WO2014197826A1

WO2014197826A1 - Compositions and methods for modulating foxp3 expression

Info

Publication number: WO2014197826A1
Application number: PCT/US2014/041345
Authority: WO
Inventors: James Barsoum; Mariano SEVERGNINI; James Mcswiggen
Original assignee: Rana Therapeutics, Inc.
Priority date: 2013-06-07
Filing date: 2014-06-06
Publication date: 2014-12-11
Also published as: EP3004354A4; AU2014274730A1; US20160122760A1; CA2914536A1; EP3004354A1; JP2016521556A; KR20160027968A

Abstract

Aspects of the invention provide single stranded oligonucleotides for activating or enhancing expression of FOXP3. Further aspects provide compositions and kits comprising single stranded oligonucleotides for activating or enhancing expression of FOXP3. Methods for modulating expression of FOXP3 using the single stranded oligonucleotides are also provided. Further aspects of the invention provide methods for selecting a candidate oligonucleotide for activating or enhancing expression of FOXP3.

Description

COMPOSITIONS AND METHODS FOR MODULATING 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. 61/832,677, entitled "COMPOSITIONS AND METHODS FOR

MODULATING FOXP3 EXPRESSION", filed June 7, 2013, the contents of which are incorporated herein by reference in its entirety. FIELD OF THE INVENTION

The invention relates to oligonucleotide based compositions, as well as methods of using oligonucleotide based compositions for treating disease.

BACKGROUND OF THE INVENTION 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. SUMMARY OF THE INVENTION

Aspects of the invention disclosed herein provide methods and compositions that are useful for upregulating FOXP3 in cells. In some embodiments, single stranded

oligonucleotides are provided that target a PRC2- associated region of an FOXP3 gene (e.g. , human FOXP3) and thereby cause upregulation of the gene. In some embodiments, single stranded oligonucleotides are provided that target a PRC2-associated region of the gene encoding FOXP3. In some embodiments, these single stranded oligonucleotides activate or enhance expression of FOXP3 by relieving or preventing PRC2 mediated repression of FOXP3. Aspects of the invention disclosed herein 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.

Further aspects of the invention provide methods for selecting oligonucleotides for activating or enhancing expression of FOXP3. In some embodiments, methods are provided for selecting a set of oligonucleotides that is enriched in candidates (e.g. , compared with a random selection of oligonucleotides) for activating or enhancing expression of FOXP3. Accordingly, the methods may be used to establish sets of clinical candidates that are enriched in oligonucleotides that activate or enhance expression of FOXP3. Such libraries may be utilized, for example, to identify lead oligonucleotides for developing therapeutics to treat FOXP3. Furthermore, in some embodiments, oligonucleotide chemistries are provided that are useful for controlling the pharmacokinetics, biodistribution, bioavailability and/or efficacy of the single stranded oligonucleotides for activating expression of FOXP3.

According to some aspects of the invention 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 of the nucleotide sequence set forth as SEQ ID NO: 1, 2, 5, 6, 7, 46, or 47. 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 has less than a threshold level of sequence identity with every sequence of nucleotides, of equivalent length to the second nucleotide sequence, that are between 50 kilobases upstream of a 5 '-end of an off-target gene and 50 kilobases downstream of a 3 '-end of the off-target gene; d) 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; and e) a sequence that has greater than 60% G-C content. 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 certain embodiments, the oligonucleotide has the sequence 5'X-Y-Z, in which the oligonucleotide is 8-50 nucleotides in length.

According to some aspects of the invention, single stranded oligonucleotides are provided that have a sequence X-Y-Z, in which X is any nucleotide, Y is a nucleotide sequence of 6 nucleotides in length that is not a seed sequence of a human microRNA, and Z is a nucleotide sequence of 1 to 23 nucleotides in length, in which the single stranded oligonucleotide is complementary with a PRC2- associated region of a FOXP3 gene, e.g. , a PRC2-associated region of the nucleotide sequence set forth as SEQ ID NO: 1, 2, 5, 6, 7, 46, or 47. In some aspects of the invention, single stranded oligonucleotides are provided that have a sequence 5' -X-Y-Z, in which X is any nucleotide, Y is a nucleotide sequence of 6 nucleotides in length that is not a seed sequence of a human microRNA, and Z is a nucleotide sequence of 1 to 23 nucleotides in length, in which the single stranded

oligonucleotide is complementary with at least 8 consecutive nucleotides of a PRC2- associated region of FOXP3 gene, e.g. , a PRC2- associated region of the nucleotide sequence set forth as SEQ ID NO: 1, 2, 5, 6, 7, 46, or 47. In some embodiments, Y is a sequence selected from Table 1. In some embodiments, the PRC2-associated region is a sequence listed in any one of SEQ ID NOS: 8-45 or 48-59.

In some embodiments, the single stranded oligonucleotide comprises a nucleotide sequence as set forth in any one of SEQ ID NOS: 60-45713, or a fragment thereof that is at least 8 nucleotides. In some embodiments, the single stranded oligonucleotide comprises a nucleotide sequence as set forth in any one of SEQ ID NOS: 60-45713, in which the 5' end of the nucleotide sequence provided is the 5' end of the oligonucleotide. In some embodiments, the region of complementarity (e.g. , the at least 8 consecutive nucleotides) is also present within the nucleotide sequence set forth as SEQ ID NO: 3 or 4.

In some embodiments, the PRC2-associated region is a sequence listed in any one of SEQ ID NOS: 8-45. In some embodiments, the single stranded oligonucleotide comprises a nucleotide sequence as set forth in any one of SEQ ID NOS: 16426-45713 or a fragment thereof that is at least 8 nucleotides. In some embodiments, the single stranded

oligonucleotide comprises a nucleotide sequence as set forth in any one of SEQ ID NOS: 16426-45713, wherein the 5' end of the nucleotide sequence provided in any one of SEQ ID NOS: 16426-45713 is the 5' end of the oligonucleotide. In some embodiments, the at least 8 consecutive nucleotides are also present within the nucleotide sequence set forth as SEQ ID NO: 4.

In some embodiments, the PRC2-associated region is a sequence listed in any one of

SEQ ID NOS: 48-59. In some embodiments, the single stranded oligonucleotide comprises a nucleotide sequence as set forth in any one of SEQ ID NOS: 60- 16461 or a fragment thereof that is at least 8 nucleotides. In some embodiments, the single stranded oligonucleotide comprises a nucleotide sequence as set forth in any one of SEQ ID NOS: 60-16461, wherein the 5' end of the nucleotide sequence provided in any one of SEQ ID NOS: 60- 16461 is the 5' end of the oligonucleotide. In some embodiments, the at least 8 consecutive nucleotides are also present within the nucleotide sequence set forth as SEQ ID NO: 3.

In some embodiments, a single stranded oligonucleotide comprises a nucleotide sequence as set forth in any one of SEQ ID NOS: 60- 16461. In some embodiments, the oligonucleotide is up to 50 nucleotides in length. In some embodiments, a single stranded oligonucleotide comprises a fragment of at least 8 nucleotides of a nucleotide sequence as set forth in any one of SEQ ID NOS: 60- 16461.

In some embodiments, a single stranded oligonucleotide comprises a nucleotide sequence as set forth in Table 4. In some embodiments, the single stranded oligonucleotide comprises a fragment of at least 8 nucleotides of a nucleotide sequence as set forth in Table 4. In some embodiments, a single stranded oligonucleotide consists of a nucleotide sequence as set forth in Table 4.

In some embodiments, a single stranded oligonucleotide, when delivered to a cell, is capable of increasing the level of CTLA4, GITR, and/or IL- 10 expression in the cell (e.g. , results in a level of expression of CTLA4, GITR, and/or IL- 10 that is at least 30% greater than a level of expression of CTLA4, GITR, and/or IL-10 in a control cell). In some embodiments, the cell is a T cell. In some embodiments, the single stranded oligonucleotide, when delivered to a population of T cells, is capable of increasing the number of

CD4+CD25+FOXP3+ T cells in the population of T cells (e.g. , results in a number of CD4+CD25+FOXP3+ T cells in the population that is at least 30% greater than a number of CD4+CD25+FOXP3+ T cells in a control population). In some embodiments, the single stranded oligonucleotide does not comprise three or more consecutive guanosine nucleotides. In some embodiments, the single stranded oligonucleotide does not comprise four or more consecutive guanosine nucleotides.

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 8 to 10 nucleotides in length and all but 1, 2, or 3 of the nucleotides of the complementary sequence of the PRC2- associated region are cytosine or guanosine nucleotides.

In some embodiments, the single stranded oligonucleotide is complementary with at least 8 consecutive nucleotides of a PRC2-associated region of a FOXP3 gene, e.g. , a PRC2- associated region of a nucleotide sequence set forth as SEQ ID NO: 1, 2, 5, 6, 7, 46, or 47, in which the nucleotide sequence of the single stranded oligonucleotide comprises one or more of a nucleotide sequence selected from the group consisting of

(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, wherein "X" denotes a nucleotide analogue, (X) denotes an optional nucleotide analogue, and "x" denotes a DNA or RNA nucleotide unit.

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' -O-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 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 invention compositions are provided that comprise any of the oligonucleotides disclosed herein, and a carrier. In some embodiments, compositions are provided that comprise any of the oligonucleotides in a buffered solution. In some embodiments, the oligonucleotide is conjugated to the carrier. In some embodiments, the carrier is a peptide. In some embodiments, the carrier is a steroid. According to some aspects of the invention pharmaceutical compositions are provided that comprise any of the oligonucleotides disclosed herein, and a pharmaceutically acceptable carrier.

According to other aspects of the invention, kits are provided that comprise a container housing any of the compositions disclosed herein.

According to some aspects of the invention, methods of increasing expression of FOXP3 in a cell are provided. In some embodiments, the methods involve delivering any one or more of the single stranded oligonucleotides disclosed herein into the cell. In some embodiments, delivery of the single stranded oligonucleotide into the cell results in a level of expression of FOXP3 that is greater (e.g. , at least 50% greater) than a level of expression of FOXP3 in a control cell that does not comprise the single stranded oligonucleotide. In some embodiments, delivery of the single stranded oligonucleotide into the cell results in an increased level of CTLA4, GITR, and/or IL- 10 expression compared to an appropriate control cell that does not comprise the singled stranded oligonucleotide. In some embodiments, delivery of the single stranded oligonucleotide into the cell results in a level of expression of CTLA4, GITR, and/or IL- 10 that is greater than (e.g. , 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. In some embodiments, the cell is a T cell.

According to some aspects of the invention, methods of increasing levels of FOXP3 in a subject are provided. According to some aspects of the invention, methods of 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 in a subject are provided. In some embodiments, the methods involve administering any one or more of the single stranded oligonucleotides disclosed herein to the subject. In some embodiments, administration of the single stranded oligonucleotide to the subject results in an increased level of CTLA4, GITR, and/or IL- 10 expression the subject compared to an appropriate control subject who has not been administered the single stranded oligonucleotide. In some embodiments, administration of the single stranded oligonucleotide to the subject results in a level of expression of CTLA4, GITR, and/or IL- 10 that is greater than (e.g. , at least 30% greater than) a level of CTLA4, GITR, and/or IL- 10 in the appropriate control subject who has not been administered the single stranded oligonucleotide. In some embodiments, administration of the single stranded oligonucleotide to the subject results in an increased level of CTLA4, GITR, and/or IL- 10 in a T cell of the subject compared to a T cell in the control subject who has not been administered the single stranded oligonucleotide. In some embodiments, administration of the single stranded oligonucleotide to the subject results in a level of expression of CTLA4, GITR, and/or IL- 10 in the T cell of the subject that is greater than (e.g. , at least 30% greater than) a level of CTLA4, GITR, and/or IL- 10 in the T cell in the control subject who has not been administered the single stranded oligonucleotide. In some embodiments, administration of the of the single stranded oligonucleotide to the subject results in an increased number of CD4+CD25+FOXP3+ T cells in the subject compared to a control subject who has not been administered the single stranded oligonucleotide. In some embodiments, administration of the single stranded oligonucleotide to the subject results in a number of

CD4+CD25+FOXP3+ T cells in the subject that is greater than (e.g. , at least 30% greater than) a number of CD4+CD25+FOXP3+ T cells in the control subject who has not been administered the single stranded oligonucleotide.

Other aspects of the invention relate to a method of increasing expression of FOXP3 in a cell, 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 by inhibiting or decreasing expression of EZH1 and/or EZH2 or another component of PRC2, e.g. , Suzl2, EED1, or RbAp48. In some embodiments, the method comprises delivering an oligonucleotide having a region of complementarity that is complementary with at least 8 consecutive nucleotides of an EZH1 mRNA or EZH2 mRNA to the cell. In some embodiments, the method comprises delivering to the cell a first oligonucleotide having a region of complementarity that is complementary with at least 8 consecutive nucleotides of an EZH1 mRNA and a second oligonucleotide having a region of complementarity that is complementary with at least 8 consecutive nucleotides of an EZH2 mRNA.

In some embodiments, the oligonucleotide is is 8 to 30 nucleotides in length. In some embodiments, at least one nucleotide of the oligonucleotide is a nucleotide analogue.

In some embodiments, the oligonucleotide comprises a gapmer. In some

embodiments, the gapmer comprises a central region of at least 4 DNA nucleotides flanked one both sides by at least two nucleotide analogues. In some embodiments, the at least two nucleotide analogues comprise at least one LNA or at least one 2'-0 modified ribonucleotide.

In some embodiments, the oligonucleotide comprises at least 8 nucleotides of a nucleotide sequence as set forth in Table 8. In some embodiments, the oligonucleotide comprises a nucleotide sequence as set forth in Table 8. In some embodiments, the oligonucleotide consists of a nucleotide sequence as set forth in Table 8. In some

embodiments, the oligonucleotide (e.g. , single stranded oligonucleotide) comprises a sequence as set forth in any one of SEQ ID NO: 45714-45717 or a complement of anyone of them.

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 between all nucleotides.

Other aspects of the invention relate to a single stranded oligonucleotide having a region of complementarity that is complementary with at least 8 consecutive nucleotides of an EZH1 mRNA or EZH2 mRNA. In some embodiments, a single stranded oligonucleotide comprises a nucleotide sequence as set forth in Table 8. In some embodiments, the single stranded oligonucleotide comprises a fragment of at least 8 nucleotides of a nucleotide sequence as set forth in Table 8. In some embodiments, a single stranded oligonucleotide consists of a nucleotide sequence as set forth in Table 8.

In some embodiments, the oligonucleotide comprises a gapmer. In some

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a series of four graphs showing the levels of CD69, CD62L, CDKN1A, and IL-2RA expression in human T cells that were activated with PMA and Ionomycin.

FIG. 2 is a diagram showing the location along the human FOXP3 gene where the FOXP3 oligos in Table 4 bind.

FIG. 3 is a series of two graphs showing the levels of CD62L and CD69 mRNA in human T cells activated with different concentrations of PMA and Ionomycin (IX or 2X), compared to T cells treated with DMSO, or untreated.

FIG. 4 is a series of two graphs showing the levels of CD62L and CD25 mRNA in cells activated with dynabeads at a ratio of 2: 1 beads to cells (left bars) or 1: 1 beads to cells (right bars).

FIG. 5 is a graph showing the downregulation of GAPDH mRNA with GAPDH gapmers at concentrations 0, 0.032, 0.16, 0.8, 4 and 20 uM delivered gymnotically to activated human T cells.

FIG. 6 is a graph showing FOXP3 mRNA levels at 48 hours in PMA/Iono activated human T cells treated with 10 uM FOXP3 oligos. Bars with stars indicate oligo treatments where stable housekeeper gene Ct values were observed.

FIG. 7 is a graph showing FOXP3 mRNA levels at 96 hours in dynabead activated human T cells treated with 10 uM FOXP3 oligos. FIG. 8 is a graph showing GAPDH mRNA levels at 96 hours in dynabead activated human T cells treated with FOXP3 oligos. Black colored bars indicate oligos where housekeeper gene varied more than 1.5Cts from negative control.

FIG. 9 is a graph showing CTLA4 mRNA levels at 96 hours in dynabead activated human T cells treated with FOXP3 oligos.

FIG. 10 is a graph showing GITR mRNA levels at 96 hours in dynabead activated human T cells treated with FOXP3 oligos.

FIG. 11 is a graph showing FOXP3 mRNA levels at 96 hours in dynabead activated human T cells treated with FOXP3 oligos.

FIG. 12 is a graph showing GAPDH mRNA levels at 96 hours in dynabead activated human T cells treated with FOXP3 oligos. Black colored bars indicate oligos where housekeeper gene varied more than 1.5Cts from negative control.

FIG. 13 is a graph showing FoxP3 fluorescent intensity at 96 hours in dynabead activated in CD4+CD25+FoxP3+ human T cells treated with FOXP3 oligos.

FIG. 14 is a diagram showing flow cytometry results in activated human T cells treated with a negative control oligo (293) and an exemplary FOXP3 oligo (FOXP3-35).

FIG. 15 is a graph showing the percentage of CD4+CD25+FoxP3+ cells at 96 hours in dynabead activated in human T cells treated with FOXP3 oligos.

FIG. 16 is a graph showing IL-10 protein levels at 96 hours in dynabead activated in CD4+CD25+FoxP3+ human T cells treated with FOXP3 oligos.

FIG. 17 is a graph showing MALAT-1 mRNA levels in sorted CD4+ cells from whole blood collected from mice treated with MALAT-1 gapmer oligos.

FIG. 18 is a graph showing MALAT-1 mRNA levels in liver collected from mice treated with MALAT-1 gapmer oligos.

FIG. 19 is a graph showing EZHl mRNA levels at 3 or 5 days in activated human T cells treated with EZHl gapmers, EZH2 gapmers, or combinations of EZHl and EZH2 gapmers. The left bar in each pair of bars is 3 days. The right bar in each pair of bars is 5 days.

FIG. 20 is a graph showing EZH2 mRNA levels at 3 or 5 days in activated human T cells treated with EZHl gapmers, EZH2 gapmers, or combinations of EZHl and EZH2 gapmers. The left bar in each pair of bars is 3 days. The right bar in each pair of bars is 5 days.

FIG. 21 is a graph showing FOXP3 mRNA levels at 3 or 5 days in activated human T cells treated with EZH1 gapmers, EZH2 gapmers, or combinations of EZH1 and EZH2 gapmers. The left bar in each pair of bars is 3 days. The right bar in each pair of bars is 5 days.

FIG. 22 is a heatmap showing mRNA expression of T cell genes after EZH1/2 knockdown.

FIG. 23 is a series of graphs showing flow cytometry data of FOXP3 protein levels in activated human T cells treated with EZH1 gapmers, EZH2 gapmers, or combinations of EZH1 and EZH2 gapmers.

BRIEF DESCRIPTION OF CERTAIN TABLES

Table 1: Hexamers that are not seed sequences of human miRNAs

Table 2: Experimental evaluation of single stranded oligonucleotides. SEQ ID

(column 1) refers to the SEQ ID NO: that corresponds to the base sequence of the oligonucleotide. The formatted sequence, including any modifications, for each

oligonucleotide is provided in Table 4. Oligo name (column 2) refers to the name for a given oligonucleotide and also refers to the same formatted oligonucleotide in Table 4. RQ (column 3) and AVG RQ SD (column 4) shows the expression level of the "probe" gene in a well containing oligo relative to a control well (carrier alone or a universal negative control oligo 293) and the standard deviation for the triplicate replicates of the experiment. Target (column 5) refers to the gene that is targeted by the oligonucleotide. Probe (column 6) refers to the gene whose expression was measured in a given assay. For example, Target FOXP3 and Probe GITR refers to an experiment where are oligo that targets FOXP3 was added to a well and the level of GITR was measured by qRT-PCR. The RQ and AVG RQ SD for that experiment would be the RQ and AVG RQ SD for GITR. [Oligo] is shown in nanomolar for in vitro experiments and in milligrams per kilogram of body weight for in vivo experiments.

Table 3: A listing of oligonucleotide modifications

Table 4: Formatted oligonucleotide sequences made for testing showing nucleotide modifications. The table shows the sequence of the modified nucleotides, where InaX represents an LNA nucleotide with 3' phosphorothioate linkage, omeX is a 2'-0-methyl nucleotide, dX is a deoxy nucleotide. An s at the end of a nucleotide code indicates that the nucleotide had a 3' phosphorothioate linkage. The "-Sup" at the end of the sequence marks the fact that the 3' end lacks either a phosphate or thiophosphate on the 3' linkage. The Formatted Sequence column shows the sequence of the oligonucleotide, including modified nucleotides, for the oligonucleotides tested in Table 2.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

Aspects of the invention 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.

Aspects of the invention 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 invention relate to compositions and methods for upregulating FOXP3.

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 has a sequence as set forth in any one of SEQ ID NOS: SEQ ID NOS: 8-45 or 48-59.

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 that has a sequence as set forth in any one of SEQ ID NOS: 8-45 or 48-59. In some embodiments, single stranded oligonucleotides are provided that specifically bind to, or are complementary to, a PRC2-associated region that has a sequence as set forth in any one of SEQ ID NOS: 8-45 or 48-59 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). In some embodiments, single stranded oligonucleotides have a sequence as set forth in any one of SEQ ID NOS: 60-45713. In some embodiments, a single stranded oligonucleotide has a sequence as set forth in Table 4.

Without being bound by a theory of invention, 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.

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 throughout the description contemplates use of the compound 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, this aspect of the invention 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 invention, 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., a nucleotide sequence as set forth in any one of SEQ ID NOS: 8-45 or 48-59). 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 o/FOXP3

In one aspect of the invention, 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 single stranded oligonucleotide may comprise a region of complementarity that is complementary with a PRC2-associated region of a nucleotide sequence set forth in any one of SEQ ID NOS: 1-7, 46, or 47. 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 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. 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 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 1.

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. The threshold level of sequence identity may be 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity.

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 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 sequence as set forth in SEQ ID NO: 1, 2, 5, 6, 7, 46, or 47, which is a human genomic region encompassing or in proximity to the FOXP3 gene, and also be complementary to a sequence as set forth in SEQ ID NO: 3 or 4, 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. 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 contain 1, 2 or 3 base mismatches compared to the portion of the consecutive nucleotides of a PRC2-associated region. In some embodiments the 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 target 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, it has been found that 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, it has been found that 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 of FOXP3

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 -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 (e.g., a nucleotide sequence as set forth in any one of SEQ ID NOS: 8-45 or 48-59) that maps to a chromosomal position encompassing or in proximity to the FOXP3 gene (e.g. , a chromosomal position having a sequence as set forth in any one of SEQ ID NOS: 1-7, 46, or 47). The PRC2-associated region may map to the strand of the 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 first 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).

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. 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, T_m, 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 of the invention are synthesized chemically. Oligonucleotides used to practice this invention can be synthesized in vitro by well-known chemical synthesis techniques.

Oligonucleotides of the invention can be stabilized against nucleolytic degradation such as by the incorporation of a modification, e.g., a nucleotide modification. For example, nucleic acid sequences of the invention 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 2'-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'- O 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

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 T_m 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 T_m 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 15₅ 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 15₅ 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 of the invention 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-O-, -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 Ci-4-alkyl; Z and Z* are independently selected among an internucleoside 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(R ); 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 Ci-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 of the invention 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(R^H)-0-, o- PO(OCH₃)-0-, -0-PO(NR^H)-0-, -0-PO(OCH₂CH₂S-R)-O-, -0-PO(BH₃)-0-, -0-PO(NHR^H)- 0-, -0-P(0)₂-NR^H-, -NR^H-P(0)₂-0-, -NR^H-CO-0-, where R^H is selected from hydrogen and Ci_4-alkyl.

Specifically preferred LNA units are shown in scheme 2:

j$~0^«thio-kNA

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 Ci-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; CF ; OCF ; 0-, S-, or N-alkyl; 0-, S-, or N-alkenyl; SOCH₃; S0₂ CH₃; ON0₂; N0₂; N₃; NH2; heterocycloalkyl; heterocyclo alkaryl; 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-CH₂CH₂OCH₃, 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 hetero substituted 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-trifluoromethyl 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 oligomeric compounds of the invention. 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 of the invention 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 invention, 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 invention, include groups that improve uptake, distribution, metabolism or excretion of the compounds of the present invention. 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,

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

(d) (X)xxXXX, (X)xXxXXX, (X)xXXxXX, (X)xXXXxX, (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 gapmer, siRNA, miRNA or other oligonucleotide that inhibits expression of a target 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_001203248.1, 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, gapmer oligonucleotides are provided herein. 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 be 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 of the invention 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.

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 of the present invention 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.

Methods for Modulating Gene Expression

In one aspect, the invention 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 invention 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 invention, 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.

In another aspect of the invention 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.

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 regimes 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 in accordance with this invention. 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 of the invention) 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 of this invention 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 mediated 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 of the invention 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 of the present invention 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 pH 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, the invention features 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). 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 of the invention 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 invention, kits are provided, 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 invention 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

The invention is further described in the following examples, which do not limit the scope of the invention described in the claims. EXAMPLE 1

MATERIALS AND METHODS:

Real Time (Quantitative) PCR

RNA was harvested from the cells and converted to cDNA as described below. Real time PCR (also referred to herein as quantitative PCR or qPCR or qRTPCR) was performed in a 96 well format with a final volume of 20ul/well. Each well included 2.5ul of cDNA, 5.5ul of water, lul of target gene probe, lul of housekeeper probe and 10 ul of TaqMan® Fast Advanced master mix (Life Technologies™, Invitrogen).

Target gene probes for qPCR for detection of human specific FoxP3, IL2RA, CD69, CD62L, CDKNIA, TNFRSF18 (GITR) and B-Actin (Life Technologies) were used to detect mRNA levels. The target gene probes were labeled with Fam™ and the housekeeper probe was labeled with VIC® (Life Technologies™, Invitrogen).

An Applied Biosystems® StepOne™ Plus Real Time PCR System machine was used to read the samples and determine levels of mRNA. The following cycle program was used:

Step 1 : 1 Cycle of 95 degrees Celsius for 20 seconds

Step 2: 40 cycles of 95 degrees Celsius for 3 seconds

Step 3: 40 cycles of 60 degrees Celsius for 20 seconds

A baseline level of mRNA expression for each target gene was determined. Baseline levels were also determined for mRNA of various housekeeping genes which are

constitutively expressed. A "control" housekeeping gene with approximately the same level of baseline expression as the target gene was chosen for comparison purposes. Quantitative PCR data presented in Table 2 is represented as RQ (relative quantification): Target dCT= Target Ct - Housekeeper Ct; ddCT= Target dCT - Negative control dCT (unc-293 mOl, which is a universal negative control oligonucleotide); RQ= Log2 - ddCT. Oligonucleotide design

Oligonucleotides were designed within PRC2-interacting regions in order to upregulate FOXP3 (see, e.g., FIG. 2). The sequence and structure of each oligonucleotide is shown in Table 4. Table 3 provides a description of the nucleotide analogs, modifications and intranucleotide linkages used for certain oligonucleotides tested and described in Table 2 or Table 4.

T Cell Isolation and Culture

Human T cells were obtained from one healthy male and one healthy female donor, both donors were of similar age and health status. For each experiment, T cells were freshly isolated from the donors and sorted for CD4-positivity using fluorescent activated cell sorting (FACS). The human T cells (Stem Cells Technologies) were cultured in RPMI 1640/10% fetal bovine serum in the presence of Anti-Anti (Antibiotic- Antimycotic, Life

Technologies™, Invitrogen). Cells were thawed and cultured for one day before stimulation.

T Cell Stimulation

T-Cells stimulation was performed by using 5ng/ml of Phorbol 12-myristate 13- acetate (PMA) and luM of Ionomycin (Sigma) for 5 hours. For 2X concentrations doses of PMA and Ionomcin were double. The initial T-cell stimulation conditions were determined using T cells from the healthy male donor. The screening of the oligonucleotides (described below) was carried out with T cells from the healthy female donor (referred to as huTcell+ in Table 2).

In vitro transfection of cells with oligonucleotides

T-cells were stimulated by PMA/Ionomycin for 5 hours as described above. After stimulation, cells were plated into a 96 well V-bottom plate format with approximately 100,000 cells/well. Each well contained the adequate oligonucleotide amount to produce a final concentration of lOuM of oligonucleotide. Final volume per well was lOOul. The same method was used for dose response experiments. The oligonucleotides were delivered gymnotically. The term gymnotic (or gymnotically), with reference to delivery, refers to unassisted uptake of agents into cells without use of transfection reagents or delivery to a subject without transfection reagents. After 48 hours cells were spun down at 2000 RPM, 4C for 5 minutes, washed with ice cold PBS (Life Technologies) once and a cell lysate was generated using a Cell-to-Ct kit (Life Technologies). The amount of buffer used was

35ul/well. cDNA was generated utilizing 15ul of lysate for a total of 50ul of reaction volume. Quantitative RT-PCR was then carried out as outlined above.

RESULTS:

Use of PMA and Ionomycin to stimulate T cells

T cells were stimulated with either (a) 5ng/ml PMA and luM Ionomycin, (b) lOng/ml PMA and 2uM Ionomycin, (c) DMSO alone, or (d) no treatment. Biomarkers for activation and proliferation of the T cells were evaluated to determine whether PMA and Ionomycin treatment successfully stimulated the T cells. CDKNIA is a housekeeper gene that is upregulated upon or during active cell proliferation. CD69 and IL-2RA are known to be upregulated in activated T cells. CD69, CDKNIA, and IL-2RA were found to be upregulated upon stimulation with PMA and Ionomycin (FIG. 1). CD62L, a biomarker for naive T cells, was found to be downregulated upon stimulation with PMA and Ionomycin (FIG. 1). These results show that PMA and Inomycin were able to stimulate T cells.

In vitro delivery of single stranded oligonucleotides upregulated FOXP3 expression

Oligonucleotides were designed as candidates for upregulating FOXP3 expression. Single stranded oligonucleotides were designed to be complementary to a PRC2-interacting region within a sequence as set forth in SEQ ID NO: 1-7, 46, or 47. Multiple

oligonucleotides were tested in at least duplicate. The sequence and structural features of the oligonucleotides are set forth in Table 4. Briefly, T-cells were stimulated as described above and then gymnotically transfected in vitro with each of the oligonucleotides as described above. The unc-293 mOl oligo is a universal negative control oligo. "Cntl un" refers to a well that contained no oligonucleotide, which also served as a negative control. FOXP3 expression in stimulated T cells following treatment was evaluated by qRT-PCR.

Oligonucleotides that upregulated FOXP3 expression were identified. A subset of the oligonucleotides that upregulated FOXP3 were further tested for expression of two T cell biomarkers, GITR (also called TNFRSF18) and IL2RA. The levels of these two biomarkers were measured by qRT-PCR. GITR is a biomarker for Tregs and thus increased expression of this biomarker may indicate that the activated T cells are switching to a T-regulatory state. IL2RA is biomarker for activated T cells and thus a decrease in IL2RA may indicate a decrease in the T cell activation state (e.g., switching to a T-regulatory state). Further details regarding FOXP3 and T cell biomarker expression are outlined in Table 2.

EXAMPLE 2 METHODS: T cell activation by PMA/Ionomycin

Cells were incubated for 5 hours with two different concentrations of

PMA/Ionomycin (IX and 2X). IX and 2X concentrations of PMA/Ionomycin were the same as defined in Example 1. Naive T cells were used as control (untreated). CD62L (Naive cell marker) and CD69 (activated cell marker) mRNA levels were measured (FIG. 3).

T cell activation by Dynabeads

Human T cells were incubated with different ratios of Dynabeads (2: 1 or 1: 1 beads/cells ratios) for 2, 5, 24 and 48 hours. Naive T cells were used as control (untreated). CD62L (naive T cell marker) and CD25 (activated T cell marker) mRNA levels were measured to test T cell activation (FIG. 4).

Screening

Cells were activated with PMA/Ionomycin or Dynabeads (anti-CD3/CD28) for 5 hours prior to adding oligos. GAPDH gapmers were used to determine an optimized transfection method. The FOXP3 oligos in Table 4 were then screened by gymnotic delivery (unassisted uptake of oligonucleotides into cells without use of transfection reagents) to cells, at ΙΟμΜ for 24, 48 and 96 hours. Two separate experiments were performed with two biological replicates each. Foxp3 mRNA levels were measured by qPCR and Foxp3 protein levels were measured by flow cytometry. GITR and CTLA4 (Treg biomarkers) mRNA levels were also measured after Foxp3 oligo treatment. Anti-inflammatory cytokine IL-10 was measured in cells supernatants derived from triple positive cells: Foxp3+, CTLA4+ and GITR+. Certain FOXP3 oliogos from Table were selected as having desired properties by the following criteria: Foxp3 mRNA and protein levels, presence of Tregs biomarkers.

RESULTS:

Human T cells were activated using PMA/Ionomycin. Activation of T cells was confirmed by measuring CD62L and CD69 mRNA levels (FIG. 3). GAPDH gapmer oligos were used to show that oligos could be delivered to activated human T cells. GAPDH gapmers showed up to 70% mRNA knockdown in PMA/Iono activated T cells at 4 and 20 μΜ after 48 hours of oligo treatment (FIG. 5).

Next, human T cells were activated either using PMA/Ionomycin or dynabeads. Activation of T cells was confirmed by measuring CD62L and CD69 or CD62L and CD25 mRNA levels (FIGs. 3 and 4). FOXP3 oligos from Table 4 (FOXP3-01 to FOXP3-60) were delivered gynmnotically at ΙΟμΜ to the activated T cells. The FOXP3 oligos showed 2-6 fold Foxp3 mRNA upregulation compared to a negative control oligo (293) in

PMA/Ionomycin activated cells (FIG. 6). The FOXP3 oligos showed 2-8 fold Foxp3 mRNA upregulation compared to a negative control oligo (293) in Dynabead activated T cells (FIG. 7). In the dynabead-activated T cells, housekeeper gene (GAPDH) Ct levels were very consistent between treatments (FIG. 8).

The level of CTLA4 mRNA and GITR mRNA, both Treg biomarkers, were also measured after delivery of the FOXP3 oligos to Dynabead activated T cells. The FOXP3 oligos were shown, in general, to upregulate CTLA4 mRNA levels after 96 hours of treatment (FIG. 9). The FOXP3 oligos were shown, in general, to upregulate GITR mRNA levels after 96 hours of treatment, with over 20 oligos showing upregulation (FIG. 10). A second experiment was performed to confirm that the FOXP3 oligos upregulated FOXP3 mRNA levels Dynabead activated T cells. In general, the FOXP3 oligos upregulated FOXP3 mRNA levels by 2-10 fold in the activated T cells (FIG. 11). FOXP3 oligos that caused housekeeper gene changes of more than 1.5 Cts were not considered positives (FIG. 12).

Next, the level of Foxp3 protein in oligo treated activated human T cells was detected using flow cytometry. Foxp3 protein levels were measured in cells that were CD4+, CD25+, and FoxP3+. In general, FOXP3 oligos (e.g. , FOXP3-2 to FOXP3-60) were found to increase Foxp3 protein levels in the triple positive human T cells (FIG. 13). Raw flow cytometry data from an exemplary oligo, FOXP3-35, is shown in FIG. 14, demonstrating the difference in FOXP3 expression compared to cells treated with a negative control oligo (293).

The percentage of triple positive Treg cells (CD4+CD25+FoxP3+) compared to the total cell population was next investigated. It was found that several oligos (e.g. , FOXP3-3, FOXP3-5 to FOXP3-44, FOXP3-46 to FOXP3-50, FOXP3-52-60) increased the Treg cell population by more than 2 fold compared to a oligo control.

In some embodiments, several FOXP3 oligos were selected as possible lead molecules according to the following criteria: FoxP3 mRNA and protein levels, percent of Tregs within the total cell population and CTLA4/GITR mRNA expression. A summary of results from oligos from two experiments is provided in Tables 5 and 6. Oligos FOXP3-28, FOXP3-29, FOXP3-30 and FOXP3-57 showed positive biomarkers expression (i.e., met the criteria) in both experiments.

Table 5. Experiment #1 exemplary results

Oligo # FoxP3 Tregs % fold FoxP3 MFI CTLA4 GITR mRNA mRNA fold upregulation fold mRNA fold fold upregulation compared to upregulation upregulation upregulation compared to control oligo compared to compared to compared to control oligo 293 control oligo control oligo control oligo 293 293 293 293

293 1 1 1 1

¹

Untreated 1 1 1 1.1

¹

13 5.5 1.8 1.2 1.3 2.4

22 2.2 1.8 1.2 1.3 2.4

23 3.5 1.8 1.3 0.8 1.5

28 3 1.9 1.4 1.7 2.8

29 3 2.1 1.5 1.6 1.7

30 4.5 2.1 1.5 1.2 1.4

49 10 1.2 1.2 1 2.2

52 3.5 1.8 1.5 1.6 1.7

54 5 1.8 1.5 0.9 0.9

57 2.6 1.8 1.6 2.3 2

59 3.8 1.8 1.7 1.7 1.6

MFI = Mean F uorescence Intensity

Table 6. Experiment #2 results

293 1 1 1 1 1

Untreated 1 1 1 1 1

3 3.9 1.7 1.4 3.8 2

5 2.1 1.6 1.4 2.3 1.9

15 2.2 1.6 1.5 2.1 1.6

26 2.2 1.5 1.4 3.8 1.5

27 3.8 1.6 1.4 3.2 3.8

28 2.2 1.6 1.4 3.4 3.8

29 2.2 1.6 1.5 4.4 4.2

30 3.8 1.7 1.4 3.1 2

38 2 1.5 1.3 1.9 1.9

41 4 1.7 1.5 5.1 2.3

53 4.1 2.7 1.5 2.1 2.1

57 3.1 1.1 1.3 2.9 2.9

Oligos that were selected based on triple positive markers (CD4+/CD25+/FoxP3+) upregulated IL-10 protein, an immunosuppressive cytokine (FIG. 16). In summary, the results in this Example show that FOXP3 oligos are capable of upregulating FOXP3, as well as several Treg biomarkers and IL-10.

EXAMPLE 3 METHODS:

Mice (n=4) were treated with a single dose of 25mg/kg gapmer targeting MALAT- 1. Blood and liver were harvested after 1, 5 and 7 days from dosing. CD4+ cells were sorted from blood and MALAT- 1 mRNA was measured by qPCR. RESULTS:

MALAT- 1 gapmers were used to show that oligos can be delivered in vivo to T cells. It was shown that a single dose of MALAT- 1 gapmer oligonucleotides could reduce levels of MALAT- 1 mRNA in vivo in CD4+ T cells and in the liver (FIGs. 17 and 18). These results show that oligos can be successfully delivered to T cells in vivo. EXAMPLE 4

Potential lead oligos identified in Example 2 are tested in mouse models, as these oligos were designed to have 1 or 0 mismatches with the mouse FoxP3 gene (see Table 7 below). Exemplary mouse models include GFP/RFP Treg reporter mice, EAE (multiple sclerosis) and NOD (type 1 diabetes) mouse models, mouse inflammatory disease models, and humanized mouse models. Exemplary inflammatory disease models include graft versus host disease (GvHD) models, inflammatory bowel disease (IBD) models such as models of Crohn's disease and ulcerative colitis, rheumatoid arthritis models and psoriasis models. GvHD models include several models involving introduction of donor cells or tissues into a MHC mismatched or miHA mismatched host, e.g. , C57/B16(H2b) donor strain splenocytes or T cells into BALB/c(H2d) recipient strain or B 10.Br(H2k) donor strain bone marrow cells or T cells into BALB.K(H2k) recipient strain {see, e.g. , Schroeder et al. Dis Model Mech. May 2011 ; 4(3): 318-333). IBD models include genetic models IL- 10R2^_/~x dominant negative TGFPRII mice, SAMPl/Yit, Mdrla^"7", ΙΚΚγ^_/", and chemical agent models Dextran Sodium Sulfate, 2,4,6-trinitrobenzenesulfonic acid, and oxazolone (see, e.g., Mizoguchi. Prog Mol Biol Transl Sci. 2012; 105:263-320). Rheumatoid arthritis models include Collagen-induced arthritis, collagen-antibody induced arthritis, inflammatory arthritis primed with an antigen {e.g. methylated BSA in complete Freund's adjuvant), TNF-a transgenic mice, SKG mice, SCID mice, DR4-CD4 mice, and DNase ΙΓ^/_ IFN-IR^"7" mice (see, e.g. , Asquith et al. Eur. J. Immunol. 2009. 39: 1991-2058). Psoriasis models include Ttc7^rsVTtc7^rsn mice, K5-Stat3C mice, K14-IL-20 mice, K14-IL-6 mice, K5-latent TGFbetal mice, KlO-BMP-6 mice, K14- ILlalpha mice, K14-VEGF mice, ILl-ra knockout mice, IRF-2 knockout mice, and IKK2 knockout mice (see, e.g. , Gudjonsson et al. J Invest Dermatol. 2007 Jun; 127(6): 1292-308). Appropriate mouse models are also available, for example, from the Jackson Laboratory (Bar Harbor, Maine) or another commercial source.

Table 7. FOXP3 oligo number of mismatches with mouse FoxP3 gene.

Oligo # # of mismatches

FOXP3-03

¹

FOXP3-05

¹

FOXP3- 13

¹

FOXP3- 15

¹

FOXP3-22

¹

FOXP3-23

¹

FOXP3-26 0

FOXP3-27 0

FOXP3-28 0

FOXP3-29 1

FOXP3-30 1

FOXP3-38 1

FOXP3-41 1 FOXP3-49 1

FOXP3-50 1

FOXP3-54 1

FOXP3-57 0

EXAMPLE 5

Gapmers were designed to target and degrade human EZHl and EZH2 mRNA. Gapmers were used to evaluate the extent to which FOXP3 expression is regulated by EZHl and/or EZH2.

Table 8. EZHl and EZH2 gapmers

OligolD Base Formatted Sequence* SeqID

Sequence

EZH1- GACACGAA lnaGs;lnaAs;lnaCs;lnaAs;dCs;dGs;dAs;dAs;dAs;dT 45714 28 m08 ATCACGCA s;dCs;dAs;dCs;lnaGs;lnaCs;lnaAs;lnaT-Sup

T

EZH1- CGACACGA lnaCs;lnaGs;lnaAs;lnaCs;dAs;dCs;dGs;dAs;dAs;dA 45715 29 m08 AATCACGC s;dTs;dCs;dAs;lnaCs;lnaGs;lnaCs;lnaA-Sup

A

EZH2- GATTTTAC lnaGs;lnaAs;lnaTs;lnaTs;dTs;dTs;dAs;dCs;dAs;dCs; 45716 09 m08 ACGCTTCC dGs;dCs;dTs;lnaTs;lnaCs;lnaCs;lnaG-Sup

G

EZH2- CTTTCGAT lnaCs;lnaTs;lnaTs;lnaTs;dCs;dGs;dAs;dTs;dGs;dCs; 45717 38 m08 GCCGACAT dCs;dGs;dAs;lnaCs;lnaAs;lnaTs;lnaA-Sup A

*Table 3 provides a description of the nucleotide analogs, modifications and intranucleotide linkages used for certain oligonucleotides tested and described in Table 8.

Gapmers targeting EZHl reduced levels of EZHl mRNA levels in human T cells by 90-95% after 5 days at ΙΟμΜ (unassisted uptake) when compared to negative control (293) (FIG. 19). At 5μΜ (combination oligos) levels of EZHl were decreased between 60 and 90% depending on gapmer and time (FIG. 19). EZH2 gapmers decreased EZH2 mRNA levels up to 99% after 5 days at ΙΟμΜ when compared to negative control (293) (FIG. 20). Gapmer combinations decreased EZH2 mRNA levels by 75% (FIG. 20).

EZHl gapmers increased FoxP3 mRNA levels about 2 fold when compared to negative control (FIG. 21). FoxP3 mRNA levels increased up to 10 fold by EZH2 gapmer #9 (FIG. 21). The effect of the EZHl and EZH2 gapmer combinations (e.g., 28-9, 29-9, 28-38, and 29-38) on FoxP3 mRNA levels was far greater than either alone, appearing to be synergistic (FIG. 21).

Several T cell related genes were tested for gene expression levels after EZH1/2 gapmer treatment. Foxp3 showed a higher increase in mRNA levels after EZH1/2 KD. Again, a possible synergistic effect was observed when EZHl and EZH2 gapmers were combined (FIG 22). Treatment with EZHl and EZH2 gapmers also increased the percent of cells double positive (++) for Foxp3 and CD3z (FIG. 23).

Tables

Table 1: 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, AACU UA, AACUUC, AACU UG, 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, AAGU UU, AAUAAA, AAUAAC, AAUAAG, AAUAAU, AAUACA, AAUACC, AAUACG, AAUAGA, AAUAGC, AAUAGG, AAUAGU, AAUAUC, AAUAU U, 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, AAU UCC, AAU UCG, AAUUGA, AAUUGG, AAU UGU, AAUUUC, AAU UUG, 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, ACAGU U, ACAUAA, ACAUAC, ACAUCC, ACAUCG, ACAUCU, ACAUGA, ACAUGC, ACAUGU, ACAU UG, ACAU UU, ACCAAA, ACCAAC, ACCAAG, ACCAAU, ACCACC, ACCACG, ACCAGA, ACCAGU, ACCAUA, ACCAUG, ACCAU U, 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, ACCUU U, ACGAAA, ACGAAC, ACGAAG, ACGAAU, ACGACA, ACGACC, ACGACG, ACGACU, ACGAGA, 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, ACGU UC, ACGUUG, ACGUU U, AC U AAA, 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, ACU UAC, ACUUAU, ACU UCA, ACUUCC, ACUUCG, ACUUCU, ACUUGA, ACU UGC, ACU UGU, ACUUUA, ACU UUC, ACUU UG, 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, AGAU UA, AGAU UC, AGAUUG, AGAUU U, 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, AGCU UC, AGCUU U, 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, AGGU UC, 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, AGUCU U, AGUGAA, AGUGAC, AGUGCG, AGUGGG, AGUGUC, AGU UAA, AGU UAC, AGUUAG, AGUUCC, AGU UCG, AGUUGA, AGUUGC,

AGUUGU, AGUUUA, AGUUUC, AGUU UG, AGU UU U, 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, AUAU UG, AUAUU U, 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, AUCGU U, AUCUAA, AUCUAC, AUCUAG, AUCUAU, AUCUCC, AUCUCG, AUCUGU, AUCUUG, AUCUU U, AUGAAA, AUGAAC, AUGAAG, AUGAAU, AUGACC, AUGACU, AUGAGG, AUGAGU, AUGAUA, AUGAUC, AUGAU U, 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, AU UAAA, AUUAAC, AU UAAG, AU UAAU, AU UACA, AUUACC, AUUACG, AUUACU, AU UAGA, AUUAGC, AUUAGG, AUUAGU, AU UAUA, AUUAUC, AUUAUG, AUUCAC, AUUCCA, AU UCCG, AU UCCU, AUUCGA, AUUCGC, AUUCGG, AU UCGU, AUUCUA, AUUCUC, AUUCU U, AUUGAA, AUUGAC, AUUGAU, AU UGCC, AUUGCG, AU UGCU, AUUGGA, AUUGGC,

AUUGGG, AU UGGU, AUUGUA, AUUGUC, AUUGUG, AU UGU U, AU UUAA, AU UUAG, AU UUAU, AUU UCC, AUU UCG, AUU UCU, AU UUGA, AU UUGC, AU UUGU, AU UUUA, AU UU UC, AUU UUG, AUU UU U, 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, CAAGU U, CAAUAA, CAAUAC, CAAUAG, CAAUCC, CAAUCG, CAAUCU, CAAUGA, CAAUGC, CAAUGG, CAAUGU, CAAU UC, CAAU UG, CAAU UU, CACAAU, CACACA, CACACG, CACACU, CACAGA, CACAGC, CACAGG, CACAUA, CACAUC, CACAUU, CACCAA, CACCAC, CACCAU, CACCCA, CACCCC, CACCCG, CACCGA, CACCGC, CACCGG, CACCGU, CACCUA, CACCU U, CACGAA, CACGAC, CACGAG, CACGAU, CACGCA, CACGCC, CACGCU, CACGGA, CACGGC, CACGGG, CACGG U, CACGUA, CACGUC, CACGUG, CACGU U, CACUAA, CACUAG, CACUAU, CACUCA, CACUCG, CACUGA, CACUGC, CACUGG, CACUUA, CACU UC, CACU UU, CAGAAA, CAGAAG, CAGAAU, CAGACC, CAGACG, CAGAGC, CAGAUA, CAGAUC, CAGCCG, CAGCCU, CAGCGA, CAGCGC, CAGCGG, CAGCGU, CAGCUC, CAGCUU, CAGGAU, CAGGGG, CAGGGU, CAGGUA, CAGGUC, CAGGUU, CAGUAC, CAGUCG, CAGU UG, 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, CAU UAA, CAUUAC, CAUUAG, CAUUCA, CAU UCC, CAU UCG, CAU UCU, CAU UGA, CAU UGG, CAUU UC, CAU UUG, CAUU UU, CCAAAA, CCAAAC, CCAAAG, CCAAAU, CCAACA, CCAACC, CCAACG, CCAACU, CCAAGA, CCAAGC, CCAAGG, CCAAUC, CCAAUG, CCAAU U, 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, CCAUU U, 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, CCCGU U, CCCUAA, CCCUAG, CCCUCA, CCCUCU, CCCUGC, CCCUUA, CCCU UC, CCCU UU, CCGAAA, CCGAAC, CCGAAU, CCGACA, CCGACC, CCGACG, CCGACU, CCGAGA, CCGAGG, CCGAGU, CCGAUA, CCGAUC, CCGAUG, CCGAU U, CCGCAA, CCGCAC, CCGCAG, CCGCAU, CCGCCA, CCGCCC, CCGCCG, CCGCCU, CCGCGA, CCGCGC, CCGCGG, CCGCGU, CCGCUA, CCGCUC, CCGCUG, CCGCU U, CCGGAA, CCGGAU, CCGGCA, CCGGCC, CCGGCG, CCGGCU, CCGGGA, CCGGGC, CCGGGG, CCGGGU, CCGGUA, CCGGUC, CCGGUG, CCGUAA, CCGUAG, CCGUAU, CCGUCA, CCGUCC, CCGUCG, CCGUGA, CCGUGU, CCGUUA, CCGUUC, CCGU UG, CCGUU U, 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, CCUGU U, CCUUAA, CCUUAC, CCUUAG, CCUUAU, CCUUCG, CCUUGA, CCUUGU, CCU UUA, CCUU UC, CCU UU U, 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, CGACU U, 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, CGAU UC, CGAUUG, CGAUU U, CGCAAA, CGCAAC, CGCAAG, CGCAAU, CGCACA, CGCACC, CGCACG, CGCAGA, CGCAGC, CGCAGG, CGCAGU, CGCAUA, CGCAUC, CGCAUG, CGCAU U, 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, CGCGU U, CGCUAA, CGCUAC, CGCUAG, CGCUAU, CGCUCA, CGCUCC, CGCUCG, CGCUCU, CGCUGA, CGCUGC, CGCUGG, CGCUGU, CGCUUA, CGCU UC, CGCU UG, CGGAAA, CGGAAC, CGGAAG, CGGACA, CGGACC, CGGACG, CGGACU, CGGAGC, CGGAGG, CGGAGU, CGGAUA, CGGAU U, CGGCAA, CGGCAC, CGGCAG, CGGCCA, CGGCCC, CGGCCG, CGGCGC, CGGCGG, CGGCGU, CGGCUA, CGGCUC, CGGCUG, CGGCU U, CGGGAA, CGGGAC, CGGGAG, CGGGAU, CGGGCA, CGGGCC, CGGGCG, CGGGCU, CGGGGU, CGGGUA, CGGGUC, CGGGUG, CGGUAA, CGGUAC, CGGUAG, CGGUAU, CGGUCA, CGGUCG, CGGUCU, CGGUGA, CGGUGG, CGGUGU, CGGU UA, CGGU UC, 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, CG UCGA, CGUCGG, CGUCGU, CGUCUA, CGUCUC, CGUCUG, CGUCU U, CGUGAA, CGUGAC, CGUGAG, CGUGAU, CGUGCC, CGUGCG, CGUGCU, CGUGGA, CGUGGG, CGUGGU, CGUGUA, CGUGUG, CGUUAA, CGUUAC, CGU UAG, CGU UAU, CGUUCA, CGUUCC, CGUUCG, CGU UCU, CGUUGA, CGUUGC, CGU UGU, CGU UUA, CGUUUC, CGU U UU, 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, CUCAU U, CUCCAC, CUCCCC, CUCCCG, CUCCGA, CUCCGC, CUCCGG, CUCCUA, CUCCUC, CUCCU U, CUCGAA, CUCGAC, CUCGAG, CUCGAU, CUCGCA, CUCGCC, CUCGCG, CUCGGG, CUCGGU, CUCGUA, CUCGUC, CUCGUG, CUCGU U, CUCUAA, CUCUAC, CUCUAU, CUCUCA, CUCUCC, CUCUCU, CUCUGC, CUCUGU, CUCUUA, CUCU UG, CUGAAG, CUGACC, CUGACG, CUGAGC, CUGAUA, CUGAUC, CUGCCG, CUGCCU, CUGCGA, CUGCUA, CUGCUU, CUGGAG, CUGGAU, CUGGCG, CUGGGU, CUGUAC, CUGUCA, CUGUCC, CUGUCG, CUGUGG, CUGUGU, CUGUUA, CUGU UU, CUUAAC, CUUAAG, CU UAAU, CU UACC, CUUACG, CUUAGA, CU UAGC, CU UAGG, CUUAGU, CUUAUA, CU UAUC, CU UAUG, CUUAUU, CU UCAG, CU UCAU, CUUCCA, CUUCCC, CU UCCG, CU UCCU, CUUCGA, CUUCGC, CU UCGG, CUUCGU, CU UCUA, CUUGAC, CUUGAG, CUUGAU, CUUGCA, CUUGCC, CUUGCG, CU UGCU, CUUGGC, CUUGGU, CU UGUU, CUU UAC, CU UUAG, CU UUAU, CUU UCA, CUU UCG, CU UUCU, CUUUGA, CU UUGC, CU UUGU, CU U UUA, CUUU UC, CUU UUG, CUU UUU, 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, GAAU UA, GAAU UC, GAAUU U, GACAAA, GACAAG, GACAAU, GACACC, GACAGA, GACAGG, GACAUA, GACAUG, GACAUU, GACCAA, GACCAC, GACCAG, GACCCA, GACCCC, GACCCG, GACCGC, GACCGG, GACCGU, GACCUA, GACCUC, GACCU U, 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, GACUU U, GAGAAU, GAGAGA, GAGAGC, GAGAGG, GAGAUA, GAGAUC, GAGCAA, GAGCAU, GAGCCA, GAGCGA, GAGCGG, GAGCGU, GAGGGU, GAGGUC, GAGGUG, GAGUAA, GAGUAG, GAGUCC, GAGUUC, GAGU UU, GAUAAA, GAUAAC, GAUAAG, GAUAAU, GAUACA, GAUACC, GAUACG, GAUACU, GAUAGA, GAUAGC, GAUAGG, GAUAGU, GAUAUA, GAUCAA, GAUCAC, GAUCAU, GAUCCA, GAUCCC, GAUCCU, GAUCGC, GAUCGG, GAUCGU, GAUCUA, GAUCUG, GAUCU U, GAUGAA, GAUGAC, GAUGAG, GAUGCA, GAUGCC, GAUGCG, GAUGCU, GAUGGC, GAUGGG, GAUGGU, GAUGUG, GAUGUU, GAUUAA, GAUUAC, GAUUAG, GAUUAU, GAUUCA, GAUUCG, GAU UCU, GAUUGA, GAUUGC, GAU UUA, GAU UUC, GAUU UG, GAUUU U, 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, GCAU UA, GCAU UC, GCAUUG, GCAUU U, 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, GCCGU U, GCCUAA, GCCUAU, GCCUCA, GCCUCC, GCCUCG, GCCUGA, GCCU UA, GCCU UU, 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, GCGU UA, GCGUUC, GCGU UG, GCGUU U, 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, GCUCU U, GCUGAA, GCUGAC, GCUGAU, GCUGCA, GCUGCC, GCUGCG, GCUGCU, GCUGUG, GCUGUU, GCUUAC, GCUUAG, GCUUAU, GCU UCA, GCUUCG, GCU UGA, GCUUGG, GCUUGU, GCUU UA, GCU UUG, GGAAAG, GGAACA, GGAACC, GGAACG, GGAACU, GGAAGU, GGAAUA, GGAAUC, GGAAU U, GGACAA, GGACAC, GGACAG, GGACAU, GGACCG, GGACGA, GGACGC, GGACGU, GGACUA, GGACUC, GGACU U, 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, GGCCU U, GGCGAA, GGCGAG, GGCGAU, GGCGCA, GGCGCU, GGCGGU, GGCGUA, GGCGUC, GGCGUG, GGCGU U, GGCUAA, GGCUAC, GGCUAG, GGCUAU, GGCUCC, GGCUCG, GGCUGA, GGCU UA, GGCUUC, GGCUUG, GGGAAU, GGGACA, GGGAGA, GGGAGU, GGGAUA, GGGAU U, GGGCAA, GGGCAC, GGGCAG, GGGCCG, GGGCGG, GGGGCC, GGGGGG,

GGGGGU, GGGGUA, GGGUAC, GGGUAU, GGGUCA, GGGUCC, GGGUCG, GGGUGA, GGGUGC, GGGU UA, GGGU UG, GGUAAA, GGUAAC, GGUAAG, GGUAAU, GGUACA, GGUACC, GGUACG,

GGUACU, GGUAGC, GGUAGG, GGUAGU, GGUAUA, GGUAUC, GGUAUG, GGUCAA, GGUCAC,

GGUCAG, GGUCAU, GGUCCA, GGUCCG, GGUCCU, GGUCGA, GGUCGC, GG UCGG, GGUCGU, GGUCUC, GGUCU U, GGUGAA, GGUGAC, GGUGAU, GGUGCA, GGUGCC, GGUGGC, GGUGUA, GGUGUC, GGU UAA, GGU UAG, GGU UAU, GGUUCA, GGU UCC, GGU UCG, GGU UGC, GGU UUC, GGUU UU, 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, GUAU UG, G UAU UU, GUCAAA, GUCAAG, GUCAAU, GUCACA, GUCACC, GUCACG, GUCAGA, GUCAGC, GUCAGG, GUCAUA, GUCAUC, GUCAUG, GUCCAA, GUCCAC, GUCCAU, GUCCCC, GUCCCU, GUCCGA, GUCCGC, GUCCGG, GUCCGU, GUCCUA, GUCCUG, GUCCU U, GUCGAA, GUCGAC, GUCGAG, GUCGAU, GUCGCA, GUCGCC, GUCGCG, GUCGCU, GUCGGA, GUCGGC, GUCGGG, GUCGGU, GUCGUA, GUCGUC, GUCGU U, GUCUAA, GUCUAG, GUCUCA, GUCUCC, GUCUCG, GUCUGA, GUCUGG, GUCUGU, GUCU UC, GUCU UU, 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, GUGU UU, GU UAAA, GUUAAC, GUUAAG, GU UACA, GU UACC, GUUACG, GU UACU, GU UAGA, GUUAGC, GUUAGU, GUUAUA, GUUAUC, GUUAUG, GU UAUU, GUUCAA, GUUCAC, GUUCAG, GU UCCA, GUUCCG, GUUCGA, GU UCGC, GU UCGG, GU UCGU, GU UCUA, GUUCUG, GUUGAA, GU UGAC, GUUGAG, GUUGAU, GUUGCG, GUUGCU, GUUGGA, GU UGGC, GU UGGU, GU UGUC, GUUGUG, GUUGU U, GUU UAA, GU UUAC, GU UUAG, GUU UAU, GU UUCA, GUUUCC, GU UUCU, GU UUGA, GUU UGC, GUU UGG, GUU UGU, GU UUUA, GU UU UC, GU UU UU, UAAAAA, UAAAAC, UAAAAG, UAAAAU, UAAACA, UAAACC, UAAACG, UAAACU, UAAAGA, UAAAGG, UAAAGU, UAAAUA, UAAAUC, UAAAUG, UAAAU U, U A AC A A, 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, UAAU UC, UAAUUG, UACAAC, UACAAG, UACAAU, UACACC, UACACG, UACACU, UACAGA, UACAGC, UACAUA, UACAUC, UACAU U, 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, UACU UA, UACU UG, UACU UU, 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, UAGU UC, UAGU UG, UAGUU U, UAUAAC, UAUAAG, UAUACU, UAUAGA, UAUAGC, UAUAGG, UAUAGU, UAUAUA, UAUAUC, UAUAUG, UAUAU U, 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, UAUGU U, UAU UAG, UAUUCA, UAU UCC, UAUUCG, UAUUCU, UAUUGA, UAUUGG, UAUU UA, UAU UUC, UAU UUG, UAUUU U, 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, UCAGU U, UCAUAA, UCAUCA, UCAUCC, UCAUCG, UCAUGC, UCAUGG, UCAUGU, UCAUUA, UCAUUG, UCCAAA, UCCAAC, UCCAAG, UCCAAU, UCCACA, UCCACC, UCCACG, UCCAGC, UCCAGG, UCCAUA, UCCAUC, UCCAU U, 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, UCCU UA, UCCU UC, UCCUU U, UCGAAA, UCGAAC, UCGAAG, UCGAAU, UCGACA, UCGACC, UCGACG, UCGACU, UCGAGA, UCGAGC, UCGAGG, UCGAUA, UCGAUC, UCGAUG, UCGAU U, UCGCAA, UCGCAC, UCGCAG, UCGCAU, UCGCCA, UCGCCC, UCGCCG, UCGCCU, UCGCGA, UCGCGC, UCGCGU, UCGCUA, UCGCUC, UCGGAA, UCGGAC, UCGGAG, UCGGAU, UCGGCA, UCGGCU, UCGGGG, UCGGGU, UCGGUC, UCGGUG, UCGGU U, UCGUAA, UCGUAC, UCGUAG,

UCGUAU, UCGUCA, UCGUCC, UCGUCG, UCGUCU, UCGUGA, UCGUGU, UCGUUA, UCGU UC, UCGU UG, UCGU UU, 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, UCUGU U, UCUUAA, UCU UAC, UCUUAG, UCU UAU, UCU UCA, UCUUCC, UCUUCG, UCUUCU, UCU UGC, UCU UGG, UCUUGU, UCUU UA, UCU UUC, UCU UUG, UCU UU U, 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 2: Experimental evaluation of single stranded oligonucleotides. Seq ID Oligo Avg RQ Avg RQ Target Probe [oligo] cell line Time Assay Name SD (hr) Type

45714 unc- 1 0 F0XP3 F0XP3 10000 huTcell+ 48 q TPC

293

mOl

Ctrl Un 0.8683 0.2839 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR

1487 9334

42428 FOXP3- 3.0412 1.4156 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 01 mOl 3508 7948

42429 FOXP3- 0.8050 0.1047 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 02 mOl 441 0739

42431 F0XP3- 0.6028 0.0036 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 03 mOl 9195 5975

42427 F0XP3- 2.0087 0.5894 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 04 mOl 2837 724

42426 F0XP3- 3.6326 0.4029 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 05 mOl 7026 6967

42420 F0XP3- 3.7983 0.1878 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 06 mOl 637 1968

42381 F0XP3- 1.9322 0.7812 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 07 mOl 5341 3517

42862 F0XP3- 3.1661 0.0383 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 08 mOl 807 4323

42861 F0XP3- 1.9057 0.6671 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 09 mOl 6687 1136

42859 F0XP3- 1.8638 0.0836 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 10 mOl 0551 3845

42858 F0XP3- 3.1376 1.0860 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 11 mOl 3788 6849

42854 F0XP3- 1.9168 0.1910 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 12 mOl 3566 0954

41897 F0XP3- 3.8208 0.3243 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 13 mOl 8674 6208

41052 F0XP3- 1.7192 0.2843 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 14 mOl 4737 9737

41877 F0XP3- 1.6897 0.0533 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 15 mOl 8766 9158

43243 F0XP3- 1.3242 0.1124 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 16 mOl 2504 1901

43375 F0XP3- 2.9662 0.8060 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 17 mOl 7721 2523

43376 F0XP3- 2.4887 0.3137 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 18 mOl 9558 6938

43379 F0XP3- 1.6217 0.0703 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 19 mOl 6367 6153

43380 F0XP3- 0.9836 0.3594 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 20 mOl 4872 1408 Seq ID Oligo Avg RQ Avg RQ Target Probe [oligo] cell line Time Assay Name SD (hr) Type

43381 FOXP3- 2.4213 0.8607 F0XP3 F0XP3 10000 huTcell+ 48 q TPC 21 mOl 5072 0806

43216 FOXP3- 4.4056 0.5274 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 22 mOl 7095 3242

43215 FOXP3- 5.2594 1.2834 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 23 mOl 1152 2081

43209 FOXP3- 0.8441 0.1162 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 24 mOl 0485 5078

43207 FOXP3- 1.9700 0.7081 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 25 mOl 6843 3234

43205 FOXP3- 1.4102 0.5560 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 26 mOl 0745 462

43204 FOXP3- 2.3137 0.8893 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 27 mOl 6112 6632

43203 FOXP3- 2.3111 0.9571 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 28 mOl 4582 8326

42533 F0XP3- 2.4213 0.8569 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 29 mOl 3228 018

42535 F0XP3- 2.8420 0.7355 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 30 mOl 5507 6048

42580 F0XP3- 1.6829 0.3197 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 31 mOl 5946 4715

42579 F0XP3- 1.8023 0.3645 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 32 mOl 751 291

42578 F0XP3- 1.5049 0.3524 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 33 mOl 9095 5102

42576 F0XP3- 1.5541 0.4739 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 34 mOl 0054 9665

42575 F0XP3- 1.9417 0.7923 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 35 mOl 3584 7942

42574 F0XP3- 1.7850 0.5616 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 36 mOl 2753 8053

42572 F0XP3- 1.1849 0.6381 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 37 mOl 7696 9605

42571 F0XP3- 2.8387 0.1652 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 38 mOl 527 0034

42570 F0XP3- 2.1055 0.9999 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 39 mOl 0749 9309

42569 F0XP3- 2.1616 0.4122 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 40 mOl 7457 0644

42568 F0XP3- 2.1596 0.1463 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 41 mOl 0714 9811

42567 F0XP3- 2.0196 0.2450 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 42 mOl 1568 3396 Seq ID Oligo Avg RQ Avg RQ Target Probe [oligo] cell line Time Assay Name SD (hr) Type

43447 FOXP3- 1.4034 0.4300 F0XP3 F0XP3 10000 huTcell+ 48 q TPC 43 mOl 1903 4175

43450 FOXP3- 2.6826 0.8637 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 44 mOl 3331 6389

43452 FOXP3- 1.2972 0.0760 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 45 mOl 8052 7689

43455 FOXP3- 1.6933 0.2872 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 46 mOl 3468 7806

43456 FOXP3- 3.9881 1.1155 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 47 mOl 4452 3672

43458 FOXP3- 1.6118 0.5129 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 48 mOl 209 9822

43459 FOXP3- 2.7114 0.8349 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 49 mOl 1213 2691

36466 FOXP3- 5.6757 0.1780 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 50 mOl 0129 0894

36439 F0XP3- 0.7754 0.1134 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 51 mOl 7248 4935

36437 F0XP3- 1.6663 0.1888 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 52 mOl 5528 1864

29244 F0XP3- 4.7858 0.0573 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 53 mOl 9887 1016

29253 F0XP3- 1.4969 0.5128 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 54 mOl 7594 6873

41675 F0XP3- 1.3438 0.1669 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 55 mOl 8255 8218

43610 F0XP3- 3.1971 0.0377 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 56 mOl 9516 4779

43612 F0XP3- 1.1602 0.6360 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 57 mOl 3805 6665

43613 F0XP3- 1.3291 0.7585 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 58 mOl 3775 1478

43616 F0XP3- 0.6957 0.2899 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 59 mOl 4924 1794

40694 F0XP3- 1.4396 0.3110 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 60 mOl 5065 3627

45714 unc- 1 0 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR

293

mOl

Ctrl Un 1.0124 0.1494 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR

9113 1646

42428 F0XP3- 1.0142 0.2083 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 01 mOl 1223 8028

42429 F0XP3- 0.8054 0.1630 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 02 mOl 8151 1023 Seq ID Oligo Avg RQ Avg RQ Target Probe [oligo] cell line Time Assay Name SD (hr) Type

42431 FOXP3- 1.8537 0.4985 F0XP3 F0XP3 10000 huTcell+ 48 q TPC 03 mOl 5015 9871

42427 FOXP3- 1.0380 0.3009 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 04 mOl 3618 8857

42426 FOXP3- 1.1894 0.3374 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 05 mOl 9951 6604

42420 FOXP3- 1.4921 0.3555 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 06 mOl 3089 691

42381 FOXP3- 1.0446 0.2059 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 07 mOl 5779 1828

42862 FOXP3- 1.2084 0.3087 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 08 mOl 6974 0018

42861 FOXP3- 1.0682 0.0122 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 09 mOl 3309 3336

42859 FOXP3- 1.1899 1.0015 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 10 mOl 6991 8697

42858 F0XP3- 1.0199 0.2469 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 11 mOl 5327 2075

42854 F0XP3- 0.9671 0.3334 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 12 mOl 974 0657

41897 F0XP3- 2.0861 0.9747 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 13 mOl 8587 9621

41052 F0XP3- 1.5627 0.5077 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 14 mOl 0566 9572

41877 F0XP3- 1.2444 0.3812 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 15 mOl 8641 6836

43243 F0XP3- 1.0948 0.1286 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 16 mOl 2815 0521

43375 F0XP3- 0.7561 0.0689 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 17 mOl 1626 9775

43376 F0XP3- 1.3842 0.2324 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 18 mOl 3343 2212

43379 F0XP3- 2.2321 0.4839 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 19 mOl 9084 7258

43380 F0XP3- 0.7731 0.1246 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 20 mOl 9797 8607

43381 F0XP3- 0.9295 0.3616 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 21 mOl 1965 7181

43216 F0XP3- 2.1920 0.3335 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 22 mOl 9374 4356

43215 F0XP3- 4.6076 1.3203 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 23 mOl 9744 3965

43209 F0XP3- 0.1886 0.0778 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 24 mOl 2818 0705 Seq ID Oligo Avg RQ Avg RQ Target Probe [oligo] cell line Time Assay Name SD (hr) Type

43207 FOXP3- 0.5319 0.0250 F0XP3 F0XP3 10000 huTcell+ 48 q TPC 25 mOl 8871 394

43205 FOXP3- 0.7856 0.0515 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 26 mOl 9731 7862

43204 FOXP3- 1.4702 0.1624 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 27 mOl 824 2886

43203 FOXP3- 1.8324 0.1517 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 28 mOl 4549 0473

42533 FOXP3- 2.1467 0.6541 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 29 mOl 1471 5214

42535 FOXP3- 1.9818 0.7000 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 30 mOl 3515 0334

42580 FOXP3- 0.8055 0.3641 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 31 mOl 3315 5893

42579 FOXP3- 1.1178 0.4133 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 32 mOl 8013 0598

42578 F0XP3- 1.5216 0.8118 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 33 mOl 6183 2555

42576 F0XP3- 0.9007 0.3606 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 34 mOl 9369 8588

42575 F0XP3- 0.9535 0.3423 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 35 mOl 541 4744

42574 F0XP3- 0.7644 0.2827 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 36 mOl 1529 9382

42572 F0XP3- 1.4914 0.1581 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 37 mOl 7643 1177

42571 F0XP3- 1.6627 0.7107 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 38 mOl 2868 2917

42570 F0XP3- 2.1235 0.2007 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 39 mOl 0232 7097

42569 F0XP3- 1.2400 0.2779 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 40 mOl 1965 0611

42568 F0XP3- 1.9969 1.6955 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 41 mOl 2286 0223

42567 F0XP3- 0.7874 0.1994 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 42 mOl 3477 6904

43447 F0XP3- 0.7247 0.1884 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 43 mOl 555 6039

43450 F0XP3- 1.0890 0.0321 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 44 mOl 5435 6293

43452 F0XP3- 1.1322 0.4860 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 45 mOl 7275 5068

43455 F0XP3- 0.6668 0.1395 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 46 mOl 9557 0169 Seq ID Oligo Avg RQ Avg RQ Target Probe [oligo] cell line Time Assay Name SD (hr) Type

43456 FOXP3- 1.6205 0.2706 F0XP3 F0XP3 10000 huTcell+ 48 q TPC 47 mOl 9627 113

43458 FOXP3- 1.4206 0.4705 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 48 mOl 2488 0077

43459 FOXP3- 5.0448 0.7548 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 49 mOl 2435 6048

36466 FOXP3- 1.7578 0.2027 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 50 mOl 4419 8994

36439 FOXP3- 0.5987 0.1928 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 51 mOl 679 4557

36437 FOXP3- 1.5100 0.4988 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 52 mOl 0454 2253

29244 FOXP3- 1.5607 0.2212 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 53 mOl 9422 1849

29253 FOXP3- 1.7781 0.1253 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 54 mOl 9386 2551

41675 FOXP3- 0.7469 0.0475 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 55 mOl 4704 2939

43610 F0XP3- 1.4511 0.3049 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 56 mOl 6927 5519

43612 F0XP3- 0.6720 0.2669 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 57 mOl 4413 7083

43613 F0XP3- 1.2570 0.3751 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 58 mOl 8457 4513

43616 F0XP3- 2.2490 0.0058 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 59 mOl 5264 918

40694 F0XP3- 0.1994 0.0896 F0XP3 F0XP3 10000 huTcell+ 48 qRTPCR 60 mOl 952 9861

41897 F0XP3- 1 0 F0XP3 F0XP3 0 huTcell+ 48 qRTPCR 13 mOl

41897 F0XP3- 0.8732 0.1530 F0XP3 F0XP3 32 huTcell+ 48 qRTPCR 13 mOl 9521 4712

41897 F0XP3- 1.4179 0.0846 F0XP3 F0XP3 160 huTcell+ 48 qRTPCR 13 mOl 8849 702

41897 F0XP3- 0.8385 0.3318 F0XP3 F0XP3 800 huTcell+ 48 qRTPCR 13 mOl 0744 1934

41897 F0XP3- 2.2378 0.9376 F0XP3 F0XP3 4000 huTcell+ 48 qRTPCR 13 mOl 9843 5398

41897 F0XP3- 2.6500 0.2728 F0XP3 F0XP3 20000 huTcell+ 48 qRTPCR 13 mOl 2339 9354

43216 F0XP3- 1 0 F0XP3 F0XP3 0 huTcell+ 48 qRTPCR 22 mOl

43216 F0XP3- 0.7945 0.1061 F0XP3 F0XP3 32 huTcell+ 48 qRTPCR 22 mOl 4623 1272 Seq ID Oligo Avg RQ Avg RQ Target Probe [oligo] cell line Time Assay Name SD (hr) Type

43216 FOXP3- 1.0808 0.2371 F0XP3 F0XP3 160 huTcell+ 48 q TPC 22 mOl 0897 8635

43216 FOXP3- 0.7656 0.0008 F0XP3 F0XP3 800 huTcell+ 48 qRTPCR 22 mOl 0804 5315

43216 FOXP3- 9.5268 0.2504 F0XP3 F0XP3 4000 huTcell+ 48 qRTPCR 22 mOl 3765 5621

43216 FOXP3- 34.676 10.035 F0XP3 F0XP3 20000 huTcell+ 48 qRTPCR 22 mOl 1694 3302

43215 FOXP3- 1 0 F0XP3 F0XP3 0 huTcell+ 48 qRTPCR 23 mOl

43215 FOXP3- 2.2567 1.7897 F0XP3 F0XP3 32 huTcell+ 48 qRTPCR 23 mOl 9314 399

43215 FOXP3- 1.4137 0.8424 F0XP3 F0XP3 160 huTcell+ 48 qRTPCR 23 mOl 6849 4148

43215 FOXP3- 3.4493 0.1864 F0XP3 F0XP3 800 huTcell+ 48 qRTPCR 23 mOl 4969 5628

43215 F0XP3- 2.6047 0.2414 F0XP3 F0XP3 4000 huTcell+ 48 qRTPCR 23 mOl 2728 3156

43215 F0XP3- 21.998 7.3981 F0XP3 F0XP3 20000 huTcell+ 48 qRTPCR 23 mOl 4023 651

43203 F0XP3- 1 0 F0XP3 F0XP3 0 huTcell+ 48 qRTPCR 28 mOl

43203 F0XP3- 1.0375 0.1325 F0XP3 F0XP3 32 huTcell+ 48 qRTPCR 28 mOl 8152 2612

43203 F0XP3- 0.8822 0.3462 F0XP3 F0XP3 160 huTcell+ 48 qRTPCR 28 mOl 9811 2482

43203 F0XP3- 3.6553 3.1166 F0XP3 F0XP3 800 huTcell+ 48 qRTPCR 28 mOl 2124 2006

43203 F0XP3- 1.9607 0.8421 F0XP3 F0XP3 4000 huTcell+ 48 qRTPCR 28 mOl 7256 269

43203 F0XP3- 2.3976 0.6419 F0XP3 F0XP3 20000 huTcell+ 48 qRTPCR 28 mOl 0299 2758

42533 F0XP3- 1 F0XP3 F0XP3 0 huTcell+ 48 qRTPCR 29 mOl

42533 F0XP3- 1.1688 F0XP3 F0XP3 32 huTcell+ 48 qRTPCR 29 mOl 0692

42533 F0XP3- 1.2862 F0XP3 F0XP3 160 huTcell+ 48 qRTPCR 29 mOl 768

42533 F0XP3- 1.1965 F0XP3 F0XP3 800 huTcell+ 48 qRTPCR 29 mOl 0249

42533 F0XP3- 1.0911 F0XP3 F0XP3 4000 huTcell+ 48 qRTPCR 29 mOl 3218

42533 F0XP3- 5.3190 F0XP3 F0XP3 20000 huTcell+ 48 qRTPCR 29 mOl 0948 Seq ID Oligo Avg RQ Avg RQ Target Probe [oligo] cell line Time Assay Name SD (hr) Type

42535 FOXP3- 1 0 F0XP3 F0XP3 0 huTcell+ 48 qRTPCR 30 mOl

42535 FOXP3- 0.5703 0.3256 F0XP3 F0XP3 32 huTcell+ 48 qRTPCR 30 mOl 4656 9034

42535 FOXP3- 0.9960 0.7761 F0XP3 F0XP3 160 huTcell+ 48 qRTPCR 30 mOl 5844 3072

42535 FOXP3- 0.6841 0.4257 F0XP3 F0XP3 800 huTcell+ 48 qRTPCR 30 mOl 2094 1495

42535 FOXP3- 1.3951 0.0506 F0XP3 F0XP3 4000 huTcell+ 48 qRTPCR 30 mOl 0325 8993

42535 FOXP3- 4.5089 2.9259 F0XP3 F0XP3 20000 huTcell+ 48 qRTPCR 30 mOl 7649 5793

43459 FOXP3- 1 0 F0XP3 F0XP3 0 huTcell+ 48 qRTPCR 49 mOl

43459 FOXP3- 1.1830 0.4841 F0XP3 F0XP3 32 huTcell+ 48 qRTPCR 49 mOl 2438 8155

43459 F0XP3- 1.6376 0.1163 F0XP3 F0XP3 160 huTcell+ 48 qRTPCR 49 mOl 4765 3382

43459 F0XP3- 2.3993 1.3192 F0XP3 F0XP3 800 huTcell+ 48 qRTPCR 49 mOl 9746 8791

43459 F0XP3- 3.7357 0.7467 F0XP3 F0XP3 4000 huTcell+ 48 qRTPCR 49 mOl 8628 6582

43459 F0XP3- 2.7983 0.0985 F0XP3 F0XP3 20000 huTcell+ 48 qRTPCR 49 mOl 6436 2596

36466 F0XP3- 1 0 F0XP3 F0XP3 0 huTcell+ 48 qRTPCR 50 mOl

36466 F0XP3- 1.1387 0.0813 F0XP3 F0XP3 32 huTcell+ 48 qRTPCR 50 mOl 5449 526

36466 F0XP3- 1.0366 0.6754 F0XP3 F0XP3 160 huTcell+ 48 qRTPCR 50 mOl 5859 8254

36466 F0XP3- 1.2655 0.0481 F0XP3 F0XP3 800 huTcell+ 48 qRTPCR 50 mOl 3217 9054

36466 F0XP3- 2.1175 0.0176 F0XP3 F0XP3 4000 huTcell+ 48 qRTPCR 50 mOl 8319 3026

36466 F0XP3- 1.8174 0.8071 F0XP3 F0XP3 20000 huTcell+ 48 qRTPCR 50 mOl 7187 0599

41897 F0XP3- 1 0 F0XP3 IL2 A 0 huTcell+ 48 qRTPCR 13 mOl

41897 F0XP3- 1 0 F0XP3 GITR 0 huTcell+ 48 qRTPCR 13 mOl

41897 F0XP3- 0.5250 0.0481 F0XP3 IL2 A 4000 huTcell+ 48 qRTPCR 13 mOl 1902 4779

41897 F0XP3- 2.1846 0.0467 F0XP3 GITR 4000 huTcell+ 48 qRTPCR 13 mOl 7964 7421 Seq ID Oligo Avg RQ Avg RQ Target Probe [oligo] cell line Time Assay Name SD (hr) Type

43216 FOXP3- 1 0 F0XP3 IL2 A 0 huTcell+ 48 qRTPCR 22 mOl

43216 FOXP3- 1 0 F0XP3 GITR 0 huTcell+ 48 qRTPCR 22 mOl

43216 FOXP3- 0.5362 0.0068 F0XP3 IL2 A 4000 huTcell+ 48 qRTPCR 22 mOl 2332 3636

43216 FOXP3- 1.2428 0.5601 F0XP3 GITR 4000 huTcell+ 48 qRTPCR 22 mOl 8249 923

43215 FOXP3- 1 F0XP3 IL2RA 0 huTcell+ 48 qRTPCR 23 mOl

43215 FOXP3- 1 0 F0XP3 GITR 0 huTcell+ 48 qRTPCR 23 mOl

43215 FOXP3- 0.4977 0.0224 F0XP3 IL2RA 4000 huTcell+ 48 qRTPCR 23 mOl 5779 37

43215 FOXP3- 1.8166 0.2598 F0XP3 GITR 4000 huTcell+ 48 qRTPCR 23 mOl 2608 0483

43203 F0XP3- 1 0 F0XP3 IL2RA 0 huTcell+ 48 qRTPCR 28 mOl

43203 F0XP3- 1 0 F0XP3 GITR 0 huTcell+ 48 qRTPCR 28 mOl

43203 F0XP3- 0.5752 0.0146 F0XP3 IL2RA 4000 huTcell+ 48 qRTPCR 28 mOl 8551 2293

43203 F0XP3- 1.8044 0.4291 F0XP3 GITR 4000 huTcell+ 48 qRTPCR 28 mOl 9429 7742

42533 F0XP3- 1 0 F0XP3 IL2RA 0 huTcell+ 48 qRTPCR 29 mOl

42533 F0XP3- 1 0 F0XP3 GITR 0 huTcell+ 48 qRTPCR 29 mOl

42533 F0XP3- 0.9446 0.1468 F0XP3 IL2RA 4000 huTcell+ 48 qRTPCR 29 mOl 5714 1686

42533 F0XP3- 1.1006 0.1469 F0XP3 GITR 4000 huTcell+ 48 qRTPCR 29 mOl 6946 2655

42535 F0XP3- 1 0 F0XP3 IL2RA 0 huTcell+ 48 qRTPCR 30 mOl

42535 F0XP3- 1 0 F0XP3 GITR 0 huTcell+ 48 qRTPCR 30 mOl

42535 F0XP3- 0.6023 0.1068 F0XP3 IL2RA 4000 huTcell+ 48 qRTPCR 30 mOl 8158 1676

42535 F0XP3- 1.1649 0.0896 F0XP3 GITR 4000 huTcell+ 48 qRTPCR 30 mOl 6654 547

43459 F0XP3- 1 0 F0XP3 IL2RA 0 huTcell+ 48 qRTPCR 49 mOl

43459 F0XP3- 1 0 F0XP3 GITR 0 huTcell+ 48 qRTPCR 49 mOl Seq ID Oligo Avg RQ Avg RQ Target Probe [oligo] cell line Time Assay Name SD (hr) Type

43459 FOXP3- 0.9856 0 F0XP3 IL2 A 4000 huTcell+ 48 qRTPCR 49 mOl 3279

43459 FOXP3- 0.4263 0.1416 F0XP3 GITR 4000 huTcell+ 48 qRTPCR 49 mOl 3761 6645

36466 FOXP3- 1 0 F0XP3 IL2 A 0 huTcell+ 48 qRTPCR 50 mOl

36466 FOXP3- 1 0 F0XP3 GITR 0 huTcell+ 48 qRTPCR 50 mOl

36466 FOXP3- 0.7201 0.1418 F0XP3 IL2RA 4000 huTcell+ 48 qRTPCR 50 mOl 0675 7437

36466 FOXP3- 0.4229 0.4426 F0XP3 GITR 4000 huTcell+ 48 qRTPCR 50 mOl 1963 0683

41897 FOXP3- 1 0 F0XP3 IL10 0 huTcell+ 48 qRTPCR 13 mOl

41897 FOXP3- 0.2719 0.1474 F0XP3 IL10 4000 huTcell+ 48 qRTPCR 13 mOl 813 0902

43216 FOXP3- 1 0 F0XP3 IL10 0 huTcell+ 48 qRTPCR 22 mOl

43216 FOXP3- 0.4588 0.1131 F0XP3 IL10 4000 huTcell+ 48 qRTPCR 22 mOl 1437 6447

43215 F0XP3- 1 0 F0XP3 IL10 0 huTcell+ 48 qRTPCR 23 mOl

43215 F0XP3- 0.4485 0.2508 F0XP3 IL10 4000 huTcell+ 48 qRTPCR 23 mOl 3129 683

43203 F0XP3- 1 0 F0XP3 IL10 0 huTcell+ 48 qRTPCR 28 mOl

43203 F0XP3- 0.6462 0.6948 F0XP3 IL10 4000 huTcell+ 48 qRTPCR 28 mOl 9725 4085

42533 F0XP3- 1 0 F0XP3 IL10 0 huTcell+ 48 qRTPCR 29 mOl

42533 F0XP3- 0.7821 0.0987 F0XP3 IL10 4000 huTcell+ 48 qRTPCR 29 mOl 0159 5678

42535 F0XP3- 1 0 F0XP3 IL10 0 huTcell+ 48 qRTPCR 30 mOl

42535 F0XP3- 0.2204 0.1835 F0XP3 IL10 4000 huTcell+ 48 qRTPCR 30 mOl 8642 8706

43459 F0XP3- 1 0 F0XP3 IL10 0 huTcell+ 48 qRTPCR 49 mOl

43459 F0XP3- 0.5118 0.1457 F0XP3 IL10 4000 huTcell+ 48 qRTPCR 49 mOl 1181 1517

36466 F0XP3- 1 0 F0XP3 IL10 0 huTcell+ 48 qRTPCR 50 mOl

36466 F0XP3- 0.2278 0.0426 F0XP3 IL10 4000 huTcell+ 48 qRTPCR 50 mOl 3415 5941 Seq ID Oligo Avg RQ Avg RQ Target Probe [oligo] cell line Time Assay Name SD (hr) Type

41897 FOXP3- 1 0 F0XP3 CCDC2 0 huTcell+ 48 q TPC 13 mOl 2

41897 FOXP3- 0.9949 0.1179 F0XP3 CCDC2 4000 huTcell+ 48 qRTPCR 13 mOl 7325 4901 2

43216 FOXP3- 1 0 F0XP3 CCDC2 0 huTcell+ 48 qRTPCR 22 mOl 2

43216 FOXP3- 1.9199 0.5184 F0XP3 CCDC2 4000 huTcell+ 48 qRTPCR 22 mOl 6125 9461 2

43215 FOXP3- 1 0 F0XP3 CCDC2 0 huTcell+ 48 qRTPCR 23 mOl 2

43215 FOXP3- 2.0208 0.0466 F0XP3 CCDC2 4000 huTcell+ 48 qRTPCR 23 mOl 0353 1423 2

43203 FOXP3- 1 0 F0XP3 CCDC2 0 huTcell+ 48 qRTPCR 28 mOl 2

43203 FOXP3- 1.0871 0.3805 F0XP3 CCDC2 4000 huTcell+ 48 qRTPCR 28 mOl 9018 6095 2

42533 FOXP3- 1 0 F0XP3 CCDC2 0 huTcell+ 48 qRTPCR 29 mOl 2

42533 FOXP3- 0.9581 0.0680 F0XP3 CCDC2 4000 huTcell+ 48 qRTPCR 29 mOl 5978 6494 2

42535 F0XP3- 1 0 F0XP3 CCDC2 0 huTcell+ 48 qRTPCR 30 mOl 2

42535 F0XP3- 0.7900 0.0324 F0XP3 CCDC2 4000 huTcell+ 48 qRTPCR 30 mOl 138 5348 2

43459 F0XP3- 1 0 F0XP3 CCDC2 0 huTcell+ 48 qRTPCR 49 mOl 2

43459 F0XP3- 0.8339 0.4320 F0XP3 CCDC2 4000 huTcell+ 48 qRTPCR 49 mOl 0492 3537 2

36466 F0XP3- 1 0 F0XP3 CCDC2 0 huTcell+ 48 qRTPCR 50 mOl 2

36466 F0XP3- 0.3622 0.0633 F0XP3 CCDC2 4000 huTcell+ 48 qRTPCR 50 mOl 4221 3645 2

Table 3: A listing of oligonucleotide modifications

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 NA w/3' phosphate

rC RNA w/3' phosphate

rG RNA w/3' phosphate

rU RNA w/3' phosphate

Table 4: Foraiatted oligonucleotide sequences showing nucleotide modifications.

OligolD Base Sequence Formatted Sequence SeqID

FOXP3-01 CCTCGATGGTC lnaCs;omeCs;lnaTs;omeCs;lnaGs;omeAs;lnaTs;omeGs;lnaGs 42428 mOl TGGA ;omeUs;lnaCs;omeUs;lnaGs;omeGs;lnaA-Sup

FOXP3-02 CTCGATGGTCT lnaCs;omeUs;lnaCs;omeGs;lnaAs;omeUs;lnaGs;omeGs;lnaT 42429 mOl GGAT s;omeCs;lnaTs;omeGs;lnaGs;omeAs;lnaT-Sup

FOXP3-03 CGATGGTCTGG lnaCs;omeGs;lnaAs;omeUs;lnaGs;omeGs;lnaTs;omeCs;lnaTs 42431 mOl ATGA ;omeGs;lnaGs;omeAs;lnaTs;omeGs;lnaA-Sup

FOXP3-04 TCCTCGATGGT lnaTs;omeCs;lnaCs;omeUs;lnaCs;omeGs;lnaAs;omeUs;lnaGs 42427 mOl CTGG ;omeGs;lnaTs;omeCs;lnaTs;omeGs;lnaG-Sup

FOXP3-05 GTCCTCGATGG lnaGs;omeUs;lnaCs;omeCs;lnaTs;omeCs;lnaGs;omeAs;lnaTs 42426 mOl TCTG ;omeGs;lnaGs;omeUs;lnaCs;omeUs;lnaG-Sup

FOXP3-06 GCCTGTGTCCT lnaGs;omeCs;lnaCs;omeUs;lnaGs;omeUs;lnaGs;omeUs;lnaC 42420 mOl CGAT s;omeCs;lnaTs;omeCs;lnaGs;omeAs;lnaT-Sup

FOXP3-07 CACCTGCTCCT lnaCs;omeAs;lnaCs;omeCs;lnaTs;omeGs;lnaCs;omeUs;lnaCs 42381 mOl CGAG ;omeCs;lnaTs;omeCs;lnaGs;omeAs;lnaG-Sup

FOXP3-08 ATCAGTCACCG lnaAs;omeUs;lnaCs;omeAs;lnaGs;omeUs;lnaCs;omeAs;lnaCs 42862 mOl CAAA ;omeCs;lnaGs;omeCs;lnaAs;omeAs;lnaA-Sup

FOXP3-09 CATCAGTCACC lnaCs;omeAs;lnaTs;omeCs;lnaAs;omeGs;lnaTs;omeCs;lnaAs; 42861 mOl GCAA omeCs;lnaCs;omeGs;lnaCs;omeAs;lnaA-Sup

FOXP3-10 CTCATCAGTCA lnaCs;omeUs;lnaCs;omeAs;lnaTs;omeCs;lnaAs;omeGs;lnaTs 42859 mOl CCGC ;omeCs;lnaAs;omeCs;lnaCs;omeGs;lnaC-Sup

FOXP3-11 GCTCATCAGTC lnaGs;omeCs;lnaTs;omeCs;lnaAs;omeUs;lnaCs;omeAs;lnaGs 42858 mOl ACCG ;omeUs;lnaCs;omeAs;lnaCs;omeCs;lnaG-Sup

FOXP3-12 ACAAGCTCATC lnaAs;omeCs;lnaAs;omeAs;lnaGs;omeCs;lnaTs;omeCs;lnaAs 42854 mOl AGTC ;omeUs;lnaCs;omeAs;lnaGs;omeUs;lnaC-Sup

FOXP3-13 GCTCGGTAGTC lnaGs;omeCs;lnaTs;omeCs;lnaGs;omeGs;lnaTs;omeAs;lnaGs 41897 mOl CTCC ;omeUs;lnaCs;omeCs;lnaTs;omeCs;lnaC-Sup

FOXP3-14 AGAGCCTTCAC lnaAs;omeGs;lnaAs;omeGs;lnaCs;omeCs;lnaTs;omeUs;lnaCs 41052 mOl AACC ;omeAs;lnaCs;omeAs;lnaAs;omeCs;lnaC-Sup

FOXP3-15 CAGCGTTCTCC lnaCs;omeAs;lnaGs;omeCs;lnaGs;omeUs;lnaTs;omeCs;lnaTs 41877 mOl TGGC ;omeCs;lnaCs;omeUs;lnaGs;omeGs;lnaC-Sup

FOXP3-16 GTGTAGGCCAG lnaGs;omeUs;lnaGs;omeUs;lnaAs;omeGs;lnaGs;omeCs;lnaC 43243 mOl CCGG s;omeAs;lnaGs;omeCs;lnaCs;omeGs;lnaG-Sup

FOXP3-17 CAGCTGCTTAT lnaCs;omeAs;lnaGs;omeCs;lnaTs;omeGs;lnaCs;omeUs;lnaTs 43375 mOl AGAC ;omeAs;lnaTs;omeAs;lnaGs;omeAs;lnaC-Sup

FOXP3-18 AGCTGCTTATA lnaAs;omeGs;lnaCs;omeUs;lnaGs;omeCs;lnaTs;omeUs;lnaAs 43376 OligolD Base Sequence Formatted Sequence SeqID mOl GACC ;omeUs;lnaAs;omeGs;lnaAs;omeCs;lnaC-Sup

FOXP3-19 TGCTTATAGAC lnaTs;omeGs;lnaCs;omeUs;lnaTs;omeAs;lnaTs;omeAs;lnaGs 43379 mOl CTCC ;omeAs;lnaCs;omeCs;lnaTs;omeCs;lnaC-Sup

FOXP3-20 GCTTATAGACC lnaGs;omeCs;lnaTs;omeUs;lnaAs;omeUs;lnaAs;omeGs;lnaA 43380 mOl TCCT s;omeCs;lnaCs;omeUs;lnaCs;omeCs;lnaT-Sup

FOXP3-21 CTTATAGACCT lnaCs;omeUs;lnaTs;omeAs;lnaTs;omeAs;lnaGs;omeAs;lnaCs 43381 mOl CCTC ;omeCs;lnaTs;omeCs;lnaCs;omeUs;lnaC-Sup

FOXP3-22 TTGCCCACGAT lnaTs;omeUs;lnaGs;omeCs;lnaCs;omeCs;lnaAs;omeCs;lnaGs 43216 mOl CTCC ;omeAs;lnaTs;omeCs;lnaTs;omeCs;lnaC-Sup

FOXP3-23 GTTGCCCACGA lnaGs;omeUs;lnaTs;omeGs;lnaCs;omeCs;lnaCs;omeAs;lnaCs 43215 mOl TCTC ;omeGs;lnaAs;omeUs;lnaCs;omeUs;lnaC-Sup

FOXP3-24 CCGGATGTTGC lnaCs;omeCs;lnaGs;omeGs;lnaAs;omeUs;lnaGs;omeUs;lnaT 43209 mOl CCAC s;omeGs;lnaCs;omeCs;lnaCs;omeAs;lnaC-Sup

FOXP3-25 TTCCGGATGTT lnaTs;omeUs;lnaCs;omeCs;lnaGs;omeGs;lnaAs;omeUs;lnaG 43207 mOl GCCC s;omeUs;lnaTs;omeGs;lnaCs;omeCs;lnaC-Sup

FOXP3-26 GCTTCCGGATG lnaGs;omeCs;lnaTs;omeUs;lnaCs;omeCs;lnaGs;omeGs;lnaAs 43205 mOl TTGC ;omeUs;lnaGs;omeUs;lnaTs;omeGs;lnaC-Sup

FOXP3-27 TGCTTCCGGAT lnaTs;omeGs;lnaCs;omeUs;lnaTs;omeCs;lnaCs;omeGs;lnaGs 43204 mOl GTTG ;omeAs;lnaTs;omeGs;lnaTs;omeUs;lnaG-Sup

FOXP3-28 CTGCTTCCGGA lnaCs;omeUs;lnaGs;omeCs;lnaTs;omeUs;lnaCs;omeCs;lnaGs 43203 mOl TGTT ;omeGs;lnaAs;omeUs;lnaGs;omeUs;lnaT-Sup

FOXP3-29 CCCCTCACCTC lnaCs;omeCs;lnaCs;omeCs;lnaTs;omeCs;lnaAs;omeCs;lnaCs; 42533 mOl GTGC omeUs;lnaCs;omeGs;lnaTs;omeGs;lnaC-Sup

FOXP3-30 CCTCACCTCGT lnaCs;omeCs;lnaTs;omeCs;lnaAs;omeCs;lnaCs;omeUs;lnaCs; 42535 mOl GCAG omeGs;lnaTs;omeGs;lnaCs;omeAs;lnaG-Sup

FOXP3-31 AGCATCGTCCT lnaAs;omeGs;lnaCs;omeAs;lnaTs;omeCs;lnaGs;omeUs;lnaCs 42580 mOl TCTT ;omeCs;lnaTs;omeUs;lnaCs;omeUs;lnaT-Sup

FOXP3-32 CAGCATCGTCC lnaCs;omeAs;lnaGs;omeCs;lnaAs;omeUs;lnaCs;omeGs;lnaTs 42579 mOl TTCT ;omeCs;lnaCs;omeUs;lnaTs;omeCs;lnaT-Sup

FOXP3-33 ACAGCATCGTC lnaAs;omeCs;lnaAs;omeGs;lnaCs;omeAs;lnaTs;omeCs;lnaGs 42578 mOl CTTC ;omeUs;lnaCs;omeCs;lnaTs;omeUs;lnaC-Sup

FOXP3-34 GAACAGCATCG lnaGs;omeAs;lnaAs;omeCs;lnaAs;omeGs;lnaCs;omeAs;lnaTs 42576 mOl TCCT ;omeCs;lnaGs;omeUs;lnaCs;omeCs;lnaT-Sup

FOXP3-35 CGAACAGCATC lnaCs;omeGs;lnaAs;omeAs;lnaCs;omeAs;lnaGs;omeCs;lnaAs 42575 mOl GTCC ;omeUs;lnaCs;omeGs;lnaTs;omeCs;lnaC-Sup

FOXP3-36 CCGAACAGCAT lnaCs;omeCs;lnaGs;omeAs;lnaAs;omeCs;lnaAs;omeGs;lnaCs 42574 mOl CGTC ;omeAs;lnaTs;omeCs;lnaGs;omeUs;lnaC-Sup

FOXP3-37 TTCCGAACAGC lnaTs;omeUs;lnaCs;omeCs;lnaGs;omeAs;lnaAs;omeCs;lnaAs 42572 mOl ATCG ;omeGs;lnaCs;omeAs;lnaTs;omeCs;lnaG-Sup

FOXP3-38 CTTCCGAACAG lnaCs;omeUs;lnaTs;omeCs;lnaCs;omeGs;lnaAs;omeAs;lnaCs 42571 mOl CATC ;omeAs;lnaGs;omeCs;lnaAs;omeUs;lnaC-Sup

FOXP3-39 CCTTCCGAACA lnaCs;omeCs;lnaTs;omeUs;lnaCs;omeCs;lnaGs;omeAs;lnaAs 42570 mOl GCAT ;omeCs;lnaAs;omeGs;lnaCs;omeAs;lnaT-Sup

FOXP3-40 GCCTTCCGAAC lnaGs;omeCs;lnaCs;omeUs;lnaTs;omeCs;lnaCs;omeGs;lnaAs 42569 mOl AGCA ;omeAs;lnaCs;omeAs;lnaGs;omeCs;lnaA-Sup OligolD Base Sequence Formatted Sequence SeqID

FOXP3-41 GGCCTTCCGAA lnaGs;omeGs;lnaCs;omeCs;lnaTs;omeUs;lnaCs;omeCs;lnaGs 42568 mOl CAGC ;omeAs;lnaAs;omeCs;lnaAs;omeGs;lnaC-Sup

FOXP3-42 AGGCCTTCCGA lnaAs;omeGs;lnaGs;omeCs;lnaCs;omeUs;lnaTs;omeCs;lnaCs 42567 mOl ACAG ;omeGs;lnaAs;omeAs;lnaCs;omeAs;lnaG-Sup

FOXP3-43 GATCTCTGCCA lnaGs;omeAs;lnaTs;omeCs;lnaTs;omeCs;lnaTs;omeGs;lnaCs; 43447 mOl GCCG omeCs;lnaAs;omeGs;lnaCs;omeCs;lnaG-Sup

FOXP3-44 CTCTGCCAGCC lnaCs;omeUs;lnaCs;omeUs;lnaGs;omeCs;lnaCs;omeAs;lnaGs 43450 mOl GTCG ;omeCs;lnaCs;omeGs;lnaTs;omeCs;lnaG-Sup

FOXP3-45 CTGCCAGCCGT lnaCs;omeUs;lnaGs;omeCs;lnaCs;omeAs;lnaGs;omeCs;lnaCs 43452 mOl CGAG ;omeGs;lnaTs;omeCs;lnaGs;omeAs;lnaG-Sup

FOXP3-46 CCAGCCGTCGA lnaCs;omeCs;lnaAs;omeGs;lnaCs;omeCs;lnaGs;omeUs;lnaCs 43455 mOl GAAG ;omeGs;lnaAs;omeGs;lnaAs;omeAs;lnaG-Sup

FOXP3-47 CAGCCGTCGAG lnaCs;omeAs;lnaGs;omeCs;lnaCs;omeGs;lnaTs;omeCs;lnaGs 43456 mOl AAGA ;omeAs;lnaGs;omeAs;lnaAs;omeGs;lnaA-Sup

FOXP3-48 GCCGTCGAGAA lnaGs;omeCs;lnaCs;omeGs;lnaTs;omeCs;lnaGs;omeAs;lnaGs 43458 mOl GATT ;omeAs;lnaAs;omeGs;lnaAs;omeUs;lnaT-Sup

FOXP3-49 CCGTCGAGAAG lnaCs;omeCs;lnaGs;omeUs;lnaCs;omeGs;lnaAs;omeGs;lnaA 43459 mOl ATTC s;omeAs;lnaGs;omeAs;lnaTs;omeUs;lnaC-Sup

FOXP3-50 CTTCGAAGAGC lnaCs;omeUs;lnaTs;omeCs;lnaGs;omeAs;lnaAs;omeGs;lnaAs 36466 mOl CAGA ;omeGs;lnaCs;omeCs;lnaAs;omeGs;lnaA-Sup

FOXP3-51 CTGCAAGTGGC lnaCs;omeUs;lnaGs;omeCs;lnaAs;omeAs;lnaGs;omeUs;lnaG 36439 mOl CCGG s;omeGs;lnaCs;omeCs;lnaCs;omeGs;lnaG-Sup

FOXP3-52 GTCTGCAAGTG lnaGs;omeUs;lnaCs;omeUs;lnaGs;omeCs;lnaAs;omeAs;lnaG 36437 mOl GCCC s;omeUs;lnaGs;omeGs;lnaCs;omeCs;lnaC-Sup

FOXP3-53 TGT AC AC AG CT lnaTs;omeGs;lnaTs;omeAs;lnaCs;omeAs;lnaCs;omeAs;lnaGs 29244 mOl GGCG ;omeCs;lnaTs;omeGs;lnaGs;omeCs;lnaG-Sup

FOXP3-54 CTGGCGTTTAA lnaCs;omeUs;lnaGs;omeGs;lnaCs;omeGs;lnaTs;omeUs;lnaTs 29253 mOl TAAT ;omeAs;lnaAs;omeUs;lnaAs;omeAs;lnaT-Sup

FOXP3-55 GCTGGACTATC lnaGs;omeCs;lnaTs;omeGs;lnaGs;omeAs;lnaCs;omeUs;lnaAs 41675 mOl ACCC ;omeUs;lnaCs;omeAs;lnaCs;omeCs;lnaC-Sup

FOXP3-56 GAGGTGGCGG lnaGs;omeAs;lnaGs;omeGs;lnaTs;omeGs;lnaGs;omeCs;lnaG 43610 mOl TACTC s;omeGs;lnaTs;omeAs;lnaCs;omeUs;lnaC-Sup

FOXP3-57 GGTGGCGGTA lnaGs;omeGs;lnaTs;omeGs;lnaGs;omeCs;lnaGs;omeGs;lnaT 43612 mOl CTCAG s;omeAs;lnaCs;omeUs;lnaCs;omeAs;lnaG-Sup

FOXP3-58 GTGGCGGTACT lnaGs;omeUs;lnaGs;omeGs;lnaCs;omeGs;lnaGs;omeUs;lnaA 43613 mOl CAGC s;omeCs;lnaTs;omeCs;lnaAs;omeGs;lnaC-Sup

FOXP3-59 GCGGTACTCAG lnaGs;omeCs;lnaGs;omeGs;lnaTs;omeAs;lnaCs;omeUs;lnaCs 43616 mOl CGAG ;omeAs;lnaGs;omeCs;lnaGs;omeAs;lnaG-Sup

FOXP3-60 GTGGACCGTG lnaGs;omeUs;lnaGs;omeGs;lnaAs;omeCs;lnaCs;omeGs;lnaT 40694 mOl GATGA s;omeGs;lnaGs;omeAs;lnaTs;omeGs;lnaA-Sup

BRIEF DESCRIPTION OF THE SEQUENCE LISTING SeqID Chrom gene Chr.Start Chr.End strand Organism

1 chrX F0XP3 49094896 49133288 - Homo sapiens

2 chrX F0XP3 49094896 49133288 + Homo sapiens

3 chrX Foxp3 7567675 7607243 + Mus musculus

4 chrX Foxp3 7567675 7607243 - Mus musculus

5 chrX F0XP3 49091852 49146158 + Homo sapiens

6 chrX F0XP3 49105387 49126985 + Homo sapiens

7 chrX F0XP3 49105442 49121156 + Homo sapiens

8 chrX F0XP3 49131266 49131313 + Homo sapiens

9 chrX F0XP3 49131123 49131172 + Homo sapiens

10 chrX F0XP3 49127994 49128033 + Homo sapiens

11 chrX F0XP3 49127843 49127890 + Homo sapiens

12 chrX F0XP3 49127628 49127670 + Homo sapiens

13 chrX F0XP3 49124798 49124897 + Homo sapiens

14 chrX F0XP3 49123918 49123965 + Homo sapiens

15 chrX F0XP3 49120701 49120753 + Homo sapiens

16 chrX F0XP3 49118531 49118555 + Homo sapiens

17 chrX F0XP3 49115652 49115685 + Homo sapiens

18 chrX F0XP3 49112995 49113044 + Homo sapiens

19 chrX F0XP3 49112863 49112906 + Homo sapiens

20 chrX F0XP3 49112637 49112717 + Homo sapiens

21 chrX F0XP3 49107522 49107575 + Homo sapiens

22 chrX F0XP3 49106607 49106653 + Homo sapiens

23 chrX F0XP3 49106128 49106175 + Homo sapiens

24 chrX F0XP3 49105839 49105886 + Homo sapiens

25 chrX F0XP3 49105669 49105701 + Homo sapiens

26 chrX F0XP3 49105241 49105285 + Homo sapiens

27 chrX F0XP3 49129266 49133313 + Homo sapiens

28 chrX F0XP3 49129123 49133172 + Homo sapiens

29 chrX F0XP3 49125994 49130033 + Homo sapiens

30 chrX F0XP3 49125843 49129890 + Homo sapiens

31 chrX F0XP3 49125628 49129670 + Homo sapiens

32 chrX F0XP3 49122798 49126897 + Homo sapiens

33 chrX F0XP3 49121918 49125965 + Homo sapiens

34 chrX F0XP3 49118701 49122753 + Homo sapiens

35 chrX F0XP3 49116531 49120555 + Homo sapiens

36 chrX F0XP3 49113652 49117685 + Homo sapiens

37 chrX F0XP3 49110995 49115044 + Homo sapiens

38 chrX F0XP3 49110863 49114906 + Homo sapiens 39 chrX F0XP3 49110637 49114717 + Homo sapiens

40 chrX F0XP3 49105522 49109575 + Homo sapiens

41 chrX F0XP3 49104607 49108653 + Homo sapiens

42 chrX F0XP3 49104128 49108175 + Homo sapiens

43 chrX F0XP3 49103839 49107886 + Homo sapiens

44 chrX F0XP3 49103669 49107701 + Homo sapiens

45 chrX F0XP3 49103241 49107285 + Homo sapiens

46 chrX F0XP3 49091852 49146158 - Homo sapiens

47 chrX F0XP3 49105387 49126985 - Homo sapiens

48 chrX F0XP3 49127432 49127481 - Homo sapiens

49 chrX F0XP3 49127343 49127398 - Homo sapiens

50 chrX F0XP3 49117756 49117794 - Homo sapiens

51 chrX F0XP3 49100610 49100635 - Homo sapiens

52 chrX F0XP3 49100129 49100194 - Homo sapiens

53 chrX F0XP3 49099553 49099595 - Homo sapiens

54 chrX F0XP3 49125432 49129481 - Homo sapiens

55 chrX F0XP3 49125343 49129398 - Homo sapiens

56 chrX F0XP3 49115756 49119794 - Homo sapiens

57 chrX F0XP3 49098610 49102635 - Homo sapiens

58 chrX F0XP3 49098129 49102194 - Homo sapiens

59 chrX F0XP3 49097553 49101595 - Homo sapiens

Single Strand Oligonucleotides (Antisense Strand of Target Gene)

SeqID range: 60-16461

SeqIDs w/o G Runs:

60-81, 96- 201, 215-232 , 255 -378 f 392- 600, 616-654 , 678 -685, 699- 745,

759- 781, 795-802 , 816- 876, 902- 915, 930-950, 970- 1006, 1021- 1044 r

1058 -1196, 1216- 1248, 1264- 1307 f 1321 -1322, 1342- 1448, 1463- 1938 r

1952 -2189, 2204- 2289, 2303- 2315 r 2330 -2416, 2430- 2439, 2453- 2646 r

2671 -2717, 2731- 2846, 2868- 2889 r 2903 -2912, 2926- 2995, 3009- 3055 r

3075 -3127, 3141- 3142, 3156- 3161 r 3175 -3182, 3196- 3311, 3325- 3345 r

3360 -3399, 3413- 3455, 3469- 3520 r 3534 , 3554- ^■3603, 3618 -3756, 3770-

3803 , 3817 -3895, 3909- 3973, 3987- -4087 , 4101- ^■4147, 4163 -4223, 4237,

4251 -4304, 4318- 4374, 4409- 4488 r 4502 -4540, 4556- 4567, 4587- 4633 r

4647 -4692, 4707- 4864, 4894- 4957 r 4972 -4997, 5011- 5075, 5089- 5100 r

5124 -5167, 5185- 5201, 5238- 5245 r 5259 -5291, 5305- 5382, 5397- 5449 r

5470 -5512, 5537- 5616, 5630- 5669 r 5683 -5743, 5768- 5794, 5822- 5889 r

5915 -5937, 5961- 5998, 6012- 6016 r 6030 -6065, 6079- 6087, 6127- 6167 r

6182 -6186, 6200- 6227, 6244- 6289 r 6313 -6328, 6343- 6351, 6365- 6367 r

6395 -6418, 6433- 6444, 6458- 6488 r 6502 -6771, 6785- 6967, 6981- 6984 r

6998 -7543, 7554- 7561, 7575- 7578 r 7592 -7611, 7625- 7760, 7774- 7840 r 7855-8099, 8130-8275, 8289-8420, 8434-8548, 8563-8617, 8631-8632,

8659-8844, 8858-8862, 8877-8895, 8909-8937, 8957-9073, 9087-9091,

9106-9114, 9138-9158, 9174-9185, 9208-9230, 9244-9261, 9276-9342,

9357-9361, 9376-9384, 9399-9443, 9469-9491, 9507-9565, 9579-9609, 9623-9707, 9722-9801, 9825-9872, 9886-9950, 9974-10033, 10047-10065,

10079- 10136, 10163 -10174, 10201- 10207, 10221- 10226, 10240- 10254,

10278- 10280, 10296 -10304, 10318- 10346, 10360- 10632, 10648- 10691,

10706- 10743, 10763 -10772, 10786- 10808, 10824- 10854, 10868- 10881,

10897- 10922, 10959 -10971, 10985- 11116, 11131- 11148, 11162- 11208,

11222- 11246, 11260 -11428, 11443, 11458- 11517, 11531, 11547 , 11563

11602, 11617 -11685 , 11699- 11800, 11814- 12163, 12175- ^■12237, 12251-

12853, 12856 -12906 , 12920- 12952, 12966- 12992, 13006- 13017, 13031-

13521, 13533 -13588 , 13597- 13635, 13648- ^■13660, 13674- 13816, 13830-

13841, 13856 -13893 , 13907- 14033, 14047- ^■14096, 14139- ^■14174, 14188-

14237, 14251 -14272 , 14286- 14300, 14318- ^■14320, 14340- ^■14410, 14424-

14483, 14506 -14578 , 14592- 14599, 14634- ^■14703, 14717- 14730, 14746-

14864, 14883 -15085 , 15108- 15152, 15172- ^■15196, 15233- 15273, 15293-

15314, 15387 -15506 , 15520- 15560, 15574- ^■15593, 15607- 15615, 15651-

15652, 15666 -15860 , 15874- 16122, 16141- ^■16163, 16177- ^■16246, 16272-

16285, 16299 -16370 , 16384- 16420, 16426- ^■16443, 16451- ^■16453, 16455-

16461

SeqIDs w/o miR Seeds :

60-65, 68-69 , 74, 77, 79, 88-91, 93-94, 97, 99-100, 103, 105-106, 108, 110-112, 115-127, 130, 134-141, 144-145, 148-149, 152, 154-157, 159, 161-165, 167-169, 172-173, 176, 180-183, 185, 187-190, 193-195, 202-205, 207-210, 212-216, 218, 220, 222-225, 228, 230-234, 236, 238-239, 241, 245, 248-259, 262-264, 266-270, 273-274, 277-280, 282- 286, 290-296, 298-301, 303-309, 312-314, 316-320, 322, 324, 326-329, 334, 336, 338-339, 341, 343-349, 351, 354-360, 362-368, 370-373,

376, 378-379, 382-386, 389-392, 394, 396, 399-400, 402-403, 405-411, 413-421, 423, 426, 429, 431-432, 434-435, 437, 441, 443, 446-447, 449-457, 459-462, 464-468, 470, 472-478, 480-484, 487-489, 492-494, 497, 499-504, 506-509, 511-512, 514-517, 520-525, 527, 530, 534, 538, 540-541, 545-550, 552, 557-570, 575, 577-580, 582-588, 592-594, 596, 599-600, 602, 604, 607, 609-610, 612, 614, 618-624, 627-628, 630-634, 636, 638, 641-645, 648-651, 653-654, 656-657, 659-661, 663- 664, 666, 674-676, 678-680, 683-687, 689, 696-699, 702-712, 716-718, 722, 728-733, 736, 741-742, 745-746, 751, 753, 756-757, 760, 762- 764, 766-767, 769-775, 778, 781-782, 785-786, 788-789, 791-792, 794- 796, 799-800, 802, 807-811, 813-814, 817-822, 824, 827-830, 833-835, 837-841, 843, 846-853, 860, 862, 866-873, 875-877, 881, 883-884, 886-891, 893-894, 896-898, 902, 905-913, 915, 919-920, 923-927, 929, 931, 934, 938-942, 944, 947-951, 956, 959, 963, 965-973, 975, 979, 981-983, 986, 990, 999-1001, 1003-1004, 1006-1007, 1012-1013, 1016- 1018, 1021, 1023, 1025, 1028-1029, 1031-1037, 1040, 1042-1044, 1048, 1051-1053, 1056, 1058, 1060-1065, 1070-1074, 1076, 1079-1081, 1084- 1095, 1097-1108, 1110, 1112-1113, 1115-1118, 1121-1128, 1130, 1132, 1138, 1140-1143, 1151, 1153-1154, 1159, 1162, 1165-1166, 1168-1169, 1171, 1177, 1179-1181, 1183-1185, 1187-1188, 1192-1194, 1196-1199, 1201-1202, 1204-1205, 1209-1211, 1213-1214, 1216-1218, 1221, 1225- 1231, 1234, 1236-1237, 1239-1241, 1246-1250, 1252, 1256-1265, 1267, 1269, 1271, 1273-1275, 1277-1287, 1291, 1295-1301, 1303-1307, 1312- 1321, 1325, 1329, 1332-1333, 1335, 1338, 1344, 1346-1347, 1349, 1351, 1354-1363, 1367, 1370, 1373, 1375-1378, 1380-1387, 1389, 1393 1395, 1397-1398, 1400-1403, 1405, 1408, 1411-1413, 1415-1420, 1422, 1426, 1428-1429, 1433, 1435, 1437-1441, 1443-1444, 1453-1456, 1458, 1460-1461, 1464-1466, 1469-1470, 1472-1476, 1478-1479, 1481, 1484- 1486, 1488, 1491-1493, 1495, 1497, 1499-1502, 1504, 1506-1513, 1515 1522, 1524-1527, 1530-1531, 1533, 1535, 1542-1544, 1550, 1555, 1558 1559, 1562-1565, 1567, 1569-1572, 1576, 1579, 1582-1584, 1586, 1590 1592, 1594, 1596, 1601-1603, 1605-1610, 1612-1615, 1617-1618, 1620, 1622-1623, 1625, 1627-1630, 1632, 1635, 1638, 1641, 1643, 1645-1656 1660, 1665, 1668-1669, 1672, 1677-1679, 1681-1682, 1684, 1687-1690, 1694-1700, 1702-1703, 1705, 1707-1709, 1711, 1714, 1719, 1721-1726, 1732, 1734, 1739-1740, 1743-1744, 1746, 1749-1750, 1752, 1755, 1757 1759, 1761, 1763-1767, 1769, 1771, 1776, 1779-1780, 1784, 1786, 1790, 1793-1794, 1797-1800, 1803-1805, 1807-1809, 1811-1814, 1816, 1820, 1823, 1825, 1837-1838, 1840-1845, 1847-1852, 1854-1856, 1858- 1859, 1862, 1868-1869, 1873-1876, 1878, 1881, 1887-1888, 1890-1898, 1900, 1902-1903, 1905, 1912-1915, 1921-1924, 1926, 1928, 1931-1933, 1935-1937, 1940-1942, 1947, 1951, 1955, 1960-1962, 1964, 1967, 1971 1973, 1977, 1979, 1981, 1983, 1986-1988, 1990-1991, 1994-1997, 1999 2000, 2004-2006, 2008, 2011-2013, 2015-2020, 2022, 2024, 2026, 2028 2029, 2032, 2036, 2038-2044, 2046, 2048-2049, 2051-2052, 2054, 2056 2059-2064, 2066-2071, 2073, 2075-2076, 2078, 2080, 2083, 2085, 2087 2090-2091, 2093-2097, 2099, 2101, 2103, 2105-2109, 2112, 2114, 2119 2120, 2122, 2124, 2126-2127, 2130-2131, 2133-2135, 2138-2142, 2144- 2145, 2148, 2150-2152, 2154-2160, 2162-2166, 2169, 2172-2173, 2175- 2176, 2178, 2181-2184, 2186-2191, 2193, 2195-2201, 2203-2215, 2217- 2218, 2223-2224, 2226-2229, 2231-2236, 2238, 2240-2246, 2248-2250, 2252-2257, 2260-2262, 2266-2272, 2274, 2277-2278, 2280-2281, 2284, 2290, 2292, 2294-2296, 2300, 2303-2311, 2319-2320, 2323, 2326-2327, 2329, 2333-2336, 2338, 2341, 2343-2345, 2347, 2349-2350, 2352, 2358 2360, 2362-2363, 2365-2368, 2373-2374, 2377-2383, 2385, 2387-2388, 2395, 2398-2405, 2408, 2411-2414, 2416-2417, 2424-2425, 2429-2433, 2435-2439, 2443-2447, 2450-2455, 2457-2467, 2469-2471, 2473, 2476, 2478-2479, 2482-2488, 2492, 2494, 2496, 2498-2499, 2501, 2503-2504, 2506-2507, 2509, 2512, 2515-2517, 2519, 2521, 2523-2527, 2531-2534, 2536-2539, 2541, 2543, 2545, 2547-2549, 2552-2560, 2563-2565, 2567- 2570, 2572-2574, 2578-2581, 2584-2585, 2587-2588, 2590, 2592-2593, 2595, 2597, 2600, 2603, 2605-2606, 2609-2613, 2617, 2619-2620, 2622 2624, 2626, 2629-2631, 2633-2636, 2643-2646, 2655, 2662-2663, 2665- 2667, 2670-2674, 2677-2679, 2681-2683, 2685, 2687-2688, 2690-2691, 2693-2694, 2696, 2703, 2705, 2709-2711, 2713-2718, 2721-2722, 2729, 2737, 2740, 2743-2752, 2756-2757, 2759, 2763-2768, 2770-2774, 2776- 2777, 2780, 2782-2783, 2785, 2787-2790, 2795-2799, 2801, 2805, 2810 2811, 2813, 2819, 2823-2824, 2828-2832, 2839-2841, 2843, 2852, 2854 2855, 2859-2860, 2862-2864, 2868-2875, 2882-2884, 2886-2887, 2889- 2890, 2900-2905, 2907, 2910, 2912-2915, 2918-2919, 2921, 2923, 2926 2927, 2929-2931, 2934-2939, 2941, 2943-2952, 2954-2956, 2958, 2960- 2961, 2963-2964, 2971, 2975-2979, 2981-2982, 2984, 2988, 2993-2997, 2999, 3007-3009, 3016, 3018, 3020-3024, 3026, 3028-3030, 3032, 3034- 3041, 3043-3045, 3047, 3049-3054, 3057, 3059-3061, 3069, 3071-3073, 3075-3077, 3080-3083, 3085-3089, 3091-3092, 3094-3097, 3102, 3108- 3110, 3113-3114, 3120, 3123-3124, 3126, 3128, 3132, 3134, 3137, 3148, 3150-3155, 3158-3162, 3164-3165, 3168, 3171, 3175, 3182, 3185, 3188-3189, 3191, 3194-3198, 3200-3201, 3204, 3206-3207, 3209-3210, 3213, 3215-3216, 3218, 3220-3221, 3226-3227, 3231-3235, 3237, 3240, 3243-3245, 3247, 3249, 3251, 3253-3254, 3256-3262, 3264, 3271, 3273, 3276-3280, 3282-3283, 3285-3288, 3290-3291, 3294-3300, 3302-3303, 3306-3312, 3315-3323, 3328, 3330-3331, 3334-3336, 3338, 3340-3342, 3344, 3349, 3351-3353, 3355-3364, 3367-3379, 3382, 3384-3389, 3391, 3394, 3396-3397, 3399-3400, 3402, 3404-3412, 3415, 3417, 3420-3424, 3427-3429, 3431, 3434-3439, 3441-3442, 3445, 3447, 3450, 3454-3455, 3457, 3461, 3463-3466, 3472-3474, 3476-3478, 3482-3483, 3485-3486, 3488, 3491-3504, 3506, 3508-3517, 3519-3523, 3525-3526, 3529-3531, 3533, 3541, 3545-3547, 3549, 3552, 3556-3557, 3559-3562, 3565-3567, 3569, 3571, 3573-3574, 3577, 3581-3582, 3584-3585, 3587-3589, 3591, 3594-3597, 3599, 3601, 3605, 3607, 3609, 3613-3616, 3618-3620, 3622- 3630, 3632-3633, 3635-3637, 3639-3640, 3642-3648, 3650-3651, 3653- 3654, 3656-3657, 3661, 3664, 3666-3668, 3670-3672, 3674-3681, 3684, 3686-3691, 3693-3694, 3697, 3699-3702, 3704-3707, 3710, 3715, 3720- 3722, 3725-3727, 3732-3736, 3739-3745, 3747, 3749-3753, 3758-3761, 3763-3766, 3771, 3773-3779, 3781, 3783, 3785-3786, 3788, 3790-3793, 3795, 3797, 3799, 3801, 3803, 3808, 3810, 3812, 3814, 3819-3824, 3827-3829, 3831, 3834-3835, 3840-3842, 3844, 3846-3848, 3850-3851, 3853-3860, 3862-3863, 3865-3866, 3868, 3870, 3872, 3875, 3880-3882, 3884, 3887-3890, 3892-3893, 3895, 3901-3905, 3909, 3911-3912, 3914- 3919, 3922, 3926-3927, 3930-3934, 3939, 3941, 3943, 3945-3947, 3949- 3950, 3952-3954, 3956, 3960, 3962, 3965-3976, 3978-3983, 3985-3994, 3996-4005, 4008, 4010, 4012-4014, 4017, 4019, 4021-4023, 4025-4032, 4034-4037, 4039-4041, 4043-4045, 4048-4049, 4052, 4054-4055, 4058, 4060-4061, 4064-4065, 4067-4069, 4073-4078, 4081, 4087-4088, 4091, 4096-4098, 4100-4103, 4106-4107, 4109, 4111, 4118-4119, 4121-4123, 4127-4128, 4130-4136, 4139-4141, 4147, 4154-4158, 4161-4163, 4165, 4171-4178, 4184-4186, 4189, 4191, 4193-4195, 4198-4199, 4201, 4203- 4206, 4211, 4215, 4218-4224, 4227, 4234, 4237-4238, 4240, 4243, 4245-4246, 4248-4257, 4259, 4263-4265, 4267-4268, 4274-4275, 4277, 4280-4285, 4288-4291, 4293-4295, 4300, 4304, 4308, 4310, 4312-4316, 4319-4320, 4322-4323, 4325, 4334, 4336-4340, 4343-4348, 4350, 4352, 4358, 4360, 4364-4365, 4367, 4369-4372, 4374, 4377-4378, 4381, 4385, 4389, 4391-4392, 4404, 4406, 4411-4415, 4417, 4419-4421, 4423-4424, 4426-4427, 4431-4433, 4435, 4437-4438, 4440-4441, 4443, 4446, 4450- 4452, 4454-4465, 4467, 4469-4474, 4476, 4478-4479, 4482-4483, 4492- 4493, 4495, 4497-4498, 4501-4505, 4507-4517, 4519-4521, 4523-4527, 4530-4533, 4536-4539, 4541, 4543, 4546, 4549-4552, 4554-4555, 4558, 4561-4563, 4565, 4569, 4572-4576, 4579-4580, 4584-4587, 4590, 4592- 4595, 4597-4598, 4601-4602, 4604-4610, 4613-4614, 4616-4617, 4619- 4621, 4624, 4626-4628, 4632-4635, 4637-4639, 4642-4645, 4647-4648, 4651-4653, 4656, 4658-4659, 4663-4664, 4669, 4671-4675, 4680-4681, 4683-4686, 4688, 4690-4691, 4693, 4698, 4700-4701, 4705, 4707, 4709- 4712, 4714-4717, 4719, 4721-4725, 4729-4734, 4736, 4739-4741, 4744, 4746-4747, 4752, 4755, 4757, 4759, 4762-4763, 4766-4768, 4770-4773, 4776, 4779-4780, 4782, 4784, 4789, 4791, 4793-4795, 4802, 4804, 4806, 4808, 4812, 4816, 4818, 4820, 4822, 4825, 4829, 4832, 4834, 4844-4845, 4849-4851, 4855-4857, 4859-4860, 4863, 4867-4868, 4872, 4875, 4878-4880, 4884-4888, 4895-4897, 4899, 4902-4904, 4908-4910,

4913, 4916, 4921, 4923-4924, 4926, 4935-4939, 4942, 4944-4947, 4950, 4952, 4954, 4965-4967, 4970, 4976, 4980-4981, 4992-4995, 5000-5002, 5004-5007, 5009-5011, 5013-5014, 5016-5017, 5019-5021, 5024, 5026- 5029, 5031-5033, 5041-5046, 5048-5052, 5055-5058, 5068-5070, 5072- 5073, 5080-5083, 5085-5088, 5091-5092, 5095-5099, 5102, 5108, 5116- 5123, 5125-5126, 5128, 5131-5133, 5135, 5137, 5139, 5141, 5143, 5145-5150, 5153, 5155, 5157, 5164-5165, 5167-5168, 5172, 5176-5179, 5181-5183, 5185-5188, 5190-5192, 5195, 5197, 5199-5201, 5206, 5210, 5212, 5214-5215, 5217, 5219, 5221-5226, 5231-5232, 5235, 5237-5241, 5244-5248, 5251, 5253-5267, 5269-5271, 5273-5278, 5280-5286, 5288,

5293, 5295, 5297-5298, 5300, 5302-5303, 5307-5308, 5310, 5312, 5316, 5318-5324, 5326-5332, 5334, 5336-5337, 5340-5344, 5349-5350, 5352- 5353, 5360-5364, 5366-5369, 5371-5372, 5374, 5377-5383, 5388, 5390- 5396, 5399-5400, 5404, 5406-5413, 5415-5418, 5423-5424, 5428, 5430- 5433, 5435, 5437, 5439, 5441, 5444, 5446-5447, 5449-5454, 5461-5463, 5467, 5470-5471, 5476, 5479, 5481-5483, 5485-5487, 5492, 5495-5496, 5498-5500, 5502-5506, 5518, 5522-5523, 5529-5530, 5532-5536, 5538- 5540, 5542-5547, 5553-5561, 5563-5564, 5566, 5568, 5571-5575, 5578- 5579, 5583, 5586-5588, 5590-5592, 5600, 5603, 5606, 5611-5612, 5614- 5615, 5618, 5620, 5627-5628, 5630, 5633-5636, 5638, 5640-5641, 5643, 5646-5651, 5664-5665, 5667, 5669, 5673, 5675, 5677-5681, 5683-5684, 5687-5689, 5691-5692, 5695-5706, 5708-5712, 5715-5717, 5719, 5721, 5723-5724, 5726, 5728-5740, 5742-5743, 5749-5756, 5765-5766, 5770- 5771, 5773-5775, 5778-5780, 5782-5784, 5786-5790, 5800-5802, 5807, 5817-5819, 5821, 5825, 5831, 5834, 5836-5838, 5840-5842, 5844, 5846- 5853, 5855-5870, 5872, 5874, 5876-5877, 5879-5880, 5884-5888, 5891- 5893, 5896, 5901, 5905, 5907, 5909-5910, 5914, 5916-5918, 5920-5922, 5924, 5933-5935, 5940-5941, 5943-5944, 5950, 5952, 5954-5956, 5961- 5965, 5967, 5969-5976, 5978-5979, 5981, 5983-5990, 5992-5997, 5999, 6005-6012, 6015-6016, 6019, 6021-6029, 6032-6033, 6038-6044, 6046-

6051, 6053, 6055-6062, 6064-6065, 6067, 6070, 6072, 6074-6076, 6079- 6082, 6085-6086, 6088-6090, 6098-6099, 6101, 6107-6113, 6115, 6117, 6120-6121, 6124-6128, 6130-6134, 6136, 6138-6141, 6143-6147, 6151, 6153, 6156-6157, 6159-6167, 6174-6177, 6179-6181, 6183-6184, 6186- 6192, 6195, 6198-6199, 6201-6204, 6206-6214, 6217, 6219, 6221, 6223- 6225, 6227-6228, 6231, 6237, 6239-6246, 6248-6251, 6254-6259, 6261- 6262, 6266, 6269-6273, 6275-6276, 6282-6283, 6293, 6297-6298, 6304, 6310-6311, 6313-6321, 6324-6331, 6335, 6338, 6341, 6343, 6347-6348, 6350, 6353-6356, 6358-6359, 6361, 6364, 6368, 6371, 6374, 6376, 6379, 6382, 6386, 6388-6399, 6401-6408, 6410-6411, 6413-6418, 6427, 6430-6440, 6442-6444, 6446-6450, 6452-6453, 6455-6457, 6460-6461, 6463-6464, 6467, 6469-6477, 6479-6484, 6487-6488, 6490-6491, 6493- 6494, 6496-6503, 6505-6507, 6509-6510, 6512, 6515-6517, 6520-6521, 6523-6528, 6533-6534, 6536, 6541, 6543-6549, 6551, 6553-6554, 6556- 6560, 6562-6563, 6565-6566, 6569-6571, 6573-6580, 6582-6585, 6587-

6588, 6590, 6594-6597, 6599-6600, 6602, 6605, 6610-6612, 6614, 6617- 6618, 6621, 6623, 6626-6633, 6636-6637, 6640-6643, 6645-6646, 6648, 6650-6651, 6654-6655, 6657-6663, 6665-6666, 6668-6675, 6679, 6681- 6684, 6686, 6688-6690, 6692-6693, 6696-6699, 6703-6707, 6709, 6711, 6715-6716, 6718, 6721, 6723-6726, 6728-6731, 6733, 6735-6740, 6742- 6743, 6745-6749, 6755-6757, 6759, 6762-6767, 6769-6770, 6772, 6774- 6775, 6777-6779, 6781, 6783-6791, 6793-6794, 6796-6797, 6799-6802, 6804-6809, 6811, 6813-6818, 6821, 6826-6827, 6829-6833, 6836-6839, 6841-6846, 6848, 6851, 6853-6857, 6859-6864, 6869, 6875-6883, 6885- 6886, 6888, 6890, 6893-6896, 6898-6899, 6902, 6904-6905, 6907-6914, 6917, 6919-6920, 6922-6923, 6926-6930, 6932-6933, 6938-6942, 6944, 6948, 6950-6954, 6956-6962, 6964-6966, 6971-6973, 6975, 6977-6978, 6983, 6985, 6987-6990, 6992-7000, 7002-7007, 7009-7019, 7022-7027, 7032, 7035-7039, 7042-7043, 7045-7046, 7048-7050, 7052, 7056-7064, 7066-7068, 7070, 7073-7074, 7076-7085, 7087-7093, 7097-7098, 7100- 7103, 7107-7111, 7113-7118, 7121-7124, 7127, 7129, 7132-7139, 7142- 7151, 7154-7161, 7163-7165, 7167-7169, 7173, 7175-7177, 7181-7182, 7184, 7186-7189, 7192-7198, 7200-7201, 7203-7209, 7211-7212, 7215- 7216, 7218-7225, 7227-7230, 7232-7234, 7237-7238, 7240-7250, 7252- 7253, 7255-7257, 7263-7264, 7268-7273, 7276-7279, 7281-7282, 7284- 7285, 7287-7288, 7290, 7292, 7295-7300, 7302-7305, 7307-7311, 7314, 7316-7327, 7330-7334, 7336-7338, 7340, 7342, 7344, 7347-7349, 7351, 7353-7355, 7357-7363, 7367-7370, 7372-7373, 7375, 7378-7382, 7384- 7385, 7387, 7389-7400, 7403, 7406, 7409-7411, 7413-7429, 7431-7434, 7438, 7440-7441, 7445-7451, 7453-7454, 7456, 7458-7459, 7462, 7464, 7466-7467, 7470-7471, 7473-7484, 7486, 7490, 7495-7496, 7498-7501, 7506-7507, 7512-7515, 7517-7523, 7525-7528, 7536-7544, 7546-7549, 7551, 7555-7556, 7558-7561, 7566, 7571-7573, 7577-7580, 7582-7587, 7590-7591, 7595-7596, 7598-7599, 7601-7609, 7611, 7614, 7617, 7619- 7623, 7625-7642, 7645, 7647-7650, 7653, 7656, 7658-7659, 7661-7663, 7665-7666, 7669, 7671-7674, 7677-7683, 7685-7689, 7692, 7695-7697, 7699-7700, 7702-7710, 7712-7716, 7718, 7720-7728, 7730-7736, 7739- 7742, 7744, 7746, 7748-7752, 7754-7756, 7758, 7760, 7765-7771, 7774, 7776, 7779-7781, 7783-7784, 7786, 7788, 7790-7796, 7798-7803, 7805, 7807, 7811-7813, 7815-7816, 7820-7821, 7825-7826, 7828-7840, 7846- 7847, 7850-7852, 7854, 7857, 7859, 7861-7869, 7871-7872, 7874-7881, 7883-7884, 7886, 7888, 7891-7892, 7894-7899, 7901-7902, 7904, 7907, 7910, 7914, 7916-7919, 7921-7925, 7927-7928, 7931-7941, 7943, 7945- 7948, 7951, 7953-7955, 7959, 7961-7966, 7968-7972, 7974-7975, 7977, 7979-7981, 7988-7989, 7991-7992, 7994-7997, 7999, 8001-8008, 8011- 8015, 8017-8018, 8021-8022, 8024-8025, 8027-8028, 8030-8034, 8037- 8038, 8042-8043, 8047, 8049-8059, 8061-8066, 8068-8072, 8074, 8076, 8080-8082, 8084-8086, 8088, 8092-8094, 8096, 8098-8099, 8103, 8105- 8111, 8116-8117, 8122, 8124, 8126, 8128, 8133-8137, 8145-8148, 8151- 8152, 8156-8158, 8164, 8166, 8171-8174, 8176-8182, 8185, 8187-8192, 8194-8199, 8210-8212, 8215-8216, 8218-8220, 8222-8228, 8233-8237,

8239-8240, 8243-8244, 8246-8252, 8254, 8256, 8258, 8260, 8263-8265, 8268, 8271, 8273-8275, 8279, 8281-8286, 8289, 8291-8295, 8297, 8299- 8306, 8308-8309, 8311-8312, 8315-8321, 8323-8326, 8328, 8330-8331, 8333-8336, 8338-8339, 8342-8343, 8345-8346, 8348, 8350-8355, 8357- 8360, 8362-8365, 8368, 8371, 8373, 8375-8378, 8380-8382, 8384-8386, 8389-8391, 8393, 8395-8399, 8402, 8405, 8407-8409, 8411, 8413, 8415, 8422, 8426-8430, 8432-8433, 8436, 8440-8442, 8444, 8446, 8448-8449, 8454-8455, 8457, 8461-8462, 8465, 8469, 8473, 8478-8479, 8485-8492, 8495-8497, 8501-8503, 8506, 8510-8512, 8514-8518, 8521, 8524-8525, 8529, 8533-8543, 8547-8548, 8554-8559, 8561-8562, 8566-8570, 8572, 8582, 8584-8585, 8594-8595, 8597-8599, 8602, 8604, 8607-8608, 8610- 8611, 8614-8617, 8621, 8626-8628, 8630-8635, 8641-8642, 8647, 8649, 8652, 8654, 8656-8658, 8660-8666, 8668, 8672, 8674-8676, 8678-8679, 8681-8687, 8694, 8698-8699, 8702-8707, 8709, 8713-8714, 8716, 8719- 8721, 8723-8725, 8732-8734, 8736, 8738, 8740, 8742, 8744, 8749-8750, 8752-8754, 8759, 8762-8763, 8767, 8769-8770, 8772, 8776-8778, 8780- 8783, 8785-8788, 8791-8796, 8798-8800, 8804-8806, 8810-8816, 8818- 8819, 8822, 8824, 8827, 8829-8830, 8832-8835, 8837-8838, 8840, 8842- 8847, 8850-8855, 8858, 8862, 8866-8867, 8869, 8871-8877, 8879-8881, 8883-8886, 8888-8889, 8891, 8895-8896, 8903, 8907-8909, 8911-8913, 8915, 8917-8921, 8923-8924, 8928-8934, 8936, 8944, 8947-8948, 8950- 8953, 8955-8956, 8958-8961, 8964-8965, 8968-8969, 8971, 8973-8982, 8984, 8987-8993, 8995-8998, 9000, 9005-9007, 9015, 9018-9024, 9027- 9032, 9034, 9037-9039, 9042, 9044, 9047-9050, 9052-9057, 9061-9063, 9065-9070, 9075, 9077, 9080-9082, 9084-9085, 9087-9091, 9093, 9099- 9102, 9105, 9108, 9110-9111, 9113, 9115, 9117-9118, 9124, 9126,

9128, 9130, 9133, 9135-9136, 9138, 9140, 9142, 9145-9148, 9150-9155, 9157, 9167, 9169, 9176-9187, 9195, 9197, 9200, 9202, 9205-9213, 9215, 9217-9221, 9225-9228, 9231, 9239, 9241, 9244-9248, 9250-9258, 9260-9261, 9264, 9266, 9268-9270, 9274-9278, 9281, 9285, 9290, 9292- 9294, 9298, 9301, 9303-9306, 9308-9310, 9312-9314, 9316-9318, 9320- 9322, 9333-9334, 9336-9340, 9343-9344, 9351-9352, 9361, 9366-9371, 9373, 9376-9377, 9379-9382, 9385, 9387, 9390, 9392, 9396-9397, 9399, 9401, 9403-9404, 9406-9408, 9410, 9412, 9415-9418, 9420-9425, 9428, 9430-9437, 9442, 9451-9453, 9455, 9461, 9463, 9465, 9468-9469, 9472, 9474, 9480, 9482, 9484, 9497-9499, 9501, 9503, 9505-9506, 9508,

9510-9513, 9515-9516, 9518, 9520, 9522-9523, 9526-9530, 9532-9533, 9535-9543, 9549, 9551-9553, 9559-9560, 9562, 9564-9565, 9570-9573, 9575-9579, 9582-9583, 9586-9587, 9594, 9599-9601, 9603, 9605-9607, 9615, 9617-9619, 9621-9625, 9628, 9633-9635, 9638, 9641, 9643-9644, 9646-9650, 9654-9662, 9664-9665, 9667, 9670-9671, 9673, 9676-9677,

9680, 9682-9685, 9687, 9691-9693, 9697, 9699, 9703-9706, 9714, 9716, 9719-9721, 9723, 9727, 9732-9734, 9736, 9738-9740, 9743-9749, 9752- 9758, 9762-9763, 9766-9767, 9770, 9772-9774, 9776-9778, 9780, 9785, 9787-9794, 9796, 9800-9802, 9811, 9816-9821, 9823-9829, 9831, 9843, 9848, 9850, 9852-9854, 9857-9858, 9861, 9863, 9866, 9869, 9871-9872, 9874, 9876, 9878, 9882, 9890, 9892, 9894-9895, 9898-9903, 9906-9910, 9912-9916, 9918, 9921, 9923-9928, 9930-9937, 9939, 9941, 9946, 9949- 9950, 9952, 9954-9955, 9957, 9960-9961, 9966, 9968, 9970, 9973-9977, 9979, 9985-9987, 9989, 9995, 10001-10002, 10004-10012, 10014, 10016, 10018-10020, 10022-10024, 10027-10034, 10038-10040, 10044-10045, 10050-10066, 10068-10071, 10073, 10075-10076, 10078-10079, 10081- 10082, 10086-10089, 10091-10099, 10102-10107, 10110-10121, 10125- 10126, 10128-10132, 10134, 10143-10145, 10147, 10152-10157, 10159- 10160, 10162-10163, 10165-10169, 10172-10173, 10175-10176, 10180- 10183, 10185, 10194, 10196-10197, 10199-10202, 10204-10208, 10211- 10213, 10218-10221, 10223-10227, 10232, 10234-10235, 10238, 10240, 10243- 10244, 10246, 10248- 10254, 10257, 10262- 10264, 10266, 10269,

10271- 10274, 10276, 10283- 10284, 10288, 10291- 10295, 10297, 10304-

10307, 10309- 10310, 10313, 10315- 10318, 10320- 10329, 10332, 10336,

10338- 10339, 10341- 10346, 10354, 10357- 10360, 10362, 10364, 10367,

10369- 10370, 10373- 10375, 10377, 10379- 10381, 10383- 10384, 10387,

10389, 10395- 10396, 10399, 10401- 10402, 10405- 10407, 10409, 10411-

10413, 10416- 10417, 10422, 10427, 10434 -10435, 10437 , 10441 , 10443-

10445, 10447- 10450, 10452, 10456, 10459 , 10462 , 10464-10474 , 10476-

10477, 10479- 10481, 10483, 10486- 10487, 10490, 10493 -10504, 10507-

10512, 10515- 10516, 10519, 10522- 10523, 10525- 10526, 10529, 10531-

10539, 10541, 10543 -10545, 10548- 10550, 10552, 10555 -10556, 10558,

10560, 10564, 10568 -10574, 10576- 10577, 10579- 10580, 10582, 10588-

10593, 10595, 10598 -10601, 10604, 10606 -10611, 10618 -10619, 10624-

10625, 10628, 10631 -10632, 10634, 10640 , 10642 -10646 , 10648 -10653, 10659-10665, 10667-10669, 10671, 10673-10675, 10677-10680, 10684- 10685, 10687-10693, 10697-10698, 10700, 10702-10703, 10705, 10708- 10715, 10717, 10720, 10722, 10729-10730, 10732-10733, 10735, 10737- 10740, 10743, 10745, 10750, 10753-10754, 10756-10758, 10760-10764, 10768, 10770-10772, 10775-10776, 10782, 10785-10786, 10788, 10791- 10794, 10796-10800, 10803, 10806-10809, 10819-10820, 10822-10825, 10829-10831, 10833, 10836-10838, 10840-10843, 10845-10847, 10851- 10853, 10856, 10863, 10865-10870, 10872, 10874-10878, 10880, 10885, 10892, 10894-10897, 10899-10910, 10912-10913, 10915-10920, 10930- 10931, 10935, 10938-10942, 10944-10945, 10949, 10951, 10954, 10958- 10959, 10964, 10967-10975, 10979, 10981-10984, 10987, 10990-10991, 10993-10994, 10996-11002, 11004-11007, 11010-11011, 11014, 11016, 11018, 11020, 11022, 11025-11026, 11028-11030, 11032-11038, 11040- 11051, 11055, 11057, 11059-11066, 11068-11070, 11072, 11074, 11076, 11079-11083, 11085, 11087-11091, 11095-11109, 11115-11117, 11120, 11124-11127, 11129-11130, 11133-11140, 11142-11145, 11147, 11151,

11158, 11160, 11162-11163, 11165, 11171-11172, 11175, 11177, 11179, 11181, 11183-11184, 11186-11187, 11189-11191, 11193-11195, 11198, 11201, 11204-11208, 11219, 11221, 11224, 11226-11229, 11231-11233, 11237-11238, 11243, 11245, 11247, 11249-11250, 11252-11255, 11258- 11264, 11268-11270, 11272, 11274, 11276-11277, 11279-11280, 11284, 11286-11287, 11289-11290, 11292, 11296, 11298, 11300, 11302-11306, 11308-11314, 11321, 11324-11325, 11330-11332, 11334, 11336-11338, 11340, 11344-11347, 11349, 11351, 11353-11356, 11360-11363, 11365, 11370-11375, 11380-11393, 11395-11396, 11398, 11400-11403, 11408- 11409, 11412-11414, 11419, 11421-11425, 11429, 11431, 11435, 11437- 11439, 11441, 11444, 11449-11450, 11452-11453, 11455, 11461-11462, 11465-11466, 11468-11469, 11471, 11473, 11475-11477, 11482-11483, 11485-11486, 11488, 11490-11491, 11493-11497, 11501, 11504, 11506- 11511, 11516, 11519, 11524, 11527-11528, 11531, 11535, 11537-11539, 11541, 11544-11545, 11557, 11564, 11568-11569, 11571-11572, 11574- 11579, 11583-11585, 11588-11589, 11591-11593, 11595, 11597-11600, 11602-11603, 11606-11609, 11612, 11615-11623, 11625-11626, 11628- 11633, 11635-11637, 11644, 11646-11648, 11652-11655, 11659-11660, 11666-11670, 11672, 11676-11683, 11685-11686, 11689, 11693-11696, 11698-11700, 11703-11704, 11707, 11709, 11711-11712, 11714-11716, 11718-11723, 11726-11727, 11734, 11738-11740, 11744-11745, 11747- 11750, 11752, 11755-11757, 11759, 11761-11763, 11765-11769, 11771- 11774, 11776-11784, 11786-11791, 11793-11795, 11797-11801, 11804, 11806, 11808-11812, 11815-11816, 11818-11819, 11821-11823, 11826- 11832, 11834-11837, 11839-11841, 11848, 11851-11855, 11862-11863, 11865-11866, 11868, 11870-11871, 11873-11880, 11882-11885, 11888- 11893, 11898, 11900-11901, 11903-11905, 11907, 11909-11912, 11915, 11918-11921, 11923, 11926, 11928, 11930, 11933, 11935, 11937, 11940- 11945, 11948-11949, 11952-11954, 11956-11964, 11966-11970, 11973- 11974, 11976-11981, 11983-11985, 11987-11989, 11991-11994, 11997- 12001, 12003-12006, 12008-12009, 12011, 12013-12014, 12016, 12019- 12022, 12024-12027, 12029-12031, 12033, 12035-12040, 12042-12043, 12048, 12051-12055, 12058-12061, 12063-12070, 12072-12076, 12086- 12090, 12092-12096, 12098-12100, 12105, 12107, 12109-12113, 12116- 12120, 12122-12123, 12125, 12127-12128, 12130-12131, 12133, 12137- 12139, 12141-12145, 12148, 12151, 12153-12155, 12157-12159, 12161, 12163, 12166-12167, 12170-12171, 12174-12180, 12182-12189, 12193- 12194, 12196, 12201-12202, 12206-12210, 12213-12215, 12218-12221, 12223-12225, 12227-12231, 12233, 12237-12238, 12241, 12246-12248, 12250-12252, 12255-12256, 12259, 12262-12264, 12266-12270, 12275, 12277-12278, 12280, 12282, 12290-12296, 12298-12303, 12309, 12311- 12314, 12316-12319, 12322, 12325, 12328, 12330, 12332-12348, 12350- 12353, 12357-12361, 12363-12364, 12366-12367, 12370, 12372-12374, 12377, 12379, 12382-12391, 12393-12394, 12396-12400, 12402, 12404, 12409-12410, 12412, 12415-12417, 12419-12423, 12425-12427, 12430- 12434, 12437-12438, 12440, 12446-12447, 12450-12456, 12459-12463, 12465-12466, 12469-12470, 12472-12473, 12475-12481, 12485-12486, 12491, 12495, 12497-12499, 12501-12503, 12505, 12507-12508, 12510- 12512, 12515-12517, 12519-12525, 12527, 12529, 12531-12532, 12535- 12557, 12561, 12563-12565, 12567-12568, 12570, 12574-12575, 12577- 12578, 12581-12585, 12587-12592, 12594-12595, 12597-12598, 12601, 12603, 12605-12607, 12609, 12611, 12613-12614, 12620-12622, 12624, 12626, 12628, 12630, 12633-12636, 12641-12643, 12645-12649, 12651, 12654-12658, 12660-12684, 12687, 12689-12690, 12692-12697, 12699, 12701, 12703-12710, 12714-12715, 12718-12727, 12729-12732, 12734- 12741, 12743-12744, 12747-12755, 12757-12765, 12767-12768, 12771- 12778, 12781, 12784-12791, 12795, 12798, 12801-12805, 12807-12809, 12812, 12815, 12817-12820, 12824, 12827, 12830-12831, 12835, 12837- 12845, 12847-12848, 12850, 12853-12857, 12860-12863, 12866-12867, 12869-12870, 12872-12874, 12876-12877, 12879, 12881, 12885, 12888- 12889, 12893-12896, 12899, 12901, 12904-12905, 12907, 12912-12913, 12915, 12917, 12919-12920, 12922-12926, 12928-12929, 12931-12939, 12941-12944, 12946, 12951, 12955-12956, 12958-12963, 12967-12970, 12972, 12974-12975, 12977, 12980-12982, 12984-12986, 12988-12992, 12995-12996, 12998-12999, 13001, 13003, 13006-13010, 13016-13017, 13023, 13025-13027, 13033-13035, 13037-13038, 13049-13051, 13053-

13056, 13058, 13060-13062, 13064-13070, 13072, 13075, 13078, 13080, 13083-13087, 13091-13097, 13101, 13103, 13105, 13108-13110, 13112- 13116, 13119-13122, 13126, 13130-13131, 13133-13137, 13139-13147, 13149, 13151, 13154-13155, 13159-13163, 13166-13168, 13170-13171, 13173-13174, 13176-13179, 13182-13186, 13188, 13190-13197, 13199- 13200, 13202-13205, 13207-13216, 13218-13227, 13229-13231, 13234- 13235, 13237, 13239-13240, 13242-13243, 13245-13247, 13250-13252, 13254, 13262, 13264-13266, 13269-13281, 13283-13284, 13286-13287, 13291, 13294-13298, 13300, 13303, 13305, 13307-13311, 13316, 13318- 13319, 13322, 13324-13332, 13334, 13336-13337, 13340-13343, 13345, 13347, 13349-13351, 13355-13358, 13361-13368, 13370-13371, 13373- 13374, 13376, 13378, 13380-13386, 13389-13390, 13392, 13394-13398, 13400-13405, 13407, 13409-13415, 13419-13420, 13422-13424, 13428, 13431-13439, 13441, 13443-13444, 13446-13452, 13455-13461, 13463, 13465-13468, 13470-13471, 13474-13475, 13477, 13479, 13482, 13484- 13489, 13492-13500, 13504-13505, 13507-13514, 13516-13517, 13519, 13525-13526, 13531, 13533, 13535-13537, 13540-13541, 13543-13544, 13548-13552, 13554, 13556, 13559-13564, 13566-13568, 13573, 13575, 13577-13580, 13582, 13584, 13587-13588, 13591-13593, 13596-13599, 13601-13604, 13608-13610, 13615, 13617, 13619-13622, 13625-13626, 13628-13629, 13633, 13635-13637, 13640-13643, 13647-13648, 13650- 13658, 13660-13661, 13664, 13666-13667, 13669-13672, 13674-13675, 13678-13679, 13682-13686, 13689-13690, 13693-13699, 13703, 13712- 13714, 13719-13720, 13722, 13724, 13726, 13728, 13730, 13732, 13735- 13740, 13742, 13745-13748, 13751-13752, 13754-13761, 13763, 13768- 13770, 13772, 13774-13775, 13778, 13781, 13786, 13788, 13790-13793, 13798-13802, 13809-13812, 13816, 13823-13824, 13826-13827, 13829- 13830, 13832, 13835-13836, 13838-13842, 13844, 13847, 13849, 13852- 13854, 13856, 13860, 13862-13864, 13867, 13872, 13874, 13876-13878, 13881, 13883, 13885, 13889, 13891-13895, 13901-13902, 13905, 13907- 13916, 13920, 13922-13923, 13925, 13929, 13933, 13935, 13939-13950, 13953, 13955, 13957-13963, 13966-13969, 13971-13972, 13974, 13976, 13979, 13981-13983, 13985-13989, 13994, 13996-13997, 13999-14000, 14004-14006, 14008-14009, 14012-14017, 14019, 14021, 14024, 14027, 14030-14031, 14033, 14039, 14043, 14046, 14049-14051, 14057-14058, 14061, 14064-14070, 14072-14075, 14079-14080, 14082, 14084-14087, 14089-14090, 14092-14102, 14107-14108, 14112-14113, 14116-14121, 14123-14125, 14127-14130, 14132, 14134, 14141, 14143-14144, 14148- 14149, 14152-14157, 14159-14165, 14167-14171, 14173, 14175, 14177- 14178, 14182-14184, 14186, 14189, 14191-14194, 14196-14197, 14199, 14203-14204, 14206-14207, 14210, 14213, 14216-14218, 14221, 14223- 14224, 14226-14230, 14233, 14235-14237, 14239-14240, 14242-14244, 14246-14247, 14249, 14252-14255, 14257, 14260, 14262-14263, 14266, 14268-14269, 14273, 14279-14285, 14289-14293, 14296, 14298, 14300- 14301, 14310, 14322, 14324, 14326, 14332-14333, 14337-14339, 14341- 14344, 14346-14347, 14349, 14351, 14353, 14356-14360, 14362, 14364- 14367, 14370, 14373-14374, 14380, 14386-14388, 14391, 14393-14394, 14396, 14398-14400, 14403, 14411, 14418, 14423-14424, 14431-14432, 14436-14437, 14441, 14446-14447, 14449-14451, 14455-14457, 14459- 14460, 14462-14463, 14465, 14471-14473, 14475-14476, 14478, 14480- 14482, 14484, 14487, 14490-14494, 14496, 14498-14499, 14502-14505, 14508-14509, 14513, 14515-14517, 14520-14522, 14524-14526, 14528, 14532-14533, 14536, 14538, 14540-14541, 14543-14544, 14546, 14552, 14555-14558, 14560, 14562, 14564-14570, 14574-14575, 14577, 14580- 14582, 14586-14592, 14595, 14599, 14602, 14604-14606, 14608-14612, 14617-14620, 14623-14624, 14626-14627, 14629, 14632-14633, 14635- 14636, 14638-14639, 14641-14642, 14644-14651, 14653-14656, 14659, 14661, 14664-14671, 14673, 14675-14676, 14680-14682, 14687, 14689- 14690, 14692, 14694-14698, 14701, 14703-14704, 14706-14709, 14711, 14713-14715, 14723, 14727, 14729-14732, 14734, 14737, 14745-14747, 14749-14751, 14753-14755, 14760, 14762-14765, 14767-14768, 14771- 14775, 14781-14786, 14788-14792, 14794-14796, 14798-14799, 14801- 14807, 14809-14810, 14812, 14814, 14816-14817, 14819-14822, 14827- 14828, 14830-14831, 14833-14834, 14837-14839, 14842-14846, 14848- 14849, 14851-14853, 14855, 14858-14864, 14867, 14880-14887, 14891, 14894, 14896-14899, 14901-14904, 14907-14909, 14911, 14913, 14915, 14917-14919, 14921-14923, 14925, 14928, 14930, 14935-14936, 14938- 14939, 14941, 14948, 14952-14953, 14955-14956, 14960, 14962, 14966- 14972, 14974-14975, 14977, 14979-14982, 14985, 14987-14989, 14992, 14994-14997, 15000, 15002-15005, 15008-15010, 15012-15017, 15019- 15023, 15027-15028, 15030-15031, 15033, 15035, 15037-15041, 15043- 15044, 15046-15048, 15051-15053, 15056, 15058-15060, 15062-15066, 15068-15074, 15076-15077, 15081-15083, 15085, 15087, 15089, 15094, 15098, 15102, 15104-15108, 15110-15116, 15119-15123, 15125-15126, 15128, 15132, 15139-15142, 15144-15145, 15147, 15151-15152, 15155, 15158, 15163-15164, 15166-15168, 15170-15172, 15174-15175, 15177- 15178, 15181-15183, 15185-15186, 15188-15190, 15192, 15194, 15197, 15202, 15209, 15215, 15219, 15224-15226, 15228, 15230, 15232-15233, 15235-15238, 15249-15250, 15252-15253, 15255, 15257-15259, 15262, 15266, 15268-15271, 15273-15274, 15277, 15279-15280, 15283-15287, 15289-15290, 15292-15297, 15300, 15302, 15308-15312, 15315, 15319, 15328-15331, 15334, 15338, 15342, 15344, 15346, 15351, 15353-15354, 15360, 15365, 15371, 15374, 15377-15383, 15387, 15390-15391, 15393, 15395, 15398-15400, 15407, 15409, 15411-15416, 15418-15419, 15423- 15426, 15428, 15430, 15432-15434, 15437, 15444, 15446, 15448, 15450- 15456, 15459-15460, 15463-15464, 15468-15476, 15478-15482, 15484, 15487-15493, 15497-15498, 15504-15507, 15515, 15517, 15519-15521, 15523-15527, 15529-15531, 15533-15534, 15536-15539, 15542, 15545, 15549, 15551, 15555, 15567, 15570-15571, 15573, 15575-15576, 15581, 15583-15584, 15586, 15589-15594, 15598-15599, 15601-15602, 15605, 15609-15611, 15614-15615, 15618-15620, 15626, 15635, 15645, 15648, 15650-15652, 15659, 15661-15666, 15668, 15670-15671, 15674, 15676- 15677, 15679-15691, 15694, 15700-15701, 15703-15713, 15715-15716, 15718, 15720-15722, 15727-15730, 15732-15733, 15736-15740, 15742, 15746, 15749-15765, 15768, 15770, 15773-15774, 15777, 15779-15784, 15786-15802, 15804-15809, 15811, 15813-15828, 15830-15835, 15837- 15838, 15840-15846, 15848-15858, 15860-15861, 15864, 15866, 15868- 15878, 15880, 15882-15886, 15892-15893, 15897-15898, 15900-15902, 15904, 15906-15907, 15910-15923, 15926-15927, 15929-15933, 15935- 15936, 15942-15943, 15945-15947, 15949-15950, 15953, 15956-15963, 15965-15966, 15968, 15970-15971, 15973-15974, 15976, 15978-15981, 15983, 15986, 15988-15989, 15991-15995, 15997-15998, 16004-16005, 16007-16012, 16015-16016, 16018-16020, 16023-16032, 16034, 16037- 16038, 16044, 16046-16050, 16053, 16057, 16059, 16061, 16063-16064, 16069, 16071-16079, 16082-16083, 16085-16088, 16094-16096, 16098, 16101, 16109-16110, 16112-16115, 16117, 16119-16120, 16127-16129, 16131, 16137, 16139-16141, 16143-16145, 16149, 16151, 16154-16155, 16159-16162, 16166-16174, 16178, 16180-16182, 16185-16189, 16191, 16197-16204, 16206-16209, 16216-16217, 16219, 16223-16227, 16229, 16233-16234, 16236-16239, 16241-16244, 16246-16247, 16249, 16252, 16254, 16257, 16259-16260, 16263-16264, 16266-16269, 16273, 16276, 16279-16284, 16288-16290, 16292, 16295, 16298, 16302-16303, 16305- 16309, 16318, 16321, 16327, 16330-16334, 16336-16341, 16343, 16347, 16350, 16352-16353, 16356, 16358-16359, 16363, 16365-16370, 16374, 16376, 16381, 16383, 16385-16392, 16394-16401, 16403-16404, 16407, 16409, 16412-16414, 16416-16423, 16425-16426, 16430-16433, 16435, 16437, 16439-16441, 16444-16447, 16450, 16452-16455, 16457-16459

Single Strand Oligonucleotides (Sense Strand of Target Gene)

SeqID range : 16426- 45713

SeqIDs w/o G Runs :

16426- 16443, 16451- 16453, 16455- 16465, 16480- 16511, 16554- 16810,

16824- 16869, 16884- 16919, 16934- 17023, 17037- 17389, 17403- 17628,

17643- 17720, 17734- 17741, 17765- 17790, 17814- 17818, 17843- 17880,

17894- 17927, 17941- 17950, 17964- 17998, 18012- 18082, 18098, 18114-

18140, 18155, 18174 -18189, 18216 -18220, 18234 -18264, 18278 -18336,

18350- 18354, 18368- 18377, 18391- 18402, 18421- 18428, 18444- 18445,

18459- 18578, 18592- 18628, 18642- 18660, 18674- 18687, 18702- 18703,

18718- 18722, 18737- 18744, 18758- 18770, 18791- 18808, 18823- 18926,

18956- 19009, 19023- 19040, 19054- 19078, 19092- 19133, 19147- 19205,

19220- 19401, 19415- 19417, 19431- 19497, 19511- 19539, 19553- 19555,

19569- 19591, 19605- 19606, 19620- 19846, 19861- 20036, 20050- 20056,

20084- 20099, 20114- 20130, 20144- 20172, 20186- 20261, 20275- 20454,

20469- 20683, 20697- 20848, 20862- 21014, 21049- 21165, 21180- 21242,

21256- 21261, 21275- 21280, 21294- 21361, 21398- 21446, 21460- 21521,

21535- 21603, 21617- 21984, 21998- 22155, 22174- 22182, 22196- 22318,

22341- 22450, 22464- 22465, 22479- 22500, 22515- 22522, 22548- 22598,

22613- 22657, 22671- 22699, 22713- 22723, 22737- 22753, 22767- 22793,

22816- 22817, 22831- 22919, 22933- 22954, 22980- 23032, 23046- 23050,

23065- 23077, 23098, 23119- ^■23211, 23225- ^■23290, 23305- ^■23318, 23333-

23340, 23357- ^■23362, 23389- ^■23425, 23480- ^■23483, 23513- ^■23535, 23557-

23602, 23648- ^■23668, 23684- ^■23744, 23765- ^■23780, 23802- ^■23815, 23841-

23889, 23903- ^■23920, 23935- ^■23954, 23969- ^■24010, 24033- ^■24053, 24067-

24088, 24110- ^■24135, 24149- ^■24169, 24191- ^■24198, 24213- ^■24219, 24235-

24242, 24256- ^■24309, 24323- ^■24369, 24389- ^■24511, 24525- ^■24717, 24739-

24799, 24814- ^■24913, 24928- ^■24930, 24944- ^■24992, 25006- ^■25072, 25086-

25182, 25198- ^■25248, 25262- ^■25274, 25288- ^■25487, 25502- ^■25823, 25838-

25932, 25947- ^■26034, 26061- ^■26135, 26149, 26164 -26220, 26254 -26297,

26311- 26322, 26345, 26359- ^■26361, 26375- ^■26392, 26407, 26422 -26521,

26535- 26544, 26559- 26603, 26617- 26670, 26694- 26793, 26807, 26821-

26848, 26862, 26905 -26979, 26993 -27012, 27043 -27072, 27088 -27264,

27279- 27295, 27309- 27325, 27339- 27405, 27420- 27539, 27554- 27573,

27587- 27614, 27653- 27671, 27697, 27729- ^■27790, 27812- ^■27832, 27858-

27897, 27928- ^■27935, 27949- ^■27951, 27966- ^■27976, 27990- ^■27997, 28024-

28139, 28153- ^■28272, 28287- ^■28306, 28320- ^■28627, 28641- ^■28650, 28664-

28666, 28691- ^■28694, 28708- ^■28794, 28814- ^■29339, 29354- ^■29395, 29423-

29515, 29535- ^■29839, 29873- ^■30111, 30125- ^■30439, 30453- ^■30461, 30475- 30691, 30715 -30731, 30763-^■30884, 30898-^■30941, 30955-^■30996, 31010-

31244, 31259 -31304, 31318- ^■31352, 31366- ^■31378, 31392- ^■31439, 31453-

31501, 31531 -31596, 31611- ^■31618, 31632- ^■31750, 31765- ^■31776, 31790-

31899, 31913 -31923, 31937- ^■31940, 31954- ^■32036, 32050- ^■32066, 32080-

32176, 32191 -32227, 32241- ^■32250, 32264- ^■32270, 32285- ^■32337, 32351-

32359, 32373 -32388, 32408- ^■32478, 32503, 32517 -32589, 32603 -32729,

32744- 32795, 32809- 32953, 32967- 32968, 32993- 33318, 33332- 33367,

33382- 33464, 33478- 33507, 33531- 33590, 33604- 33606, 33633- 33639,

33653- 33690, 33704- 33709, 33723, 33739- ^■33749, 33776, 33797 -34293,

34308- 34364, 34389- 34414, 34430- 34457, 34471- 34472, 34486- 34506,

34531- 34562, 34599- 34804, 34818- 34832, 34876- 34881, 34896- 34945,

34985- 35012, 35047- 35053, 35067- 35091, 35105- 35240, 35255- 35263,

35277- 35352, 35368- 35379, 35393- 35498, 35523- 35582, 35635- 35764,

35779- 35901, 35917- 36076, 36099- 36103, 36125- 36130, 36148- 36160,

36174- 36217, 36231- 36242, 36265- 36317, 36336- 36523, 36550- 36605,

36619- 36804, 36818- 37213, 37260- 37264, 37292- 37303, 37323- 37418,

37437- 37482, 37518- 37557, 37578- 37592, 37617- 37619, 37633- 37640,

37654- 37726, 37740- 37800, 37822- 37830, 37844- 37865, 37880- 37945,

37959- 37974, 38000- 38107, 38121- 38138, 38152- 38344, 38358- 38398,

38412- 38530, 38547- 38564, 38578- 38669, 38683- 38700, 38714- 38775,

38797- 38866, 38892- 38902, 38908- 38925, 38948- 38970, 38993- 39015,

39036- 39069, 39083- 39423, 39437- 40260, 40275- 40294, 40309- 40460,

40474- 40493, 40523- 40526, 40557- 40656, 40670- 40672, 40688- 40796,

40824- 40826, 40847- 40861, 40875- 40899, 40913- 40967, 40982- 41097,

41111- 41285, 41309- 41311, 41326- 41450, 41464- 41600, 41614- 41684,

41698, 41724 -41758, 41772- ^■41809, 41824- ^■41882, 41896- ^■41921, 41935-

42033, 42047 -42172, 42186- ^■42187, 42222- ^■42253, 42278- ^■42448, 42463-

42601, 42615 -42621, 42635- ^■42711, 42725- ^■42740, 42760- ^■42887, 42901-

42965, 42980 -42988, 43002- ^■43010, 43024- ^■43030, 43053- ^■43096, 43110-

43228, 43242 -43243, 43270- ^■43287, 43301- ^■43391, 43405- ^■43505, 43519-

43523, 43537 -43557, 43571- ^■43622, 43636- ^■43668, 43682- ^■43695, 43716-

43722, 43736 -43816, 43830- ^■43851, 43865- ^■44058, 44072- ^■44344, 44358-

44406, 44420 -44445, 44459- ^■44464, 44484- ^■44496, 44516- ^■44547, 44561-

44583, 44627 -44630, 44644- ^■44738, 44752- ^■44763, 44777- ^■44861, 44878-

44925, 44941 -45010, 45036- ^■45040, 45074- ^■45201, 45215- ^■45267, 45293-

45342, 45355 -45378, 45391- ^■45433, 45452- ^■45513, 45524- ^■45665, 45679-

45688, 45702 -45713

SeqIDs w/o miR Seeds :

16426, 16430 -16433, 16435, 16437 , 16439 -16441 , 16444 -16447 , 16450

16452- 16455, 16457- 16459, 16462- 16465, 16467- 16468, 16470- 16472,

16478- 16487, 16489, 16492, 16495 -16497, 16499 -16500, 16504 , 16507

16508, 16510 -16516, 16524- ^■16529, 16531- ^■16534, 16536, 16541 , 16544

16549, 16552, 16554, 16558, 16560-16565, 16567, 16570-16572, 16574, 16576, 16580, 16582, 16585-16586, 16588-16593, 16595-16597, 16599- 16602, 16604-16605, 16607-16611, 16613, 16615-16621, 16623, 16625, 16627-16636, 16638, 16640-16643, 16646, 16649-16650, 16652-16663, 16665-16668, 16670-16671, 16673, 16676-16677, 16679-16682, 16685- 16686, 16690, 16692, 16694, 16699-16701, 16703, 16705-16707, 16709- 16710, 16712, 16714-16718, 16720-16725, 16729, 16732-16735, 16737- 16742, 16747-16753, 16755, 16757-16758, 16760-16776, 16778-16779, 16781-16783, 16785-16789, 16792, 16795-16803, 16806, 16809-16811, 16813-16816, 16818-16820, 16822-16823, 16826-16827, 16830-16832, 16834-16835, 16837-16847, 16851-16852, 16854, 16858-16861, 16863, 16866, 16868-16870, 16872-16874, 16882, 16884-16894, 16896, 16898, 16900-16901, 16905-16907, 16909, 16911-16913, 16915, 16917, 16919, 16922-16923, 16925, 16928, 16930, 16932-16934, 16936-16937, 16940, 16942-16946, 16948, 16953-16954, 16956-16957, 16959-16961, 16964- 16966, 16968-16971, 16973, 16975-16984, 16987, 16989-16993, 16998- 17003, 17005, 17010-17012, 17014-17015, 17017, 17019-17021, 17023- 17025, 17027-17029, 17039, 17041, 17043, 17045, 17048, 17052-17053, 17055-17056, 17058-17059, 17061-17068, 17072, 17075, 17078, 17081, 17084-17087, 17089, 17092, 17094-17097, 17099-17101, 17104, 17107- 17108, 17112-17120, 17122, 17124-17125, 17128-17130, 17132-17133, 17139, 17141, 17143-17146, 17149, 17154-17156, 17158-17159, 17161- 17162, 17164, 17169-17176, 17179-17182, 17185, 17187-17188, 17190, 17195, 17197-17198, 17200-17210, 17213, 17217-17220, 17222-17227, 17229-17230, 17234-17236, 17238-17239, 17241, 17245-17253, 17255, 17257-17262, 17264-17265, 17267, 17270-17271, 17273-17276, 17279, 17281-17283, 17285-17288, 17292, 17294-17296, 17299, 17301-17309, 17313, 17316-17318, 17320-17321, 17324-17325, 17328, 17333, 17337, 17340-17343, 17347-17350, 17354-17355, 17357, 17359-17362, 17364- 17367, 17370, 17372-17374, 17377-17384, 17386-17387, 17391, 17396, 17398, 17402, 17405, 17407, 17410-17412, 17414, 17420-17421, 17424- 17428, 17430-17432, 17435, 17439-17447, 17449-17451, 17453, 17455, 17457-17460, 17462-17464, 17467, 17470, 17472-17475, 17478, 17481- 17483, 17490, 17492-17493, 17495-17500, 17502-17503, 17505-17506, 17510-17515, 17518-17519, 17522, 17526-17528, 17530, 17532-17535, 17537-17538, 17541, 17543, 17545, 17547-17549, 17558-17559, 17562, 17565, 17567, 17570, 17572, 17577-17581, 17583, 17586-17588, 17590- 17593, 17595, 17597-17598, 17600, 17603, 17605-17609, 17611-17613, 17615-17622, 17626, 17628, 17630, 17632-17635, 17642, 17645, 17649- 17652, 17657-17660, 17662-17668, 17670-17671, 17673-17674, 17676- 17678, 17680, 17683-17684, 17686-17687, 17689-17691, 17698-17709, 17711-17712, 17715-17716, 17718, 17721-17722, 17724-17730, 17732, 17734, 17741-17747, 17750-17751, 17753-17756, 17764-17765, 17767- 17770, 17772, 17775-17776, 17779, 17781-17783, 17785, 17790-17794, 17796, 17798-17799, 17807-17808, 17810, 17812, 17816-17819, 17823- 17824, 17826, 17829, 17831-17832, 17834, 17837, 17841, 17843-17844, 17846-17853, 17855-17857, 17860-17866, 17870, 17872-17877, 17881- 17883, 17886-17887, 17889-17890, 17894-17896, 17898, 17900-17901, 17904, 17906, 17910-17920, 17923-17925, 17927-17929, 17931-17934, 17936, 17941-17944, 17949, 17952-17953, 17955, 17960, 17963-17965, 17967, 17969, 17971-17972, 17974-17975, 17983, 17987, 17994-17998, 18000, 18004, 18007, 18012, 18014-18016, 18021-18027, 18029-18030, 18032, 18036, 18038, 18040-18042, 18044-18046, 18049, 18051, 18058, 18060-18063, 18067-18069, 18071-18072, 18076-18079, 18081, 18083- 18085, 18087-18092, 18097-18100, 18102-18108, 18110, 18112, 18114- 18116, 18118, 18125, 18129, 18131, 18133-18135, 18139, 18142, 18145- 18146, 18150-18151, 18153-18155, 18161-18162, 18166, 18169-18178, 18180, 18182-18183, 18187-18193, 18198, 18201, 18203, 18206-18210, 18214-18219, 18225-18227, 18231, 18233-18234, 18236, 18239-18242, - Ill -

18245, 18247-18248, 18251-18252, 18255-18256, 18262-18267, 18270, 18273, 18280, 18282-18283, 18286, 18288, 18295, 18297, 18300-18302, 18305-18306, 18310, 18312, 18315, 18317-18319, 18322-18325, 18327- 18328, 18330, 18332, 18335-18336, 18338, 18340-18341, 18345, 18350, 18352-18353, 18357-18359, 18363, 18366, 18369, 18371, 18374-18376, 18378-18380, 18383-18384, 18388, 18390-18399, 18401-18402, 18405, 18407-18413, 18416, 18421, 18425-18428, 18434-18435, 18441, 18443- 18445, 18447-18449, 18451-18453, 18457, 18459-18461, 18463-18471, 18473-18474, 18478-18480, 18482-18487, 18489-18491, 18493-18494, 18498, 18500, 18503-18504, 18507-18510, 18514-18517, 18521, 18523- 18527, 18529, 18531, 18534, 18536-18539, 18541, 18545, 18548-18553, 18557-18561, 18563-18564, 18566-18567, 18569-18572, 18574, 18576- 18580, 18582, 18588-18589, 18591, 18593-18601, 18603, 18605-18610, 18612-18615, 18617-18621, 18623, 18625, 18631, 18635-18638, 18640- 18642, 18644-18645, 18648-18651, 18653, 18658-18659, 18662-18668, 18673-18674, 18676-18678, 18682-18683, 18685-18693, 18697, 18699, 18702, 18704, 18708, 18710-18712, 18720-18721, 18724-18728, 18736- 18739, 18741, 18743-18746, 18748-18751, 18755, 18757-18759, 18761, 18763-18764, 18766, 18770, 18777, 18779, 18781-18786, 18788-18794, 18796-18797, 18801, 18803-18805, 18808, 18812-18813, 18818, 18821, 18823-18824, 18830-18831, 18836, 18838-18839, 18841, 18844-18846, 18848, 18853, 18856-18857, 18859-18860, 18863-18868, 18873-18878, 18880-18881, 18887, 18890-18893, 18897, 18900-18901, 18903, 18907, 18911-18915, 18917-18918, 18920, 18924, 18929-18930, 18933, 18935, 18943, 18947-18948, 18951-18954, 18958, 18960, 18962, 18966-18970, 18973, 18975, 18977-18978, 18980-18982, 18984-18991, 18993-18995, 18998, 19002-19005, 19008, 19010-19020, 19022, 19024-19025, 19027- 19028, 19036-19038, 19045-19046, 19049, 19055-19056, 19061-19062, 19064, 19067, 19071-19073, 19076-19078, 19080, 19083, 19093-19095, 19097, 19099-19102, 19104-19107, 19109, 19113-19114, 19116, 19120, 19123-19126, 19128, 19130-19134, 19137, 19139, 19141-19142, 19147, 19150-19155, 19157-19162, 19164-19169, 19172-19173, 19176-19181, 19183-19186, 19188-19189, 19194-19195, 19204-19210, 19215, 19221, 19226-19232, 19234, 19236, 19240, 19244-19247, 19249-19252, 19255- 19257, 19260-19261, 19263-19265, 19271, 19275-19276, 19278-19279,

19282, 19284, 19286, 19289-19290, 19292-19294, 19298, 19303, 19306, 19309-19315, 19317-19318, 19323-19326, 19329-19332, 19336-19337, 19341, 19343-19352, 19354, 19356-19363, 19365, 19368, 19371-19374, 19376-19379, 19381-19382, 19384-19389, 19391, 19396, 19398-19399, 19404-19406, 19414-19415, 19417, 19419, 19421-19424, 19426-19427, 19430-19432, 19434, 19440-19444, 19446, 19448-19449, 19451-19461, 19463, 19468-19472, 19475-19476, 19478, 19480-19481, 19483-19484, 19487, 19490, 19493, 19500-19503, 19506, 19510, 19512, 19514-19515, 19517-19520, 19522-19525, 19527-19529, 19532, 19538, 19541-19542, 19544-19547, 19552-19553, 19556-19558, 19560-19564, 19566-19567, 19569-19570, 19572-19573, 19578-19582, 19584-19585, 19587-19592, 19594-19597, 19599-19600, 19605-19610, 19613-19617, 19619-19621, 19623, 19626-19628, 19630, 19634, 19637-19639, 19641-19643, 19645, 19648, 19651, 19655-19659, 19661-19662, 19664, 19667-19671, 19673- 19674, 19678-19679, 19682-19683, 19685-19691, 19695-19703, 19705- 19707, 19709-19713, 19716-19717, 19719-19722, 19724-19730, 19732- 19740, 19742-19744, 19746, 19750-19752, 19755-19758, 19760, 19762, 19764-19765, 19768-19769, 19771, 19774-19776, 19778-19782, 19785, 19787, 19789-19798, 19800-19807, 19811-19812, 19814-19816, 19818- 19820, 19822, 19825, 19827-19828, 19831-19832, 19834-19839, 19842, 19844-19846, 19848-19852, 19858, 19860-19861, 19863-19869, 19871- 19878, 19880-19881, 19883-19885, 19890-19891, 19893, 19895-19900, 19903-19905, 19907-19908, 19911-19914, 19916, 19918-19919, 19922- 19923, 19925-19928, 19930-19932, 19934-19935, 19937, 19939-19940, 19942, 19944-19946, 19948-19949, 19952-19953, 19957-19958, 19960, 19963, 19965, 19967-19973, 19976-19977, 19983, 19985-19989, 19991, 19993-19994, 19996, 19998, 20000-20002, 20005-20020, 20022, 20024- 20025, 20027-20035, 20037, 20039-20041, 20044-20046, 20049, 20051- 20055, 20057, 20059-20064, 20071, 20073, 20075-20076, 20078, 20081, 20083-20084, 20086, 20089-20092, 20094, 20096-20097, 20102-20105, 20111-20115, 20121, 20129-20131, 20133-20135, 20137, 20140, 20144- 20149, 20151, 20155, 20159, 20162-20164, 20166-20167, 20169, 20171, 20173, 20176, 20180-20181, 20185-20186, 20189, 20191-20197, 20202- 20211, 20215, 20218-20219, 20223-20228, 20232, 20236-20245, 20248- 20254, 20256-20257, 20259-20267, 20269, 20272, 20274-20275, 20277- 20280, 20282, 20285-20286, 20288, 20290, 20293-20300, 20306, 20308, 20310-20312, 20314-20317, 20320-20322, 20324, 20328, 20330-20332, 20334-20346, 20348, 20350-20356, 20358, 20360-20372, 20374-20377, 20379-20380, 20391-20392, 20394-20395, 20398-20399, 20401-20403, 20406-20408, 20410, 20412-20417, 20419-20420, 20422, 20425, 20427- 20428, 20430, 20432-20433, 20437-20438, 20440-20444, 20447-20448,

20450- 20461, 20467- 20472, 20476, 20479- 20483, 20488- 20491, 20494-

20496, 20498- ^■20500, 20504, 20508 -20513, 20516 , 20518 -20519, 20521,

20523- 20525, 20527, 20529- 20532, 20534, 20537 , 20539 -20543, 20545,

20551- 20555, 20557, 20561- 20563, 20566- 20572, 20574- 20576, 20578-

20588, 20590- ^■20593, 20595- 20597, 20601- 20603, 20605, 20607, 20609-

20614, 20617- ^■20618, 20620- 20632, 20634, 20637 -20638, 20641- 20644,

20647- 20648, 20652- 20654, 20657, 20664, 20667 , 20670 -20671, 20674,

20680- 20686, 20688- 20689, 20691- 20693, 20695, 20698, 20701- 20705,

20707- 20708, 20711, 20713, 20716 -20719, 20721 , 20728 , 20732 -20734,

20738- 20741, 20743- 20744, 20746- 20748, 20750- 20751, 20754, 20757,

20759- 20764, 20766, 20768, 20772 -20779, 20781 -20787, 20789- 20790,

20792- 20796, 20799, 20802- 20803, 20806- 20826, 20831- 20833, 20835-

20837, 20839- ^■20840, 20844- 20847, 20849- 20850, 20854, 20858, 20860-

20862, 20866, 20868 , 20870 -20876 , 20878 -20887 , 20889 -20890, 20894,

20897- 20899, 20906- 20907, 20910- 20913, 20916, 20918- 20922, 20925,

20928- 20933, 20939- 20946, 20948, 20950- 20955, 20957- 20960, 20962-

20966, 20968, 20970 , 20973 -20974 , 20976 -20978 , 20982 -20985, 20987-

20989, 20994, 20996 , 20998 -20999 , 21001 -21003 , 21005 -21006, 21008,

21010- 21012, 21016- 21019, 21021- 21022, 21029, 21032, 21035, 21037,

21039, 21041, 21044 -21045, 21047 -21051, 21053 , 21057 , 21065 , 21067

21069, 21076, 21078 -21080, 21084 -21085, 21087 , 21089 -21094, 21096,

21099- 21100, 21103- 21108, 21110, 21112- 21114, 21116- 21117, 21121-

21126, 21128, 21131 -21138, 21140 -21142, 21146 , 21149 -21150, 21152-

21154, 21156, 21158 -21159, 21163 , 21165 -21166 , 21170 , 21175 , 21179

21185- 21194, 21196- 21199, 21201, 21205- 21209, 21211- 21212, 21214,

21216- 21219, 21221- 21227, 21229- 21231, 21233- 21236, 21238, 21241, 21243, 21248-^■21251, 21256- 21259, 21261, 21263- 21269, 21273, 21275-

21280, 21284, 21286 -21287, 21293 -21295, 21297- 21298, 21300, 21302-

21310, 21313- ^■21314, 21317, 21320 -21321, 21325, 21327 -21328, 21330,

21332- 21333, 21337- 21338, 21340- 21341, 21344, 21346- 21348, 21351,

21354- 21358, 21365- 21370, 21375- 21377, 21379, 21381, 21385, 21387,

21389, 21395, 21397 -21403, 21405 -21406, 21408- 21411, 21418- 21422,

21424- 21425, 21427- 21431, 21433, 21436, 21438- 21439, 21444, 21448-

21449, 21451- ^■21453, 21455, 21457 -21461, 21465- 21467, 21471- 21473,

21477, 21479, 21482 -21484, 21486 , 21490 , 21492 , 21494, 21496-21497,

21499, 21501, 21503 -21507, 21511 -21514, 21516- 21517, 21519, 21522-

21525, 21527- ^■21531, 21536, 21541 , 21543 -21545, 21547 -21552, 21556-

21557, 21560- ^■21561, 21568- 21569, 21571- 21578, 21580, 21586, 21590-

21593, 21596- ^■21598, 21604- 21607, 21609, 21611- 21613, 21615- 21617,

21619, 21625- ^■21628, 21631, 21633 -21634, 21637- 21639, 21642- 21653,

21655- 21657, 21660- 21662, 21664- 21665, 21670-21671, 21673, 21675,

21678- 21680, 21682, 21687- 21695, 21697, 21699- 21702, 21708- 21711,

21713, 21718, 21721 , 21723 , 21725-21726 , 21731 , 21733-21738 , 21740-

21745, 21747, 21750 -21752, 21757 -21758, 21760, 21762 , 21769 -21774,

21777- 21780, 21783- 21785, 21788- 21792, 21794-21796, 21798-21800,

21802- 21806, 21811- 21814, 21816, 21818, 21820- 21828, 21830- 21837,

21841, 21843, 21845 , 21847 -21848 , 21850 -21851, 21853 -21855, 21857-

21859, 21861, 21863 -21868, 21870 -21874, 21876- 21878, 21880- 21881,

21883, 21887- ^■21888, 21890- 21897, 21899- 21903, 21905, 21907- 21909,

21911- 21914, 21917- 21918, 21921, 21925, 21927, 21929 -21934, 21937,

21940- 21942, 21944- 21945, 21947- 21948, 21952, 21954- 21956, 21960-

21963, 21965- ^■21966, 21968, 21971 -21973, 21976, 21978 , 21980 -21983,

21985, 21987- ^■21988, 21991- 21993, 21995, 21997- 21998, 22001, 22003,

22005- 22007, 22009- 22010, 22017, 22020- 22021, 22023, 22025- 22026,

22029- 22031, 22034, 22037, 22039 , 22041 , 22043 , 22046, 22049, 22051,

22053- 22063, 22065- 22066, 22068- 22070, 22073, 22075- 22076, 22082-

22089, 22091- 22093, 22095, 22097 -22102, 22105- 22106, 22109, 22112-

22113, 22116- ^■22117, 22119- 22125, 22130, 22132- 22133, 22135- 22136,

22139- 22143, 22153- 22157, 22161- 22162, 22165-22166, 22168-22171,

22175, 22180- ^■22181, 22185- 22186, 22188- 22190, 22196- 22199, 22201,

22205- 22206, 22208, 22210- 22215, 22217, 22222- 22225, 22227, 22229-

22234, 22236- ^■22241, 22243, 22247 , 22249 -22250, 22252 -22255, 22257-

22261, 22263- 22265, 22267- 22269, 22271, 22273, 22275 -22278, 22287-

22288, 22290- 22292, 22296, 22298 , 22301 , 22303 , 22308, 22310, 22313,

22316, 22320, 22323 -22324, 22329 , 22332 , 22339 , 22341-22342 , 22345-

22347, 22349, 22355 , 22357 -22359 , 22361 -22363, 22365 , 22367 -22369,

22373, 22376- ^■22377, 22379- 22380, 22384, 22387- 22393, 22397- 22398,

22401, 22403- ^■22407, 22410, 22413 -22414, 22416- 22427, 22430- 22432,

22434- 22437, 22440- 22441, 22446, 22448- 22450, 22452- 22459, 22461,

22469- 22474, 22476, 22479- 22480, 22482- 22485, 22491, 22493- 22495,

22500, 22502- ^■22503, 22505- 22506, 22508, 22515, 22519 -22523, 22525,

22527, 22531- 22532, 22541- 22542, 22545- 22546, 22548- 22551, 22553-

22554, 22556, 22564 , 22566 -22569 , 22571 -22579, 22581 , 22583 , 22585-

22589, 22592- ^■22597, 22601, 22604 -22605, 22608- 22609, 22612- 22615,

22618- 22622, 22624- 22635, 22637, 22641- 22642, 22644- 22645, 22647-

22650, 22653, 22655 , 22658 -22660 , 22664 -22667, 22673 , 22680 , 22682,

22685- 22691, 22694- 22698, 22700- 22708, 22712, 22719, 22721, 22723- 22724, 22730, 22732-22734, 22737, 22742-22744, 22746, 22749, 22751- 22752, 22754-22755, 22757, 22759, 22762-22763, 22766, 22768, 22771, 22774, 22777-22778, 22780, 22784-22789, 22792, 22795-22796, 22798- 22801, 22804, 22807, 22809, 22811-22813, 22815-22816, 22818-22821, 22823-22825, 22827, 22831-22836, 22838-22841, 22845-22854, 22856- 22859, 22861, 22866-22871, 22873, 22875-22879, 22883, 22885-22887, 22891-22894, 22896-22898, 22901-22902, 22904-22906, 22908-22914, 22916-22919, 22923-22927, 22931, 22933, 22935, 22938, 22944-22948, 22950-22952, 22954-22956, 22958-22959, 22961, 22964, 22968-22974, 22976-22977, 22981, 22983-22984, 22989-22991, 22994-22995, 22999- 23008, 23010-23011, 23014-23015, 23017-23020, 23022-23025, 23028- 23029, 23031-23032, 23035-23038, 23041, 23044, 23046-23047, 23049- 23054, 23056-23057, 23065, 23067-23072, 23074-23075, 23077-23079, 23081-23082, 23085, 23092-23093, 23096-23102, 23104, 23111, 23113- 23114, 23117, 23119-23123, 23125-23126, 23129-23130, 23134, 23136- 23138, 23140, 23142-23146, 23148-23150, 23152-23158, 23160-23166, 23168-23173, 23176-23178, 23180, 23182, 23184-23185, 23187-23188, 23190-23204, 23206-23213, 23216-23218, 23221-23222, 23227-23228, 23232-23235, 23237-23241, 23243, 23245, 23247-23248, 23250-23252, 23254-23256, 23259-23262, 23264, 23268-23270, 23275-23280, 23283- 23287, 23291, 23293, 23295-23296, 23298-23299, 23302, 23304-23310, 23312, 23314-23317, 23321-23322, 23324, 23326-23327, 23333, 23335- 23337, 23339-23342, 23344-23345, 23347, 23349, 23358-23359, 23362, 23364-23370, 23377-23384, 23386-23389, 23391-23393, 23396-23403, 23405-23406, 23409, 23412, 23414-23415, 23417-23418, 23420-23422, 23424, 23426-23430, 23433, 23435-23436, 23438-23442, 23454-23455, 23457, 23463, 23466, 23469, 23471-23474, 23476-23477, 23483, 23486- 23487, 23489, 23491-23492, 23494-23501, 23503-23507, 23512-23514, 23516-23518, 23520, 23522-23523, 23525-23527, 23529, 23531-23533, 23535-23537, 23540, 23544, 23549-23551, 23553-23554, 23557, 23559- 23562, 23564, 23566-23567, 23570, 23572-23582, 23584, 23586-23589, 23591-23592, 23597-23605, 23607-23610, 23616-23620, 23622-23624, 23627, 23630-23631, 23633-23635, 23637, 23642, 23644-23645, 23649- 23650, 23652-23653, 23656-23657, 23659-23663, 23667-23668, 23670, 23672-23673, 23680, 23684-23685, 23687-23691, 23693-23694, 23696- 23697, 23699-23701, 23704-23705, 23707-23715, 23720, 23722-23724, 23726-23727, 23729, 23731-23732, 23734-23735, 23737, 23739-23746, 23748, 23750, 23756-23757, 23759, 23762, 23765, 23768, 23776-23777, 23783, 23785-23788, 23794-23799, 23801-23806, 23808-23809, 23812,

23816, 23818, 23820, 23823 -23824, 23827 , 23829 -23833 , 23835 -23836,

23839- 23849, 23851, 23854- 23872, 23876- 23878, 23880- 23884, 23888-

23890, 23892- ^■23893, 23895- 23898, 23903, 23905- 23911, 23916, 23919-

23928, 23936- ^■23938, 23941- 23942, 23944- 23946, 23948, 23950- 23952,

23954- 23962, 23965, 23969- 23971, 23975, 23978, 23980 -23981, 23984,

23986, 23988- ^■23989, 23991, 23993- ^■23996, 23998- 24005, 24007, 24011-

24013, 24015- ^■24016, 24018, 24021, 24024 -24026, 24028 , 24034 -24035,

24037- 24043, 24048, 24050, 24055- ^■24057, 24061, 24063 -24064, 24066-

24068, 24072- ^■24078, 24080, 24082, 24085 -24086, 24088 , 24091 -24096,

24102, 24106- ^■24110, 24115- 24117, 24119- 24120, 24122- 24125, 24127-

24130, 24132, 24135, 24137 -24138, 24141 -24143, 24145 , 24149 , 24151

24156, 24158- ^■24162, 24164, 24166- ^■24171, 24176- 24177, 24179- 24180, 24183-24186, 24193-24194, 24197, 24201, 24204, 24206-24207, 24213- 24216, 24218-24219, 24222, 24224-24229, 24238, 24240-24242, 24244- 24245, 24247, 24249, 24257, 24260-24265, 24267-24268, 24270-24273, 24281, 24284, 24290-24301, 24303, 24308-24309, 24312, 24316-24319, 24322, 24324, 24326, 24328-24329, 24333, 24336-24342, 24344-24345, 24347-24349, 24353, 24355-24356, 24358-24359, 24361, 24364, 24367- 24368, 24375-24376, 24380, 24383, 24387, 24389-24393, 24395, 24398- 24402, 24404, 24406, 24408-24410, 24412-24415, 24417-24419, 24421- 24423, 24425, 24429-24433, 24436-24437, 24439, 24446, 24451, 24453, 24457, 24459, 24461-24462, 24465-24467, 24469, 24471, 24473, 24475, 24477-24479, 24481-24482, 24484-24488, 24491-24492, 24494-24496, 24498-24500, 24504-24506, 24508-24516, 24518-24524, 24527, 24530, 24533-24537, 24539, 24541-24542, 24544-24549, 24552-24554, 24562- 24563, 24565-24568, 24570-24571, 24573-24574, 24576-24577, 24581, 24583, 24585-24599, 24601-24602, 24605, 24609-24619, 24621-24622, 24624-24630, 24633-24642, 24645, 24648-24655, 24657-24658, 24664- 24668, 24670-24676, 24678, 24681-24684, 24686, 24690-24692, 24694- 24695, 24700-24701, 24704, 24706, 24708, 24710, 24712-24718, 24721- 24724, 24726-24728, 24730-24733, 24735, 24738-24739, 24741, 24747- 24752, 24755, 24758, 24760-24761, 24763-24765, 24767-24768, 24773, 24775-24776, 24780-24786, 24789-24790, 24792, 24794, 24797-24799, 24801-24803, 24805-24809, 24811, 24814-24815, 24817, 24819-24820, 24823, 24826-24829, 24831-24836, 24838-24841, 24844, 24846, 24849- 24852, 24856, 24859-24860, 24862, 24865-24867, 24869, 24873-24875, 24877, 24879-24880, 24886-24888, 24891, 24893-24897, 24899-24900, 24902-24915, 24919, 24922, 24927-24928, 24931-24933, 24937-24938, 24941, 24943, 24945, 24947-24950, 24955-24956, 24958-24960, 24962- 24963, 24965-24967, 24969-24971, 24973-24979, 24981, 24983-24985, 24987-24990, 24996-24997, 24999, 25003, 25007-25009, 25011-25013, 25016-25017, 25019-25025, 25033-25035, 25037, 25040-25045, 25047- 25053, 25055-25060, 25062-25063, 25065-25066, 25069, 25072, 25075, 25077-25078, 25081, 25087-25089, 25091, 25093, 25095, 25101, 25105- 25106, 25108, 25110, 25112, 25114-25118, 25123-25129, 25133-25135, 25137-25140, 25142-25145, 25147, 25149-25150, 25152, 25154-25160, 25163, 25165-25166, 25168, 25171-25176, 25180, 25182-25183, 25185- 25186, 25188-25190, 25197, 25199, 25201-25202, 25205, 25208-25209, 25211-25225, 25228-25230, 25232-25233, 25238, 25240-25241, 25243, 25246, 25248-25249, 25251-25252, 25254, 25256, 25259, 25261-25265, 25267, 25269, 25272-25273, 25275, 25278, 25282-25285, 25288, 25290- 25291, 25293-25296, 25298-25302, 25304, 25306-25308, 25310-25312, 25314, 25316-25318, 25320, 25322-25324, 25326, 25330, 25335-25340, 25345, 25347-25348, 25350-25352, 25354-25358, 25360-25364, 25367- 25370, 25372-25373, 25376, 25378, 25382-25384, 25386-25388, 25391- 25392, 25398-25404, 25407-25408, 25410-25415, 25418-25422, 25425- 25426, 25428-25429, 25434-25435, 25437, 25440-25442, 25445, 25447- 25448, 25450, 25452, 25455, 25457-25459, 25461, 25463-25464, 25469- 25470, 25473-25478, 25480-25481, 25483-25486, 25488, 25492-25493, 25495, 25497, 25501, 25503, 25505-25507, 25509-25511, 25514-25519, 25521-25523, 25525, 25528-25532, 25535-25538, 25540, 25543-25547, 25552-25556, 25558, 25561, 25565-25567, 25569-25570, 25572-25579, 25582-25585, 25588-25601, 25603, 25605, 25609-25612, 25615-25619, 25621-25624, 25626-25632, 25634-25635, 25637-25640, 25645-25647, 25649, 25651-25652, 25656-25657, 25663, 25666-25668, 25670-25673, 25675-25678, 25680, 25682-25684, 25686, 25688, 25691, 25695, 25698, 25701-25702, 25704-25705, 25707, 25709-25711, 25713, 25717-25723, 25725, 25727, 25730, 25732-25733, 25735, 25737, 25739-25740, 25742- 25744, 25752-25758, 25761-25762, 25765-25766, 25769-25770, 25773- 25774, 25776-25782, 25785-25787, 25789, 25791, 25793-25794, 25796, 25798-25799, 25801-25802, 25807, 25809-25817, 25819, 25822, 25828, 25837-25838, 25840-25842, 25845, 25847-25848, 25850-25860, 25862, 25864-25866, 25869-25870, 25873-25878, 25880-25882, 25887, 25889- 25891, 25893-25894, 25896-25897, 25899-25901, 25903-25904, 25906, 25908, 25912, 25915-25916, 25918-25921, 25924, 25926-25929, 25931, 25936-25938, 25941, 25943, 25949-25951, 25953-25961, 25963-25964, 25966, 25968, 25977, 25979-25984, 25989-25992, 25995, 25997, 25999, 26002-26008, 26011, 26013, 26017-26024, 26026-26028, 26031-26032,

26034, 26039, 26047, 26051-26055, 26058, 26060, 26063-26065, 26068, 26070-26073, 26076-26080, 26082, 26084-26086, 26088-26093, 26095- 26096, 26098-26099, 26101, 26103-26107, 26111, 26114, 26116, 26118- 26119, 26122-26126, 26133, 26136, 26138, 26142-26144, 26146-26149, 26152-26155, 26157, 26167-26171, 26173, 26175-26177, 26179, 26181, 26183-26186, 26188, 26190-26191, 26193-26194, 26196-26197, 26201- 26204, 26207, 26210, 26213-26214, 26217, 26219-26223, 26225, 26229, 26234-26236, 26239, 26241-26242, 26253-26257, 26260, 26262-26263, 26267-26271, 26273-26274, 26278-26279, 26281, 26285-26287, 26289- 26296, 26302-26304, 26306, 26310-26311, 26314-26318, 26320-26322,

26324, 26326, 26328, 26336, 26339, 26341, 26343-26346, 26348-26353, 26355-26356, 26358-26360, 26362, 26368, 26370, 26372, 26374-26375, 26377-26385, 26388, 26392-26393, 26396, 26399, 26401, 26403, 26405- 26406, 26408-26413, 26419, 26422-26424, 26426-26427, 26430-26431, 26435-26439, 26441, 26446-26450, 26452, 26455, 26458-26460, 26463- 26466, 26468-26470, 26472-26473, 26475-26476, 26478, 26483-26486, 26488, 26490, 26493-26494, 26496-26502, 26506-26510, 26512-26515, 26517, 26519, 26522-26523, 26525-26527, 26529-26531, 26533, 26535, 26537-26538, 26541, 26545-26546, 26548-26551, 26554-26555, 26559- 26560, 26562-26564, 26568-26569, 26572-26575, 26577-26581, 26589- 26595, 26598-26609, 26611-26614, 26616-26618, 26623-26624, 26627, 26631-26636, 26639-26641, 26643-26650, 26653-26657, 26659-26660, 26662-26665, 26667-26669, 26671, 26673-26678, 26684-26685, 26687, 26690, 26694-26719, 26721-26722, 26724-26731, 26733, 26735-26746, 26748, 26750-26766, 26768, 26770, 26772-26773, 26775, 26778, 26780- 26781, 26788-26792, 26797-26799, 26801, 26808-26810, 26813-26814, 26817-26819, 26821, 26824-26825, 26828, 26830-26831, 26836-26837, 26839-26840, 26847-26851, 26853-26857, 26861-26865, 26868-26869, 26880, 26882, 26887, 26889-26891, 26893, 26895-26897, 26901, 26906- 26912, 26914-26916, 26918, 26921-26929, 26931, 26933-26935, 26937, 26939-26943, 26946-26947, 26949-26956, 26960-26961, 26964, 26966- 26968, 26970-26972, 26975, 26977-26978, 26980, 26982, 26985, 26988, 26994, 27000, 27002-27005, 27008-27012, 27016-27017, 27026-27027, 27029, 27032-27034, 27036-27037, 27044, 27048-27049, 27051-27053, 27055, 27057-27063, 27066-27069, 27071, 27074, 27076, 27090, 27093- 27098, 27101, 27103-27105, 27110, 27112-27113, 27115-27116, 27118, 27130-27135, 27137-27138, 27140-27142, 27144-27146, 27148-27154, 27160, 27162, 27164-27165, 27167-27174, 27178-27179, 27181-27182, 27186, 27188-27189, 27192-27195, 27197, 27199-27200, 27203-27204, 27207, 27209, 27212, 27214, 27216, 27220, 27224-27227, 27229-27230, 27232, 27234, 27236-27238, 27240-27243, 27245, 27247, 27253-27254, 27256-27257, 27259, 27262-27264, 27267-27268, 27272, 27277, 27279, 27283, 27285-27289, 27294-27297, 27299-27301, 27303, 27305-27306, 27309-27319, 27322-27323, 27325-27326, 27328-27333, 27335-27337, 27339-27340, 27342-27344, 27346-27351, 27353-27356, 27360-27361, 27364-27366, 27369-27370, 27372, 27374-27375, 27379-27383, 27386-

27389, 27391, 27393-27397, 27399-27400, 27402, 27404, 27407, 27410- 27412, 27414, 27417, 27420-27424, 27430-27441, 27443, 27445, 27448- 27449, 27452-27459, 27462-27463, 27467-27469, 27473-27474, 27476- 27477, 27482, 27488-27489, 27492-27496, 27498, 27500, 27502-27506, 27508, 27510, 27513-27514, 27518-27519, 27522-27523, 27525-27526, 27528-27529, 27531-27532, 27534-27535, 27542, 27548, 27555, 27557- 27559, 27562-27566, 27569-27572, 27574-27575, 27577, 27579, 27587- 27588, 27590-27595, 27597-27599, 27603-27605, 27607-27610, 27616, 27618-27620, 27622-27624, 27626-27628, 27630, 27632, 27635-27638, 27640, 27645, 27652, 27654-27659, 27668-27669, 27672-27675, 27677- 27680, 27687, 27689, 27691-27692, 27694, 27697-27698, 27700-27703, 27706, 27709-27710, 27712, 27715, 27719-27723, 27727-27730, 27732- 27735, 27738, 27740-27741, 27743-27751, 27753-27756, 27758-27759, 27761-27763, 27765-27766, 27769, 27771, 27773, 27775, 27777, 27779, 27783-27788, 27795-27796, 27798, 27804, 27809-27810, 27812-27813, 27815-27818, 27821, 27823, 27827-27830, 27832-27834, 27836, 27838- 27840, 27843, 27847, 27849, 27851, 27853, 27858-27861, 27863, 27866-

27867, 27870, 27872 , 27875 -27877 , 27880 , 27882 -27885 , 27887 , 27889-

27892, 27894- ^■27896, 27898, 27900 -27905, 27910, 27912 , 27916 , 27919,

27922, 27924, 27926 -27927, 27932 , 27934 -27938, 27940 -27946, 27948,

27950, 27952, 27954 -27956, 27958 -27959, 27967- 27969, 27971- 27976,

27978- 27984, 27992- 27993, 27995, 27998, 28001, 28003 -28006, 28010,

28012, 28017, 28021 , 28023 -28024 , 28027 -28028, 28033 , 28035 , 28038,

28040, 28044, 28054 -28056, 28058 -28062, 28064- 28066, 28068, 28070,

28072- 28073, 28075- 28077, 28081- 28083, 28087-28091, 28096-28098,

28100, 28102, 28104 -28106, 28109 -28115, 28117, 28120 -28121, 28125-

28128, 28130- ^■28133, 28135, 28138 , 28140 , 28144 -28149 , 28151 , 28153-

28154, 28161- ^■28173, 28176- 28179, 28184- 28196, 28201- 28202, 28205-

28206, 28209- 28212, 28214- 28223, 28227, 28229- 28230, 28233- 28238,

28240, 28242, 28247 -28248, 28250 -28252, 28254- 28261, 28264, 28266,

28269, 28271- ^■28278, 28283, 28288 , 28290 , 28292 , 28296-28298 , 28304-

28308, 28310- ^■28316, 28318, 28320 -28321, 28323- 28327, 28330, 28332,

28334, 28338, 28342 , 28346 -28349 , 28351 -28356, 28358 -28374, 28376,

28379, 28381- ^■28384, 28387- 28392, 28395- 28401, 28404- 28408, 28412-

28414, 28416- ^■28419, 28423- 28433, 28437- 28443, 28445- 28457, 28461,

28463, 28465- ^■28467, 28469- 28473, 28475- 28483, 28486, 28488- 28491,

28493, 28495- ^■28496, 28498- 28500, 28502- 28509, 28512- 28516, 28518,

28520- 28522, 28524- 28526, 28529- 28530, 28532, 28534, 28538- 28542,

28544- 28552, 28554, 28556, 28558 -28559, 28561, 28563 -28564, 28566-

28568, 28574- ^■28576, 28578, 28580 -28584, 28586- 28588, 28590, 28592-

28593, 28595- ^■28597, 28599, 28601 -28603, 28606, 28608 , 28611 , 28613- 28614, 28616, 28620-28621, 28623-28625, 28627-28630, 28634, 28640, 28642-28647, 28651, 28654-28655, 28657, 28666-28667, 28672-28674, 28680, 28682-28684, 28686, 28688, 28692-28693, 28695, 28698-28699, 28701, 28703, 28709-28711, 28714, 28717-28725, 28727, 28730, 28734- 28739, 28741-28744, 28746-28750, 28752, 28754, 28756-28757, 28760- 28767, 28771-28772, 28774, 28776, 28778-28780, 28782-28783, 28786- 28788, 28791, 28793-28797, 28802-28805, 28809, 28812, 28817, 28819- 28821, 28823-28824, 28826, 28829-28841, 28843, 28845-28846, 28851- 28853, 28856-28864, 28866-28871, 28874, 28877-28878, 28880-28886, 28888, 28890, 28894, 28896, 28898-28909, 28911, 28913-28915, 28917, 28919-28920, 28922-28923, 28925-28926, 28928, 28930-28931, 28934- 28935, 28938, 28941-28944, 28947-28949, 28951-28956, 28958, 28962- 28966, 28969-28976, 28978-28981, 28983, 28986-28989, 28992, 28997- 28998, 29000, 29002-29003, 29005, 29009, 29011, 29019-29021, 29023, 29025-29026, 29028-29029, 29031-29032, 29034-29036, 29039, 29041- 29042, 29045, 29048-29050, 29053-29054, 29056-29062, 29065-29071, 29073-29076, 29079, 29081, 29083-29084, 29087, 29091-29097, 29099- 29100, 29106, 29109, 29113, 29116-29118, 29120-29122, 29125-29126, 29129-29130, 29132-29133, 29135, 29137, 29139-29142, 29144-29147, 29149-29151, 29155, 29157-29160, 29162, 29164-29166, 29168, 29171, 29173-29175, 29177-29178, 29180-29186, 29189-29192, 29194-29195, 29197, 29200, 29202, 29206, 29208, 29210-29211, 29214-29218, 29224, 29226, 29229-29231, 29233-29236, 29238-29242, 29244, 29246-29248, 29250, 29252-29261, 29264-29277, 29280, 29282-29295, 29297, 29302- 29305, 29307, 29309, 29312, 29314, 29316-29319, 29321-29323, 29325- 29327, 29336-29337, 29339-29340, 29344-29349, 29352, 29354-29355, 29357, 29359-29360, 29362-29369, 29373-29378, 29380, 29383-29386, 29389-29391, 29396, 29399, 29401, 29408, 29424-29427, 29429, 29432- 29433, 29435, 29437-29439, 29441-29442, 29444-29446, 29448-29456, 29463-29469, 29479, 29481, 29483, 29485-29491, 29494-29498, 29501, 29503-29504, 29508-29509, 29511-29514, 29516-29517, 29519, 29521- 29523, 29533, 29535-29536, 29538-29543, 29546, 29549, 29551-29554, 29557-29563, 29565-29566, 29568-29570, 29572, 29575-29578, 29581, 29583-29588, 29593-29608, 29612-29624, 29626, 29628-29629, 29631- 29634, 29636-29639, 29642, 29644-29645, 29647, 29650, 29652-29658, 29660-29661, 29663-29664, 29666, 29672, 29674-29676, 29678-29680, 29682, 29685, 29687-29691, 29694-29704, 29707-29716, 29718, 29720, 29722, 29724-29727, 29730-29732, 29735-29736, 29739, 29741, 29745, 29747-29748, 29750-29767, 29772-29776, 29778-29781, 29784-29785, 29787-29788, 29790, 29792, 29794-29809, 29811-29812, 29814-29819, 29823, 29827-29828, 29831-29832, 29837, 29840, 29845-29848, 29851, 29854-29857, 29862, 29864-29866, 29872-29873, 29875, 29877-29884, 29886-29893, 29895-29896, 29898, 29900, 29902, 29904, 29906, 29908, 29911-29912, 29914-29919, 29921-29922, 29924, 29927, 29929, 29931- 29934, 29936-29939, 29941-29945, 29947-29949, 29951-29956, 29958- 29963, 29965-29966, 29968-29969, 29972, 29974-29976, 29978-29983, 29986-29988, 29990-29997, 29999-30000, 30002-30005, 30007-30009, 30011-30013, 30015, 30018, 30020-30021, 30023-30024, 30026-30027, 30029-30030, 30032, 30034-30035, 30037-30042, 30045-30051, 30053, 30055-30058, 30060, 30062-30066, 30069-30071, 30073-30077, 30079- 30087, 30089-30090, 30092, 30094-30095, 30097-30098, 30101-30105, 30107-30109, 30111-30117, 30119, 30121-30122, 30126, 30128, 30130, 30132-30138, 30142-30152, 30155-30161, 30163-30167, 30169-30170, 30174, 30176-30179, 30181-30191, 30195-30196, 30198, 30200-30201, 30203, 30205-30208, 30211-30218, 30222-30225, 30228-30230, 30233- 30237, 30239-30244, 30246, 30248-30255, 30257-30258, 30260-30263, 30265, 30268-30277, 30279-30280, 30283-30287, 30289, 30291, 30293- 30294, 30296, 30298-30300, 30302-30304, 30306-30314, 30316-30317, 30319-30321, 30325, 30327, 30331, 30334, 30336, 30338, 30341-30347, 30349-30353, 30355, 30358, 30360-30361, 30363-30365, 30368, 30370- 30371, 30373-30376, 30378-30387, 30389-30393, 30395-30397, 30400, 30404-30409, 30411-30415, 30417-30422, 30424-30425, 30428-30430, 30432-30433, 30436, 30439-30440, 30442-30443, 30449, 30451-30454, 30456-30457, 30459, 30461-30462, 30465-30466, 30469, 30473-30476, 30478, 30481, 30483-30495, 30499-30502, 30504-30513, 30520-30526, 30528, 30530, 30533-30535, 30538-30539, 30541-30545, 30547, 30549, 30551-30552, 30554-30557, 30561-30563, 30565-30568, 30572-30574, 30576, 30578, 30580, 30582-30583, 30585, 30587, 30590-30598, 30601- 30605, 30609-30615, 30617, 30621-30624, 30626-30627, 30629, 30632- 30633, 30639-30641, 30643, 30646, 30648, 30650-30654, 30656-30659, 30667-30669, 30671-30672, 30676-30678, 30680-30683, 30685-30688, 30691-30692, 30694, 30696-30698, 30702, 30705-30709, 30711-30712, 30714-30718, 30720, 30723-30727, 30730-30732, 30737, 30740, 30742, 30749, 30751, 30753, 30755-30757, 30761-30767, 30773-30775, 30777- 30781, 30783-30794, 30796, 30801-30803, 30805, 30807-30809, 30811, 30813, 30815, 30818-30823, 30825-30827, 30829-30834, 30838-30842, 30845, 30848, 30850-30853, 30855-30856, 30858, 30860-30862, 30864, 30866-30869, 30872-30873, 30877-30878, 30883-30884, 30887-30891, 30894-30898, 30901-30907, 30909, 30911-30912, 30914, 30916, 30919, 30926-30927, 30930-30931, 30934-30938, 30942-30945, 30947-30948, 30954-30958, 30961-30962, 30964-30966, 30968, 30970, 30972-30973, 30975-30979, 30981, 30983-30985, 30987-30989, 30992-30996, 30999- 31000, 31002-31006, 31009, 31011-31012, 31017, 31019-31020, 31023- 31024, 31027, 31030-31036, 31039-31042, 31044-31045, 31048-31049, 31055, 31057, 31059-31066, 31068, 31071-31072, 31074, 31077, 31082, 31084, 31086, 31089-31095, 31097, 31099-31102, 31104, 31107-31108, 31111-31116, 31119-31120, 31123, 31127, 31129, 31131-31132, 31136- 31137, 31140, 31142, 31145-31151, 31153-31154, 31156-31159, 31161, 31163-31164, 31167-31171, 31174-31176, 31181-31182, 31184-31193, 31195-31196, 31200, 31202, 31205, 31209-31213, 31216-31218, 31220- 31221, 31223, 31225, 31228-31229, 31231-31234, 31238-31239, 31242- 31243, 31250, 31253, 31257-31264, 31266-31269, 31271, 31274, 31277- 31278, 31283-31301, 31305-31311, 31314, 31326, 31328-31329, 31331- 31333, 31335-31339, 31343-31344, 31346, 31348-31349, 31356-31359, 31362, 31364, 31366-31371, 31373, 31376-31378, 31380-31382, 31385, 31392-31393, 31395-31401, 31403, 31405-31410, 31413, 31415-31417, 31419, 31421-31427, 31429-31432, 31435, 31437, 31440, 31444-31447, 31456, 31460-31462, 31464, 31466-31469, 31471, 31473-31477, 31479, 31485-31486, 31488, 31490-31494, 31496-31499, 31503-31504, 31506- 31508, 31519, 31522-31523, 31530-31533, 31535-31536, 31539-31540, 31542, 31547-31548, 31552, 31556-31559, 31561, 31565-31574, 31576- 31578, 31580-31581, 31583, 31586-31590, 31592-31598, 31601, 31608, 31611-31617, 31621, 31623-31624, 31626, 31632-31635, 31638, 31641- 31642, 31644, 31646-31648, 31651-31659, 31665, 31667-31669, 31671- 31672, 31674-31676, 31678-31687, 31690, 31696, 31698-31701, 31703, 31705, 31707, 31709-31711, 31715-31716, 31718, 31721-31722, 31724- 31726, 31728, 31730, 31733, 31735-31736, 31739, 31741-31746, 31749- 31750, 31752-31755, 31757-31758, 31762, 31764, 31766-31768, 31771- 31773, 31777-31779, 31781, 31784-31786, 31791, 31794-31802, 31804, 31806, 31809, 31811, 31814-31816, 31820-31825, 31827-31832, 31836- 31839, 31841-31844, 31848-31849, 31851, 31854-31860, 31863, 31865, 31868, 31870-31873, 31876, 31882-31884, 31888-31889, 31893, 31900, 31902-31906, 31908, 31910, 31912-31917, 31919-31920, 31923, 31930- 31932, 31935, 31940, 31942, 31947-31949, 31959, 31962-31967, 31969, 31973, 31975, 31977-31979, 31981, 31985-31986, 31992-31995, 31997- 31998, 32003-32004, 32007-32008, 32010-32011, 32013-32016, 32021- 32022, 32025-32030, 32032, 32034-32037, 32040-32042, 32047-32049, 32051-32052, 32055-32056, 32058-32059, 32062-32067, 32071-32073, 32075, 32079-32084, 32086-32088, 32090-32091, 32093, 32095-32099, 32102, 32104-32107, 32109-32115, 32117-32118, 32120-32121, 32123- 32130, 32132, 32134-32136, 32138-32139, 32142, 32144-32149, 32154- 32158, 32160, 32162, 32165-32171, 32173-32176, 32178, 32180-32183, 32186-32187, 32189, 32192-32193, 32196-32201, 32203-32204, 32210, 32215-32217, 32219-32221, 32223, 32225, 32227-32228, 32233, 32237- 32240, 32242, 32244, 32246-32248, 32250-32251, 32253-32257, 32260, 32263, 32265-32271, 32273, 32275, 32277-32279, 32284-32285, 32287- 32289, 32291-32292, 32297-32298, 32301, 32303, 32305, 32308-32315, 32317-32320, 32322-32323, 32329-32330, 32332, 32334-32340, 32343, 32352-32354, 32359-32360, 32366-32368, 32373, 32376-32377, 32381- 32382, 32384-32392, 32394, 32396, 32405, 32407-32409, 32415, 32418, 32423-32424, 32427, 32429, 32431-32441, 32444, 32446-32449, 32451- 32452, 32454-32459, 32461-32465, 32467-32468, 32470-32473, 32478- 32479, 32481, 32484-32485, 32489-32490, 32492-32497, 32503, 32505, 32507, 32511-32512, 32517-32518, 32521-32523, 32526-32533, 32535- 32538, 32540-32541, 32543-32550, 32553, 32555-32559, 32561-32566, 32568-32571, 32573-32576, 32584-32586, 32589-32590, 32592-32596, 32598, 32600, 32602-32605, 32607-32609, 32611-32612, 32616, 32626- 32630, 32632, 32634-32637, 32640-32642, 32646-32647, 32649-32653, 32655-32660, 32663-32665, 32668, 32670, 32672, 32675-32676, 32681- 32684, 32690, 32694-32711, 32717-32719, 32723-32725, 32728-32729, 32731, 32733-32738, 32742, 32744-32746, 32748-32754, 32757, 32765, 32767, 32769, 32775-32778, 32780-32782, 32784-32788, 32790-32791, 32795, 32797, 32800-32801, 32804-32805, 32808, 32813-32815, 32820- 32821, 32827-32836, 32838, 32840-32846, 32848, 32850, 32852-32859, 32862-32864, 32868-32875, 32877, 32880, 32882-32883, 32885, 32887- 32890, 32895-32900, 32903-32904, 32906-32908, 32910-32916, 32919, 32925, 32928-32929, 32931, 32936-32938, 32941-32946, 32949-32950,

32954, 32956, 32963, 32965, 32967, 32970, 32972-32973, 32976, 32981, 32984-32988, 32991, 32994-32995, 32998, 33000-33001, 33003, 33010, 33012-33013, 33015-33016, 33018-33019, 33022-33023, 33025, 33028- 33032, 33035-33050, 33052-33057, 33059, 33065-33066, 33068-33076, 33080, 33082-33086, 33088, 33090, 33092-33096, 33099-33101, 33104- 33106, 33108, 33114, 33118, 33120-33121, 33124, 33132-33133, 33135- 33139, 33142-33157, 33159, 33161-33167, 33169-33174, 33176-33178, 33180, 33182, 33184-33187, 33191, 33195, 33198-33201, 33204-33209, 33211, 33213, 33217, 33219-33221, 33225-33229, 33231-33232, 33235- 33238, 33243, 33245-33249, 33254, 33256-33261, 33264, 33271-33272, 33274, 33279-33280, 33282-33283, 33285-33286, 33293-33294, 33296- 33298, 33300-33303, 33305-33307, 33309-33310, 33312-33313, 33315, 33317, 33319-33325, 33328-33329, 33332, 33335-33338, 33344-33346, 33348, 33350-33356, 33362-33364, 33366, 33368, 33370, 33373-33377, 33380, 33383, 33389, 33392, 33394, 33396, 33398, 33400, 33403, 33405-33406, 33408, 33410-33415, 33418, 33420-33421, 33423-33427, 33429-33431, 33434, 33436-33439, 33441, 33444, 33446-33448, 33450- 33451, 33453, 33455, 33458, 33461, 33463, 33465, 33467, 33469-33472, 33474, 33478-33480, 33482-33484, 33486-33487, 33489, 33491-33493, 33496-33499, 33501-33503, 33505, 33512, 33514-33515, 33520, 33522- 33523, 33528, 33531, 33534, 33536, 33538, 33542-33543, 33546, 33548- 33549, 33551, 33553, 33555-33556, 33558-33559, 33561, 33564, 33566- 33572, 33574-33576, 33580-33584, 33586, 33593, 33595-33597, 33602, 33610-33611, 33613-33614, 33622, 33627, 33630, 33633-33634, 33636, 33638-33641, 33643-33644, 33646, 33648, 33654, 33660, 33666-33667, 33669-33672, 33674-33675, 33680-33682, 33684-33688, 33690-33693,

33697-33699, 33703-33704, 33706, 33709, 33715, 33718, 33720, 33723- 33726, 33728, 33733, 33735, 33738-33743, 33745, 33747-33748, 33753- 33756, 33762-33765, 33767-33770, 33774-33776, 33782, 33784-33785, 33793, 33798, 33801-33802, 33804, 33806-33808, 33814, 33816-33817, 33819-33823, 33825-33826, 33828-33834, 33836-33837, 33839, 33842- 33843, 33845, 33847-33850, 33852, 33854-33855, 33858-33860, 33862, 33865-33866, 33869, 33872, 33875-33879, 33881-33882, 33884, 33890- 33894, 33896, 33898, 33901-33902, 33908-33911, 33914-33917, 33919- 33923, 33925, 33927-33928, 33931, 33933, 33935-33942, 33944, 33946- 33947, 33949-33950, 33954, 33957-33961, 33964, 33966-33967, 33969, 33971, 33973-33977, 33979, 33981-33988, 33990-33991, 33994-33995, 33997-33998, 34000, 34002-34017, 34019-34021, 34023-34027, 34029- 34032, 34034, 34036-34048, 34050-34051, 34053-34055, 34057, 34063, 34066-34074, 34076, 34079, 34081-34083, 34085-34091, 34093-34094, 34097-34098, 34100-34105, 34107, 34109-34117, 34120-34121, 34124, 34126-34129, 34131, 34135-34136, 34138, 34142-34145, 34149-34153, 34155-34157, 34159-34166, 34168, 34170-34172, 34174-34178, 34184, 34187-34188, 34191-34192, 34195, 34197, 34199-34201, 34204, 34212, 34214, 34216-34217, 34219, 34221-34222, 34230, 34232-34236, 34238- 34239, 34241-34243, 34245-34247, 34249-34250, 34252-34253, 34258- 34259, 34263, 34267, 34270, 34273-34284, 34286-34288, 34290-34291, 34293, 34295, 34297-34298, 34301-34302, 34306, 34310, 34312-34313, 34316-34321, 34324, 34327-34328, 34330-34335, 34338, 34341-34347, 34350, 34352-34353, 34355-34356, 34358-34365, 34370, 34374-34377, 34384, 34389-34390, 34392, 34395-34396, 34399, 34401-34403, 34405- 34406, 34408-34414, 34416, 34419, 34421, 34423, 34431-34435, 34437, 34439-34440, 34443, 34445, 34447, 34453, 34456-34458, 34460-34461, 34463, 34471, 34474, 34477-34481, 34483, 34486, 34488, 34490-34492, 34495, 34497, 34499-34500, 34505-34509, 34511-34515, 34521, 34523, 34527, 34530, 34533, 34535, 34537-34543, 34545-34546, 34551-34554, 34556, 34558, 34560, 34563-34564, 34566-34568, 34570, 34575-34576, 34578, 34580-34583, 34587, 34589, 34591, 34593-34594, 34596-34603, 34605-34607, 34609-34612, 34618-34627, 34631, 34634, 34636-34641, 34643-34644, 34648, 34650, 34652-34655, 34657, 34659-34661, 34663- 34664, 34666-34670, 34673-34675, 34677-34685, 34687-34692, 34696- 34697, 34699-34700, 34702-34704, 34706-34712, 34724-34725, 34727- 34729, 34731-34745, 34750, 34754, 34756-34758, 34762, 34766, 34769, 34771-34772, 34775-34776, 34782-34783, 34787-34790, 34792, 34799- 34806, 34809, 34811, 34813, 34818, 34821-34823, 34825, 34827-34831, 34833-34835, 34837, 34840, 34845, 34848, 34850, 34852, 34855, 34858, 34863-34866, 34868, 34870, 34874-34881, 34883-34887, 34889, 34893, 34897-34899, 34901-34902, 34904-34905, 34908, 34911-34913, 34915, 34917-34926, 34928-34930, 34933-34936, 34940, 34945, 34948, 34959, 34962, 34965, 34971, 34976-34977, 34982-34986, 34992-34994, 34999- 35011, 35014, 35017-35021, 35024, 35026-35027, 35029, 35045-35048, 35052, 35054, 35058-35061, 35063-35065, 35068, 35072, 35074-35075, 35077-35079, 35081, 35083-35086, 35088-35089, 35091, 35094, 35098, 35106-35110, 35113-35114, 35118, 35120, 35129-35130, 35132, 35134, 35136, 35138-35139, 35143, 35145-35146, 35148-35156, 35158-35162, 35168-35170, 35172, 35174, 35176, 35185-35190, 35192, 35194-35195, 35199-35202, 35204-35207, 35209-35211, 35213-35215, 35218-35219, 35221-35225, 35227-35228, 35232, 35234-35236, 35238, 35242-35243, 35248-35249, 35252, 35259-35262, 35265, 35267-35268, 35270-35272, 35276, 35279, 35282, 35285-35286, 35288-35289, 35291, 35293-35312, 35314-35317, 35320-35328, 35330, 35334-35335, 35337, 35339-35341, 35343, 35345-35347, 35349-35361, 35369-35371, 35375-35384, 35392, 35394-35396, 35404, 35406-35411, 35413-35418, 35420-35422, 35425, 35427, 35434, 35436-35438, 35441-35443, 35447, 35450-35452, 35454, 35456, 35458-35463, 35467-35469, 35473, 35475-35482, 35484, 35486- 35488, 35490-35497, 35499, 35501, 35503, 35505-35506, 35508, 35511, 35514-35516, 35522, 35527-35529, 35533-35535, 35537, 35542-35543, 35545-35546, 35548, 35550, 35554-35555, 35559-35564, 35566-35569, 35575-35576, 35578-35581, 35584-35587, 35589, 35592-35593, 35603, 35612, 35616, 35619-35621, 35623, 35629, 35632, 35634-35636, 35638- 35639, 35641-35643, 35646-35647, 35649-35651, 35653-35655, 35657- 35660, 35662, 35666-35667, 35669, 35671-35672, 35674-35677, 35679, 35681-35684, 35688-35692, 35694-35699, 35701, 35703, 35710-35712, 35717-35719, 35721-35722, 35724, 35728, 35731-35732, 35734-35737, 35739-35744, 35748-35751, 35755-35760, 35762-35769, 35772-35773, 35778-35780, 35785-35788, 35794-35796, 35798, 35802, 35804-35805, 35809, 35812, 35814-35815, 35817, 35819, 35821-35823, 35827-35828,

35831- 35837, 35839- 35841, 35843, 35845- 35846, 35848- 35850, 35853-

35854, 35856 -35857, 35859, 35861- 35862, 35864- 35866, 35870- 35871,

35874, 35876 -35878, 35884, 35886- 35890, 35892, 35895 , 35901 -35904,

35906- 35907, 35913, 35915- 35919, 35922- 35923, 35925- 35932, 35934,

35936, 35938 -35940, 35942, 35946- 35947, 35949- 35951, 35953, 35955,

35957- 35958, 35960, 35963- 35964, 35966- 35968, 35970- 35971, 35976-

35980, 35982 -35984, 35986- 35988, 35991- 35998, 36003, 36006, 36010,

36012- 36013, 36016, 36018- 36022, 36024, 36027- 36029, 36031- 36034,

36037- 36041, 36045, 36047, 36050- 36052, 36055- 36058, 36060- 36062,

36064, 36066 , 36068 , 36073 , 36077 -36080 , 36082 , 36089-36091 , 36093

36097- 36098, 36102, 36105, 36109, 36115 , 36118 -36119 , 36121 , 36124 36130, 36132, 36136, 36139-36141, 36145, 36148, 36150-36151, 36153, 36157, 36159-36161, 36164, 36166, 36168, 36175-36176, 36178-36179, 36182, 36184, 36186-36189, 36191-36200, 36202-36209, 36214-36216, 36220-36226, 36228, 36230-36232, 36234-36236, 36240, 36242-36243, 36252, 36255, 36257, 36259, 36266-36273, 36277-36278, 36281-36283, 36285-36287, 36289, 36292, 36294-36295, 36299, 36301-36302, 36310, 36312-36313, 36315-36317, 36324-36326, 36333, 36339-36345, 36350, 36353-36357, 36359-36361, 36364, 36366-36368, 36370-36379, 36381- 36382, 36386, 36388-36395, 36397, 36400-36401, 36403-36405, 36407, 36409, 36411, 36413-36414, 36416, 36419-36420, 36422, 36425, 36427, 36430-36433, 36437, 36439-36440, 36442, 36447-36449, 36452, 36454- 36455, 36461-36472, 36474-36475, 36480, 36482-36483, 36485-36487, 36490, 36492, 36499, 36504, 36511-36512, 36514, 36516-36520, 36527- 36529, 36531, 36536, 36538-36541, 36543-36549, 36552-36554, 36557- 36561, 36564, 36566-36572, 36574-36575, 36577-36580, 36583-36587, 36589-36592, 36595-36596, 36598-36602, 36611, 36613, 36615, 36618- 36619, 36624, 36626-36627, 36629-36631, 36633-36635, 36639, 36642, 36647-36648, 36650-36652, 36654, 36660-36668, 36670-36671, 36673, 36677, 36680-36685, 36687, 36689-36697, 36700, 36702, 36705-36707, 36709-36714, 36716, 36718, 36720-36727, 36729-36731, 36734-36738, 36740-36743, 36745-36746, 36748, 36750, 36755, 36757, 36761-36763, 36765-36766, 36769-36772, 36774, 36779-36784, 36787, 36789, 36792- 36794, 36796, 36804, 36806-36807, 36810, 36812, 36816, 36818-36823, 36826, 36828, 36830-36831, 36837, 36843-36846, 36848, 36851, 36853, 36855, 36857-36862, 36865, 36867, 36869-36874, 36876-36879, 36881- 36887, 36890-36891, 36893-36898, 36905, 36907-36908, 36910, 36915- 36920, 36922-36924, 36927-36930, 36934, 36936-36937, 36939-36949, 36951, 36954-36956, 36958-36963, 36965, 36967-36977, 36980-36982, 36984-36991, 36995, 36997, 36999, 37001, 37003, 37006-37010, 37014- 37015, 37017-37018, 37021-37022, 37025, 37028-37029, 37031-37034, 37040-37044, 37046-37047, 37049-37051, 37053-37054, 37056, 37061, 37064-37065, 37067, 37074-37075, 37077, 37079-37082, 37085-37091, 37093-37097, 37099-37112, 37114, 37116-37117, 37120, 37122-37123, 37132-37136, 37138-37141, 37145, 37147-37156, 37160-37163, 37169- 37170, 37174, 37177, 37181, 37183-37184, 37187, 37189-37190, 37192- 37194, 37196-37198, 37201-37203, 37206-37211, 37213-37216, 37218, 37228, 37230-37231, 37234, 37236, 37240-37244, 37246, 37250-37255, 37258, 37263, 37265, 37270-37273, 37275, 37278, 37280, 37285, 37294, 37298, 37300, 37303, 37305-37306, 37308-37310, 37312-37313, 37317, 37325-37326, 37328-37331, 37334, 37337, 37339-37340, 37342-37344, 37346-37348, 37350, 37355, 37358-37359, 37362-37363, 37365-37367, 37369-37370, 37373, 37377-37379, 37381, 37386, 37388, 37390, 37392- 37393, 37396-37399, 37405, 37407-37408, 37412-37413, 37415, 37417, 37422, 37428, 37435-37441, 37443-37444, 37446, 37448, 37451, 37453- 37457, 37459-37460, 37463-37464, 37466, 37468, 37473-37474, 37482- 37485, 37488, 37494-37495, 37497, 37499, 37501-37503, 37505-37507, 37509-37510, 37512, 37515-37516, 37518-37519, 37521, 37524-37525, 37529-37531, 37535-37542, 37549-37553, 37555-37556, 37558, 37561- 37563, 37570-37573, 37580-37582, 37585-37587, 37591, 37594-37595, 37599-37600, 37603-37606, 37608, 37610, 37617, 37619-37625, 37630- 37631, 37633-37635, 37638, 37642-37643, 37650, 37653, 37658, 37661, 37665, 37667-^■37669, 37671- 37672, 37674, 37678- 37680, 37683, 37685-

37686, 37688, 37691 , 37693 -37694 , 37696 , 37703 , 37706-37707 , 37709,

37717- 37720, 37722, 37726, 37728 , 37737 , 37740 , 37745-37748 , 37751-

37754, 37756- ^■37761, 37763- 37769, 37771, 37773, 37775 , 37777 -37779,

37785- 37786, 37788, 37790- 37792, 37794- 37795, 37798- 37799, 37802,

37806, 37808, 37825 -37826, 37829 , 37831 , 37833 -37834 , 37836 , 37843-

37849, 37851- ^■37852, 37855, 37857 , 37859 -37864, 37868 -37870, 37872-

37874, 37877, 37879 , 37884 -37886 , 37888 -37889, 37891 -37893, 37895-

37908, 37915- ^■37923, 37925, 37927 -37929, 37931- 37932, 37934, 37936,

37938, 37940- ^■37942, 37946- 37948, 37950- 37952, 37958- 37965, 37967-

37971, 37977- ^■37980, 37982, 37988 -37992, 37994, 37997 , 38001 -38013,

38015, 38017, 38019 , 38021 -38022 , 38025 , 38027 -38034 , 38036 -38037,

38039- 38047, 38052, 38055, 38057 -38062, 38065- 38067, 38069, 38071,

38074- 38077, 38084- 38089, 38091, 38095- 38096, 38099, 38102- 38103,

38107- 38110, 38112, 38114, 38123 , 38126 , 38129 , 38133-38134 , 38137-

38138, 38140, 38142 -38144, 38153 , 38156 , 38158 -38160 , 38162 -38163,

38167- 38168, 38170, 38173- 38174, 38176, 38179- 38181, 38184, 38187,

38192- 38194, 38196, 38198- 38199, 38203, 38205, 38207 -38210, 38212-

38214, 38216, 38218 -38219, 38221 -38235, 38238, 38240 -38242, 38246,

38249- 38252, 38254- 38256, 38258, 38260- 38262, 38264, 38268- 38269,

38271- 38272, 38276, 38278- 38280, 38282- 38283, 38286, 38288, 38290,

38292, 38294- ^■38295, 38297, 38301 -38302, 38304- 38307, 38310, 38313-

38315, 38317, 38321 -38322, 38324 , 38334 , 38336 , 38340, 38342, 38345

38347, 38349- ^■38350, 38357, 38361 -38362, 38364- 38365, 38369- 38370,

38374- 38375, 38377- 38378, 38380, 38382- 38384, 38386- 38387, 38391,

38393- 38398, 38400, 38403- 38411, 38414- 38421, 38423- 38424, 38426-

38431, 38433- ^■38437, 38439- 38442, 38445, 38448, 38454 , 38459 , 38464-

38465, 38468- ^■38471, 38474, 38477 -38478, 38480- 38484, 38487, 38490-

38492, 38495- ^■38503, 38505, 38507 -38515, 38517- 38518, 38521- 38523,

38526- 38531, 38534, 38537, 38540 , 38543 , 38547 -38548 , 38550 -38552,

38554- 38556, 38559- 38562, 38565- 38567, 38570-38571, 38574, 38579-

38582, 38584- ^■38585, 38590, 38592 , 38594 -38601, 38605 -38609, 38612-

38613, 38616, 38618 , 38625 -38634 , 38636 -38642, 38644 , 38648 , 38651-

38658, 38660- ^■38663, 38665- 38668, 38670- 38673, 38675, 38677, 38681,

38684, 38686- ^■38687, 38689- 38690, 38692, 38695, 38699 -38700, 38702-

38705, 38707- ^■38709, 38713- 38716, 38719- 38727, 38729- 38730, 38732-

38734, 38736- ^■38741, 38743, 38745 , 38747 , 38749 , 38751-38752 , 38754,

38759, 38761- ^■38762, 38766- 38767, 38769- 38772, 38776- 38777, 38779,

38786- 38788, 38790- 38791, 38794, 38798- 38801, 38804- 38812, 38814,

38816- 38821, 38824, 38826- 38827, 38832- 38835, 38837, 38839- 38852,

38855- 38860, 38863, 38865, 38869 -38874, 38879- 38881, 38884- 38890,

38892, 38894, 38897 -38898, 38902 -38904, 38906, 38909 , 38912 , 38915,

38917, 38919, 38921 -38925, 38927 , 38929 -38932, 38940 , 38944 , 38946-

38947, 38949- ^■38950, 38954- 38955, 38957- 38960, 38962- 38963, 38965,

38968, 38970, 38975 -38976, 38983 -38986, 38994- 38995, 39000, 39002-

39005, 39015, 39017 -39021, 39023 -39024, 39037, 39039 -39042, 39044,

39046- 39050, 39052, 39055- 39056, 39059- 39061, 39068, 39071- 39072,

39074, 39078- ^■39079, 39082, 39086 , 39088 -39090, 39092 -39097, 39100-

39102, 39106- ^■39109, 39112- 39114, 39116- 39122, 39124, 39126- 39128,

39132- 39133, 39135- 39138, 39140- 39141, 39143-39145, 39147, 39150-

39152, 39154- ^■39158, 39160, 39162 , 39164 , 39166 -39172 , 39175 -39178, 39180-39182, 39184-39185, 39187-39190, 39192-39193, 39195, 39199- 39201, 39204-39208, 39210-39212, 39219, 39221-39222, 39224-39231, 39234-39235, 39237, 39244-39248, 39250-39252, 39257-39260, 39262- 39266, 39268-39269, 39271, 39279, 39281-39283, 39285-39286, 39288, 39290-39292, 39297-39307, 39310-39311, 39314-39315, 39318-39319, 39322, 39324-39328, 39330-39334, 39336-39340, 39342-39343, 39345- 39347, 39349-39352, 39354, 39357-39360, 39362-39365, 39368, 39370- 39372, 39374-39384, 39386-39387, 39389, 39396, 39398-39403, 39405- 39409, 39412, 39414-39424, 39427-39430, 39432, 39434-39435, 39438, 39440-39441, 39443-39446, 39448-39450, 39452-39453, 39455, 39457- 39458, 39460, 39462-39463, 39465-39470, 39472, 39474-39492, 39494- 39501, 39503-39504, 39506, 39508-39509, 39511-39515, 39517-39520, 39523, 39527-39528, 39530, 39532-39535, 39537-39539, 39541, 39543- 39550, 39552-39560, 39562, 39564, 39566, 39569-39570, 39572-39573, 39575-39576, 39578, 39580-39581, 39583-39590, 39592-39593, 39596, 39599-39605, 39607, 39611, 39614-39615, 39618-39620, 39624, 39627- 39631, 39634-39636, 39638, 39640, 39644-39647, 39649-39652, 39655, 39659, 39661, 39663-39664, 39666-39668, 39670-39671, 39673, 39676, 39680-39682, 39684-39688, 39693-39699, 39701-39706, 39708, 39710, 39715, 39717, 39719-39722, 39724-39727, 39729, 39732-39734, 39736- 39738, 39740-39743, 39745-39746, 39748-39749, 39752-39753, 39755, 39757-39758, 39763-39764, 39769-39770, 39772-39786, 39788-39791, 39794, 39796, 39798-39803, 39805-39815, 39822, 39824-39825, 39829, 39831-39832, 39839-39840, 39844-39846, 39849, 39851-39854, 39856- 39859, 39861-39863, 39865-39866, 39868-39870, 39873-39874, 39876, 39880, 39882, 39886-39896, 39898-39899, 39901, 39903-39908, 39910, 39912-39913, 39916, 39918, 39922, 39924, 39926-39931, 39934-39937, 39944-39949, 39952, 39954-39957, 39963-39968, 39971-39973, 39978, 39980-39981, 39984-39987, 39989, 39992-39995, 40002-40008, 40010- 40015, 40020-40028, 40031-40034, 40037-40039, 40041-40049, 40051- 40052, 40054-40055, 40057-40061, 40066-40069, 40074-40078, 40080- 40082, 40085, 40087, 40089-40091, 40094-40095, 40097, 40099-40100, 40102-40103, 40105, 40108-40117, 40119-40121, 40124-40132, 40134- 40136, 40138-40139, 40141-40147, 40150, 40153-40162, 40164-40166, 40168-40170, 40173-40177, 40179-40200, 40202, 40204-40207, 40209- 40215, 40218-40220, 40222-40224, 40227, 40229-40234, 40237, 40239, 40241, 40243-40247, 40250-40251, 40254-40255, 40257-40260, 40263- 40264, 40266-40267, 40269-40272, 40274-40279, 40283, 40286-40289, 40291-40297, 40299-40303, 40309-40310, 40313-40314, 40317-40322, 40325-40326, 40328, 40330-40336, 40338, 40343-40344, 40346, 40349, 40353-40355, 40358, 40362-40364, 40366-40371, 40373-40374, 40376- 40379, 40381-40388, 40391-40392, 40395-40397, 40399-40405, 40407- 40409, 40411, 40416-40417, 40419-40422, 40424, 40426, 40428-40429, 40433-40434, 40443, 40445-40446, 40448, 40450-40452, 40456, 40458- 40460, 40462, 40464-40465, 40468-40469, 40474-40476, 40478-40480, 40487, 40489-40492, 40494-40496, 40498, 40501-40502, 40504, 40507, 40510, 40513-40514, 40517, 40523-40524, 40526, 40530, 40532-40535, 40543-40545, 40547, 40550, 40553, 40555, 40557-40562, 40564-40567, 40570, 40572, 40575, 40577-40578, 40580, 40582, 40584, 40586-40592, 40594, 40596, 40599-40600, 40605-40608, 40610-40611, 40613, 40616, 40618-40621, 40623-40624, 40627-40631, 40633-40636, 40638, 40641, 40646, 40648, 40650-40651, 40653, 40658-40660, 40662, 40664-40665,

40667, 40670, 40673 -40678, 40688- 40689, 40695- 40697, 40699, 40703,

40705, 40707, 40709 , 40712 -40714, 40716 -40718, 40720- 40723, 40726-

40727, 40729, 40731 -40732, 40734, 40738 -40739, 40745, 40748 -40753,

40755, 40757- 40760, 40766, 40770, 40773 -40778, 40781- 40782, 40784-

40785, 40787, 40789 , 40797 , 40799 -40806 , 40808 , 40811 -40813 , 40816,

40818, 40826, 40829 , 40831 , 40833 -40834 , 40844 , 40848 -40850 , 40861,

40864- 40870, 40872, 40875- 40880, 40882, 40885, 40888- 40890, 40895,

40899- 40900, 40912, 40916, 40918, 40921 -40926, 40929, 40931 , 40934-

40936, 40938- 40939, 40941- 40943, 40945- 40948, 40951-40953, 40955,

40957- 40960, 40962- 40964, 40966, 40969, 40974, 40976, 40979 , 40981-

40982, 40985, 40987 -40988, 40993, 40995 -40997, 41004, 41006 -41008,

41010- 41011, 41013- 41015, 41017, 41021- 41022, 41024, 41026- 41033,

41037- 41038, 41041, 41043- 41048, 41051, 41057, 41060- 41062, 41064,

41067- 41071, 41074, 41079- 41084, 41087, 41089- 41095, 41098- 41101,

41103- 41105, 41115, 41119- 41120, 41122- 41125, 41127, 41130, 41133,

41136-41140, 41142-41144, 41146-41147, 41149-41154, 41156-41157, 41159, 41161-41162, 41165, 41167-41170, 41172, 41174-41178, 41181- 41183, 41188-41191, 41194-41199, 41203-41206, 41209-41210, 41212, 41217, 41219, 41221, 41223-41225, 41227, 41229-41230, 41233, 41235, 41237, 41240-41246, 41251, 41253, 41255-41256, 41258-41261, 41264- 41269, 41274-41275, 41277-41284, 41288-41292, 41299-41304, 41307- 41309, 41311-41313, 41316-41322, 41326, 41328-41332, 41334, 41337, 41339-41340, 41342-41348, 41350-41354, 41357, 41359-41363, 41365- 41366, 41368-41369, 41373-41375, 41378-41393, 41396, 41398, 41400, 41402-41403, 41405, 41407-41414, 41416-41418, 41420, 41422-41423, 41425-41427, 41430-41438, 41440-41444, 41446-41449, 41453, 41455- 41456, 41459-41461, 41463, 41466, 41468-41469, 41471, 41474-41475, 41482, 41485, 41487-41490, 41493-41495, 41497, 41499-41504, 41507, 41510-41511, 41514-41522, 41525-41526, 41528-41529, 41531, 41536- 41537, 41540, 41542-41543, 41545, 41547, 41549-41552, 41558-41559, 41564-41565, 41567-41568, 41570-41571, 41573-41578, 41580-41582, 41594-41595, 41597-41601, 41604-41607, 41609, 41613-41617, 41621, 41624-41627, 41629-41631, 41634, 41636-41641, 41643-41644, 41646- 41649, 41651, 41655-41658, 41660, 41662-41664, 41667-41668, 41670- 41671, 41673, 41678-41689, 41691-41694, 41696-41703, 41707, 41709, 41713-41714, 41718, 41720-41726, 41729-41733, 41743-41744, 41749, 41751-41753, 41755, 41759, 41762-41767, 41772, 41778-41780, 41782, 41785-41790, 41796-41799, 41801, 41804, 41807, 41809, 41811, 41814, 41822, 41824-41825, 41827-41831, 41833-41837, 41839-41841, 41843-

41845, 41847, 41851-41855, 41857, 41859, 41861-41862, 41864, 41866- 41867, 41873, 41876-41879, 41886, 41890-41891, 41895, 41897, 41899- 41901, 41903, 41906-41910, 41912-41913, 41915, 41920-41926, 41931, 41934-41936, 41939-41944, 41946-41947, 41949-41950, 41952-41953, 41955-41958, 41962, 41964-41968, 41971-41974, 41977-41980, 41982- 41986, 41989-41991, 41993-41996, 42001-42002, 42005-42007, 42009- 42010, 42013, 42017, 42022, 42027-42031, 42033-42034, 42038, 42046, 42048, 42050, 42054-42059, 42062-42063, 42065-42067, 42074-42077, 42079-42083, 42085, 42091-42095, 42097-42099, 42102, 42105-42110, 42112, 42114, 42117-42118, 42122-42128, 42134, 42137, 42143-42145, 42147-42153, 42155, 42157, 42159-42161, 42164-42165, 42167-42168, 42171, 42173- 42176, 42188, 42192, 42196 , 42213 , 42215 , 42222, 42228

42229, 42232, 42234 , 42236 -42237, 42242 -42243, 42245, 42247 -42251,

42253, 42257- 42260, 42262, 42274, 42277 , 42279 , 42282 , 42284-42285,

42287- 42290, 42292- 42293, 42295, 42299- 42309, 42311-42316, 42319-

42323, 42325, 42331 -42340, 42342- 42343, 42348- 42349, 42352- 42357,

42359- 42361, 42363- 42364, 42367, 42370, 42376- 42377, 42379, 42381,

42384, 42386- 42395, 42397- 42400, 42402- 42408, 42410, 42412- 42416,

42421, 42426- 42433, 42435, 42440, 42442 , 42444 , 42449 , 42451-42452,

42455- 42456, 42461- 42462, 42466, 42472, 42474, 42477- 42479, 42481-

42482, 42484- 42485, 42487- 42488, 42494- 42496, 42498-42499, 42502-

42503, 42507, 42513 -42516, 42521, 42524 -42525, 42527- 42528, 42530-

42534, 42536, 42538 -42540, 42552- 42563, 42567, 42570- 42572, 42574-

42576, 42579- 42586, 42590- 42591, 42594, 42597, 42599, 42601 -42604,

42606, 42608- 42610, 42612, 42615- 42617, 42619- 42620, 42624- 42625,

42630, 42635, 42640 -42643, 42646- 42648, 42652- 42654, 42657- 42659,

42661, 42666- 42668, 42670- 42671, 42673, 42678- 42683, 42686- 42687,

42689, 42691- 42695, 42697, 42699, 42702 -42705, 42707, 42709 , 42711-

42712, 42715, 42721 , 42724 -42725, 42731 , 42733 , 42735 -42736 , 42739-

42749, 42753, 42756 -42757, 42760, 42765 -42769, 42773, 42775 -42777, 42779-42780, 42783-42784, 42786-42787, 42789-42790, 42800, 42802- 42809, 42811-42813, 42816-42817, 42820-42821, 42823-42824, 42826- 42830, 42833, 42836, 42839-42840, 42842-42843, 42845, 42847-42850, 42853-42854, 42856-42860, 42862, 42864-42879, 42881, 42885-42886, 42888-42891, 42893-42895, 42899, 42902-42910, 42914-42915, 42917, 42919, 42921-42926, 42928, 42931, 42933-42936, 42938-42939, 42941, 42943-42944, 42947-42953, 42955, 42957, 42959-42962, 42964, 42969, 42971, 42980, 42986, 42989, 42995, 42997-42998, 43000-43001, 43003, 43005-43006, 43008-43009, 43011-43015, 43018-43019, 43021, 43024, 43026, 43029, 43034, 43037-43038, 43040-43041, 43043-43044, 43047- 43048, 43055-43056, 43058, 43061, 43063-43064, 43066-43068, 43070, 43073-43075, 43077-43078, 43080, 43084-43085, 43088, 43092, 43094- 43096, 43098, 43100, 43102, 43106-43107, 43110-43111, 43113-43115, 43117, 43123-43130, 43132-43134, 43138-43139, 43141, 43143, 43145- 43148, 43150-43155, 43157, 43159, 43161, 43164-43166, 43173, 43179- 43180, 43184-43189, 43194-43197, 43202, 43205, 43207-43208, 43211, 43215-43217, 43219-43222, 43224, 43227, 43229, 43231-43232, 43234- 43236, 43238, 43241-43242, 43244, 43247, 43249-43256, 43259-43261, 43267, 43269, 43274-43275, 43280-43281, 43288-43291, 43298-43299, 43301-43304, 43307, 43310-43313, 43315, 43317, 43321, 43325-43329, 43333, 43335, 43338-43346, 43350-43352, 43354-43357, 43360-43362, 43366-43371, 43376-43383, 43385-43387, 43389, 43392-43393, 43395, 43397, 43400, 43402, 43408-43410, 43414-43416, 43418-43421, 43423- 43424, 43426, 43430, 43432-43433, 43435-43436, 43441, 43444-43445, 43449, 43454-43462, 43466-43467, 43469, 43475, 43478-43480, 43482, 43484-43488, 43492, 43494, 43496, 43500, 43503-43504, 43506, 43510- 43513, 43516, 43519-43520, 43522, 43524-43532, 43534, 43536, 43540, 43555-43561, 43563-43564, 43570, 43575-43581, 43583, 43585, 43587- 43588, 43591-43592, 43594-43596, 43600-43601, 43603-43607, 43610- 43612, 43614-43619, 43621-43624, 43628, 43631, 43636, 43639, 43652, 43655-43657, 43659-43660, 43664, 43669-43676, 43679-43684, 43686- 43687, 43690-43693, 43695, 43697-43698, 43700, 43703, 43711-43712, 43714-43716, 43718-43722, 43724-43726, 43738, 43740-43744, 43747- 43749, 43752-43754, 43757, 43760, 43763, 43765-43766, 43768-43770, 43772-43775, 43778, 43781-43782, 43786-43791, 43794, 43796-43797, 43801, 43803-43804, 43806, 43810-43811, 43813, 43815, 43817, 43821- 43822, 43824-43825, 43827, 43834-43843, 43845-43847, 43849, 43851- 43854, 43856, 43858-43859, 43864, 43869-43872, 43874, 43876, 43879, 43881-43887, 43891-43894, 43897-43904, 43907, 43909, 43912, 43914, 43919, 43921-43923, 43927-43929, 43932-43933, 43935-43936, 43939- 43941, 43943-43946, 43951-43953, 43956, 43959-43964, 43967-43969, 43971, 43973, 43975-43978, 43983, 43987, 43991-43993, 44000, 44002- 44016, 44019-44020, 44024-44027, 44029-44031, 44033, 44036, 44038, 44040-44048, 44052-44054, 44056-44058, 44061-44063, 44065, 44067- 44068, 44070-44074, 44076-44078, 44081-44091, 44093, 44095-44098, 44101, 44103, 44105-44113, 44115, 44118, 44120-44123, 44126-44129, 44131-44132, 44135-44137, 44139-44151, 44153-44155, 44157, 44163- 44166, 44168-44173, 44179-44180, 44182-44186, 44188-44189, 44193- 44196, 44198-44200, 44202-44206, 44209-44211, 44213, 44218-44219, 44221-44222, 44229, 44233, 44237-44243, 44245, 44251, 44253, 44255- 44259, 44261-44266, 44270-44273, 44277-44280, 44282-44283, 44290- 44291, 44298-44307, 44310-44313, 44317, 44320, 44322-44323, 44325- 44326, 44329-44335, 44337-44338, 44340, 44344-44351, 44354, 44357- 44366, 44370-44373, 44376, 44378-44384, 44386-44387, 44389-44390, 44392-44395, 44397-44400, 44406, 44408-44412, 44414, 44417, 44419- 44424, 44426, 44429-44430, 44432, 44437-44438, 44441, 44443-44444, 44446-44450, 44462, 44467-44469, 44471-44472, 44474, 44476, 44483, 44485-44487, 44491-44492, 44496-44502, 44505-44506, 44509, 44514- 44518, 44520-44522, 44524, 44526-44527, 44530, 44532, 44535, 44539, 44541, 44543-44544, 44546-44549, 44551-44552, 44554-44558, 44562- 44564, 44569-44570, 44572-44574, 44578-44580, 44582, 44589-44590, 44596, 44603, 44605-44607, 44609, 44611, 44613, 44616-44621, 44626, 44629, 44635, 44649, 44651, 44653-44658, 44663-44670, 44672-44677, 44684-44687, 44689, 44692-44693, 44696-44699, 44702-44703, 44706- 44710, 44713, 44715, 44717-44718, 44720-44728, 44731-44733, 44736, 44739, 44741-44745, 44748, 44751-44753, 44759, 44761-44762, 44764, 44766-44767, 44770-44772, 44774, 44776-44778, 44786-44790, 44792- 44793, 44798-44799, 44801, 44806, 44808, 44813-44815, 44817-44820, 44822, 44824-44825, 44827, 44829, 44831, 44834, 44836-44839, 44841- 44842, 44844-44850, 44854, 44856-44857, 44859-44861, 44870, 44875, 44879-44880, 44882, 44886-44892, 44894-44895, 44899-44902, 44906, 44908-44910, 44912, 44914-44917, 44922-44924, 44926, 44932, 44934, 44937, 44941, 44944-44945, 44947-44950, 44953, 44955-44956, 44960- 44961, 44963-44965, 44967, 44969, 44971-44972, 44975, 44977, 44980- 44982, 44984-44985, 44987-44994, 44999, 45001-45002, 45004-45005, 45007-45019, 45021, 45025, 45027-45029, 45032, 45034-45036, 45039- 45041, 45044-45045, 45047, 45049, 45054, 45056, 45060, 45063-45064, 45069, 45071, 45073-45077, 45081-45082, 45084, 45086, 45092-45093, 45095, 45098, 45101-45103, 45105, 45107, 45109, 45113, 45115-45118, 45120-45121, 45123, 45125, 45127-45130, 45136-45140, 45142-45144, 45146-45148, 45150-45152, 45154-45158, 45161-45162, 45164, 45166, 45168-45172, 45174, 45176, 45179, 45186-45188, 45190-45192, 45201- 45206, 45208-45209, 45211-45212, 45218-45220, 45229-45230, 45232, 45234-45236, 45238, 45240-45241, 45245-45248, 45253, 45255, 45261, 45268, 45270-45272, 45275, 45277-45278, 45280-45281, 45285, 45290, 45292, 45294-45298, 45300, 45303, 45305-45306, 45308-45310, 45312- 45314, 45316-45318, 45320-45322, 45329-45332, 45334-45336, 45339, 45347, 45350, 45352-45353, 45355-45359, 45361-45362, 45364, 45367, 45376, 45381, 45383, 45388-45389, 45391-45395, 45398-45399, 45402- 45404, 45406-45409, 45411-45417, 45420-45426, 45431-45434, 45439- 45442, 45444, 45448-45449, 45451, 45455, 45460-45465, 45467-45468, 45470-45476, 45482, 45485, 45488, 45494, 45496-45498, 45502, 45504, 45506, 45509, 45513, 45515-45516, 45521-45524, 45527-45529, 45533, 45535, 45537-45541, 45543-45545, 45547-45550, 45553-45570, 45572- 45575, 45577-45589, 45592-45595, 45597, 45600, 45602-45604, 45606- 45610, 45612, 45615-45616, 45618-45621, 45624, 45627, 45632-45633, 45636-45637, 45639, 45642, 45644, 45646-45648, 45651, 45653-45655, 45658-45660, 45662, 45665, 45667, 45669, 45671-45675, 45678-45681, 45685-45687, 45689-45695, 45697-45698, 45701-45704, 45706, 45709- 45713

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 having a sequence 5'-X-Y-Z, wherein X is any nucleotide, Y is a nucleotide sequence of 6 nucleotides in length that is not a seed sequence of a human microRNA, and Z is a nucleotide sequence of 1-23 nucleotides in length, wherein the single stranded oligonucleotide is complementary with at least 8 consecutive nucleotides of a PRC2-associated region of a FOXP3 gene.

2. The single stranded oligonucleotide of claim 1, wherein the oligonucleotide does not comprise three or more consecutive guanosine nucleotides.

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

oligonucleotide does not comprise four or more consecutive guanosine nucleotides.

4. The single stranded oligonucleotide of any one of claims 1 to 3, wherein the oligonucleotide is 8 to 30 nucleotides in length.

5. The single stranded oligonucleotide of any one of claims 1 to 4, wherein the oligonucleotide is 8 to 10 nucleotides in length and all but 1, 2, or 3 of the nucleotides of the complementary sequence of the PRC2-associated region are cytosine or guanosine nucleotides.

6. The single stranded oligonucleotide of any one of claims 1 to 5, wherein at least one nucleotide of the oligonucleotide is a nucleotide analogue.

7. The single stranded oligonucleotide of claim 6, 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.

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

9. The single stranded oligonucleotide of any one of claims 1 to 8, wherein each nucleotide of the oligonucleotide comprises a 2' O-methyl.

10. The single stranded oligonucleotide of any one of claims 1 to 9, wherein the oligonucleotide comprises at least one ribonucleotide, at least one deoxyribonucleotide, or at least one bridged nucleotide.

11. The single strand oligonucleotide of claim 10, wherein the bridged nucleotide is a LNA nucleotide, a cEt nucleotide or a ENA modified nucleotide.

12. The single stranded oligonucleotide of any one of claims 1 to 7, wherein each nucleotide of the oligonucleotide is a LNA nucleotide.

13. The single stranded oligonucleotide of any one of claims 1 to 7, wherein the nucleotides of the oligonucleotide comprise alternating deoxyribonucleotides and 2'-fluoro- deoxyribonucleotides.

14. The single stranded oligonucleotide of any one of claims 1 to 7, wherein the nucleotides of the oligonucleotide comprise alternating deoxyribonucleotides and 2'-0- methyl nucleotides.

15. The single stranded oligonucleotide of any one of claims 1 to 7, wherein the nucleotides of the oligonucleotide comprise alternating deoxyribonucleotides and ENA nucleotide analogues.

16. The single stranded oligonucleotide of any one of claims 1 to A 7, wherein the nucleotides of the oligonucleotide comprise alternating deoxyribonucleotides and LNA nucleotides.

17. The single stranded oligonucleotide of any one of claims 13 to 16, wherein the 5' nucleotide of the oligonucleotide is a deoxyribonucleotide.

18. The single stranded oligonucleotide of any one of claims 1 to 7, wherein the nucleotides of the oligonucleotide comprise alternating LNA nucleotides and 2' -O-methyl nucleotides.

19. The single stranded oligonucleotide of claim 18, wherein the 5' nucleotide of the oligonucleotide is a LNA nucleotide.

20. The single stranded oligonucleotide of any one of claims 1 to 8, 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.

21. The single stranded oligonucleotide of any one of claims 1 to 20, further comprising phosphorothioate internucleotide linkages between at least two nucleotides.

22. The single stranded oligonucleotide of claim 21, further comprising phosphorothioate internucleotide linkages between all nucleotides.

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

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

25. The single stranded oligonucleotide of any one of claims 1 to 24, further comprising a biotin moiety conjugated to the 5' nucleotide.

26. A single stranded oligonucleotide comprising a region of complementarity that is complementary with at least 8 consecutive nucleotides of a PRC2-associated region of a FOXP3 gene, wherein the oligonucleotide has at least one of: 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 has less than a threshold level of sequence identity with every sequence of nucleotides, of equivalent length to the second nucleotide sequence, that are between 50 kilobases upstream of a 5 '-end of an off-target gene and 50 kilobases downstream of a 3 '-end of the off-target gene; d) 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; and/or e) a sequence that has greater than 60% G-C content.

27. The single stranded oligonucleotide of claim 26, wherein the oligonucleotide has the sequence 5 'X-Y-Z and wherein the oligonucleotide is 8-50 nucleotides in length.

28. The single stranded oligonucleotide of any one of claims 1 to 27, wherein the single stranded oligonucleotide, when delivered to a cell, is capable of increasing the level of CTLA4, GITR, and/or IL-10 expression in the cell.

29. The single stranded oligonucleotide of claim 28, wherein the cell is a T cell.

30. The single stranded oligonucleotide of any one of claims 1 to 29, wherein the single stranded oligonucleotide, when delivered to a population of T cells, is capable of increasing the number of CD4+CD25+FOXP3+ T cells in the population of T cells.

31. A composition comprising a single stranded oligonucleotide of any one of claims 1 to 30 and a carrier.

32. A composition comprising a single stranded oligonucleotide of any one of claims 1 to 30 in a buffered solution.

33. A composition of claim 32, wherein the oligonucleotide is conjugated to the carrier.

34. The composition of claim 33, wherein the carrier is a peptide.

35. The composition of claim 33, wherein the carrier is a steroid.

36. A pharmaceutical composition comprising a composition of any one of claims 31 to 35 and a pharmaceutically acceptable carrier.

37. A kit comprising a container housing the composition of any one of claims 31 to 36.

38. A method of increasing expression of FOXP3 in a cell, the method comprising delivering the single stranded oligonucleotide of any one of claims 1 to 30 into the cell.

39. The method of claim 38, 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.

40. The method of claim 38 or 39, wherein delivery of the single stranded oligonucleotide into the cell results in an increased level of CTLA4, GITR, and/or IL-10 expression compared to an appropriate control cell that does not comprise the singled stranded oligonucleotide.

41. The method of claim 40, wherein delivery of the single stranded

oligonucleotide into the cell results in a level of expression of CTLA4, GITR, and/or IL-10 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.

42. The method of any one of claims 38 to 41, wherein the cell is a T cell.

43. A method increasing levels of FOXP3 in a subject, the method comprising administering the single stranded oligonucleotide of any one of claims 1 to 30 to the subject.

44. The method of claim 43, wherein administration of the single stranded oligonucleotide to the subject results in an increased level of CTLA4, GITR, and/or IL-10 expression the subject compared to an appropriate control subject who has not been administered the single stranded oligonucleotide.

45. The method of claim 44, wherein administration of the single stranded oligonucleotide to the subject results in a level of expression of CTLA4, GITR, and/or IL-10 that is at least 30% greater than a level of CTLA4, GITR, and/or IL-10 in the appropriate control subject who has not been administered the single stranded oligonucleotide.

46. The method of claim 43, wherein administration of the single stranded oligonucleotide to the subject results in an increased level of CTLA4, GITR, and/or IL-10 in a T cell of the subject compared to a T cell in the control subject who has not been administered the single stranded oligonucleotide.

47. The method of claim 46, wherein administration of the single stranded oligonucleotide to the subject results in a level of expression of CTLA4, GITR, and/or IL-10 in the T cell of the subject that is at least 30% greater than a level of CTLA4, GITR, and/or IL-10 in the T cell in the control subject who has not been administered the single stranded oligonucleotide.

48. The method of any one of claims 43 to 47, wherein administration of the of the single stranded oligonucleotide to the subject results in an increased number of

CD4+CD25+FOXP3+ T cells in the subject compared to a control subject who has not been administered the single stranded oligonucleotide.

49. The method of claim 48, wherein administration of the single stranded oligonucleotide to the subject results in a number of CD4+CD25+FOXP3+ T cells in the subject that is at least 30% greater than a number of CD4+CD25+FOXP3+ T cells in the control subject who has not been administered the single stranded oligonucleotide.

50. A method of treating a condition or disease associated with decreased levels of FOXP3 in a subject, the method comprising administering the single stranded oligonucleotide of any one of claims 1 to 30 to the subject.

51. The method of claim 50, wherein the condition or disease is associated with aberrant immune cell activation.

52. A method of increasing expression of FOXP3 in a cell, the method comprising delivering an oligonucleotide having a region of complementarity that is complementary with at least 8 consecutive nucleotides of a EZH1 mRNA or EZH2 mRNA to the cell.

53. The method of claim 52, wherein the oligonucleotide is is 8 to 30 nucleotides in length.

54. The method of claim 52 or 53, wherein at least one nucleotide of the oligonucleotide is a nucleotide analogue.

55. The method of any one of 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 55, wherein the at least two nucleotide analogues comprise at least one LNA or at least one 2'-0 modified ribonucleotide.