyaluronic acid, chondroitin sulfate, hydroxypropyl methylcellulose or poly(vinyl alcohol), preservatives such as sorbic acid, EDTA or benzylchronium chloride, and the usual quantities of diluents and/or carriers. The single stranded oligonucleotide can also be administered to the interior of the eye, and can be introduced by a needle or other delivery device which can introduce it to a selected area or structure.

Pulmonary delivery compositions can be delivered by inhalation by the patient of a dispersion so that the composition, preferably single stranded oligonucleotides, within the dispersion can reach the lung where it can be readily absorbed through the alveolar region directly into blood circulation. Pulmonary delivery can be effective both for systemic delivery and for localized delivery to treat diseases of the lungs.

Pulmonary delivery can be achieved by different approaches, including the use of nebulized, aerosolized, micellular and dry powder-based formulations. Delivery can be achieved with liquid nebulizers, aerosol-based inhalers, and dry powder dispersion devices. Metered-dose devices are preferred. One of the benefits of using an atomizer or inhaler is that the potential for contamination is minimized because the devices are self-contained. Dry powder dispersion devices, for example, deliver agents that may be readily formulated as dry powders. A single stranded oligonucleotide composition may be stably stored as lyophilized or spray-dried powders by itself or in combination with suitable powder carriers. The delivery of a composition for inhalation can be mediated by a dosing timing element which can include a timer, a dose counter, time measuring device, or a time indicator which when incorporated into the device enables dose tracking, compliance monitoring, and/or dose triggering to a patient during administration of the aerosol medicament.

The term "powder" means a composition that consists of finely dispersed solid particles that are free flowing and capable of being readily dispersed in an inhalation device and subsequently inhaled by a subject so that the particles reach the lungs to permit penetration into the alveoli. Thus, the powder is said to be "respirable." Preferably the average particle size is less than about 10 μιη in diameter preferably with a relatively uniform spheroidal shape distribution. More preferably the diameter is less than about 7.5 μ m and most preferably less than about 5.0 μ m. Usually the particle size distribution is between about 0.1 μ m and about 5 μ m in diameter, particularly about 0.3 μ m to about 5 μ m.

The term "dry" means that the composition has a moisture content below about 10% by weight (% w) water, usually below about 5% w and preferably less it than about 3% w. A dry composition can be such that the particles are readily dispersible in an inhalation device to form an aerosol.

The types of pharmaceutical excipients that are useful as carrier include stabilizers such as human serum albumin (HSA), bulking agents such as carbohydrates, amino acids and polypeptides; pH adjusters or buffers; salts such as sodium chloride; and the like. These carriers may be in a crystalline or amorphous form or may be a mixture of the two. Suitable H adjusters or buffers include organic salts prepared from organic acids and bases, such as sodium citrate, sodium ascorbate, and the like; sodium citrate is preferred. Pulmonary administration of a micellar single stranded oligonucleotide formulation may be achieved through metered dose spray devices with propellants such as tetrafluoroethane, heptafluoroethane, dimethylfluoropropane, tetrafluoropropane, butane, isobutane, dimethyl ether and other non-CFC and CFC propellants.

Exemplary devices include devices which are introduced into the vasculature, e.g., devices inserted into the lumen of a vascular tissue, or which devices themselves form a part of the vasculature, including stents, catheters, heart valves, and other vascular devices. These devices, e.g., catheters or stents, can be placed in the vasculature of the lung, heart, or leg.

Other devices include non-vascular devices, e.g., devices implanted in the

peritoneum, or in organ or glandular tissue, e.g., artificial organs. The device can release a therapeutic substance in addition to a single stranded oligonucleotide, e.g., a device can release insulin.

In one embodiment, unit doses or measured doses of a composition that includes single stranded oligonucleotide are dispensed by an implanted device. The device can include a sensor that monitors a parameter within a subject. For example, the device can include pump, e.g., and, optionally, associated electronics.

Tissue, e.g., cells or organs can be treated with a single stranded oligonucleotide, ex vivo and then administered or implanted in a subject. The tissue can be autologous, allogeneic, or xenogeneic tissue. E.g., tissue can be treated to reduce graft v. host disease . In other embodiments, the tissue is allogeneic and the tissue is treated to treat a disorder characterized by unwanted gene expression in that tissue. E.g., tissue, e.g., hematopoietic cells, e.g., bone marrow hematopoietic cells, can be treated to inhibit unwanted cell proliferation. Introduction of treated tissue, whether autologous or transplant, can be combined with other therapies. In some implementations, the single stranded oligonucleotide treated cells are insulated from other cells, e.g., by a semi-permeable porous barrier that prevents the cells from leaving the implant, but enables molecules from the body to reach the cells and molecules produced by the cells to enter the body. In one embodiment, the porous barrier is formed from alginate. In one embodiment, a contraceptive device is coated with or contains a single stranded oligonucleotide. Exemplary devices include condoms, diaphragms, IUD

(implantable uterine devices, sponges, vaginal sheaths, and birth control devices. Dosage

In one aspect, a method of administering a single stranded oligonucleotide (e.g., as a compound or as a component of a composition) to a subject (e.g., a human subject) is provided herein. In one embodiment, the unit dose is between about 10 mg and 25 mg per kg of bodyweight. In one embodiment, the unit dose is between about 1 mg and 100 mg per kg of bodyweight. In one embodiment, the unit dose is between about 0.1 mg and 500 mg per kg of bodyweight. In some embodiments, the unit dose is more than 0.001, 0.005, 0.01, 0.05,

0.1, 0.5, 1, 2, 5, 10, 25, 50 or 100 mg per kg of bodyweight.

The defined amount can be an amount effective to treat or prevent a disease or disorder, e.g., a disease or disorder associated with FOXP3. The unit dose, for example, can be administered by injection (e.g., intravenous or intramuscular), an inhaled dose, or a topical application.

In some embodiments, the unit dose is administered daily. In some embodiments, less frequently than once a day, e.g., less than every 2, 4, 8 or 30 days. In another embodiment, the unit dose is not administered with a frequency (e.g., not a regular frequency). For example, the unit dose may be administered a single time. In some embodiments, the unit dose is administered more than once a day, e.g., once an hour, two hours, four hours, eight hours, twelve hours, etc.

In one embodiment, a subject is administered an initial dose and one or more maintenance doses of a single stranded oligonucleotide. The maintenance dose or doses are generally lower than the initial dose, e.g., one-half less of the initial dose. A maintenance regimen can include treating the subject with a dose or doses ranging from 0.0001 to 100 mg/kg of body weight per day, e.g., 100, 10, 1, 0.1, 0.01, 0.001, or 0.0001 mg per kg of bodyweight per day. The maintenance doses may be administered no more than once every

1, 5, 10, or 30 days. Further, the treatment regimen may last for a period of time which will vary depending upon the nature of the particular disease, its severity and the overall condition of the patient. In some embodiments, the dosage may be delivered no more than once per day, e.g., no more than once per 24, 36, 48, or more hours, e.g., no more than once for every 5 or 8 days. Following treatment, the patient can be monitored for changes in his condition and for alleviation of the symptoms of the disease state. The dosage of the oligonucleotide may either be increased in the event the patient does not respond significantly to current dosage levels, or the dose may be decreased if an alleviation of the symptoms of the disease state is observed, if the disease state has been ablated, or if undesired side-effects are observed.

The effective dose can be administered in a single dose or in two or more doses, as desired or considered appropriate under the specific circumstances. If desired to facilitate repeated or frequent infusions, implantation of a delivery device, e.g., a pump, semi- permanent stent (e.g., intravenous, intraperitoneal, intracisternal or intracapsular), or reservoir may be advisable.

In some embodiments, the oligonucleotide pharmaceutical composition includes a plurality of single stranded oligonucleotide species. In another embodiment, the single stranded oligonucleotide species has sequences that are non-overlapping and non-adjacent to another species with respect to a naturally occurring target sequence (e.g., a PRC2-associated region). In another embodiment, the plurality of single stranded oligonucleotide species is specific for different PRC2-associated regions. In another embodiment, the single stranded oligonucleotide is allele specific.

In some cases, a patient is treated with a single stranded oligonucleotide in

conjunction with other therapeutic modalities.

Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the compound, molecule, or agent is administered in maintenance doses, ranging from 0.0001 mg to 100 mg per kg of body weight.

The concentration of the single stranded oligonucleotide composition is an amount sufficient to be effective in treating or preventing a disorder or to regulate a physiological condition in humans. The concentration or amount of single stranded oligonucleotide administered will depend on the parameters determined for the agent and the method of administration, e.g. nasal, buccal, pulmonary. For example, nasal formulations may tend to require much lower concentrations of some ingredients in order to avoid irritation or burning of the nasal passages. It is sometimes desirable to dilute an oral formulation up to 10-100 times in order to provide a suitable nasal formulation. Certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a single stranded oligonucleotide can include a single treatment or, preferably, can include a series of treatments. It will also be appreciated that the effective dosage of a single stranded oligonucleotide used for treatment may increase or decrease over the course of a particular treatment. For example, the subject can be monitored after administering a single stranded oligonucleotide composition. Based on information from the monitoring, an additional amount of the single stranded

oligonucleotide composition can be administered.

Dosing is dependent on severity and responsiveness of the disease condition to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of disease state is achieved. Optimal dosing schedules can be calculated from measurements of FOXP3 expression levels in the body of the patient.

Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual compounds, and can generally be estimated based on EC50s found to be effective in in vitro and in vivo animal models. In some embodiments, the animal models include transgenic animals that express a human FOXP3. In another embodiment, the composition for testing includes a single stranded oligonucleotide that is complementary, at least in an internal region, to a sequence that is conserved between FOXP3 in the animal model and the FOXP3 in a human.

In one embodiment, the administration of the single stranded oligonucleotide composition is parenteral, e.g. intravenous (e.g., as a bolus or as a diffusible infusion), intradermal, intraperitoneal, intramuscular, intrathecal, intraventricular, intracranial, subcutaneous, transmucosal, buccal, sublingual, endoscopic, rectal, oral, vaginal, topical, pulmonary, intranasal, urethral or ocular. Administration can be provided by the subject or by another person, e.g., a health care provider. The composition can be provided in measured doses or in a dispenser which delivers a metered dose. Selected modes of delivery are discussed in more detail below.

Kits In certain aspects of the disclosure, kits are provided herein, comprising a container housing a composition comprising a single stranded oligonucleotide. In some embodiments, the composition is a pharmaceutical composition comprising a single stranded oligonucleotide and a pharmaceutically acceptable carrier. In some embodiments, the individual components of the pharmaceutical composition may be provided in one container. Alternatively, it may be desirable to provide the components of the pharmaceutical composition separately in two or more containers, e.g., one container for single stranded oligonucleotides, and at least another for a carrier compound. The kit may be packaged in a number of different configurations such as one or more containers in a single box. The different components can be combined, e.g., according to instructions provided with the kit. The components can be combined according to a method described herein, e.g., to prepare and administer a pharmaceutical composition. The kit can also include a delivery device.

The present disclosure is further illustrated by the following Examples, which in no way should be construed as further limiting. The entire contents of all of the references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference.

EXAMPLES

Various aspects of the invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

Example 1. Downregulation of CCDC22 expression upregulates FOXP3 expression

CD4+ T cells were incubated with CCDC22 gapmers at a concentration of 10 μΜ for seven days. For example, a CCDC22 gapmer 1, was designed to target the first exon of

CCDC22. A CCDC22 gapmer 2 was designed to target the second exon of CCDC22. RNA was isolated from the cells after incubation. cDNA was created from the RNA samples and FOXP3 and CCDC22 expressions were measured using TaqMan or Droplet digital method.

As shown in FIG. 1, knocking down CCDC22 in T cells with CCDC22 gapmers resulted in the upregulation of FOXP3 compared to untreated control. Example 2. Design ofFOXP3 oligonucleotides (e.g., mixmers) to target FOXP3/CCDC22 region

FOXP3 mixmers Oligo-1 through Oligo-10 were designed against a region that includes part of the 3' UTR of CCDC22 and a further sequence that is antisense to the 3 'UTR of FOXP3 (FIG. 2). Their sequences are given in Table 1 below.

Table 1. Sequences of FOXP3 Mixmers

b = LNA; m = 2'Ome; s = phosphorothioate FIG. 8 shows a genome browser view (e.g., using Integrative Genomics Viewer

(IGV)) of the RIPseq against the EZH2 subunit of PRC2, and illustrates RIP reads that overlapped with the 3' UTR of CCDC22 and a region that was located a bit downstream of CCDC22 (antisense to FOXP3). The data were obtained from two donor samples RIP compared to the background input from those donors. The region that oligos were designed against is highlighted and the sequence corresponding to the highlighted region is set forth in SEQ ID NO: 3. SEQ ID NO: 3 shows the plus strand sequence that includes part of the 3' UTR of CCDC22 and a region that is located a bit downstream of CCDC22 and is antisense to the 3' UTR of FOXP3. The specific regions against which individual oligos were designed to target are shown at the bottom of FIG. 8. Example 3. Modulation ofFOXP3 expression with FOXP3 mixmer oligonucleotides

To isolate CD4+ T cells from fresh whole blood, peripheral blood mononuclear cells (PBMCs) were isolated from whole blood (e.g., collected from adult donors) using FICOLL® Paque and CD4+ T cells were then isolated from PBMCs using a negative selection kit from Miltenyi Biotech (Cat. No. 130-096-533). The isolated CD4+ T cells were cultured in RPMI with 10% heat inactivated fetal bovine serum (HI FBS) and IX Antibiotic-antimycotic (Anti- anti). CD4+ T cells were then activated using anti-CD3/anti-CD28 dynabeads (Thermo Cat. No. 11132D). Individual FOXP3 oligonucleotides (Table 1) were added approximately five hours after activation at a concentration of 1 μΜ to 20 μΜ. (e.g., at a concentration of 3μΜ, 10μΜ, or 20μΜ). Then, cells were incubated with the oligonucleotides for a range of time points between 4 and 11 days. RNA was isolated from the cells after incubation. cDNA was created from the RNA samples and FOXP3 expression was measured using TaqMan or Droplet digital method. An EZH2 gapmer was used as a positive control.

The results of initial screens of the FOXP3 mixmers in modulating FOXP3 expression are shown in FIG. 3. A threshold level for fold change in FOXP3 expression was selected to identify FOXP3 mixmer candidates, e.g., ones that are more effective in increasing FOXP3 expression in T cells, for further analysis. Generally, a threshold level of at least two fold change in FOXP3 expression or higher (e.g., at least three fold change in FOXP3 expression) was used. As shown in FIG. 3, based on the selected threshold level, five oligos were selected for further analysis: Oligo-2, Oligo-3, Oligo-5, Oligo-7, and Oligo-8.

Summaries of the additional analyses of the selected oligos are provided in

FIGs 4-7B. In FIGs. 4-7B, activated CD4+ T cells from various donors were incubated with individual FOXP3 oligonucleotides at a concentration of 10 μιη or 20 μΜ for 4 days or longer (e.g., 7 days or 11 days). For samples with longer incubation time, cell culture media were replenished at day 4 (for 7-day samples) and, if necessary, also at day 7 (for 11-day samples) and fresh FOXP3 oligonucleotides were added during each medium replenishment in order to maintain the concentration of the oligonucleotides constant during incubation. FIGs. 7A-7B show that FOXP3mixmer TaqMan data was confirmed using droplet digital PCR.

Example 4. Modulation ofFOXP3 protein levels Donor T cells were isolated and activated as described in Example 3. The isolated cells were then incubated with FOXP3 oligonucleotide Oligo-5 described in Example 2 or control oligonucleotides, each at a concentration of 10 μΜ or 20 μΜ for 4 days or 7 days. FOXP3 protein levels were measured by flow cytometry, gating on CD4+, CD25+ populations and measuring FOXP3 expression within that population at 4 and 7 days in culture.

FIGs. 9A and 9B show an increased number of FOXP3+ activated T cells after 4 and 7 days in culture when treated with Oligo-5 or with an EZH2 gapmer (positive control, GATTTTACACGCTTCCG, SEQ ID NO: 14) compared to both a negative control mixmer oligonucleotide (CGCTCCGCCCTCCAG, SEQ ID NO: 15) and untreated cells. The negative control oligonucleotide treatment also showed modest increases in FOXP3 expression but was thought to be a by-product of activation of the T cells with CD3/CD28 beads. These data confirm that FOXP3 protein levels can also be increased using single stranded oligonucleotides such as FOXP3 mixmers.

While several embodiments of the present invention have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the functions and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the teachings of the present invention is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, and/or methods, if such features, systems, articles, materials, and/or methods are not mutually inconsistent, is included within the scope of the present invention.

The indefinite articles "a" and "an," as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean "at least one." The phrase "and/or," as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined, e.g. , elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements may optionally be present other than the elements specifically identified by the "and/or" clause, whether related or unrelated to those elements specifically identified unless clearly indicated to the contrary. Thus, as a non-limiting example, a reference to "A and/or B," when used in conjunction with open-ended language such as "comprising" can refer, in one embodiment, to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and/or" as defined above. For example, when separating items in a list, "or" or "and/or" shall be interpreted as being inclusive, e.g. , the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (e.g. "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently "at least one of A and/or B") can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another

embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

In the claims, as well as in the specification above, all transitional phrases such as "comprising," "including," "carrying," "having," "has," "containing," "involving,"

"holding," and the like are to be understood to be open-ended, e.g. , to mean including but not limited to. Only the transitional phrases "consisting of and "consisting essentially of shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

Use of ordinal terms such as "first," "second," "third," etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements.

ADDITIONAL SEQUENCES OF THE DISCLOSURE:

>SEQ ID NO: 1 - >CCDC22-001 cdna:KNOWN_protein_coding. This sequence corresponds to the cDNA sequence of CCDC22 with exons only. CTCACATCCGGCATGCGCCGTGCTCGCTCACAGAACTACACTTTCCAACTCTCCCCACAC GACCCGTGACACTCTGTGGACCGCGAGCACGGAGCAGGGTTTCTACAGCTGCTCCCCACT TTCTCGGACCCGGTCCTGGACCCAGCCCCCGACTCCGACACGGCTCCACCATGGAGGAGG CGGACCGAATCCTCATCCATTCGCTGCGCCAGGCCGGCACGGCAGTTCCTCCAGATGTGC AGACCTTGCGCGCCTTCACCACTGAGCTGGTTGTAGAGGCTGTGGTCCGCTGCCTGCGTG TGATCAACCCTGCGGTGGGCTCTGGCCTCAGCCCTCTGCTGCCTCTTGCCATGTCTGCCC GGTTCCGCCTGGCCATGAGCCTGGCTCAGGCCTGCATGGACCTGGGCTATCCCTTGGAGC TTGGCTATCAGAACTTCCTCTACCCCAGTGAGCCTGACCTCCGAGACCTGCTTCTCTTCT TGGCTGAGCGTCTGCCCACCGATGCCTCTGAGGATGCAGACCAGCCTGCAGGTGACTCAG CTATTCTCCTCCGGGCCATTGGGAGCCAAATTCGGGACCAGCTGGCACTGCCTTGGGTCC CGCCCCACCTTCGCACTCCCAAGCTGCAGCACCTCCAGGGCTCGGCCCTCCAGAAGCCTT TCCATGCCAGCAGGCTGGTCGTGCCAGAATTGAGTTCCAGAGGTGAGCCACGGGAGTTCC AGGCGAGTCCCCTGCTGCTTCCAGTCCCTACCCAGGTGCCTCAGCCTGTTGGAAGGGTGG CCTCGCTCCTCGAACACCATGCCCTGCAGCTCTGCCAGCAGACGGGCCGGGACCGGCCAG GGGATGAGGACTGGGTCCACCGGACATCCCGCCTCCCACCCCAGGAGGACACACGGGCTC AGCGGCAGCGGCTGCAAAAGCAACTGACTGAGCATCTGCGCCAAAGCTGGGGCCTGCTTG GGGCCCCCATACAAGCCCGGGACCTGGGAGAACTGCTGCAGGCCTGGGGTGCTGGGGCCA AGACTGGTGCTCCTAAGGGCTCCCGCTTCACGCACTCAGAGAAGTTCACCTTCCATCTGG AGCCCCAGGCCCAGGCCACTCAGGTGTCAGATGTGCCAGCCACCTCCCGGCGGCCTGAAC AGGTCACGTGGGCAGCTCAGGAACAGGAGCTCGAGTCCCTTCGGGAGCAGCTGGAAGGAG TGAACCGCAGCATTGAGGAGGTTGAGGCCGACATGAAGACCCTGGGCGTCAGCTTTGTGC AGGCAGAGTCTGAGTGCCGGCACAGCAAGCTCAGTACAGCAGAGCGTGAGCAGGCCCTGC GCCTGAAGAGCCGCGCGGTGGAGCTGCTGCCCGATGGGACTGCCAACCTTGCCAAGCTGC AGCTTGTGGTGGAGAATAGTGCCCAGCGGGTCATCCACTTGGCGGGTCAGTGGGAGAAGC ACCGGGTCCCACTCCTCGCTGAGTACCGCCACCTCCGAAAGCTGCAGGATTGCAGAGAGC TGGAATCTTCTCGACGGCTGGCAGAGATCCAAGAACTGCACCAGAGTGTCCGGGCGGCTG CTGAAGAGGCCCGCAGGAAGGAGGAGGTCTATAAGCAGCTGATGTCAGAGCTGGAGACTC TGCCCAGAGATGTGTCCCGGCTGGCCTACACCCAGCGCATCCTGGAGATCGTGGGCAACA TCCGGAAGCAGAAGGAAGAGATCACCAAGATCTTGTCTGATACGAAGGAGCTTCAGAAGG AAATCAACTCCCTATCTGGGAAGCTGGACCGGACGTTTGCGGTGACTGATGAGCTTGTGT TCAAGGATGCCAAGAAGGACGATGCTGTTCGGAAGGCCTATAAGTATCTAGCTGCTCTGC ACGAGAACTGCAGCCAGCTCATCCAGACCATCGAGGACACAGGCACCATCATGCGGGAGG TTCGAGACCTCGAGGAGCAGATCGAGACAGAGCTGGGCAAGAAGACCCTCAGCAACCTGG AGAAGATCCGGGAGGACTACCGAGCCCTCCGCCAGGAGAACGCTGGCCTCCTAGGCCGGG TCCGGGAGGCCTGAGGAGCCGCCGGCAGAGGTCTCTCCCCAGCCTCAGGCAGGGATTTGG GGTGCTGGAGGCAGTGGCCAAGCACATGCCCTAGCTACTTCCTCCGCTGTCCAGTTCCTC CTGCTGCGGCCTTGGACCCAGACCCCTGCCCACTGACCGCAACCCTTATATGGGGTGATA GTCCAGCATGTGGGGAGCTCGGCTGCAGTTTATTGGGGACGGTACTGTGGGTTGGGGGCC TTGGATCCCAAATAAATGAGTAGTTCCTCTGCAGTCTAA

>SEQ ID NO: 2 - dna: chromosome chromosome: GRCh38:X: 49235467: 49250526:1. This sequence corresponds to the full DNA sequence of CCDC22 (with exons and introns) CTCACATCCGGCATGCGCCGTGCTCGCTCACAGAACTACACTTTCCAACTCTCCCCACACGACCCGTGACACTCT GTGGACCGCGAGCACGGAGCAGGGTTTCTACAGCTGCTCCCCACTTTCTCGGACCCGGTCCTGGACCCAGCCCCC GACTCCGACACGGCTCCACCATGGAGGAGGCGGACCGAATCCTCATCCATTCGCTGCGCCAGGCCGGCACGTAAG GACAGAGCCCCCGCCCACCCCCGAAGCCCACATCCGGGACTCTAAAGCCCAGGACCCCGTTTCCCGGGAACCTTA AAACCCGGGATCCTGACATTCAGGGCTCCAACTCCAGGATCCTAAGACCCTCACCCCCTTACACACACACACACA CACACACACACACACACACACACGCACACACACACCGCCCTCCCTGACACCGACATCAGAGGCCTCCCAAATCCT TTAACCATGATATTTGGTACACCCAAAGCTCTGGGACCCAGACACCTTGAGACGTTACAACCTTGAAACCTCAAG ACCCGGAACCCTGTAATCTGGGGAATCTTAACATCAGTTCCCTGAGACCCTGTTATCTGGAGACCCTAAGACACC TGTGCCCTAAGAACCAGGGAGCGTAAAACCCCAGGACCCTGGCACTCGGGGACTCCAAAAAATCCCTGGACCAGA TACTTGGGATCCTTCAAACTCCAGTTCCCCAAACACCTGGGGCTTAAAAAAACCCAGGATTCTTTTTTTTTTTTT TCCTTTTCCGAGATGGAGTCTCGCTCTGTCGCCTAGGCTGGAGTGCAGTGGGGTGATCTCAGCTCACTGCAGCCT CCGCCTCCCAGGTTCAAGTGATTCTCCTGCCTCAGCCTCCCAAGTAGCTGGTATTACAGCCGTGCGCCACCTTGC CCGGCTAATTTTTGTATTTTTAGCAGAGACAGGGTTTCACCATGTTGGTCAGGCTGGTCTCCAACTCCTGACCTC AAGTGATCTGCCCGCCTCCGCCTCCCAAAGTGCTGGGATTACAGGTGTGAGCCACCACGCCCAGCCAAAAAAACC CAGAATTCTAAAATCTGACATCAGCATTTGGTCACCCTGACGCACGAAGACTCCTCCGTTCAGAGGCCCTAAAAA CCCAGGATGCCAACATCTAGGGAGTCTGATTTTTGTATTGCTTGAAACTAGGTGGTCTTGATATTCAGGGATCTT GATACCCAGGGACCCTGACATTTGGAACTTCTGAAAACAGCAGCCTCCTAAAATCTAGTCTTCTCAAAACTCTAG CAGCTCAATATGTATGCAAAGAACATACTATCTGGGAACCAAGAAACCCAGAGATTGTGCCACCTGGACCCTTTC ACCTTCCCAGACTCCCAAATTTTGATATTTTTCTAAACTGAGTTCTCTTCCACCCACCATTCTCCTACTCCAAGA TCTTAGGACTTGAGGATTTCCCTAGTTCCTGGTTTTCCAAATGGAAGTCACAGTCACCAACATCCAGGATCTGTC TGAACTGTAGGCATCCTCCCTGCCCCGACCCTGGTACTAGTGTTTCCAAAAGGCTGCAGGGCTCAGCCTGATCCC TGTTGCCTGACTATTCCTGCGATCAAGCTGGTCCCCTTCTTCAGGGCAGTTCCTCCAGATGTGCAGACCTTGCGC GCCTTCACCACTGAGCTGGTTGTAGAGGCTGTGGTCCGCTGCCTGCGTGTGATCAACCCTGCGGTGGGCTCTGGC CTCAGCCCTCTGCTGCCTCTTGCCATGTCTGCCCGGTTCCGCCTGGCCATGAGCCTGGCTCAGGCCTGCATGGTG AGTGGCCCTCCTCCTAATGCACACATCCTCTTTCTTCCTCTTTTACAAAGTAACCAAGTTCCTTTGCAACACTTG CCTTTCCTCTGCAACACTTGCCTTTCCTCTGCAACACTTGCCTTTCCTCTGTCATTTTTCCTGCGGCTTAGTTTT CATTAGTGGTTAAGGCTGCTGACTGTACTGCCAAACTGCCTGAGTAGTTTAGATCCTAGCCCCACCACTTGGTAA CTGTGTGACCCTAACCCTTCTGGGCCCCAGTTTTCCTATTTGTGAAATGGAGATGATAAATGTAGTGCTTTTATG AGGAGCAAATGAGTTTATCCAGTTTGCACCTGGGCTGCTCCTGGCTTCTATGTTTTAAATTTAGCTGCAAAATAC CACCCCCACCCCATAGTATCCATTTCCTAGTTCCAGAAAATACTCTGTCTGGGTCATATACTCCTAAACTAATCA CTCTGTGCTCTGATTGGCCCAGTCTGGATGACATGGTCATTCCTTTTTTTTTTTTTTTTTTGAGATGGAGTCTCA CTTTGTCGCCCAAGCTGGAGTGCAGTGGTGCGATCTTGGCTCACTGCAACCTCTGCCTCCCGGGTTCAAGCAATT CTCCTGCCTCAGCCTCCCGAGTAGCTGGGACTACAGGCGTGTGCCACCACACCCAACTAATTTTCATGTTTTTAG TAGAGACGGGGTTTCACCATGTTGGCCAGGATGGTTTTAATCTCTTGACCTCATGATCCACCTGCCTTGGCCTCC CAAAGTGCTGGGATTACAGGAGTGAGCCACCGTGCCCAGCCTTTTCTTTTCTTTTTTTCTTTTTTTTTTTTTTTT GATGTGAAGTCTCTCTCTGTCACCCAAGCTGGAATGCAGTGGCGTGATCTCAGCTCACGGCTCACTGCAACCTCT GTCTCCTGGGTTCAAGTGATTCTCCTGCCTCAGCCTCCCGAGTAGCTGGGACTACAGGCATGCACCACCATGCTC AGTTAATTTTTGTATTTTTAGTACAGACGGGGTTTCACTATGTTGGCTAGGCTGGTCTTGAACTCCTGACCTCGT GATCTGCCCACCTCGGCCTCCCAAAGTGCTGAGATTACAGGCGTGAGCCACTGCGCCCGGCCGACACGGTCATTC CTATAGGACACTGTGATTAGCCACTCCTTCAGGATCCTGTGGAGTTGGGATAAGGATGATTACCCAAAGAAAGGC ATGCTGGTTACCCAAAAGAGTGTCTGCTATATTACCTCTGTGGGAACCACATATCCTGCCTCTGCTAAGAGCAAT TCCAACAATGTCTCTGTGACAGAATAAATAAAGTGCTTTTCTTTTAAAAAATATTTTAATTTTTTTAGAGGTAAG TGTCTTGCTATATTGCCCAGGCTGGTCTTGAACTCCTGGCCTCAAGAGATTCTCCTGCCTCTGCCTCACTAGTAG CTAGGACTATAGGCACATGCCACCCTGCCCGAGAATTTTTAAATTTTTCGTAGAGACGGGGTCTCGCTTTTGTAG AGATGTTGCCCAGGCTGGTCTTAAGCTCCTGGCCTCAAGCAATCTTCCTGCCTTGGCCTCCTGAGTAGTTGGGAC TACAGGCGTGCACCACTGTGCCTGGTAAAGAGCCATTCTGATGAAACACTCACCCATTCCCAGTATAGAGCTGAG TCCAGGAGTCCAGTTCCTGTATTCAAGAGCTGCAAGTAATGCCACTCCCCCTGGCTCACTCACTCTGTGACTTTG GGCCAGTCTTTTTTTTTTTTGAAGCCCAGGCTGGAGTGCAGTGGCACAATCTCGGCTTACTGCAACCTCCTCCGC CTCCTGGGTTCCAGCAATTCTCCTGCCTCAGCCTGCCGAGTGGCTGGGATTACAGGTGTCTGCCACTGTGCCCGG CTAATTTTTTTGTATTTTTAGTGGAGACAGGGTTTCACCATCTTGGCCAGGCTGGTCTCGAACTCCTGACCTTGT GATCCACCCACCTCAGCCTCCCAAAGTGTTGGGATTACAGGTGTGAGCCACCGCGTCTGGCTCTCTTAACCTTTT TGAGGCTAAGTTTCCACATGTGTAAAATGGGTATAAGAATTGTAGCTACTGTATAGGGTTGCTGTGAGGATTAAA CATGAGTTAATGTGTGAAAAGCTGGTTATAATAAGCTTTGCATAAATGGGATTACTATTATTGGATAGGTCCGAT CTGGAACCTGTGAATACATAGTGAATGGAAACACTTTGAACTGACCCAGGAAGTATATGGTGGTGGAGGGACGAT AGAGTAACTACCGTGAAAACTTTCATTTAGATATAGGGGACTGGGTGGCTAGAGTTGTTAAATTTGGGCCTTGCT TATGCAGTCTCTGTCTCTTAGCAACAGGTCTCAGAGCTCCATCCATCCCTTCGCTCTCAGGTTCACCCAGCTCTC AGGAGTTGTCACATTGTTCTCTCTGGGGCTCTTGGTGGCCTTATGAGGCAGGCAGTCTGTCCCCTGGCCCAGGAC TGTATGTATTCTTAAGGTTAGCACTTAATAGGGGGGAAGTTATGTCTTCTGTTTGCAGAGGAGAGTACACAGCAG GAGGTGTTGAGGTGGGGGCTCAGGCTTCCTGCAGTTCTCTGTTCTTCCCTCAGTGCTGTCTCTCTTGGATTTTGT TCACCTGCTTTTGCTTACATTGATTTTAGTGGGGGTTAGTGACTATGGCTTTTCCAGTGGCCAGGAGGTACATGT GGGCTGGGCACGTTGGCTCTTGCATGTAATCCCAGCACTTTGGGAGTCTGAGGTGGGAGGATCAGTTGAGCCCAG GAGCTCGAGATCAGCCTGGGCAACATAGTGAGACCCCCATCTCTACAAAAAATAAAAAAAAAATAGCTGGGCATG GTGGCACACGCCTGTGGTCCCAGCTATGTGGGAGGCTGAGGTAGGAGGATTGCTTGAGCCTGGGAGGTCCAGGCT GCAGTGGGCTGTGATGCGCCACTGCACTCTAGCTTGGGAAACAGAGTGAAACCCCATCTCCAAAAAAAAAAAAAA AAAAAAGACTGGGGACGGTGGCTCCTGTAATTCCAGCACTTTGGGAGGTGGAGGCGGGCAGATCTCACCAGAGGT CAGGAGTTTGAGACCAGCCTGGCCAACCTGGCGAAACCCTGTCTCTACTAAAAATACAAAAATTAGCCTGGTGTG GTGGTGTGTGCCTGTAATCCCAGCTACTCAGGAGGCTGAGGCAGGAGAATCGCTTGAACCCAGGAGGCGGAGGTT CCAGTGAGTCGAGATTGCACCACTGCACTCCAGCCTGAGCGACAGAGCAAGACTCTTGTTTCCAAAAAAATAAAA AACAGGTACATATGGTTGTCTGGCCCCCAGCAGCCTTGGTTTATCAGCAGCAGGCAAAAGGAGTTCTCTTAATCC AGCTGTGTGCTGTCCCTGTAGCCCCCCCGCAACTCAGCACTGCCATGTTCTGGCATCTTTTCTTCATATGCCCTG CTCCTGGCAACAGTTTCTGCACCTTAGCCACCTCCATATTTTGGCACCTTCCCCACTCCTGGAATGAGTTTCTAT ATCAGTCACAGCTCTTTGATTGCATATTGTGGAAAAAATCCAAAGTAAAATACCTTAAAAGCCAGTGGTTATACT TATTTGACCTTGAGCCTCCAAAATAAATATATCTTGCATTGTGACCCAGTATACTCCTAAGTATAGAATAATGAT GGCTTCTAAGTGTACTCTGTGCCAGGCCCTGTGGTAAGTAACATGGTGTTAACTCATTTAATTCTCAGGACAACC TTCTGCTATATGTACTGTTGGTATACCCTCTTTCAGATGAAGAAAGTGAAGCACAGAGGGATTAAGTGATCTGCC TGAGGTCGTATAGTTGGTAAGAGGCAAAGCTTGGGTTTGAATCCAGGAAGTCTGCTTTCAGAGTCTATGCAGTTC CAAAGCAGTGGCAACTCTGGAAGAAAAAGGCTTTCCTAGGCCGGGTGTGGTGATTCCCACCTGTAATCCCAGCAC TTTGGGTGGCTGAGGTGGGCAGATTGCTTGAGCCCAGGAGTTTGAGACCAGCCTGGCCAACATGGTGTAGCCCTG TCTACACTGAAAATACAAAAATTAGCCGGGTGTGGTGGCATGCACCTGTAATCCCAGCTACTCGGGAGGCTGCGT GGGAGGATCACTTGAGCCCAGGAGGCAGAGGTTCCAGTGAGCCAAGATCACGCTACTGCACTCCAGCCTGAGCGA CAGAGCGAGACCCTGTCTCAAAAAAAAAAAAAAAAAAAAAGGAAAAAGGCTTTCTTTCTCTCCTGGCCTGTTTCC TCAGTCCCCATCAGAACCCTCATGAGTCCTTTTGTTCTGAGCTATTGTGCTTCTGGTCTTTTTGTCCTGTTTATT TTTAGGCACTCTTTCTATAATAGGTAGATTTGCTCTTTACCTATGATATGGGCTCCAAATATTTTTTCCCAACTT GTCATTAACTTTTGACTTTGCTTATTCTATTTTCTTTAAATATTTTCTTTCTTTTCATGTATTCAAAAGTACCCA TCTTTTCTTTTATGGCCTCTGGATTTTGAGTCATAGAAAGGCCAGAGCTTTAATCTTGTTTCTTGTTCTCTGTAG CGTTGACTATCATCACTCTGTGACTCCCCCCAGGGCCCAGAGGCCTTGGGGAGCTGGGGGAGGTTGGGAGGGTGG TGGTTAGTGAGAAAGTGGGAGCGTTTTCAGCCTAGGCCCAAGTCTCCCAGGGCAGGAGGACCCTGCCTGCTTCCT GATAGCCGCCCACCAACCCTCAGGACCTGGGCTATCCCTTGGAGCTTGGCTATCAGAACTTCCTCTACCCCAGTG AGCCTGACCTCCGAGACCTGCTTCTCTTCTTGGCTGAGCGTCTGCCCACCGATGCCTCTGAGGATGCAGACCAGC CTGCAGGTACTGGGTGTCTGGGGTGTGGGCGGGGGCGGTGAGGGGAGAGGAGGGCCTTCTAGGGGCTGTAGGGCT GAGAGAGGTAGAGGTGGTAAAAAGGTTGATGGGTAGGGGTGGCGGTATGTGCCTTCAGGAGAGCTAGGCAGGAGG ATTAGGCATCAGAGAGGGGCTACAGGGCAGGGGGCAGGGGGCTGAGGTTGAGCTGACCCCGGTGGTGCGTTGGTG CGGGGAGGGGCCTCCCTGACTCAACCCCTGTGCCCTCCCTCTCTCTCACTTCGCTCTAACCACTTGGGCCTCCTG GGTGCTACTCAAACATACCCCAGTACACTCCCGCCTCTGCACCTTTGTATCGTTGGTTACCTTTCCCCTAGGTAC CCTCATGGTTCACTCCCTTACCTCTTTGAGGTTAACCTCCTCTCCGCAGCCCTCTCCTGATCCCCACCTGGCTGC AGTCTGGATGGTATTCAGGATCCTATTCATGATCTCTCTCCTCTGCTAGCTTGCCGGCTCCCTGAGGGCAGGGAC CTTTATCCAGTTTGTTCCACGAAGTAGCCCTAGTGTCTAGAACAGCGACCTGTACATAGTAGGTGCTCGGTGTTT GTTGAATGACTGAACGTGAGAACGCAAGCTGGATAGATGTGTATCTCTGGATGGGGGTCAGGAGTGGAGGCTGGT CTCCTAGGGTATGCCCTTTTGGATTTGCAGCATTGACATCTGATTCACTTCCTCCCTATCCCCCATAGGTGACTC AGCTATTCTCCTCCGGGCCATTGGGAGCCAAATTCGGGACCAGCTGGCACTGCCTTGGGTCCCGCCCCACCTTCG CACTCCCAAGCTGCAGCACCTCCAGGTGAGACCCCTGACTCCCATGGATCTTCTCTTGTCCCCGTCTGGGTGCCC AGGGTTTTGGCCCCCTACCCCTGGCAACCCTCATCCCACTTCACCCTGGAGATTCTGAGCCTGCTCTCCCACCAG GGCTCGGCCCTCCAGAAGCCTTTCCATGCCAGCAGGCTGGTCGTGCCAGAATTGAGTTCCAGAGGTGGTGAGCAT GAGGCTGTGGGGAGGGGTGAGGAGGAAGGTGGGGGGGAACCTCATAGCGTTGCCATGCGGCAGGGCCAGCTGACT CTGTTCCTGCCTCCAGAGCCACGGGAGTTCCAGGCGAGTCCCCTGCTGCTTCCAGTCCCTACCCAGGTGCCTCAG CCTGTTGGAAGGGTGGCCTCGCTCCTCGAACACCATGCCCTGCAGCTCTGCCAGCAGACGGGCCGGGACCGGCCA GGGGATGAGGACTGGGTCCACCGGACATCCCGCCTCCCACCCCAGGTACAGCCAGATGCCTGGCTCCCTGCTGTC TGGGCTGCTGCTCACTGACACTCCCGCTGGTCCTCTGCTCTCCTTCCCCACTTTGTCCCTCCCTTCCATTGTTTC CCCTCTGTGTGTGCTTACCTAGCTCCCCACCTGAAAGAACACTGGAGTCAGAAAAAAGGAGAACCTGGGACAAGT CAGTATCCCTCCTCAGAGCCTTGGTTTCCTGTGCTAAAATTTGGGGTAGTAATAGTGCTCTCCTCTCAGGGCAGT AGTTAGACTGAATAATGTGCTTGAGATTCCTGGCAACTGGAGCAATCCAGATTGGCTACCTGCCTTCATCATTCA TTAATTCATTCATTCATTTGGAGTCTTGCTCTGTCACCCAGGCTGGAGTGCAGTGGCGTGATCTCAGCACACTGC AACCTCCATCTCCCGGGTTCAAGCGATTCTCCTGCCTCAGCCTCCCAAGTAGCTGGGATTACAGGCTTGCACCAC CACACCTGGCTAATTTTTATATTTTTAGTAGAGACAGGGTTTTGCCATGTTGGCTAGGCTGGTCTCGAATTCCTG ACCTCAGGTGATCTGCCCACCTTGGCCTCCCAAAGTGTTGGGATTACAGGCATGAGCCACCGCACCCGGCCCTAA TCTTTCTCTGTTTCTGCACTTGTCACTGATGTTCATTTTTCTGGTTTCTAGGTTTTTGCACAATTTCTGCCTGTC CCCATTATTCTAGTTCTGTGTTTTTGCTGCTCCTCTTTCTTACCTCTCTGTCTCCTCATTTCTGTACTGAATTCC TCTCTTGCTCTGTCTATCTATCTGTTCCCCCTTCTCCTCTGTCTACTTCTTTATCTGTCCCCTCTTCTTCTCTGT GCACCTTTTTATCTGTGTCCCCTTCTGCTCTGTCCACCTCTGTATCTCTCCCCTTCCTTGCTGTCCACCTATATA TCTGTCACCCCTCTGCCTCTGTTTACTTCTTAATCTCTCTCCTCTTCCTCTCTGTCCACCTCTGTATCTGTTGCC CCTTCCCTCTGTCCCCTTATCTCTCCCTTCTTCCTCTCTGTCCACCTCTGTATTTGTCCCTCCTCCCCTTCTGTC CATCTCTTTATCTGTCCTCTCTTCTTCTTTTTTTTTGAGACGGTGTCTCGCTCTGTCACCCAGGCTGGACTACAA TGGCATGATCAGGGCTCAAGGCAGCCTCAAATTCCCGGGCTCAAGCAATCTTCCCACCTCAGGCATCTGAGTAGC TGGGTCTACAGGTGCGTGCGCCCGGCTAATTTTTGTATTTTTTGTAGAGATGGGGTTTTGCCATGTTGACCAGGC TGGTCTCGAACTCCTGACCTGAAGCAATCCACCCACCTTGGCCTTCCAAAGTGCTGGGATTACAGGCATGAGCCA CCATGCCGAGCCCCCTCTTCTTCTCTGTCAACCTCTTTGTCCCCTCTTCTTCTCTATCCATCTCTGTATCTGTCC CCTCTTCCCCTCTGTCCACTTATCTGTCCCCTCTTCCTCTGTCCACCTCTGCATCTGTCCCCTCCTTCTCTCTGT CCACCTCTCTGTCACCCCTCTCCCTCTGTCCATTTCTTTATCTGTCCCCTTTTCCTCTCTGTCCACTTCTCTTTT TCCCTCCCTCCATTCTGCTCTCCTATTTCTGTTCCCTCTTCCTCTGTGTTCACCCAGGTATCAGTACCCCTCCCC TTCTGTCCACCTTTATATCTGTCCCCTCTTCCTCTCTGTCCACCTCTGTATTTGGCCCCCCTCCCCTTCTGTCTG CCTCTTTATCTGTCTCCTCTTCCTCTGTGTCCACATCTCTGTCTGCCCCTCTTTTTCTCCACCTCTGTGTCGGCC CCCTCCTGGTCTGTCCACTTTTTTATCTGTCCTCTCTTTCTGTCTTCACCTCTGTGTCTTTTTACATTTTTATTT TTTTATCATTATTATTATTTTTTGAGATGGAGTCTCGCTCTGTCACCCAGGCTGGAGTACAGTGGTACGATCTCA GCTCACTGCAACCTCTGCCTCCTGGGTTCAAGTGATTCTCCTGCCTCAGCCTCCCACATAGCTAGGACTACAGGC ATGCACCACCACGCCCAGCTAATTTTTGTATTTTTAGTAGAGACAGGGTTTCACCATGTTGGCCAGGATGGTCTC GATCTCTTGACCTCATGATCCATCTGCCTCGGCCTCCCAAAGTGTTGTGATTACAGGCGTGAGCCACCACGCCTG GCTGTCTGTCTTTGTTTTTATATCTGTAGTAATTTTCAAACATAAATGTAGAGAGAATATTCTAGTGAATCCTAT GTACCATTTTGCCAACTTTTCTTCATCTTTTCTCTCCCAACTTTTTCTTTGTTGCTGTATTATTTTAAAGCAAAT CTCAGACATCATGTCATTTCAGCTCTAAATACTTAGGACTACATCTCTTAACTCATAAGGACATTCAGTTTTCAA GGTAACCACTGGACCATTTTCATGGCTAATGAAGTTAACAATAATATCTTGTGGGTTTTTTTTTGTTTTGTTTTG TTTTGTTTTTTTTTTGTTTTTTTTTGTTTTGTTTTTGAGACGGAGTCTCTCTCTGTTGCCCAGGCTGGAGTGCAA TGGCGTGATGTCGCTTCACTGCAACCTCCACTTCCTGGGTTCAAGCCATTCTCCTGCCTCAGCCCCCAAGTAGCT GGAATTACAGGAGCACACTAAGTTTTGTATTTTTAGTAGAGTCGGGGTTTTACCATGTTGGCCAGGCTGGTCTTG ATCTCCTGACCTCAGGTGATCTACCTACCTTGGCCTTTCAAAGGGCTAGGATTATAGGCATGAGCCACTGTACCC GGCCAACAATGATATCTTAATACCATCCAATACTTGAGTTCATAATCAGATTTTCCCATTATCTTGAAACTCTGT TGTCCCTCTCTTGCCTCTCTCTCTCTGCCTCTTTCTGTGCTTGGTACCTTTGATTCCCTGTCTCTGCCCTGTCCC CCGATATCCTGTCTATGCTTCTCTTGGATTTGGGGGCTCTAGGCCCACCCCCCTCTCTTCCCACATCCTTCTCCA GCATGGGGATCTAGTGGGTGGAGAGAAGTATGTCTGTGAGCAAGAGGAGACCCCTGTCCTCGAGGAGATCCCAGG CTGGTGGAGCAGGAGAGTAGAGCAGGGCCTGCCTTAGTGGGAAGGCTGGAGGGTGGGGGTGACTTGTCAGTATAC TCTTGTCAGGGGGTCCTTGGAGGAGGCAGGGCCTTGGGTGCTAGGTGGGCCCCTACACCTTCCTGCTTCCCCCGC CTTTTCTCCCCAGGAGGACACACGGGCTCAGCGGCAGCGGCTGCAAAAGCAACTGACTGAGCATCTGCGCCAAAG CTGGGGCCTGCTTGGGGCCCCCATACAAGCCCGGGACCTGGGAGAACTGCTGCAGGCCTGGGGTGCTGGGGCCAA GACTGGTGCTCCTAAGGGCTCCCGCTTCACGCACTCAGAGAAGTTCACCTTCCATCTGGTGGGTGCGCCTGAGGA CATGAGATGTGTGGATGGGCGTGGCAGGCTTGGAGGGTGGCTTTTTGTGTCAGCACCACACCTTTATCCACACAG GTTCCTGTGTTCCCACTGGACAGGCCCTCGCCCCACTGTGGGATGGGTACCCAGAGGGGTCCCCTAGCTTATTAG GGACTTGACTGGAGAAAATGTGGATAGGTGGGAACCATGCAAAGGTGTGGGGGTGTTCTTGGGGCAACACCCCCT TTCCTCAGGCAGTTTCCTTTGAGCATATCTTCTGTCCTAAGATGTTCACTCTGAGGGCCCCCCATCCTTCTCATG GGCATTTGAGGCTTGAGAGATGGGGCAGGTGGGTAGGAGCTGTGACAGGGTCAGGGAGATTTCTCCACGAGCAAG CACTCTGGCCCGAGGTTGCAGATGGTGCCTTTACCCACATAGTCACAGTCTGGCCACCATCGGTGCTTCAGTGGG CATGCATGCCGCACTGGGGGCAGTTCTCAGGGGAGGCTGAGGCTGGGCCACGTGAGGAAGGGCCTTCCCTGGCAG CCAGGATGCCCCTCGTCACTCCCCTTAGGAGCCCCAGGCCCAGGCCACTCAGGTGTCAGATGTGCCAGCCACCTC CCGGCGGCCTGAACAGGTGAGCAGAGTGGTTTGGAGGGGGGTGTCCCAGGCCCTTGCTTGTCTACTGGGCCTGAC ACCCCAACCCTGACTGGCCTGGGCCTCCCAGGTCACGTGGGCAGCTCAGGAACAGGAGCTCGAGTCCCTTCGGGA GCAGCTGGAAGGAGTGAACCGCAGCATTGAGGAGGTTGAGGCCGACATGAAGACCCTGGGCGTCAGCTTTGTGCA GGTAAGGGGCGGAGGAGGGGCTGCGCGTTGGGCTAGGTCAGAAGGAGGGCCTCGGGGTGTGAGGGACTAGATGGG GCAAGAGGTGCTCTGTAGAGGTCTGCACATGGCAGAAGGGTTCCTGGGAGCCATTAGGGATCTGTGGGCCTCTTG AGGGTGGCTATGAGAATCAGGCCAGGGGAGGGTCTGTGGGCATCTATGGGGCACCGCGGGTCTGCATGGGCAGGG GATTGAGGGGTCCTCGGAGGGTCTGTGGGCATCTGTGGGGTACCCACGGGTCTGCATGGACAGGGGATTAGGGGG TCCTCGGGGTCCTTGGCACCAGCGTGGAGCTGTTAGAGAGGCCTGTGGGGGCCACAGGGGTGTACAGTCATCTGT GGAGCTCCATGGGGGCTGTGGCATGTGACTGGGTATCCACCGGCCAGGCAGAGTCTGAGTGCCGGCACAGCAAGC TCAGTACAGCAGAGCGTGAGCAGGCCCTGCGCCTGAAGAGCCGCGCGGTGGAGCTGCTGCCCGATGGGACTGCCA ACCTTGCCAAGCTGCAGGTGGGGTTGGGGCTGTAGCTGGGCGGAGAGGGGCAGGGTGGGGTGGGGTGGGGTTGGA GGGCCCAGCCTGTGTGACATGTACCCATCCCCCACCAGCTTGTGGTGGAGAATAGTGCCCAGCGGGTCATCCACT TGGCGGGTCAGTGGGAGAAGCACCGGGTCCCACTCCTCGCTGAGTACCGCCACCTCCGAAAGCTGCAGGATTGCA GAGAGGTAAGCAGTGGGGCCCTGGGCTGTGGGCGGGCCAGGGCAGGCTCGGTCCCTCTCTAGGGGGCCATCCCTA TGCTCTGCTCACTGTCTTCTGCCTGTGGGCTCATGGCAGCTGGAATCTTCTCGACGGCTGGCAGAGATCCAAGAA CTGCACCAGAGTGTCCGGGCGGCTGCTGAAGAGGCCCGCAGGAAGGAGGAGGTCTATAAGCAGCTGGTAAGGCCT GTGTGAGGGACCTGGGTAGCTTAGGAGGGTGGGGGGATGGTCCTGGGGCAGTGCCTGCTATATCCCTGCCTAGAT GTCAGAGCTGGAGACTCTGCCCAGAGATGTGTCCCGGCTGGCCTACACCCAGCGCATCCTGGAGATCGTGGGCAA CATCCGGAAGCAGAAGGAAGAGATCACCAAGGTACACTGCCAGGGCCATGGAGGGTGGGTCATGTGGGCTGTCAG GCATAGTGTGGCCGCACAGGGACCTCACACCCTCAGGCAGAGCTGTCCAGTCACACTCTAACACAGAATAGTCAC ACACAATCCATCCCAGTCACCCCTGACACAGTGACACAGTCCCTGTCTGGTACACATGAGGACCCTCCACTGCTA GCCAGCCTGCCCCAGGCAGGGGCTCATGGCTGCCATGGTGTCTGCCAGATCTTGTCTGATACGAAGGAGCTTCAG AAGGAAATCAACTCCCTATCTGGGAAGCTGGACCGGACGTTTGCGGTGACTGATGAGCTTGTGTTCAAGGTGTGG GGCAGGTTGGGCGGGGGTGAGTGGGGTGAGGCTGGGCTGCTGCCTTGTGCATCTGCTAATTGGCTGGCTGGGGTC CAGACCCAGGCCCTGTGCGAGGCTGGAGGTGCACTGATACCCAGGGCTGGCTTTGTTTCATGGAGGATGAAGCTA GTGGGGTGGTGGGAGAGGGTGGCCTTCTTAGGGCATGGAGATGGTCAAGGGCAGCCCACTGATACCTTTGAGGTC CCTGTGTCTGGTCAGGATGCCAAGAAGGACGATGCTGTTCGGAAGGCCTATAAGTATCTAGCTGCTCTGCACGAG GTGAGGGGAGACATGTGCCTGGGGTGGGGCTGCTGGGGGTGGGTGGGACTGGGTGCAAGCCTTCTGCTCCTGTTG TCCCCAGAACTGCAGCCAGCTCATCCAGACCATCGAGGACACAGGCACCATCATGCGGGAGGTTCGAGACCTCGA GGAGCAGGTGAGGCCTGGGGGCAGGATGGGGAGCCAAGGCGGGCCGGGGGGACAGTTCCTCAGGTTATGCTGACA GAGGCTGTGGAGCCACACACAGCCGATGGCTGGACACCCAGCCCTGCCCCTTAGTGCCTGTGACCTGGGACAGGC AAGTGGCCTACTGTGAGCCCCAGCTTCCACCCCAAGGGCCCTCCTGTCTGCCTCCCAGGGCCATGGGCAGAGGCT TCAGCTTAAAGATGTAGGGGGAATCCTGCCACATGGCGAAGGATGCTTTGGGTAGAGGGAACACCACACGAGGCC TGGCCATGGGACAGAGCAGGCTGTTGGAGTTGGTGGGAGGGGCCCAGAGTGGCTGTGATGGGGGCTGGTGAGCAG GAGCTGGGAAAGGGGCTGTGTGTGCTGAGGGGGCATGTGTTCACATTGCCTCAGATCGAGACAGAGCTGGGCAAG AAGACCCTCAGCAACCTGGAGAAGATCCGGGAGGACTACCGAGCCCTCCGCCAGGAGAACGCTGGCCTCCTAGGC CGGGTCCGGGAGGCCTGAGGAGCCGCCGGCAGAGGTCTCTCCCCAGCCTCAGGCAGGGATTTGGGGTGCTGGAGG CAGTGGCCAAGCACATGCCCTAGCTACTTCCTCCGCTGTCCAGTTCCTCCTGCTGCGGCCTTGGACCCAGACCCC TGCCCACTGACCGCAACCCTTATATGGGGTGATAGTCCAGCATGTGGGGAGCTCGGCTGCAGTTTATTGGGGACG GTACTGTGGGTTGGGGGCCTTGGATCCCAAATAAATGAGTAGTTCCTCTGCAGTCTAA

>SEQ ID NO: 3 - Plus strand sequence including part of the 3' UTR of CCDC22 and a region downstream of CCDC22 that is antisense to the 3' UTR of FOXP3.

Oligonucleotides as shown in the Examples were designed antisense to this sequence.

CGAGACAGAGCTGGGCAAGAAGACCCTCAGCAACCTGGAGAAGATCCGGGAGGACTACCGAGCCCTCCGCCAGGA GAACGCTGGCCTCCTAGGCCGGGTCCGGGAGGCCTGAGGAGCCGCCGGCAGAGGTCTCTCCCCAGCCTCAGGCAG GGATTTGGGGTGCTGGAGGCAGTGGCCAAGCACATGCCCTAGCTACTTCCTCCGCTGTCCAGTTCCTCCTGCTGC GGCCTTGGACCCAGACCCCTGCCCACTGACCGCAACCCTTATATGGGGTGATAGTCCAGCATGTGGGGAGCTCGG CTGCAGTTTATTGGGGACGGTACTGTGGGTTGGGGGCCTTGGATCCCAAATAAATGAGTAGTTCCTCTGCAGTCT AAGCTGAGGCATGGATCAGGGCTCAGGGAATGGGAGTGAGGTGAGTGGCAGGGGAGACACGGGGTATTTTTGGCA AGGCAGTGTGTGTGGCTGTGTGTGTCTGCACGGGACTCAAGAGACCCACTGGGGGGCTGTGCGTGTGCATATGCG TGAGATACACAGGTGAATTCTAACAGGCCGTGTGTGTGAGCGAGCACGTGTTGGGACCTCAGATCCTGAGGGTAC TGACGCTGCTTCTGTGTAGGCCTCTGGGCACACCCCTGTGTTGACAGTGCCCCTGTGGGCCCTGAGGCTGGCTGT GGGTGCGTGCCTTGGGGTGTGTGGGTTGTCAGGGCTGTGCTTGTGTGTGATTGTGTGATGATGCAGCTTTGAGGT TGTTTGAGTGTACTGAGGCAGGCTCTCTGTGTTTTGGGGTTTGTGTTGAGTGAGGGACAGGATTGTGACATTTTG TGTGTCTGTGTGACTTTTCCAGCCCTGAAGTAATCTGTGCGAGCAGCTGAGGCAGGCTCTGTGTGGCTGGTTGTG AAGGCTCTGTTTGGCTGCAG

Additional Tables Table 2: Hexamers that are not seed sequences of human miRNAs

AAAAAA , AAAAAG , AAAACA, AAAAGA, AAAAGC , AAAAGG, AAAAUA , AAACAA, AAACAC ,

AAACAG , AAACAU , AAACCC, AAACCU, AAACGA, AAACGC , AAACGU, AAACUA, AAACUC,

AAACUU, AAAGAU , AAAGCC , AAAGGA, AAAGGG, AAAGUC , AAAUAC , AAAUAU , AAAUCG,

AAAUCU, AAAUGC , AAAUGU, AAAUUA, AAAUUG, AACAAC , AACAAG , AACAAU , AACACA,

AACACG, AACAGA, AACAGC , AACAGG, AACAUC, AACAUG, AACCAA, AACCAC, AACCAG,

AACCAU, AACCCC, AACCCG, AACCGA, AACCGC, AACCGG, AACCUA, AACCUU, AACGAA,

AACGAC , AACGAG, AACGAU, AACGCU, AACGGG, AACGGU, AACGUA, AACGUC, AACGUG,

AACGUU, AACUAU, AACUCA, AACUCC, AACUCG, AACUGA, AACUGC, AACUGU, AACUUA,

AACUUC, AACUUG, AACUUU, AAGAAA, AAGAAG , AAGAAU , AAGACG, AAGAGA, AAGAGC ,

AAGAGG, AAGAGU, AAGAUU, AAGCAA, AAGCAC , AAGCAG, AAGCAU, AAGCCA, AAGCCC,

AAGCCG, AAGCCU, AAGCGA, AAGCGG, AAGCGU, AAGCUA, AAGGAA, AAGGAC , AAGGCU,

AAGGGC, AAGGGU, AAGGUU, AAGUAA, AAGUAC , AAGUAU, AAGUCC, AAGUCG, AAGUGA,

AAGUGG, AAGUUA, AAGUUU, AAUAAA , AAUAAC , AAUAAG , AAUAAU , AAUACA, AAUACC,

AAUACG, AAUAGA, AAUAGC , AAUAGG, AAUAGU, AAUAUC, AAUAUU, AAUCAA, AAUCAU,

AAUCCA, AAUCCC, AAUCCG, AAUCGA, AAUCGC, AAUCGU, AAUCUA, AAUCUG, AAUCUU, AAUGAA, AAUGAC , AAUGAG, AAUGAU, AAUGCG, AAUGCU, AAUGGA, AAUGGU, AAUGUA,

AAUGUC, AAUGUG, AAUUAA, AAUUAC , AAUUAG, AAUUCC, AAUUCG, AAUUGA, AAUUGG,

AAUUGU, AAUUUC, AAUUUG, ACAAAA, ACAAAC , ACAAAG, ACAAAU, ACAACC, ACAACG,

ACAACU, ACAAGA, ACAAGC, ACAAGU, ACAAUC, ACAAUG, ACAAUU, ACACAG, ACACCA,

ACACCC, ACACCG, ACACCU, ACACGA, ACACGC, ACACGU, ACACUC, ACACUG, ACACUU,

ACAGAA, ACAGAC, ACAGCC, ACAGCG, ACAGCU, ACAGGG, ACAGUC, ACAGUG, ACAGUU,

ACAUAA, ACAUAC, ACAUCC, ACAUCG, ACAUCU, ACAUGA, ACAUGC, ACAUGU, ACAUUG,

ACAUUU, ACCAAA, ACCAAC, ACCAAG, ACCAAU, ACCACC, ACCACG, ACCAGA, ACCAGU,

ACCAUA, ACCAUG, ACCAUU, ACCCAA, ACCCAC, ACCCCA, ACCCCG, ACCCGA, ACCCGC,

ACCCUA, ACCCUC, ACCCUU, ACCGAA, ACCGAC, ACCGAU, ACCGCA, ACCGCC, ACCGCG,

ACCGCU, ACCGGA, ACCGGC, ACCGGU, ACCGUA, ACCGUC, ACCGUG, ACCGUU, ACCUAA,

ACCUAC, ACCUAG, ACCUAU, ACCUCA, ACCUCC, ACCUCG, ACCUCU, ACCUGA, ACCUGC,

ACCUGU, ACCUUA, ACCUUC, ACCUUU, ACGAAA, ACGAAC, ACGAAG, ACGAAU, ACGACA,

ACGACC, ACGACG, ACGACU, AC GAGA, ACGAGC, ACGAGG, ACGAGU, ACGAUA, ACGAUC,

ACGAUG, ACGAUU, ACGCAA, ACGCAG, ACGCAU, ACGCCC, ACGCCG, ACGCCU, ACGCGA,

ACGCGG, ACGCGU, ACGCUA, ACGCUG, ACGCUU, ACGGAA, ACGGAC, ACGGAG, ACGGAU,

ACGGCC, ACGGCG, ACGGCU, ACGGGC, ACGGGG, ACGGGU, ACGGUA, ACGGUC, ACGGUG,

ACGGUU, ACGUAA, ACGUAC, ACGUAU, ACGUCC, ACGUCG, ACGUCU, ACGUGA, ACGUGC,

ACGUGG, ACGUGU, ACGUUA, ACGUUC, ACGUUG, ACGUUU, ACUAAA, ACUAAG, ACUAAU,

ACUACA, ACUACC, ACUACG, ACUACU, ACUAGG, ACUAUC, ACUAUG, ACUAUU, ACUCAU,

ACUCCC, ACUCCG, ACUCCU, ACUCGA, ACUCGC, ACUCGG, ACUCUC, ACUCUU, ACUGAG,

ACUGAU, ACUGCC, ACUGCG, ACUGCU, ACUGGG, ACUGGU, ACUGUC, ACUUAA, ACUUAC,

ACUUAU, ACUUCA, ACUUCC, ACUUCG, ACUUCU, ACUUGA, ACUUGC, ACUUGU, ACUUUA,

ACUUUC, ACUUUG, AGAAAA, AGAAAC , AGAAAG, AGAACC, AGAACG, AGAACU, AGAAGC,

AGAAGU, AGAAUA, AGAAUC, AGAAUG, AGAAUU, AGACAA, AGACAC, AGACAU, AGACCA,

AGACCC, AGACCG, AGACCU, AGACGA, AGACGC, AGACGU, AGACUA, AGACUC, AGACUU,

AGAGAC , AGAGAG, AGAGAU, AGAGCC, AGAGCG, AGAGCU, AGAGGC, AGAGGG, AGAGGU,

AGAGUA, AGAGUU, AGAUAC , AGAUAG, AGAUAU, AGAUCC, AGAUCG, AGAUCU, AGAUGA,

AGAUGC, AGAUGG, AGAUUA, AGAUUC, AGAUUG, AGAUUU, AGCAAC, AGCACA, AGCACG,

AGCACU, AGCAGA, AGCAUA, AGCAUC, AGCAUG, AGCCAA, AGCCAU, AGCCCA, AGCCGA,

AGCCGC, AGCCGG, AGCCGU, AGCCUA, AGCCUC, AGCGAA, AGCGAG, AGCGAU, AGCGCA,

AGCGCC, AGCGCG, AGCGCU, AGCGGA, AGCGGC, AGCGGU, AGCGUA, AGCGUC, AGCGUG,

AGCGUU, AGCUAA, AGCUAC, AGCUAG, AGCUAU, AGCUCA, AGCUCC, AGCUCG, AGCUCU,

AGCUGA, AGCUGG, AGCUGU, AGCUUC, AGCUUU, AGGAAU, AGGACC, AGGACG, AGGAGA,

AGGAGU, AGGAUA, AGGCAA, AGGCAU, AGGCCG, AGGCGA, AGGCGC, AGGCGG, AGGCUA,

AGGCUC, AGGCUU, AGGGAC, AGGGAU, AGGGGA, AGGGGU, AGGGUA, AGGGUG, AGGUAA,

AGGUAC, AGGUCA, AGGUCC, AGGUCU, AGGUGA, AGGUGC, AGGUGG, AGGUGU, AGGUUC,

AGGUUG, AGUAAA, AGUAAG, AGUAAU, AGUACA, AGUACG, AGUAGC, AGUAGG, AGUAUA,

AGUAUC, AGUAUG, AGUAUU, AGUCAA, AGUCAC, AGUCAG, AGUCAU, AGUCCA, AGUCCG, AGUCCU, AGUCGA, AGUCGC, AGUCGG, AGUCGU, AGUCUA, AGUCUC, AGUCUG, AGUCUU,

AGUGAA, AGUGAC, AGUGCG, AGUGGG, AGUGUC, AGUUAA, AGUUAC, AGUUAG, AGUUCC,

AGUUCG, AGUUGA, AGUUGC, AGUUGU, AGUUUA, AGUUUC, AGUUUG, AGUUUU, AUAAAC ,

AUAAAU , AUAACA, AUAACC, AUAACG, AUAACU, AUAAGA, AUAAGC , AUAAGG, AUAAGU,

AUAAUC, AUAAUG, AUAAUU, AUACAC, AUACAG, AUACAU, AUACCA, AUACCC, AUACCG,

AUACGA, AUACGC, AUACGG, AUACGU, AUACUA, AUACUC, AUACUG, AUACUU, AUAGAA,

AUAGAC , AUAGAU, AUAGCA, AUAGCG, AUAGCU, AUAGGA, AUAGGU, AUAGUA, AUAGUC,

AUAGUG, AUAGUU, AUAUAC , AUAUAG, AUAUCC, AUAUCG, AUAUCU, AUAUGA, AUAUGC,

AUAUGG, AUAUGU, AUAUUC, AUAUUG, AUAUUU, AUCAAA, AUCAAC, AUCAAG, AUCAAU,

AUCACA, AUCACC, AUCACG, AUCAGC, AUCAGG, AUCCAA, AUCCAU, AUCCCC, AUCCCG,

AUCCGA, AUCCGC, AUCCGG, AUCCUA, AUCCUC, AUCCUG, AUCGAA, AUCGAC, AUCGAG,

AUCGAU, AUCGCA, AUCGCC, AUCGCG, AUCGCU, AUCGGC, AUCGGG, AUCGGU, AUCGUC,

AUCGUG, AUCGUU, AUCUAA, AUCUAC, AUCUAG, AUCUAU, AUCUCC, AUCUCG, AUCUGU,

AUCUUG, AUCUUU, AUGAAA, AUGAAC, AUGAAG, AUGAAU, AUGACC, AUGACU, AUGAGG,

AUGAGU, AUGAUA, AUGAUC, AUGAUU, AUGCAA, AUGCAG, AUGCCA, AUGCCC, AUGCCG,

AUGCGA, AUGCGG, AUGCGU, AUGCUC, AUGCUU, AUGGAC, AUGGCC, AUGGGA, AUGGGC,

AUGGGU, AUGGUC, AUGGUG, AUGUAC, AUGUAU, AUGUCA, AUGUCC, AUGUCG, AUGUGU,

AUGUUA, AUGUUC, AUUAAA, AUUAAC , AUUAAG, AUUAAU, AUUACA, AUUACC, AUUACG,

AUUACU, AUUAGA, AUUAGC, AUUAGG, AUUAGU, AUUAUA, AUUAUC, AUUAUG, AUUCAC,

AUUCCA, AUUCCG, AUUCCU, AUUCGA, AUUCGC, AUUCGG, AUUCGU, AUUCUA, AUUCUC,

AUUCUU, AUUGAA, AUUGAC, AUUGAU, AUUGCC, AUUGCG, AUUGCU, AUUGGA, AUUGGC,

AUUGGG, AUUGGU, AUUGUA, AUUGUC, AUUGUG, AUUGUU, AUUUAA, AUUUAG, AUUUAU,

AUUUCC, AUUUCG, AUUUCU, AUUUGA, AUUUGC, AUUUGU, AUUUUA, AUUUUC, AUUUUG,

AUUUUU, CAAAAG , CAAACA, CAAACC, CAAACG, CAAACU, CAAAGA, CAAAGG, CAAAUA,

CAAAUU, CAACAC , CAACAU, CAACCA, CAACCC, CAACCG, CAACGA, CAACGC, CAACGG,

CAACGU, CAACUA, CAACUC, CAACUG, CAACUU, CAAGAA, CAAGAC , CAAGAU, CAAGCA,

CAAGCC, CAAGCG, CAAGCU, CAAGGA, CAAGGG, CAAGUC, CAAGUG, CAAGUU, CAAUAA,

CAAUAC , CAAUAG, CAAUCC, CAAUCG, CAAUCU, CAAUGA, CAAUGC, CAAUGG, CAAUGU,

CAAUUC, CAAUUG, CAAUUU, CACAAU, CACACA, CACACG, CACACU, CACAGA, CACAGC,

CACAGG, CACAUA, CACAUC, CACAUU, CACCAA, CACCAC, CACCAU, CACCCA, CACCCC,

CACCCG, CACCGA, CACCGC, CACCGG, CACCGU, CACCUA, CACCUU, CACGAA, CACGAC,

CACGAG, CACGAU, CACGCA, CACGCC, CACGCU, CACGGA, CACGGC, CACGGG, CACGGU,

CACGUA, CACGUC, CACGUG, CACGUU, CACUAA, CACUAG, CACUAU, CACUCA, CACUCG,

CACUGA, CACUGC, CACUGG, CACUUA, CACUUC, CACUUU, CAGAAA, CAGAAG, CAGAAU,

CAGACC, CAGACG, CAGAGC, CAGAUA, CAGAUC, CAGCCG, CAGCCU, CAGCGA, CAGCGC,

CAGCGG, CAGCGU, CAGCUC, CAGCUU, CAGGAU, CAGGGG, CAGGGU, CAGGUA, CAGGUC,

CAGGUU, CAGUAC, CAGUCG, CAGUUG, CAUAAA , CAUAAC , CAUAAG, CAUAAU, CAUACA,

CAUACC, CAUACG, CAUACU, CAUAGA, CAUAGG, CAUAGU, CAUAUA, CAUAUC, CAUAUG,

CAUCAA, CAUCAC, CAUCAG, CAUCAU, CAUCCA, CAUCCC, CAUCCG, CAUCGA, CAUCGC, CAUCGG, CAUCGU, CAUCUA, CAUCUC, CAUCUG, CAUCUU, CAUGAA, CAUGAC, CAUGAG,

CAUGAU, CAUGCA, CAUGCC, CAUGCG, CAUGCU, CAUGGC, CAUGGG, CAUGGU, CAUGUA,

CAUGUC, CAUGUU, CAUUAA, CAUUAC, CAUUAG, CAUUCA, CAUUCC, CAUUCG, CAUUCU,

CAUUGA, CAUUGG, CAUUUC, CAUUUG, CAUUUU, CCAAAA, CCAAAC, CCAAAG, CCAAAU,

CCAACA, CCAACC, CCAACG, CCAACU, CCAAGA, CCAAGC, CCAAGG, CCAAUC, CCAAUG,

CCAAUU, CCACAA, CCACAC, CCACAG, CCACAU, CCACCA, CCACCC, CCACCG, CCACCU,

CCACGA, CCACGC, CCACGG, CCACGU, CCACUA, CCACUC, CCACUU, CCAGAA, CCAGAC,

CCAGAG, CCAGCC, CCAGGU, CCAGUC, CCAGUU, CCAUAA, CCAUAC, CCAUAG, CCAUAU,

CCAUCA, CCAUCC, CCAUCU, CCAUGA, CCAUGC, CCAUGG, CCAUUC, CCAUUG, CCAUUU,

CCCAAC, CCCAAG, CCCAAU, CCCACA, CCCAGA, CCCAGC, CCCAGU, CCCAUA, CCCAUC,

CCCAUG, CCCAUU, CCCCAA, CCCCAG, CCCCAU, CCCCCC, CCCCCG, CCCCCU, CCCCGA,

CCCCGC, CCCCGU, CCCCUA, CCCCUC, CCCGAA, CCCGAC, CCCGAU, CCCGCA, CCCGCU,

CCCGGA, CCCGGC, CCCGUA, CCCGUG, CCCGUU, CCCUAA, CCCUAG, CCCUCA, CCCUCU,

CCCUGC, CCCUUA, CCCUUC, CCCUUU, CCGAAA, CCGAAC, CCGAAU, CCGACA, CCGACC,

CCGACG, CCGACU, CCGAGA, CCGAGG, CCGAGU, CCGAUA, CCGAUC, CCGAUG, CCGAUU,

CCGCAA, CCGCAC, CCGCAG, CCGCAU, CCGCCA, CCGCCC, CCGCCG, CCGCCU, CCGCGA,

CCGCGC, CCGCGG, CCGCGU, CCGCUA, CCGCUC, CCGCUG, CCGCUU, CCGGAA, CCGGAU,

CCGGCA, CCGGCC, CCGGCG, CCGGCU, CCGGGA, CCGGGC, CCGGGG, CCGGGU, CCGGUA,

CCGGUC, CCGGUG, CCGUAA, CCGUAG, CCGUAU, CCGUCA, CCGUCC, CCGUCG, CCGUGA,

CCGUGU, CCGUUA, CCGUUC, CCGUUG, CCGUUU, CCUAAC, CCUAAG, CCUAAU, CCUACA,

CCUACC, CCUACG, CCUACU, CCUAGA, CCUAGC, CCUAGG, CCUAGU, CCUAUA, CCUAUC,

CCUAUG, CCUAUU, CCUCAA, CCUCAC, CCUCAG, CCUCAU, CCUCCA, CCUCCC, CCUCCG,

CCUCGA, CCUCGC, CCUCGG, CCUCGU, CCUCUA, CCUCUG, CCUGAC, CCUGAU, CCUGCA,

CCUGGG, CCUGGU, CCUGUU, CCUUAA, CCUUAC, CCUUAG, CCUUAU, CCUUCG, CCUUGA,

CCUUGU, CCUUUA, CCUUUC, CCUUUU, CGAAAA, CGAAAC , CGAAAG, CGAAAU, CGAACA,

CGAACC, CGAACG, CGAACU, CGAAGA, CGAAGC, CGAAGG, CGAAGU, CGAAUA, CGAAUC,

CGAAUG, CGAAUU, CGACAA, CGACAC, CGACAU, CGACCA, CGACCU, CGACGA, CGACGC,

CGACGG, CGACGU, CGACUA, CGACUG, CGACUU, CGAGAA, CGAGAC, CGAGAG, CGAGAU,

CGAGCA, CGAGCC, CGAGCG, CGAGCU, CGAGGC, CGAGGG, CGAGGU, CGAGUA, CGAGUC,

CGAGUG, CGAGUU, CGAUAA, CGAUAC, CGAUAG, CGAUAU, CGAUCA, CGAUCC, CGAUCG,

CGAUCU, CGAUGA, CGAUGC, CGAUGG, CGAUGU, CGAUUA, CGAUUC, CGAUUG, CGAUUU,

CGCAAA, CGCAAC, CGCAAG, CGCAAU, CGCACA, CGCACC, CGCACG, CGCAGA, CGCAGC,

CGCAGG, CGCAGU, CGCAUA, CGCAUC, CGCAUG, CGCAUU, CGCCAA, CGCCAC, CGCCAG,

CGCCAU, CGCCCA, CGCCCC, CGCCCG, CGCCGA, CGCCGC, CGCCGG, CGCCGU, CGCCUA,

CGCCUG, CGCCUU, CGCGAA, CGCGAC, CGCGAG, CGCGAU, CGCGCA, CGCGCC, CGCGCG,

CGCGCU, CGCGGA, CGCGGC, CGCGGG, CGCGGU, CGCGUA, CGCGUC, CGCGUG, CGCGUU,

CGCUAA, CGCUAC, CGCUAG, CGCUAU, CGCUCA, CGCUCC, CGCUCG, CGCUCU, CGCUGA,

CGCUGC, CGCUGG, CGCUGU, CGCUUA, CGCUUC, CGCUUG, CGGAAA, CGGAAC, CGGAAG,

CGGACA, CGGACC, CGGACG, CGGACU, CGGAGC, CGGAGG, CGGAGU, CGGAUA, CGGAUU, CGGCAA, CGGCAC, CGGCAG, CGGCCA, CGGCCC, CGGCCG, CGGCGC, CGGCGG, CGGCGU,

CGGCUA, CGGCUC, CGGCUG, CGGCUU, CGGGAA, CGGGAC, CGGGAG, CGGGAU, CGGGCA,

CGGGCC, CGGGCG, CGGGCU, CGGGGU, CGGGUA, CGGGUC, CGGGUG, CGGUAA, CGGUAC,

CGGUAG, CGGUAU, CGGUCA, CGGUCG, CGGUCU, CGGUGA, CGGUGG, CGGUGU, CGGUUA,

CGGUUC, CGGUUG, CGGUUU, CGUAAA, CGUAAC, CGUAAG, CGUAAU, CGUACA, CGUACG,

CGUACU, CGUAGA, CGUAGC, CGUAGG, CGUAGU, CGUAUA, CGUAUC, CGUAUG, CGUAUU,

CGUCAA, CGUCAC, CGUCAG, CGUCAU, CGUCCA, CGUCCC, CGUCCG, CGUCCU, CGUCGA,

CGUCGG, CGUCGU, CGUCUA, CGUCUC, CGUCUG, CGUCUU, CGUGAA, CGUGAC, CGUGAG,

CGUGAU, CGUGCC, CGUGCG, CGUGCU, CGUGGA, CGUGGG, CGUGGU, CGUGUA, CGUGUG,

CGUUAA, CGUUAC, CGUUAG, CGUUAU, CGUUCA, CGUUCC, CGUUCG, CGUUCU, CGUUGA,

CGUUGC, CGUUGU, CGUUUA, CGUUUC, CGUUUU, CUAAAA, CUAAAC, CUAAAU, CUAACA,

CUAACC, CUAACG, CUAACU, CUAAGA, CUAAGC, CUAAGU, CUAAUA, CUAAUC, CUAAUG,

CUACAC, CUACAU, CUACCA, CUACCC, CUACCG, CUACCU, CUACGA, CUACGC, CUACGG,

CUACGU, CUACUA, CUACUC, CUACUG, CUAGAA, CUAGAG, CUAGAU, CUAGCA, CUAGCC,

CUAGCG, CUAGCU, CUAGGA, CUAGGG, CUAGGU, CUAGUG, CUAGUU, CUAUAA, CUAUAG,

CUAUAU, CUAUCA, CUAUCC, CUAUCG, CUAUCU, CUAUGA, CUAUGC, CUAUGG, CUAUGU,

CUAUUA, CUAUUG, CUCAAC, CUCAAG, CUCAAU, CUCACC, CUCACG, CUCAGC, CUCAUA,

CUCAUC, CUCAUG, CUCAUU, CUCCAC, CUCCCC, CUCCCG, CUCCGA, CUCCGC, CUCCGG,

CUCCUA, CUCCUC, CUCCUU, CUCGAA, CUCGAC, CUCGAG, CUCGAU, CUCGCA, CUCGCC,

CUCGCG, CUCGGG, CUCGGU, CUCGUA, CUCGUC, CUCGUG, CUCGUU, CUCUAA, CUCUAC,

CUCUAU, CUCUCA, CUCUCC, CUCUCU, CUCUGC, CUCUGU, CUCUUA, CUCUUG, CUGAAG,

CUGACC, CUGACG, CUGAGC, CUGAUA, CUGAUC, CUGCCG, CUGCCU, CUGCGA, CUGCUA,

CUGCUU, CUGGAG, CUGGAU, CUGGCG, CUGGGU, CUGUAC, CUGUCA, CUGUCC, CUGUCG,

CUGUGG, CUGUGU, CUGUUA, CUGUUU, CUUAAC, CUUAAG, CUUAAU, CUUACC, CUUACG,

CUUAGA, CUUAGC, CUUAGG, CUUAGU, CUUAUA, CUUAUC, CUUAUG, CUUAUU, CUUCAG,

CUUCAU, CUUCCA, CUUCCC, CUUCCG, CUUCCU, CUUCGA, CUUCGC, CUUCGG, CUUCGU,

CUUCUA, CUUGAC, CUUGAG, CUUGAU, CUUGCA, CUUGCC, CUUGCG, CUUGCU, CUUGGC,

CUUGGU, CUUGUU, CUUUAC, CUUUAG, CUUUAU, CUUUCA, CUUUCG, CUUUCU, CUUUGA,

CUUUGC, CUUUGU, CUUUUA, CUUUUC, CUUUUG, CUUUUU, GAAAAA, GAAAAG, GAAAAU,

GAAACC , GAAACG, GAAAGA, GAAAGC, GAAAGU, GAAAUA, GAAAUC, GAAAUG, GAAAUU,

GAACAA, GAACAC , GAACAG, GAACAU, GAACCA, GAACCC, GAACCG, GAACCU, GAACGA,

GAACGC, GAACGG, GAACGU, GAACUA, GAACUG, GAACUU, GAAGAC , GAAGAG, GAAGCA,

GAAGCG, GAAGCU, GAAGUC, GAAUAA, GAAUAC , GAAUAG, GAAUAU, GAAUCC, GAAUCG,

GAAUCU, GAAUGA, GAAUGC, GAAUGU, GAAUUA, GAAUUC, GAAUUU, GACAAA, GACAAG,

GACAAU, GACACC, GACAGA, GACAGG, GACAUA, GACAUG, GACAUU, GACCAA, GACCAC,

GACCAG, GACCCA, GACCCC, GACCCG, GACCGC, GACCGG, GACCGU, GACCUA, GACCUC,

GACCUU, GACGAA, GACGAC, GACGAG, GACGAU, GACGCA, GACGCC, GACGCG, GACGCU,

GACGGA, GACGGC, GACGGG, GACGGU, GACGUA, GACGUC, GACGUG, GACGUU, GACUAA,

GACUAC, GACUAG, GACUAU, GACUCA, GACUCC, GACUCG, GACUGG, GACUGU, GACUUA, GACUUG, GACUUU, GAGAAU, GAGAGA, GAGAGC, GAGAGG, GAGAUA, GAGAUC, GAGCAA,

GAGCAU, GAGCCA, GAGCGA, GAGCGG, GAGCGU, GAGGGU, GAGGUC, GAGGUG, GAGUAA,

GAGUAG, GAGUCC, GAGUUC, GAGUUU, GAUAAA, GAUAAC , GAUAAG, GAUAAU, GAUACA,

GAUACC, GAUACG, GAUACU, GAUAGA, GAUAGC, GAUAGG, GAUAGU, GAUAUA, GAUCAA,

GAUCAC, GAUCAU, GAUCCA, GAUCCC, GAUCCU, GAUCGC, GAUCGG, GAUCGU, GAUCUA,

GAUCUG, GAUCUU, GAUGAA, GAUGAC, GAUGAG, GAUGCA, GAUGCC, GAUGCG, GAUGCU,

GAUGGC, GAUGGG, GAUGGU, GAUGUG, GAUGUU, GAUUAA, GAUUAC , GAUUAG, GAUUAU,

GAUUCA, GAUUCG, GAUUCU, GAUUGA, GAUUGC, GAUUUA, GAUUUC, GAUUUG, GAUUUU,

GCAAAC , GCAAAG, GCAAAU, GCAACA, GCAACC, GCAAGC, GCAAGU, GCAAUA, GCAAUC,

GCAAUG, GCAAUU, GCACAA, GCACAC, GCACAG, GCACCC, GCACCG, GCACCU, GCACGA,

GCACGC, GCACGU, GCACUA, GCACUC, GCACUG, GCACUU, GCAGAU, GCAGCC, GCAGCG,

GCAGGC, GCAGUA, GCAGUC, GCAGUG, GCAGUU, GCAUAA, GCAUAG, GCAUAU, GCAUCG,

GCAUCU, GCAUGA, GCAUGC, GCAUGG, GCAUGU, GCAUUA, GCAUUC, GCAUUG, GCAUUU,

GCCAAA, GCCAAC, GCCAAU, GCCACA, GCCACC, GCCACG, GCCAGA, GCCAGU, GCCAUA,

GCCAUC, GCCAUG, GCCAUU, GCCCAA, GCCCAC, GCCCAG, GCCCCG, GCCCGA, GCCCGG,

GCCCGU, GCCGAA, GCCGAC, GCCGAG, GCCGAU, GCCGCA, GCCGCU, GCCGGA, GCCGGC,

GCCGGG, GCCGGU, GCCGUA, GCCGUC, GCCGUG, GCCGUU, GCCUAA, GCCUAU, GCCUCA,

GCCUCC, GCCUCG, GCCUGA, GCCUUA, GCCUUU, GCGAAA, GCGAAC, GCGAAG, GCGAAU,

GCGACC, GCGACG, GCGACU, GCGAGA, GCGAGC, GCGAGG, GCGAGU, GCGAUA, GCGAUC,

GCGAUG, GCGAUU, GCGCAA, GCGCAC, GCGCAG, GCGCAU, GCGCCA, GCGCCC, GCGCCU,

GCGCGA, GCGCGU, GCGCUA, GCGCUC, GCGCUG, GCGCUU, GCGGAA, GCGGAC, GCGGAU,

GCGGCA, GCGGCC, GCGGCU, GCGGGA, GCGGUA, GCGGUC, GCGGUU, GCGUAA, GCGUAC,

GCGUAG, GCGUAU, GCGUCA, GCGUCC, GCGUCG, GCGUCU, GCGUGA, GCGUGC, GCGUGG,

GCGUGU, GCGUUA, GCGUUC, GCGUUG, GCGUUU, GCUAAA, GCUAAC, GCUAAG, GCUAAU,

GCUACC, GCUACG, GCUACU, GCUAGA, GCUAGG, GCUAGU, GCUAUA, GCUAUC, GCUAUU,

GCUCAA, GCUCAC, GCUCAG, GCUCAU, GCUCCA, GCUCCC, GCUCCG, GCUCGA, GCUCGC,

GCUCGU, GCUCUA, GCUCUC, GCUCUU, GCUGAA, GCUGAC, GCUGAU, GCUGCA, GCUGCC,

GCUGCG, GCUGCU, GCUGUG, GCUGUU, GCUUAC, GCUUAG, GCUUAU, GCUUCA, GCUUCG,

GCUUGA, GCUUGG, GCUUGU, GCUUUA, GCUUUG, GGAAAG, GGAACA, GGAACC, GGAACG,

GGAACU, GGAAGU, GGAAUA, GGAAUC, GGAAUU, GGACAA, GGACAC, GGACAG, GGACAU,

GGACCG, GGACGA, GGACGC, GGACGU, GGACUA, GGACUC, GGACUU, GGAGAC, GGAGCA,

GGAGCG, GGAGGG, GGAGUA, GGAUAA, GGAUAC, GGAUCA, GGAUCC, GGAUCG, GGAUCU,

GGAUGC, GGAUUA, GGAUUG, GGCAAU, GGCACA, GGCACU, GGCAGA, GGCAUA, GGCAUC,

GGCCAC, GGCCAG, GGCCCC, GGCCGA, GGCCGC, GGCCGU, GGCCUA, GGCCUG, GGCCUU,

GGCGAA, GGCGAG, GGCGAU, GGCGCA, GGCGCU, GGCGGU, GGCGUA, GGCGUC, GGCGUG,

GGCGUU, GGCUAA, GGCUAC, GGCUAG, GGCUAU, GGCUCC, GGCUCG, GGCUGA, GGCUUA,

GGCUUC, GGCUUG, GGGAAU, GGGACA, GGGAGA, GGGAGU, GGGAUA, GGGAUU, GGGCAA,

GGGCAC, GGGCAG, GGGCCG, GGGCGG, GGGGCC, GGGGGG, GGGGGU, GGGGUA, GGGUAC,

GGGUAU, GGGUCA, GGGUCC, GGGUCG, GGGUGA, GGGUGC, GGGUUA, GGGUUG, GGUAAA, GGUAAC, GGUAAG, GGUAAU, GGUACA, GGUACC, GGUACG, GGUACU, GGUAGC, GGUAGG,

GGUAGU, GGUAUA, GGUAUC, GGUAUG, GGUCAA, GGUCAC, GGUCAG, GGUCAU, GGUCCA,

GGUCCG, GGUCCU, GGUCGA, GGUCGC, GGUCGG, GGUCGU, GGUCUC, GGUCUU, GGUGAA,

GGUGAC, GGUGAU, GGUGCA, GGUGCC, GGUGGC, GGUGUA, GGUGUC, GGUUAA, GGUUAG,

GGUUAU, GGUUCA, GGUUCC, GGUUCG, GGUUGC, GGUUUC, GGUUUU, GUAAAA, GUAAAG,

GUAAAU, GUAACC, GUAACG, GUAACU, GUAAGA, GUAAGC, GUAAGG, GUAAGU, GUAAUA,

GUAAUC, GUAAUG, GUAAUU, GUACAA, GUACAC, GUACAG, GUACAU, GUACCA, GUACCC,

GUACCG, GUACCU, GUACGA, GUACGC, GUACGG, GUACGU, GUACUA, GUACUC, GUACUG,

GUACUU, GUAGAA, GUAGAC, GUAGCA, GUAGCC, GUAGCG, GUAGCU, GUAGGA, GUAGGC,

GUAGGG, GUAGGU, GUAGUA, GUAGUC, GUAUAA, GUAUAC , GUAUAG, GUAUAU, GUAUCA,

GUAUCG, GUAUCU, GUAUGA, GUAUGC, GUAUGG, GUAUUA, GUAUUG, GUAUUU, GUCAAA,

GUCAAG, GUCAAU, GUCACA, GUCACC, GUCACG, GUCAGA, GUCAGC, GUCAGG, GUCAUA,

GUCAUC, GUCAUG, GUCCAA, GUCCAC, GUCCAU, GUCCCC, GUCCCU, GUCCGA, GUCCGC,

GUCCGG, GUCCGU, GUCCUA, GUCCUG, GUCCUU, GUCGAA, GUCGAC, GUCGAG, GUCGAU,

GUCGCA, GUCGCC, GUCGCG, GUCGCU, GUCGGA, GUCGGC, GUCGGG, GUCGGU, GUCGUA,

GUCGUC, GUCGUU, GUCUAA, GUCUAG, GUCUCA, GUCUCC, GUCUCG, GUCUGA, GUCUGG,

GUCUGU, GUCUUC, GUCUUU, GUGAAA, GUGAAC, GUGAAG, GUGACC, GUGACG, GUGAGA,

GUGAGC, GUGAGU, GUGAUC, GUGAUG, GUGAUU, GUGCAC, GUGCAU, GUGCCC, GUGCCG,

GUGCGA, GUGCGG, GUGCGU, GUGCUA, GUGCUC, GUGCUG, GUGGAG, GUGGCG, GUGGCU,

GUGGGU, GUGGUC, GUGGUG, GUGUAA, GUGUAG, GUGUCG, GUGUGA, GUGUGC, GUGUGU,

GUGUUG, GUGUUU, GUUAAA, GUUAAC , GUUAAG, GUUACA, GUUACC, GUUACG, GUUACU,

GUUAGA, GUUAGC, GUUAGU, GUUAUA, GUUAUC, GUUAUG, GUUAUU, GUUCAA, GUUCAC,

GUUCAG, GUUCCA, GUUCCG, GUUCGA, GUUCGC, GUUCGG, GUUCGU, GUUCUA, GUUCUG,

GUUGAA, GUUGAC, GUUGAG, GUUGAU, GUUGCG, GUUGCU, GUUGGA, GUUGGC, GUUGGU,

GUUGUC, GUUGUG, GUUGUU, GUUUAA, GUUUAC, GUUUAG, GUUUAU, GUUUCA, GUUUCC,

GUUUCU, GUUUGA, GUUUGC, GUUUGG, GUUUGU, GUUUUA, GUUUUC, GUUUUU, UAAAAA,

UAAAAC , UAAAAG, UAAAAU, UAAACA, UAAACC, UAAACG, UAAACU, UAAAGA, UAAAGG,

UAAAGU, UAAAUA, UAAAUC, UAAAUG, UAAAUU, UAACAA, UAACAC , UAACAG, UAACCA,

UAACCC, UAACCG, UAACCU, UAACGA, UAACGC, UAACGG, UAACGU, UAACUA, UAACUG,

UAACUU, UAAGAG, UAAGAU, UAAGCA, UAAGCC, UAAGCG, UAAGCU, UAAGGA, UAAGGC,

UAAGGG, UAAGGU, UAAGUA, UAAGUC, UAAGUG, UAAGUU, UAAUAA, UAAUCA, UAAUCC,

UAAUCG, UAAUCU, UAAUGA, UAAUGG, UAAUGU, UAAUUA, UAAUUC, UAAUUG, UACAAC ,

UACAAG, UACAAU, UACACC, UACACG, UACACU, UACAGA, UACAGC, UACAUA, UACAUC,

UACAUU, UACCAA, UACCAC, UACCAG, UACCAU, UACCCC, UACCCG, UACCCU, UACCGA,

UACCGC, UACCGG, UACCGU, UACCUA, UACCUG, UACGAA, UACGAC, UACGAG, UACGAU,

UACGCA, UACGCC, UACGCG, UACGCU, UACGGC, UACGGG, UACGGU, UACGUA, UACGUC,

UACGUG, UACGUU, UACUAA, UACUAC, UACUAG, UACUAU, UACUCA, UACUCC, UACUCG,

UACUCU, UACUGA, UACUGC, UACUGG, UACUUA, UACUUG, UACUUU, UAGAAA, UAGAAG,

UAGAAU, UAGACA, UAGACG, UAGAGA, UAGAGC, UAGAGU, UAGAUA, UAGAUC, UAGAUG, UAGCAU, UAGCCC, UAGCCG, UAGCCU, UAGCGA, UAGCGC, UAGCGU, UAGCUA, UAGCUC,

UAGCUG, UAGGAA, UAGGAU, UAGGCG, UAGGCU, UAGGGU, UAGGUC, UAGGUG, UAGGUU,

UAGUAA, UAGUAC, UAGUAG, UAGUAU, UAGUCA, UAGUCG, UAGUGU, UAGUUA, UAGUUC,

UAGUUG, UAGUUU, UAUAAC , UAUAAG, UAUACU, UAUAGA, UAUAGC, UAUAGG, UAUAGU,

UAUAUA, UAUAUC, UAUAUG, UAUAUU, UAUCAA, UAUCAC, UAUCAU, UAUCCA, UAUCCC,

UAUCCG, UAUCCU, UAUCGA, UAUCGC, UAUCGG, UAUCGU, UAUCUA, UAUCUC, UAUCUG,

UAUCUU, UAUGAA, UAUGAC, UAUGAG, UAUGAU, UAUGCA, UAUGCG, UAUGCU, UAUGGA,

UAUGGC, UAUGUC, UAUGUG, UAUGUU, UAUUAG, UAUUCA, UAUUCC, UAUUCG, UAUUCU,

UAUUGA, UAUUGG, UAUUUA, UAUUUC, UAUUUG, UAUUUU, UCAAAA, UCAAAC, UCAAAG,

UCAACC, UCAACU, UCAAGA, UCAAGC, UCAAUA, UCAAUC, UCAAUG, UCAAUU, UCACCC,

UCACCG, UCACCU, UCACGA, UCACGC, UCACGG, UCACGU, UCACUA, UCACUC, UCACUU,

UCAGAA, UCAGAC, UCAGAG, UCAGCG, UCAGCU, UCAGGA, UCAGGC, UCAGGU, UCAGUC,

UCAGUU, UCAUAA, UCAUCA, UCAUCC, UCAUCG, UCAUGC, UCAUGG, UCAUGU, UCAUUA,

UCAUUG, UCCAAA, UCCAAC, UCCAAG, UCCAAU, UCCACA, UCCACC, UCCACG, UCCAGC,

UCCAGG, UCCAUA, UCCAUC, UCCAUU, UCCCAA, UCCCAG, UCCCAU, UCCCCC, UCCCCG,

UCCCCU, UCCCGA, UCCCGC, UCCCGG, UCCCGU, UCCCUA, UCCCUC, UCCGAA, UCCGAC,

UCCGAG, UCCGAU, UCCGCA, UCCGCC, UCCGGA, UCCGGC, UCCGGU, UCCGUA, UCCGUC,

UCCGUG, UCCUAA, UCCUCA, UCCUCG, UCCUCU, UCCUGC, UCCUGU, UCCUUA, UCCUUC,

UCCUUU, UCGAAA, UCGAAC, UCGAAG, UCGAAU, UCGACA, UCGACC, UCGACG, UCGACU,

UCGAGA, UCGAGC, UCGAGG, UCGAUA, UCGAUC, UCGAUG, UCGAUU, UCGCAA, UCGCAC,

UCGCAG, UCGCAU, UCGCCA, UCGCCC, UCGCCG, UCGCCU, UCGCGA, UCGCGC, UCGCGU,

UCGCUA, UCGCUC, UCGGAA, UCGGAC, UCGGAG, UCGGAU, UCGGCA, UCGGCU, UCGGGG,

UCGGGU, UCGGUC, UCGGUG, UCGGUU, UCGUAA, UCGUAC, UCGUAG, UCGUAU, UCGUCA,

UCGUCC, UCGUCG, UCGUCU, UCGUGA, UCGUGU, UCGUUA, UCGUUC, UCGUUG, UCGUUU,

UCUAAC, UCUAAG, UCUAAU, UCUACA, UCUACC, UCUACG, UCUACU, UCUAGC, UCUAGG,

UCUAGU, UCUAUA, UCUAUC, UCUAUG, UCUAUU, UCUCAG, UCUCAU, UCUCCG, UCUCGC,

UCUCGG, UCUCGU, UCUCUC, UCUGAA, UCUGAU, UCUGCA, UCUGCG, UCUGCU, UCUGGC,

UCUGGU, UCUGUC, UCUGUG, UCUGUU, UCUUAA, UCUUAC, UCUUAG, UCUUAU, UCUUCA,

UCUUCC, UCUUCG, UCUUCU, UCUUGC, UCUUGG, UCUUGU, UCUUUA, UCUUUC, UCUUUG,

UCUUUU, UGAAAA, UGAAAC, UGAACA, UGAACC, UGAAGG, UGAAUC, UGAAUG, UGACAA,

UGACAC, UGACAG, UGACCA, UGACCC, UGACCG, UGACGA, UGACGC, UGACGG, UGACGU,

UGACUA, UGACUC, UGACUU, UGAGAG, UGAGAU, UGAGCA, UGAGCC, UGAGCU, UGAGGC,

UGAGGU, UGAGUA, UGAGUU, UGAUAC, UGAUAG, UGAUAU, UGAUCA, UGAUCG, UGAUCU,

UGAUGA, UGAUGC, UGAUGG, UGAUGU, UGAUUA, UGAUUC, UGAUUG, UGAUUU, UGCAAC,

UGCAAG, UGCACA, UGCACG, UGCAGG, UGCAGU, UGCAUC, UGCCCA, UGCCCC, UGCCCG,

UGCCGA, UGCCGC, UGCCGG, UGCCGU, UGCCUA, UGCCUC, UGCCUG, UGCCUU, UGCGAA,

UGCGAC, UGCGAU, UGCGCC, UGCGCG, UGCGCU, UGCGGC, UGCGGG, UGCGGU, UGCGUA,

UGCGUC, UGCGUG, UGCGUU, UGCUAC, UGCUAU, UGCUCC, UGCUCG, UGCUGC, UGCUGG,

UGCUGU, UGCUUA, UGCUUU, UGGAAC, UGGAAG, UGGAGC, UGGAUC, UGGAUU, UGGCAA, UGGCAC, UGGCAG, UGGCCG, UGGCCU, UGGCGA, UGGCGC, UGGCGU, UGGCUA, UGGCUC,

UGGCUU, UGGGAA, UGGGCA, UGGGCC, UGGGGC, UGGGUC, UGGUAA, UGGUAG, UGGUAU,

UGGUCC, UGGUCG, UGGUCU, UGGUGA, UGGUGC, UGGUGG, UGGUGU, UGGUUA, UGGUUG,

UGUAAA, UGUAAC, UGUAAG, UGUACC, UGUACG, UGUACU, UGUAGA, UGUAGC, UGUAGU,

UGUAUC, UGUAUU, UGUCAA, UGUCAC, UGUCAG, UGUCAU, UGUCCA, UGUCCC, UGUCCG,

UGUCGA, UGUCGC, UGUCGG, UGUCGU, UGUCUA, UGUCUC, UGUGAC, UGUGAG, UGUGAU,

UGUGCA, UGUGGU, UGUGUA, UGUGUU, UGUUAC, UGUUAG, UGUUAU, UGUUCA, UGUUCC,

UGUUCG, UGUUGG, UGUUGU, UGUUUA, UGUUUC, UGUUUG, UGUUUU, UUAAAA, UUAAAC ,

UUAAAG, UUAAAU, UUAACC, UUAACG, UUAACU, UUAAGU, UUAAUA, UUAAUC, UUAAUG,

UUAAUU, UUACAA, UUACAC, UUACAG, UUACAU, UUACCA, UUACCC, UUACCG, UUACCU,

UUACGA, UUACGC, UUACGG, UUACGU, UUACUA, UUACUC, UUACUG, UUACUU, UUAGAA,

UUAGAC, UUAGCC, UUAGCG, UUAGCU, UUAGGC, UUAGGU, UUAGUA, UUAGUC, UUAGUU,

UUAUAA, UUAUAC , UUAUAG, UUAUAU, UUAUCC, UUAUCG, UUAUCU, UUAUGA, UUAUGG,

UUAUGU, UUAUUA, UUAUUC, UUAUUG, UUAUUU, UUCAAC, UUCAAU, UUCACA, UUCACC,

UUCACG, UUCACU, UUCAGC, UUCAGG, UUCAGU, UUCAUA, UUCAUC, UUCAUG, UUCAUU,

UUCCAA, UUCCCA, UUCCCG, UUCCGA, UUCCGU, UUCCUU, UUCGAA, UUCGAC, UUCGAG,

UUCGAU, UUCGCA, UUCGCC, UUCGCG, UUCGCU, UUCGGA, UUCGGC, UUCGGG, UUCGGU,

UUCGUA, UUCGUC, UUCGUG, UUCGUU, UUCUAC, UUCUAG, UUCUCA, UUCUCG, UUCUGG,

UUCUUA, UUCUUU, UUGAAA, UUGAAC, UUGAAG, UUGAAU, UUGACC, UUGACG, UUGACU,

UUGAGA, UUGAGC, UUGAGU, UUGAUA, UUGAUC, UUGAUG, UUGAUU, UUGCAA, UUGCAC,

UUGCAG, UUGCAU, UUGCCC, UUGCCG, UUGCGA, UUGCGC, UUGCGG, UUGCGU, UUGCUA,

UUGCUC, UUGCUG, UUGCUU, UUGGAA, UUGGAG, UUGGCC, UUGGCG, UUGGCU, UUGGGC,

UUGGGU, UUGGUA, UUGGUG, UUGUAA, UUGUAC, UUGUCA, UUGUCG, UUGUCU, UUGUGC,

UUGUGG, UUGUUA, UUGUUG, UUGUUU, UUUAAA, UUUAAC , UUUAAG, UUUAAU, UUUACA,

UUUACC, UUUACG, UUUACU, UUUAGA, UUUAGC, UUUAGG, UUUAGU, UUUAUA, UUUAUC,

UUUAUG, UUUAUU, UUUCAU, UUUCCA, UUUCCG, UUUCCU, UUUCGA, UUUCGC, UUUCGG,

UUUCGU, UUUCUA, UUUCUC, UUUCUG, UUUCUU, UUUGAA, UUUGAC, UUUGAG, UUUGAU,

UUUGCC, UUUGCU, UUUGGA, UUUGGC, UUUGGG, UUUGGU, UUUGUA, UUUGUC, UUUGUU,

UUUUAA, UUUUAG, UUUUAU, UUUUCC, UUUUCG, UUUUCU, UUUUGA, UUUUGC, UUUUGG,

UUUUGU, UUUUUA, UUUUUC, UUUUUU

Table 3. A listing of oligonucleotide modifications that can be used in the single stranded oligonucleotides described herein

Symbol Feature Description

bio 5' biotin

dAs DNA w/3' thiophosphate

dCs DNA w/3' thiophosphate

dGs DNA w/3' thiophosphate dTs DNA w/3' thiophosphate dG DNA w/3' phosphate

dT DNA w/3' phosphate

dU deoxyuridine w/3' phosphate d5mCs deoxy-5-methylcytidine w/3'

thiophosphate

enaAs ENA w/3' thiophosphate

enaCs ENA w/3' thiophosphate

enaGs ENA w/3' thiophosphate

enaTs ENA w/3' thiophosphate

fluAs 2'-fluoro w/3' thiophosphate fluCs 2'-fluoro w/3' thiophosphate fluGs 2'-fluoro w/3' thiophosphate fluUs 2'-fluoro w/3' thiophosphate

InaAs LNA w/3' thiophosphate

InaCs LNA w/3' thiophosphate

InaGs LNA w/3' thiophosphate

InaTs LNA w/3' thiophosphate

omeAs 2'-OMe w/3' thiophosphate omeCs 2'-OMe w/3' thiophosphate omeGs 2'-OMe w/3' thiophosphate omeTs 2'-OMe w/3' thiophosphate

InaAs-Sup LNA w/3' thiophosphate at 3' terminus

InaCs-Sup LNA w/3' thiophosphate at 3' terminus

InaGs-Sup LNA w/3' thiophosphate at 3' terminus

InaTs-Sup LNA w/3' thiophosphate at 3' terminus

InaA-Sup LNA w/3' OH at 3' terminus

InaC-Sup LNA w/3' OH at 3' terminus

InaG-Sup LNA w/3' OH at 3' terminus

InaT-Sup LNA w/3' OH at 3' terminus

omeA-Sup 2'-OMe w/3' OH at 3' terminus omeC-Sup 2'-OMe w/3' OH at 3' terminus omeG-Sup 2'-OMe w/3' OH at 3' terminus omeU-Sup 2'-OMe w/3' OH at 3' terminus dAs-Sup DNA w/3' thiophosphate at 3' terminus dCs-Sup DNA w/3' thiophosphate at 3' terminus dGs-Sup DNA w/3' thiophosphate at 3' terminus dTs-Sup DNA w/3' thiophosphate at 3' terminus dA-Sup DNA w/3' OH at 3' terminus dC-Sup DNA w/3' OH at 3' terminus dG-Sup DNA w/3' OH at 3' terminus dT-Sup DNA w/3' OH at 3' terminus

dU deoxyuridine w/3' OH at 3' terminus

rA RNA w/3' phosphate

rC RNA w/3' phosphate

rG RNA w/3' phosphate

rU RNA w/3' phosphate

The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The present invention is not to be limited in scope by examples provided, since the examples are intended as a single illustration of one aspect of the invention and other functionally equivalent embodiments are within the scope of the invention. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. The advantages and objects of the invention are not necessarily encompassed by each embodiment of the invention.

Claims

CLAIMS What is claimed is:

1. A single stranded oligonucleotide comprising a region of complementarity that is complementary to at least 8 consecutive nucleotides of a first sequence corresponding to nucleotide 511 to nucleotide 920 of SEQ ID NO: 3.

2. The single stranded oligonucleotide of claim 1, wherein the oligonucleotide has at least one of the following features:

a) a sequence that is 5'X-Y-Z, wherein X is any nucleotide and wherein X is anchored at the 5' end of the oligonucleotide, Y is a nucleotide sequence of 6 nucleotides in length that is not a human seed sequence of a microRNA, and Z is a nucleotide sequence of 1 to 23 nucleotides in length;

b) a sequence that does not comprise three or more consecutive guanosine

nucleotides;

c) a sequence that does not bind to an off-target sequence that is located beyond 50 kilobases upstream of a 5 '-end of a FOXP3 gene or beyond 50 kilobases downstream of a 3'- end of the FOXP3 gene;

d) a sequence comprising the region of complementarity that is complementary to the at least 8 consecutive nucleotides of the first sequence, wherein the at least 8 consecutive nucleotides encode a portion of an RNA that forms a secondary structure comprising at least two single stranded loops;

e) a sequence that has greater than 60% G-C content; and/or

f) a sequence that is not complementary to at least 8 consecutive nucleotides of a second sequence corresponding to nucleotide 1 to nucleotide 510 of SEQ ID NO: 3.

3. The single stranded oligonucleotide of claim 1 or 2, wherein the

oligonucleotide is 8 to 30 nucleotides in length.

4. The single stranded oligonucleotide of any one of claims 1 to 3, wherein at least one nucleotide of the oligonucleotide is a nucleotide analogue.

5. The single stranded oligonucleotide of claim 4, wherein the at least one nucleotide analogue results in an increase in Tm of the oligonucleotide in a range of 1 to 5 °C compared with an oligonucleotide that does not have the at least one nucleotide analogue.

6. The single stranded oligonucleotide of any one of claims 1 to 5, wherein at least one nucleotide of the oligonucleotide comprises a 2' O-methyl.

7. The single stranded oligonucleotide of any one of claims 1 to 6, wherein each nucleotide of the oligonucleotide comprises a 2' O-methyl.

8. The single stranded oligonucleotide of any one of claims 1 to 7, wherein the oligonucleotide comprises at least one ribonucleotide, at least one deoxyribonucleotide, or at least one bridged nucleotide.

9. The single strand oligonucleotide of claim 8, wherein the bridged nucleotide is a

LNA nucleotide, a cEt nucleotide or a ENA modified nucleotide.

10. The single stranded oligonucleotide of any one of claims 1 to 5, wherein each nucleotide of the oligonucleotide is a LNA nucleotide.

11. The single stranded oligonucleotide of any one of claims 1 to 5, wherein the nucleotides of the oligonucleotide comprise alternating deoxyribonucleotides and 2'-fluoro- deoxyribonucleotides .

12. The single stranded oligonucleotide of any one of claims 1 to 5, wherein the nucleotides of the oligonucleotide comprise alternating deoxyribonucleotides and 2'-0- methyl nucleotides.

13. The single stranded oligonucleotide of any one of claims 1 to 5, wherein the nucleotides of the oligonucleotide comprise alternating deoxyribonucleotides and ENA nucleotide analogues.

14. The single stranded oligonucleotide of any one of claims 1 to 5, wherein the nucleotides of the oligonucleotide comprise alternating deoxyribonucleotides and LNA nucleotides.

15. The single stranded oligonucleotide of any one of claims 11 to 14, wherein the 5' nucleotide of the oligonucleotide is a deoxyribonucleotide.

16. The single stranded oligonucleotide of any one of claims 1 to 5, wherein the nucleotides of the oligonucleotide comprise alternating LNA nucleotides and 2' -O-methyl nucleotides.

17. The single stranded oligonucleotide of claim 16, wherein the 5' nucleotide of the oligonucleotide is a LNA nucleotide.

18. The single stranded oligonucleotide of any one of claims 1 to 6, wherein the nucleotides of the oligonucleotide comprise deoxyribonucleotides flanked by at least one LNA nucleotide on each of the 5' and 3' ends of the deoxyribonucleotides.

19. The single stranded oligonucleotide of any one of claims 1 to 18, further comprising phosphorothioate internucleotide linkages between at least two nucleotides.

20. The single stranded oligonucleotide of claim 19, further comprising phosphorothioate internucleotide linkages between all nucleotides.

21. The single stranded oligonucleotide of any one of claims 1 to 20, wherein the nucleotide at the 3' position of the oligonucleotide has a 3' hydroxyl group.

22. The single stranded oligonucleotide of any one of claims 1 to 20, wherein the nucleotide at the 3' position of the oligonucleotide has a 3' thiophosphate.

23. The single stranded oligonucleotide of any one of claims 1 to 22, further comprising a biotin moiety conjugated to the 5' nucleotide.

24. A single stranded oligonucleotide comprising a region of complementarity that is complementary to at least 8 consecutive nucleotides of SEQ ID NO: 3, wherein nucleotides of the oligonucleotide comprise alternating LNA nucleotides and 2'-0-methyl nucleotides, and wherein the oligonucleotide further comprises phosphorothioate intemucleotide linkages between all nucleotides.

25. The single stranded oligonucleotide of claim 24, wherein the oligonucleotide has at least one of the following features:

a) a sequence that does not comprise three or more consecutive guanosine

nucleotides;

b) a sequence that does not bind to an off-target sequence that is located beyond 50 kilobases upstream of a 5 '-end of a FOXP3 gene or beyond 50 kilobases downstream of a 3'- end of the FOXP3 gene;

c) a sequence comprising the region of complementarity that is complementary to the at least 8 consecutive nucleotides of the first sequence, wherein the at least 8 consecutive nucleotides encode a portion of an RNA that forms a secondary structure comprising at least two single stranded loops;

d) a sequence that has greater than 60% G-C content; and/or

e) a sequence that is not complementary to at least 8 consecutive nucleotides of a second sequence corresponding to nucleotide 1 to nucleotide 510 of SEQ ID NO: 3.

26. The single stranded oligonucleotide of claim 24 or 25, wherein the

oligonucleotide is 8 to 30 nucleotides in length.

27. The single stranded oligonucleotide of any one of claims 24 to 26, wherein at least one nucleotide of the oligonucleotide is a nucleotide analogue.

28. The single stranded oligonucleotide of claim 27, wherein the at least one nucleotide analogue results in an increase in Tm of the oligonucleotide in a range of 1 to 5 °C compared with an oligonucleotide that does not have the at least one nucleotide analogue.

29. The single stranded oligonucleotide of any one of claims 24 to 28, wherein the 5' nucleotide of the oligonucleotide is a LNA nucleotide.

30. The single stranded oligonucleotide of any one of claims 1 to 29, wherein the oligonucleotide has a sequence set forth as: GCTCGGTAGTCCTCC (SEQ ID NO: 4).

31. The single stranded oligonucleotide of any one of claims 1 to 29, wherein the oligonucleotide has a sequence set forth as: GCGGAGGAAGTAGCT (SEQ ID NO: 5).

32. The single stranded oligonucleotide of any one of claims 1 to 29, wherein the oligonucleotide has a sequence set forth as: GGTTGCGGTCAGTGG (SEQ ID NO: 6).

33. The single stranded oligonucleotide of any one of claims 1 to 29, wherein the oligonucleotide has a sequence set forth as: CCCACAGTACCGTCC (SEQ ID NO: 7).

34. The single stranded oligonucleotide of any one of claims 1 to 29, wherein the oligonucleotide has a sequence set forth as: CCCTGATCCATGCCT (SEQ ID NO: 8).

35. The single stranded oligonucleotide of any one of claims 1 to 29, wherein the oligonucleotide has a sequence set forth as: ATACCCCGTGTCTCC (SEQ ID NO: 9).

36. The single stranded oligonucleotide of any one of claims 1 to 29, wherein the oligonucleotide has a sequence set forth as: CTTGAGTCCCGTGCA (SEQ ID NO: 10).

37. The single stranded oligonucleotide of any one of claims 1 to 29, wherein the oligonucleotide has a sequence set forth as: AGCAGCGTCAGTACC (SEQ ID NO: 11).

38. The single stranded oligonucleotide of any one of claims 1 to 29, wherein the oligonucleotide has a sequence set forth as: ACGCACCCACAGCCA (SEQ ID NO: 12).

39. The single stranded oligonucleotide of any one of claims 1 to 29, wherein the oligonucleotide has a sequence set forth as: AGCCAAACAGAGCCT (SEQ ID NO: 13).

40. A composition comprising a single stranded oligonucleotide of any one of claims 1 to 39 and a carrier.

41. A composition comprising a single stranded oligonucleotide of any one of claims 1 to 39 in a buffered solution.

42. The composition of claim 41, wherein the oligonucleotide is conjugated to the carrier.

43. The composition of claim 42, wherein the carrier is a peptide.

44. The composition of claim 42, wherein the carrier is a steroid.

45. A pharmaceutical composition comprising a composition of any one of claims 40 to 45 and a pharmaceutically acceptable carrier.

46. A method of increasing expression of FOXP3 in a cell, the method comprising delivering to the cell the single stranded oligonucleotide of any one of claims 1 to 39.

47. The method of claim 46, wherein delivery of the single stranded

oligonucleotide into the cell results in a level of expression of FOXP3 that is at least 50% greater than a level of expression of FOXP3 in a control cell that does not comprise the single stranded oligonucleotide.

48. A method of treating a condition or disease associated with decreased levels of FOXP3 in a subject, the method comprising administering to the subject the single stranded oligonucleotide of any one of claims 1 to 39 or the composition of any one of claims 40 to 45.

49. The method of claim 48, wherein the condition or disease is associated with aberrant immune cell activation.

50. A method of increasing expression of FOXP3 in a cell, the method comprising delivering to the cell an inhibitor of CCDC22 in an amount effective for increasing expression of FOXP3 in the cell.

51. The method of claim 50, wherein delivery of the inhibitor of CCDC22 into the cell results in (i) a level of expression of CCDC22 that is at least 50% lower than a level of expression of CCDC22 in a control cell that does not comprise the inhibitor of CCDC22; and (ii) a level of expression of FOXP3 that is at least 50% higher than a level of expression of FOXP3 in the control cell.

52. The method of claim 50 or 51, wherein the inhibitor of CCDC22 is an oligonucleotide having a region of complementarity that is complementary with at least 8 consecutive nucleotides of a CCDCC22 mRNA.

53. The method of claim 52, wherein the region of complementarity is complementary with at least 8 consecutive nucleotides of exon 1 of the CCDCC22 mRNA.

54. The method of claim 52, wherein the region of complementarity is complementary with at least 8 consecutive nucleotides of exon 2 of the CCDCC22 mRNA.

55. The method of any one claims 52 to 54, wherein the oligonucleotide comprises a gapmer.

56. The method of claim 55, wherein the gapmer comprises a central region of at least 4 DNA nucleotides flanked one both sides by at least two nucleotide analogues.

57. The method of claim 56, wherein the at least two nucleotide analogues comprise at least one LNA or at least 2' O modified ribonucleotide.

58. The method of any one of claims 46 to 57, wherein the cell is a T cell.

59. A method of increasing FOXP3 expression in a T cell ex vivo for

administration to a subject, the method comprising:

a) providing a population of T cells comprising a FOXP3 gene;

b) contacting the T cells ex vivo with the single stranded oligonucleotide of any one of claims 1 to 39; and

c) administering the contacted T cells to the subject.

60. The method of claim 59, wherein the population of T cells comprises activated

T cells.

61. The method of claim 60, wherein the activated T cells are produced by contacting CD4-positive T cells with an activating agent.

62. The method of claim 61, wherein the activating agent is an anti-CD3 and/or anti-CD28 antibody.

63. The method of any one of claims 60-62, wherein the activated T cells express CD69 or IL-2RA.

64. The method of any one of claims 59-63, wherein contacting the T cells with the oligonucleotide increases the number of CD4+CD25+FOXP3+ T cells in the population.

65. The method of any one of claims 59-64, wherein the method further comprises transfecting the T cells with an expression construct encoding a chimeric antigen receptor (CAR) before, after or simultaneously with step b).

66. The method of any one of claims 59-64, wherein the method further comprises transfecting the T cells with an expression construct encoding a chimeric antigen receptor (CAR) after step b) and before step c).

67. The method of claim 65 or 66, wherein the CAR is specific for a self-antigen or an antigen that causes an inflammatory response.

68. The method of any one of claims 59-67, wherein the subject has an autoimmune or inflammatory disease or disorder.

69. The method of any one of claims 59-68, wherein the method further comprises:

d) transplanting a cell, tissue or organ into the subject.

70. The method of claim 69, wherein the method alleviates or prevents development of graft-versus-host disease in the subject.

71. The method of claim 69 or 70, wherein the cell, tissue or organ is allogeneic to the subject.

72. The method of any one of claims 59-71, wherein delivery of the

oligonucleotide into the T cells results in a level of expression of FOXP3 in the T cells that is at least 50% greater than a level of expression of FOXP3 in a control cell that does not comprise the oligonucleotide.

73. The method of any one of claims 59-72, wherein delivery of the

oligonucleotide into the T cells results in an increased level of CTLA4, GITR, and/or IL-10 expression in the T cells compared to an appropriate control cell that does not comprise the oligonucleotide.

74. The method of claim 73, wherein delivery of the oligonucleotide into the T cells results in a level of expression of CTLA4, GITR, and/or IL-10 in the T cells that is at least 30% greater than a level of expression of CTLA4, GITR, and/or IL-10 in a control cell that does not comprise the oligonucleotide.

75. A method of increasing FOXP3 expression in a T cell ex vivo for

administration to a subject, the method comprising:

a) providing a population of T cells comprising a FOXP3 gene; b) contacting the T cells ex vivo with an inhibitor of CCDC22; and c) administering the contacted T cells to the subject.

76. The method of claim 75, wherein the population of T cells comprises activated

77. The method of claim 76, wherein the activated T cells are produced by contacting CD4-positive T cells with an activating agent.

78. The method of claim 77, wherein the activating agent is an anti-CD3 and/or anti-CD28 antibody.

79. The method of any one of claims 76-78, wherein the activated T cells express CD69 or IL-2RA.

80. The method of any one of claims 75-79, wherein contacting the T cells with the inhibitor of CCDC22 increases the number of CD4+CD25+FOXP3+ T cells in the population.

81. The method of any one of claims 75-80, wherein the method further comprises transfecting the T cells with an expression construct encoding a chimeric antigen receptor (CAR) before, after or simultaneously with step b).

82. The method of any one of claims 75-80, wherein the method further comprises transfecting the T cells with an expression construct encoding a chimeric antigen receptor (CAR) after step b) and before step c).

83. The method of claim 81 or 82, wherein the CAR is specific for a self-antigen or an antigen that causes an inflammatory response.

84. The method of any one of claims 75-83, wherein the subject has an

autoimmune or inflammatory disease or disorder.

85. The method of any one of claims 75-84, wherein the method further comprises:

d) transplanting a cell, tissue or organ into the subject.

86. The method of claim 85, wherein the method alleviates or prevents development of graft-versus-host disease in the subject.

87. The method of claim 85 or 86, wherein the cell, tissue or organ is allogeneic to the subject.

88. The method of any one of claims 75-87, wherein delivery of the inhibitor of CCDC22 into the T cells results in a level of expression of FOXP3 in the T cells that is at least 50% greater than a level of expression of FOXP3 in a control cell that does not comprise the inhibitor of CCDC22.

89. The method of any one of claims 75-88, wherein delivery of the inhibitor of CCDC22 into the T cells results in an increased level of CTLA4, GITR, and/or IL- 10 expression in the T cells compared to an appropriate control cell that does not comprise the inhibitor of CCDC22.

90. The method of claim 89, wherein delivery of the inhibitor of CCDC22 into the T cells results in a level of expression of CTLA4, GITR, and/or IL-10 in the T cells that is at least 30% greater than a level of expression of CTLA4, GITR, and/or IL-10 in a control cell that does not comprise the inhibitor of CCDC22.

91. The method of any one of claims 75-90, wherein delivery of the inhibitor of CCDC22 into the T cells results in (i) a level of expression of CCDC22 that is at least 50% lower than a level of expression of CCDC22 in a control cell that does not comprise the inhibitor of CCDC22; and (ii) a level of expression of FOXP3 that is at least 50% higher than a level of expression of FOXP3 in the control cell.

92. The method of any one of claims 75-91, wherein the inhibitor of CCDC22 is an oligonucleotide having a region of complementarity that is complementary with at least 8 consecutive nucleotides of a CCDCC22 mRNA.

93. The method of claim 92, wherein the region of complementarity is complementary with at least 8 consecutive nucleotides of exon 1 of the CCDCC22 mRNA.

94. The method of claim 92, wherein the region of complementarity is complementary with at least 8 consecutive nucleotides of exon 2 of the CCDCC22 mRNA.