THEREOF

[001] This application claims priority of U.S. Provisional Application No.

61/011,571, filed February 16, 2010, the contents of which are hereby incorporated by reference.

[002] The research leading to the present invention was supported, in part by grant number HL073437, awarded by the National Institutes of Health. Accordingly, the U.S. Government has certain rights in this invention.

[003] Throughout this application, certain publications are referenced in brackets. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to describe more fully the state of the art to which this invention relates.

Technical Field

[004] The present invention broadly relates to the field of peptide nucleic acids (PNAs) and more particularly novel compounds sometimes referred to herein as embedded chimeric peptide nucleic acid molecules (ecPNAs) which comprise a PNA structure to each end of which an oligopeptide structure is attached, directly or by means of a chemical linker. Such compounds may be used to detect specific target DNA sequences present within gene promoters. Moreover, certain of these compounds may be used to regulate expression of genes of interest such as the Y -globin gene in order to treat a β- globin disorder such as sickle cell anemia or β-thalassemia.

Background of Invention

[005] Detection of DNA sequences such as DNA sequences within genes has been used for various purposes including research and disease diagnosis. Compounds useful for detecting DNA sequences include DNA and RNA oligonucleotide probes, typically labeled with some detectable marker such as a radioisotope, a fluorescent dye, or an immunogenic peptide. Nevertheless, there is a continuing need for compounds which can be employed for such purposes. The compounds of the present invention fulfill this need.

[006] Certain of the compounds of this invention may also be used to regulate transcription and expression of genes including genes the regulated expression of which is implicated in, or can be useful in, the treatment or amelioration of disease.

[007] Correct regulation of gene expression is required for fundamental processes in differentiation and development. [See Davidson, E.H., Gene Activity in Early Development, Edn. Third. (Academic Press, Inc.Orlando; 1986)]. In a number of cases, the transcriptional onset and decline of a series of closely related genes are tightly and sequentially controlled, a process that is critical for attaining the correct genotypic readout and proper phenotypic effect. [See Bresnick, E.H., Martowicz, M.L., Pal, S. & Johnson. K.D., Developmental control via GATA factor interplay at chromatin domains. J Cell Physiol 205, 1-9 (2005); Noordermeer D., de Laat W Joining the loops: β-globin gene regulation. IUMMB Life 60, 824-833 (2008); and rumlauf, R., Hox genes in vertebrate development. Cell 78, 191-201 (1994).] A particularly well-studied example of this process is the developmental control of the hemoglobin proteins, particularly those encoded by the genes within the β-like globin locus. [See Stamatoyannopoulos, G., The molecular basis of blood diseases, Edn. 3rd. (W.B. Saunders,Philadelphia; 2001); and Schechter, A.N., Hemoglobin research and the origins of molecular medicine. Blood 112, 3927-3938 (2008).] These variants exhibit a sequential erythroid-restricted pattern of expression during development, beginning with the yolk sac ε-globin, switching to the fetal γ-globin, and ending with the adult B- globin.

[008] The critical requirement for correct regulation of this locus is demonstrated by the moderate to life-threatening clinical manifestations exhibited by the B- thalassemias. β thalassemia is primarily caused by mutations in the β globin gene that lead to reduced or complete loss of β globin expression. Along with other hemoglobinopathies (such as sickle celt disease), they give rise to the most common single gene genetic disorder worldwide. [See Weatherall. D.J. in The Molecular Bases of Blood Diseases, (eds. G. STamatoyannopoulos, A.W. Nienhuis, P.W. Majerus & H. Varmus) 207-205 (W.B. Saunders Co., Philadelphia; 1994). | Pharmacological reactivation of the silent fetal (γ) globin chain provides a therapeutic benefit to these patients by compensating for absent adult β-globin chains (in β-thalassemia) or by interfering with the polymerization of mutant hemoglobins (in sickle cell disease); however, these are not always free from complications. [See Weatherall and Bunn, H.F. in The Molecular Bases of Blood Diseases, (eds. G. Stamatoyannopoulos, A.W. Nienhuis, P.W. Majerus & H. Varmus) 157-256 (W. B. Saunders Co., Philadelphia: 1994): and Atweh, G.F. & Schechter, A.N., Pharmacologic induction of fetal hemoglobin: raising the therapeutic bar in sickle cell disease. Curr Opin Hematol 8, 123- 130 (2001).] As a result, there remain compelling reasons to search for novel approaches and reagents that achieve reactivation with low toxicity and high penetrance.

[009] Peptide nucleic acids (PNAs) are oligonucleotide analogues in which the phosphodiester backbone of DNA is replaced by an achiral uncharged polyamide backbone. [See Nielsen, P.E., Egholm, M., Berg, R.H. & Buchardt, O., Sequence- selective recognition of DNA by strand displacement with a thymine-substituted polyamide. Science 254, 1497-1500 (1991).] They are true DNA mimics, as they form Watson-Crick bonds with DNA and RNA, but are of higher thermal stability than natural duplexes due to the lack of electrostatic repulsion. [See aihatsu, K. Janowski, B.A. & Corey, D.R., Recognition of chromosomal DNA by PNAs. Chem Biol 11, 749-758 (2004).] They are also resistant to proteases and nucleases, and thus afford a significantly greater biological stability in culture and in vivo. [See Pooga, M., Land, T., Bartfai, T. & Langel, U., PNA oligomers as tools for specific modulation of gene expression. Biomol Eng 17, 183-192 (2001).] A unique aspect of PNAs is that amino acids can be covalently added to the peptide backbone at either end of the sequence of bases. The PNA DNA interaction may occur through triple helical (Hoogsteen) base paring (PNA DNA/PNA) or via a single strand invasion. [See Kaihatsu, K., Janowski, B.A. & Corey, D.R., Recognition of chromosomal DNA by PNAs. Chem Biol 11, 749-758 (2004); and Zhang, X., Ishihara, T. & Corey, D.R., Strand invasion by mixed base PNAs and a PNA-peptide chimera. Nucleic Acids Res 28, 3332-3338 (2000).] In a single strand invasion, PNA, which can be of mixed sequence design, hybridizes with one strand of DNA through Watson-Crick base pairing and simply replaces the other strand of the double helix.

[0010] PNA molecules are thus promising candidates for clinical use as agents to modulate gene expression. For the most part, PNAs have been used as an antigene agent because they have the capacity for down-regulating gene expression in cultured cells and in animals. [See Nielsen, P.E., Peptide nucleic acids as antibacterial agents via the antisense principle. Expert Opin Investig Drugs 10, 331-341 (2001); Cutrona, G. et al., Effects in live cells of a c-myc anti-gene PNA linked to a nuclear localization signal. Nat Biotechnol 18, 300-303 (2000); Hu, J. et al., AUele-specific silencing of mutant huntingtin and ataxin-3 genes by targeting expanded CAG repeats in mRNAs. Nat Biotechnol 27, 478- 484 (2009); and Nielsen, P.E. PNA Technology. Mol Biotechnol 26, 233-248 (2004).]

[0011 ] PNA molecules have also been used as probes for targeted nucleic acid binding, and to follow the subcellular trafficking of plasmid DNA. Additionally, conjugation of markers such as fluorophores to PNA molecules has been described for these purposes. [See also Zhilina et al., Peptide Nucleic Acid Conjugates: Synthesis, Properties and Applications. Current Topics in Medicinal Chemistry 4:1119-1131 (2005)]. PNA molecules have also been utilized for single base pair mutation analysis by PNA directed PCR clamping. [See also Orum et al., Single base pair mutation analysis by PNA directed PCR clamping. Nucleic Acids Research 21(23):5332-5336 (1993)] However, there is a need for improved PNA structures capable of efficiently detecting the presence of DNA within the nucleus of celts.

[0012] PNAs have been modified to maximize cellular/nuclear entry in order to increase the efficiency for in vivo applications. [See Cutrona G. et al.. Effects in live cells of a c-myc anti-gene PNA linked to a nuclear localization signal. Nat Biotechnol 18, 300-303 (2000): Nielsen, P.E., Addressing the challenges of cellular delivery and bioavailability of peptide nucleic acids (PNA). Q Rev Biophys 38, 345-350 (2005); and Braun, K. et al., A biological transporter for the delivery of peptide nucleic acids (PNAs) to the nuclear compartment of living cells. J Mol Biol 318, 237-243 (2002).] However, currently there is no established PNA conjugated system that combines these varied modifications and has been demonstrated to stably and efficiently enter, target, and transcriptionally activate an endogenous chromosomal locus in living cells.

Summary of Invention

[0013] The present invention relates to a compound having the general structure: and uses thereof, wherein X is A or C and Y is A or C with the proviso that when X is A, Y is C, and when X is C, Y is A; A represents an oligopeptide structure, the sequence of which comprises a sequence which renders the compound able to enter the nucleus of a cell; B represents a peptide nucleic acid (PNA) structure at least 12 nucleotides in length, the sequence of which is capable of hybridizing with a DNA within the nucleus of the cell, which DNA is within a promoter region of a gene; C represents an oligopeptide structure; and each represents a chemical linkage between the structures at each side thereof which may be the same as or different from each other such linkage.

Brief Description of the Drawings

[0014] Figure 1 depicts in vitro target binding specificity of four PNAs designed to interact with the human γ-globin promoter. In Figure 1(a), PNA150, PNA116, PNA78, and PNA7 were designed to target the proximal promoter region (-202 to +33) of the human fetal γ-globin gene at specific sites indicated by the filled in boxes. The relative location of known promoter elements within this region is also indicated. Orientation of the PNA molecule relative to the promoter sequence is shown above, with a lys at its 3' -end and a biotin molecule (circle) attached at the S'-end. In Figure 1(b), different concentrations of PNAs (0-10 μΜ) were incubated with a constant amount of radioactively labeled DNA oligonucleotide (10 nM) containing either the wild type (wt) or mutated (mut) target sequence followed by incubation with streptavidin Dynabeads and collection with a magnetic particle concentrator. Bound/input ratios were obtained after scintillation counter analysis. In Figure 1(c), permanganate probing confirms the specificity of PNA78 by showing that oxidized and cleaved T bases (lane 3) are accessible within the designated target sequence compared to 'no PNA ^' (--) and PNA7 (as non-interacting PNA) controls. The A+G sequence ladder is shown in lane 1, along with the nucleotide sequence of the γ- globin promoter aligned on the left.

[0015] Figure 2 depicts cellular and nuclear localization of PNA78 and its derivatives. All PNA molecules contained a biotin tag at their 5'-end and were visualized with streptavidin-FITC. Figure 2(a) depicts fluorescent microscopy showing uptake of PNA78 by 562 cells exposed to different concentrations as indicated. PNA signals appear as bright punctate dots. Figure 2(b) depicts fluorescent microscopy showing uptake of PNA78 TAT or PNA78 NLS (at final concentrations of lOuM) by COS7 cells. Live cell (unfixed) images: left, DIC (brightfield) image; right, fluorescence (FITC) indicating positive PNA signals. Figure 2(c) depicts confocal microscopy showing uptake of PNA78 TAT, PNA78 NLS, or 'TAT alone ^* (positive control) (all at a final concentration of 10 uM) into 562 cells that had been attached to poly-L-lysine coated slides. Live cell (unfixed) images: left, DIC image; right, green fluorescence (FITC) indicating positive PNA signals. [0016] Figure 3 depicts in vivo target binding of PNA78/TAT in K562 cells.

Specifically, Figure 3(a) depicts steps of modified chromatin association assay using streptavidin-conjugated magnetic beads and the magnetic particle concentrator. During the actual experiment, a portion of the treated cells were simultaneously analyzed for PNA uptake by confocal microscopy. Figure 3(b), left panel, depicts a confocal image showing nuclear-entering efficiency of WT PNA78/TAT and MUT PNA78/TAT. ^* No PNA' and 'TAT alone ¹ were included as negative and positive controls. DRAQ5 was used as a nuclear stain for live (unfixed) cells. Figure 3(b), right panel, depicts quantitative chromatin association analysis of K562 cells after exposure to WT PNA78/TAT or MUT PNA78/TAT. 'No PNA' and 'TAT alone' served as controls. The results of quantitative analysis of the γ-globin promoter DNA in the precipitated material is shown.

[0017] Figure 4 depicts in vivo transcriptional activity of chimeric PNA78 derivatives. In Figure 4(a), a transient assay was performed in K562 cells that had been transfected with plasmid containing the γ-globin promoter (-299*- +36) upstream of the luciferase reporter (shown below) followed by exposure to PNA78/TAT, PNA78/NLS, TAT alone, or buffer. Activity values were normalized to a cotransfected Renilla control. In Figure 4(b), K.562 cells were transiently transfected with a luciferase reporter containing either wild type (wt) γ-globin promoter sequence or one that was mutated at the PNA target region (mut) (AATAGTTTTATAGTA) (SEQ ID NO:5), followed by exposure to PNA78/TAT or buffer. Values were normalized to a cotransfected Renilla control. In Figure 4(c), a reporter plasmid bearing 4 repeats of the PNA78 target sequence and the minimal SV40 promoter upstream of luciferase gene (schematic shown below; PNA78 sequence is underlined) was transfected into either K562 or 293T cells and followed by exposure to PNA78/TAT, VΡ2/ΡΝΑ78ΠΆΤ, or buffer. Values were normalized to a cotransfected Renilla control.

[0018] Figure 5 shows that PNA78 conjugated to a minimal activation domain reactivates dormant γ globin expression in vivo in mouse adult bone marrow cells (MBC) containing the human βYAC. Cells were treated with each of four modified PNA78 molecules (PNA78/TAT, VP2/PNA78/TAT, B io- ATF/PNA78/TAT, ATF Βio/ΡΝΑ78ΛΓΑΤ), 'TAT alone', or buffer ('no PNA') and analyzed by confocal microscopy (a) or by quantitative RT-PCR (b) 16 hours after PNA treatment. Figure 5(a) depicts a confocal image of MBC- YAC showing nuclear-entering efficiency. Figure 5(b) shows an expression profile of γ globin levels in BC-PYAC cells in (a) analyzed by quantitative RT-PCR with 2 sets of primers as described in Methods. Each experiment is the average of triplicate sample analyses. In Figure 5(c) MBC- YAC cells were treated with buffer ('no PNA') or ATF-Bio/PNA78 TAT and analyzed by confocal microscopy 48 hours later by fixing and staining with DAPI (blue) and an anti-human γ-globin protein antibody linked to Alexa 568 (red). K562 cells were also stained and served as a positive control for γ-globin protein detection.

[0019] Figure 6 shows the analysis of human peripheral blood CD34+ cells at different time points after induction of erythrotd differentiation by erythropoietin (Epo). Figure 11(a) is a line graph showing the percent of the cell culture expressing the cell surface markers CD34, CD235a. CD36, and CD71 at the indicated time points (days, "D ^* ). Figure 6(b) contains images showing the morphological (Giemsa) analyses performed on human peripheral blood CD34+ samples removed at the indicated day of culture. The progressive changes toward a more terminally differentiated state such as later stage erythroblasts are indicated by arrows and mature enucleated red cells are indicated by asterisks.

[0020] Figure 7 shows the analysis of human peripheral blood CD34+ cells during the SCF/Epo phase of culture. Figure 7(a) contains dot plots showing flow cytometric profiles of CD36 and CD235a expression in human peripheral blood (H-BP) samples at Day 2 and Day 4 of incubation in SCF Epo, showing increases in their expression during culture. Figure 7(b) is a fluorescent image showing beta globin expression in H-PB CD34+ cells and Figure 7(c) is an expression analysis by semiquantitative RT-PCR of β-globin mRNA in H-PB cells on Day 0, 2, 4, , and 7 as indicated.

[0021] Figure 8 is an analysis of the effect of PNA78 conjugated to a minimal activation domain on activating V globin expression in differentiating adult human peripheral blood (H-PB) CD34+ cells. Figure 8(a) is a bar graph showing the mRNA level of □ globin in cultured CD34+ cells 16 hours after treatment with PNA78 TAT, ATF- Bio MUT-PNA78/TAT, or ATF-Bio/WT-PNA78 TAT. Analysis of triplicates was performed by quantitative RT-PCR (*p<0.005). Figure 8(b) contains images showing immunostaining and analysis by confocal microscopy of Y globin protein in cultured CD34+ cells from the same experiment as in Figure 8(a), performed after treatment with buffer ( no PNA') or chimeric PNA78 molecules. 562 cells served as a positive control. Figure 8(c) contains dot plots and histograms from toxicity analysis by flow cytometry of cultured CD34+ cells after PNA treatment. The effectiveness of PNA78 entry was monitored with streptavidin-FTTC.

[0022] Figure 9 is an expression profile of erythroid markers in human peripheral blood CD34+ cells after PNA treatment. Human peripheral blood (H-PB) CD34+ cells from the same experiment as in Fig. 8 (day 2 of culture) were analyzed by flow cytometry 24 hours after treatment with buffer ('no PNA'), PNA78/TAT, ATF- Bio MUT-PNA78/TAT, or ATF-Bio WT- PNA78 TAT. Figure 9(a) shows forward scatter ("FSC") and side scatter ("SSC") profiles of the indicated cells. Figure 9(b) shows the CD34 and CD36 expression profiles of the cells and Figure 9(c) shows the CD36 and CD235a expression profiles of the indicated cells.

Detailed Description of the Invention

[0023] The present invention relates to a compound having the general structure: and uses thereof, wherein X is A or C and Y is A or C with the proviso that when X is A, Y is C, and when X is C, Y is A; A represents an oligopeptide structure, the sequence of which comprises a sequence which renders the compound able to enter the nucleus of a cell; B represents a peptide nucleic acid (PNA) structure at least 12 nucleotides in length, the sequence of which is capable of hybridizing with a DNA within the nucleus of the cell, which DNA is within a promoter region of a gene; C represents an oligopeptide structure; and each represents a chemical linkage between the structures at each side thereof, which may be the same as or different from each other such linkage.

[0024] In some embodiments, X is a and Y is C. In other embodiments, X is

In some embodiments, the gene is a gene, the regulation of the transcription and expression of which is desired. In certain embodiments the oligopeptide structure C is detectable when the compound is bound to the DNA.

In some embodiments, the sequence of C comprises a sequence which renders the compound able to regulate the transcription and expression of the gene.

In certain embodiments, each may be a chemical bond or a chemical linker.

In some embodiments, at least one is a chemical bond.

In certain embodiments, the chemical bond is a covalent bond, an amide bond, or a peptide bond.

In some embodiments, at least one is a chemical linker.

In some embodiments of the invention, the chemical linker comprises an amino acid, biotin, an ether (O), a stable polyether (OO), AEEA (2-aminoethoxy-2-ethoxyacetic acid), or a cleavable disulfide linkage.

In some embodiments of the invention, the sequence of C renders the compound able to regulate the transcription and expression of the gene by activating transcription of the gene.

In some embodiments of the invention, the sequence of C renders the compound able to regulate the transcription and expression of the gene by upregulating transcription of the gene.

In some embodiments, the sequence of C renders the compound able to regulate the transcription and expression of the gene by downregulating the transcription of the gene.

In some embodiments, the sequence of C renders the compound able to regulate the transcription and expression of the gene by repressing transcription of the gene.

In some embodiments of the invention, A comprises a sequence selected from the group consisting of the following sequences: YGRKKRRQRRR (SEQ ID NO:6), GR KRRQRRRPPQ (SEQ ID NO:7), YARKARRQARR (SEQ ID NO:8), YARAAARQARA (SEQ ID NO:9), YARAARRAARR (SEQ ID NO: 10), YARAARRAARA (SEQ ID NO: 11), PKKKRKV (SEQ ID NO: 12). RQIKIWFQNRRMKWKK (SEQ ID NO.13), KKWKMRRNQFWIKIQR (SEQ ID NO: 14), RQIKIWFQNRRMKWKK (SEQ ID NO: 15), RQIKIWFPNRRMKWKK (SEQ ID NO: 16), RQPKIWFPNRRMPWKK (SEQ ID NO: 17), RQIKIWFQNMRRKWKK (SEQ ID NO: 18), RQIRIWFQNRRMRWRR (SEQ ID NO: 19), RRWRRWWRRWWRRWR (SEQ ID NO:20), RQILIWFQNRRMKWKK (SEQ ID NO:22), LLIILRRRIRKQAHAHSK (SEQ ID NO:23), KLALKLALKALKAALKLA (SEQ ID NO:24), and AGYLLGKINLKALAALAKKIL (SEQ ID NO:25).

In certain embodiments of the invention, C comprises a sequence selected from the group consisting of the following sequences: DFDLDMLGDFDLDMLG (SEQ ID NO:26) MLGDFDLDMLGDFDLD (SEQ ID NO:30). CGSDALDDFDLDML (SEQ ID NO:27). PEFPGIELQELQELQALLQQ (SEQ ID NO:28). and RHGEKWFLDDFTNNQMDQDY (SEQ ID NO:29).

In some embodiments, C comprises a sequence selected from the group consisting of the sequence of an engrailed repression domain, the sequence of the HID (HDAC interaction domain) of the Sin3A protein and MSRRKQSKPRQIL (SEQ ID NO:21).

In some embodiments of the invention there is a chemical linker between A and B or between B and C and the chemical linker is selected from the group consisting of a stable polyether, AEEA (2-aminoethoxy-2-ethoxyacetic acid), and a cleavable disulfide linkage.

In certain embodiments of the invention, the gene is a γ-globin gene.

In some embodiments, the PNA structure comprises the sequence TACTCTAAGACTATT (PNA78) (SEQ ID NO: 1).

In some embodiments, the compound has the structure H ₂ N-CGSDAUDDFDLDML-Biotin- O-B -O- YGRKKRRQRRR.

In other embodiments, the compound has the structure H ₂ N-CGSDALDDFDLDML-Biotin- O-TACTCTAAGACTATT-O- YGRKKRRQRRR. In certain embodiments, the compound has the structure Biotin-OO- DFDLDMLGDFDLDMLG-O-TACTCTAAGACTATT-O-YGR RRQRRR.

In some embodiments, at least one amino acid is in the form of a D- isomer

A composition comprising the any compound disclosed herein and a carrier is also included as an embodiment of the present invention.

Certain embodiments include methods of detecting the presence of a DNA within the nucleus of a ceil which comprises contacting the cell with the compound of claim 1 under conditions such that the compound enters the cell and the PNA structure B hybridizes to the DNA to form a hybridization product, and then detecting the resulting hybridization product.

Embodiments of the invention include methods for upregulating transcription of a γ- glob in gene in a mammalian bone marrow cell comprising contacting the cell with a compound of the invention comprised of a sequence that is capable of upregulating or activating the transcription of a gene

In some embodiments, the mammalian bone marrow cell is an adult erythroid cell

Embodiments of the invention include methods for treating a β-globin disorder in a mammal comprising administering to said mammal a therapeutically effective amount of any compound disclosed herein.

In some emodiments of the invention, the β-globin disorder is sickle cell anemia or β- thalassemia

Some embodiments further comprise administering to an animal a second agent selected from the group consisting of hydroxyurea a short chain fatty acid (SCFA) inducer. 5- azacytidine and a histone deacetylase inhibitor

In some embodiments of the invention, the short chain fatty acid (SCFA) inducer is butyrate In some embodiments of the invention, the histone deacetylase inhibitor is suberoylanilide hydroxamic acid (SAHA).

In some embodiments of the invention B is from 12 to 15 nucleotides in length.

Certain embodiments of the present invention are based on the demonstration herein that peptide nucleic acid (PNA) molecules can be used to effectively modulate specific gene expression by conjugating them to two peptide sequences: (1) a cell and or nuclear entry sequence and (2) a transcription activation or repression domain. The embedded chimeric peptide nucleic acid (ecPNA) molecules of the present invention are designed to bind to a specific DNA sequence in a chromosome, thereby providing increased specificity. The presence of cell and/or nuclear entry sequence facilitates greater efficiency of ecPNA cell and/or nuclear entry. Rather than relying on a triplex structure to passively lead to transcriptional increase, as described in the prior art, the ecPNAs of the present invention include a transcriptional activation domain which directly activates transcription of a target gene. Furthermore, the novel ecPNAs of the present invention can include a transcriptional repression domain, which directly represses transcription of a target gene, a function not provided by the triplex structures of the prior art.

As disclosed in the Examples section, below, two specific ecPNA constructs of the invention ATF-Bio/PNA78/TAT and VP2/PNA78/TAT produced therapeutically significant increases (7 -fold and 2· fold, respectively) in γ-globin gene expression in mouse bone marrow cells engineered to carry a yeast artificial chromosome that contains the complete human B-like globin locus (β-YAC).

[0025] In certain embodiments, and without limitation, the invention provides the advantage that neither viral vectors nor oncogenes are introduced, thereby providing a method with increased efficacy and safety compared to other methods. Specifically, there is no transduction or transfection protocol involved, as the ecPNA molecules of the invention, while not intending to be bound by one particular theory or mechanism, are thought to enter the cell and/or nucleus by macropinocytosis. Mammalian cells take up ecPNA molecules efficiently (50-95% after four hours of incubation). In addition, effects on gene expression can be seen within 24 hours. [0026] While not intending to be bound by a specific mechanism or theory, the PNAs within ecPNAs of the present invention are believed to function via a single strand invasion by hybridizing with one strand of DNA of the target promoter sequence through Watson-Crick base pairing and replacing the other strand of the double helix. This way the PNAs of the present invention avoid the limitations that follow from a triplex- forming design of the PNAs of the prior art (e.g., requirement for homopurine in the target sequence and requirement for the use of base analogues, which limits the number of potential DNA binding sites).

[0027] By providing the novel ecPNA molecules, the present invention provides a sequence-specific and efficient (i.e., low-toxic) gene-activating/repressing tool which can be used in vitro, ex vivo or in vivo to repress the expression of harmful genes (e.g., oncogenes), or to upregulate the expression of useful genes (e.g., tumor suppressor genes), or to reactivate the expression of normally repressed genes (e.g., fetal-specific genes in adult cells). The ecPNAs of the present invention can thus be used to treat various diseases treatable by transcriptional modulation, which include, among others, various cancers and hemoglobin disorders. For example, diseases treatable by the molecules of the present invention include, but are not limited to, β-thalassemia (Cooley's anemia), sickle cell disease, chronic myelogenous leukemia (CML) and other myeloproliferative diseases, and acute myeloid leukemia.

Definitions

[0028] The term "upregulate transcription" and is used to mean enhance transcription and expression of a gene, the expression of which occurs, but at levels less than desired. The term "activate expression" is used to mean activate a gene which otherwise being transcribed and expressed. As specified herein, the use of the ecPNAs of the present invention leads to at least about 2· fold increase of transcription, more preferably at least about 5-fold increase of transcription, and most preferably at least about 7-fold increase of transcription of a gene as measured by any suitable assay (e.g., quantitative mRNA analyses [e.g., qPCR or Northern blot analysis], and/or quantitative protein analyses [e.g.. Western blot or immunofluorescence analysis]). [0029] Similarly, the term "downregulate transcription" is used interchangeably to mean reduce the transcription and expression of a gene, the expression of which occurs at levels greater than desired. The term "repress transcription" is used to mean eliminate transcription and expression of a gene, the expression of which results in an undesired effect. As specified herein, the use of the ecPNAs of the present invention leads to at least about 2-fold decrease of transcription more preferably at least about 5-fold decrease of transcription, and most preferably at least about 7-fold decrease of transcription of a gene as measured by any suitable assay (e.g., quantitative mRNA analyses [e.g., qPCR or Northern blot analysis], and/or quantitative protein analyses [e.g., Western blot or immunofluorescence analysis]).

[0030] As used herein the term "detecting the presence" means marking the presence of a DNA with an oligopeptide of a compound of the invention in any way which may be detected. In certain embodiments of the invention, an oligopeptide of a compound of the invention may be detected through the use of an antibody.

[0031] As used herein, the term 'target gene" means the gene for which the transcription is to be activated, upregulated, downregulated, or repressed by a compound of the present invention.

[0032] Within the meaning of the present invention, the term "coadministration" is used to refer to administration of an ecPNA and a second agent simultaneously in one composition, or simultaneously in different compositions, or sequentially within a certain time period.

[0033] The term "about" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within an acceptable standard deviation, per the practice in the art.

[0034] In the context of the present invention insofar as it relates to any of the disease conditions recited herein, the terms "treat ^* , "treatment", and the like mean to relieve or alleviate at least one symptom associated with such condition, or to slow or reverse the progression of such condition. For example, in relation to β-globin disorders, the symptoms include anemia, tissue hypoxia, organ dysfunction, abnormal hematocrit values, ineffective erythropoiesis, abnormal reticulocyte count, abnormal iron load, splenomegaly, hepatomegaly, impaired peripheral blood flow, dyspnea, increased hemolysis, jaundice, anemic crises and pain such as angina pectoris, etc. Within the meaning of the present invention, the term "treat" also denotes to arrest, delay the onset (i.e., the period prior to clinical manifestation of a disease) and or reduce the risk of developing or worsening a disease. The term "protect" is used herein to mean prevent, delay or treat, or all, as appropriate, development or continuance or aggravation of a disease in a subject.

[0035] As used herein the term "therapeutically effective" applied to dose or amount refers to that quantity of a compound or pharmaceutical composition that is sufficient to result in a desired activity upon administration to an animal in need thereof. Within the context of the present invention, the term "therapeutically effective" refers to that quantity of a compound or pharmaceutical composition that is sufficient to reduce or eliminate at least one symptom of a disease or disorder (e.g., a cancer or a β-globin disorder). Note that when a combination of active ingredients is administered the effective amount of the combination may or may not include amounts of each ingredient that would have been effective if administered individually.

[0036] The phrase "pharmaceutically acceptable", as used in connection with compositions of the invention, refers to molecular entities and other ingredients of such compositions that are physiologically tolerable and do not typically produce untoward reactions when administered to a mammal (e.g., a human). Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more particularly in humans.

[0037] In accordance with the present invention, there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. [See, e.g., Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, 2nd ed.. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989 (herein "Sambrook et al., 1989"); DNA Cloning: A Practical Approach, Volumes I and II (Glover ed. 1985); Oligonucleotide Synthesis (Gait ed. 1984) ^* Nucleic Acid Hybridization (Hames and Higgins eds. 1985); Transcription And Translation (Hames and Higgins eds. 1984); Animal Cell Culture (Freshney ed. 1986); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal. A Practical Guide To Molecular Cloning (1984)· Ausubel et al. eds., Current Protocols in Molecular Biology, John Wiley and Sons, Inc. 1994; among others. |

Embedded Chimeric Peptide Nucleic Acid Molecules of the Invention

[0038] Any PNA sequence which corresponds to a sequence comprised in the promoter sequence of a target gene can be used within the ecPNA molecules of the invention. In preferred embodiments, the PNA sequences are complementary to a sequence within the 200 base pair (bp) region that is upstream of the transcription start site of a target gene. While not intending to be bound by theory, this 200 bp region is important for transcriptional regulation of gene expression.

[0039] In other embodiments, the PNA sequence is complementary to a sequence within the 400, 350, 300, or 250 base pair (bp) region that is upstream of the transcription start site of a target gene. In some embodiments, the PNA sequence can comprise a nucleic acid sequence that is complementary to a sequence that is downstream of the transcription start site of a target gene.

[0040] In certain embodiments, the PNA sequence can comprise one or more nucleic acid substitutions. For example, a nucleic acid residue can be substituted, e.g. with a different nucleic acid, for example, and without limitation, inosine, pseduoisocytosine, 2- thiouracil, and/or diaminopurine. PNA sequences containing such substitutions preferably although not necessarily retain similar activity (i.e., ability to activate or repress transcription of a target gene), compared to the PNA molecule without the one or nucleic acid substitutions. In certain embodiments, a PNA molecule comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to a sequence that is complementary to a sequence within the 200 base pair (bp) region that is upstream of the transcription start site of a target gene.

[0041] In some embodiment the PNA structure is from 12 nt to 15 nt in length, however, shorter or longer sequences may be possible. In certain embodiments, the PNA structure will be between 12 and 30 nucleotides in length, e.g. between 12 and 25 nucleotides, often between 12 and 30 nucleotides in length.

[0042] The sequence of the PNA structure may be capable of hybridizing with a DNA within the nucleus of a cell. In some embodiments, the sequence of the PNA structure will be may be complementary to a sequence within the promoter region of a gene. Non-limiting examples of gene promoters with respect to which complementary PNA sequences have been described are listed in Table 1. The references describing these PNAs are also cited in Table 1 and are hereby incorporated by reference into the present disclosure. In additional embodiments, the sequence of the PNA structure may be complementary to a sequence within a promoter region bound by a polymerase, such as T7, SP6, or T3 RNA polymerase. [See also Hamilton et al., Specific and nonspecific inhibition of transcription by DNA, PNA. and phosphorothioate promoter analog duplexes. Bioorganic & Medicinal Chemistry Letters 6(23):2897-2900 (1996); and Larsen and Nielsen, Transcription- mediated binding of peptide nucleic acid (PNA) to double-stranded DNA: sequence-specific suicide transcription. Nucleic Acids Res. 24:458-463 (1996).]

Table 1. Genes with Promoters for which PNAs have been Targeted

[0043] Any oligopeptide may be used as a component of the compound of the invention since in principle any oligopeptide can be detectable. The oligopeptide structures of the compounds of this invention may be or include epitope tags such as VS- tag, Myc-tag, HA-tag, FLAG-tag, GST-tag, and His-tags or any other amino acid sequence for which antibodies with suitable specificity and affinity are generated. [See also Huang and Honda, CED: a conformational epitope database. BMC Immunology 7:7 http://www.biornedcentral.com/1471-2172/7/7#Bl. Retrieved February 16, 2011 (2006); and Walker and Rapley, Molecular biomethods handbook. Pg. 467 (Humana Press, 2008).] One of ordinary skill in the art will understand essentially any oligopeptide may be used as a marker in the invention and thus be a component of the compounds of the present invention.

[0044] In general the oligopeptide of the invention will range from 5 to 50 amino acids in length, most typically from 5 to 30 amino acids in length, e.g. from 10-30 or from 12-25 amino acids in length. [0045] Those skilled in the art will be aware of how to produce antibody molecules when provided with an oligonucleotide of the present invention. For example, polyclonal antisera or monoclonal antibodies can be made using standard methods. A mammal, (e.g., a mouse, hamster, or rabbit) can be immunized with an immunogenic form of the oligonpeptide which elicits an antibody response in the mammal. Techniques for conferring immunogenicity on a oligopeptide include conjugation to carriers or other techniques well known in the art. For example, the oligopeptide can be administered in the presence of an adjuvant. The progress of immunization can be monitored by detection of antibody titers in plasma or serum. Standard ELISA or other immunoassay can be used with the immunogen as antigen to assess the levels of antibodies. Following immunization, antisera can be obtained, and, if desired IgG molecules corresponding to the polyclonal antibodies may be isolated from the sera.

[0046] To produce monoclonal antibodies, antibody producing cells

(lymphocytes) can be harvested from an immunized animal and fused with myeloma cells by standard somatic cell fusion procedures thus immortalizing these cells and yielding hybridoma cells. Such techniques are well known in the art. Hybridoma cells can be screened immunochemically for production of antibodies which are specifically reactive with the oligopeptide, and monoclonal antibodies isolated.

[0047] To enhance their immunogenicity, it is well-known to conjugate small oligopeptide fragments to a hapten, such as, for example, dinitrophenyl (DNP), m- maleimidobenzoyl-N-hydroxyl- N-hybroxysuccinimide ester (MBS), or m-amino benzene sulphonate. A "hapten" is a non-immunogenic molecule that will react with a preformed antibody induced by an antigen or carrier molecule. Alternatively, the immunogenicity of oligopeptides may be enhanced by conjugating the oligopeptide to a carrier molecule, such as, for example, an antigenic oligopeptide, that may be conjugated to a hapten. As will be known to those skilled in the art, a "carrier" is generally an antigenic molecule. Preferred carrier molecules for this purpose include ovalbumin, KLH, and PHA.

[0048] The term "antibody" as used herein, is intended to include fragments thereof which are also specifically reactive to an oligopeptide as described herein. [0049] Immunoassays are useful in detecting the presence of an oligopeptide as disclosed herein, in a cell. Such an immunoassay is of particular use in detecting the presence of a DNA in a cell in certain embodiments of the invention. Immunoassays are also useful for the quantitation of said DNA in a cell. The invention described herein extends to all such uses of immunointeractive molecules and diagnostic assays requiring said immunoassays for their performance.

[0030] A wide range of immunoassay techniques may be such as those described in U.S. Pat. Nos. 4,016,043, 4,424, 279 and 4,018,653. These methods may be employed for detecting the presence of a D A in some embodiments the invention.

[0051] Any cell or nuclear entry sequence can be used within the ecPNA molecules of the invention. In a preferred embodiment, the cell or nuclear entry sequence comprises at least one sequence selected from the group consisting of: (a) a TAT peptide having the sequence YGR KRRQRRR (SEQ ID NO:6), (b) a TAT peptide variant having the sequence selected from GRKKRRQRRRPPQ (SEQ ID NO:7), YARKARRQARR (SEQ ID NO:8) YARAAARQARA (SEQ ID NO:9), YARAARRAARR (SEQ ID NO: 10), and YARAARRAARA (SEQ ID NO:l 1), (c) an NLS peptide having the sequence P KKRKV (SEQ ID NO: 12), (d) a penetratin peptide having the sequence selected from RQI IWFQNRRMKWK (SEQ ID NO: 13), KWKMRRNQFWIKIQR (SEQ ID NO: 14), RQIKIWFQNRRMKWKK (SEQ ID NO: 15), RQIKIWFPNRRMKWKK (SEQ ID NO: 16), RQPKIWFPNRRMPWKK (SEQ ID NO: 17), RQIKIWFQNMRRKWKK (SEQ ID NO: 18), RQIRIWFQNRRMRWRR (SEQ ID NO: 19), and RRWRRWWRRWWRRWR (SEQ ID NO:20), (e) an Antennapedia domain pAntp(43-58) having the sequence RQDLIWFQNRRMKWKK (SEQ ID NO:22), (f) pVEC(615-632) having the sequence LLIILRRRIR QAHAHSK (SEQ ID NO:23), (g) a model amphipathic peptide (MAP) having the sequence KLALKLALKALKAALKLA (SEQ ID NO:24); and (h) a transportan 10 peptide having the sequence AGYLLGKINLKALAALA KIL (SEQ ID NO:25).

[0052] Any transcription activation or repression domain can be used within the ecPNA molecules of the invention. In a preferred embodiment, the transcription activation domain comprises at least one sequence selected from the group consisting of: (a) a VP2 domain having the sequence DFDLDMLGDFDLDMLG (SEQ ID NO:26) or the sequence MLGDFDLDMLGDFDLD (SEQ ID NO:30), (b) an ATF-14 domain having the sequence CGSDALDDFDLDML (SEQ ID NO: 27), (c) an AH domain having the sequence PEFPG IELQELQELQ ALLQQ (SEQ ID NO.28), and (d) a Gal80BP domain having the sequence RHGEKWFLDDFTNNQMDQDY (SEQ ED NO:29). Preferred repression domains include the engrailed repression domain, the HID (HDAC interaction domain) from the Sin3A protein, and MSRRKQSKPRQIL (SEQ ID NO:21). [See, Manwani and Bieker, Exp. Hem. 35:39-47 (2007); Lin et al.. The N termini of Friend of GATA (FOG) proteins define a novel transcriptional repression motif and a superfamily of transcriptional repressors. J Biol Chem 279(53):55017-23 (2004): and Lauberth and Ranchman, A conserved 12-amino acid motif in SA111 recruits the nucleosome remodeling and deacetylase corepressor complex. J Biol Chem 281(33)23922-31 (2006)]

[0053] In one specific embodiment, the ecPNA molecule is ATF-

Bio/PNA/TAT having the structure N^H-CGSD ALDDFDLDML-B iotin-O-PN A-O- YGRKKRRQRRR. In another specific embodiment, the ecPNA molecule is ATF- B io/PN A78/T AT having the structure N ₂ H-CGSDALDDFDLDML-Biotin-0- TACTCTAAGACTATT-O-YGRKKRRQRRR. In yet another specific embodiment, the ecPNA molecule is VP2/PNA78/TAT having the structure Biotin-OO- DFDLDMLGDFDLDMLG-O-TACTCTAAGACTATT-O-YGRKKRRQRRR. In the above structures, "O" represents the stable polyether linker, AEEA (2-aminoethoxy-2-ethoxyacetic acid). In a preferred embodiment, all TAT amino acids in such ecPNA molecules are in D- isomer form. In any of these embodiments, the inclusion of Biotin (Bio) is optional. Other markers useful for visualizing an ecPNA molecule, such as but not limited to GFP and other fluorescent markers, may also be optionally included in the ecPNA molecules.

[0054] In certain embodiments, the ecPNA molecule has TAT in the D or L form. In certain embodiments, the transactivating domain is in the D or L form.

[0055] Methods for producing PNA-peptide conjugates of the present invention are well known in the art and include, among others, solid-phase synthesis and fragment ligation [see, e.g., de Koning et al., Current Opinion in Chemical Biology, 2003, 7(6):734-740]. Linkers useful in the PNA-peptide conjugates of the present invention include, for example, stable polyether, AEEA (2-aminoethoxy-2-ethoxyacetic acid), cleavable disulfide linkages, or other linkers that are generally known in the art.

Compositions

[00S6] While it is possible to use a composition provided by the present invention for therapy as is, it may be preferable to administer it in a pharmaceutical formulation, e.g., in admixture with a suitable pharmaceutical excipient, diluent, or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.

[0057] Accordingly, in one aspect, the present invention provides a pharmaceutical composition or formulation comprising at least one ecPNA molecule of the invention, or a pharmaceutically acceptable derivative thereof, in association with a pharmaceutically acceptable excipient, diluent, and/or carrier. The excipient, diluent and/or carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. Preferably, the at least one ecPNA molecule is present in such compositions in a therapeutically effective amount.

[0058] The compositions of the invention can be formulated for administration in any convenient way for use in human or veterinary medicine.

Carrier

[0059] The term ''carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. In some embodiments, carriers are pharmaceutical carriers. Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Alternatively, the carrier can be a solid dosage form carrier, including but not limited to one or more of a binder (for compressed pills), a glidant, an encapsulating agent, a flavorant, and a colorant. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin (1990, Mack Publishing Co., Easton, PA 18042). Formulations

[0060] The compositions and formulations of the present invention may comprise pharmaceutically or otherwise acceptable diluents, preservatives, sotubilizers, emulsifiers, adjuvants and/or carriers. Such compositions include diluents of various buffer content (e.g., Tris-HCl, acetate, phosphate) pH and ionic strength; additives such as detergents and solubilizing agents (e.g., Tween 80, Polysorbate 80), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol); incorporation of the material into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc. or into liposomes. Hylauronic acid may also be used. See, e.g., Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, PA 18042) pages 1435 1712 which are herein incorporated by reference.

[0061] Preparations according to this invention for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, or emulsions. Examples of non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and com oil, gelatin, and injectable organic esters such as ethyl oleate. Such dosage forms may also contain adjuvants, preserving, wetting, emulsifying, and dispersing agents. The pharmaceutical compositions may be sterilized by, for example, filtration through a bacteria retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions. They can also be manufactured using sterile water, or some other sterile injectable medium, immediately before use.

Methods of the Invention

[0062] In conjunction with ecPNA molecules, the present invention provides methods for using such molecules, or compositions containing such molecules, to activate or repress transcription of a target gene. Important genes that may be targeted by the ecPNA molecules of the present invention include, for example, activation of γ-globin (for treatment of □ -thalassemia or sickle cell disease, activation of the plS tumor suppressor gene for treatment of cancer, and repression of the BCR/ABL oncogene (for treatment of leukemia).

[0063] In one specific embodiment, the present invention provides a method for upregulating transcription of a γ-globin gene in a bone marrow mammalian cell comprising contacting said cell with an ecPNA molecule having the structure N ₂ H- CGSDALDDFDLDML-Biotin-0-TACTCTAAGACTATT-0-YGRKKRRQRRR (ATF- Bio PNA78 TAT). In another specific embodiment, the present invention provides a method for upregulating transcription of a γ-globin gene in a bone marrow mammalian cell comprising contacting said cell with an ecPNA molecule having the structure Biotin-OO- DFDLDMLGDFDLDMLG-O-TACTCTAAGACTATT-0-YGRKKRRQRRR

(VP2 PNA78 TAT).

[0064] Since the PNA portion of the ecPNA molecules of the present invention can be directed to any promoter region of a target gene of interest, the present invention provides a potentially powerful yet low-toxic treatment for any disorder that can be alleviated by activation or repression of a target gene.

[0065] In certain specific embodiments, the invention provides a method for treating a β-globin disorder in a mammal comprising administering to the mammal a therapeutically effective amount of an ecPNA molecule which upregulates the transcription of a γ-globin gene. Non-limiting examples of encompassed β-globin disorders include sickle cell anemia and Π -thalassemia.

[0066] The human γ-globin (HBG1) mRNA sequence has GenBank

Accesion No. NM_000559. The human γ-globin amino acid sequence has GenBank Accession No. NP_000550. The human β-globin mRNA sequence has GenBank Accesion No. NM_000518. The human β-globin amino acid sequence has GenBank Accession No. NP_000509. The gene region on chromosome 11 containing the human epsilon, gamma G, gamma A, beta 1 pseudogene, delta, and beta -3' globin genes has GenBank Accession No. NG_000007. Dosage and Administration

[0067] ecPNA-containing compositions of the invention can be directly or indirectly administered to a subject (e.g. patient). Indirect administration is performed, for example, by administering the composition to cells (e.g., bone marrow cells) ex vivo and subsequently introducing the treated cells to the patient. The cells may be obtained from the patient to be treated or from a genetically related or unrelated patient. Related patients offer some advantage by lowering the immunogenic response to the cells to be introduced. For example, using techniques of antigen matching, immunologically compatible donors can be identified and utilized. Following treatment of the cells with a composition of the invention, the cells may be administered to a patient in need of such treatment by any suitable route, such as oral, parenteral, sublingual, rectal such as suppository or enteral administration, or by pulmonary absorption or topical application. In a preferred embodiment, the cells are administered intravenously.

[0068] Direct administration of an ecPNA-containing composition to a subject may also be by oral, parenteral, sublingual, rectal such as suppository or enteral administration, or by pulmonary absorption or topical application.

[0069] Parenteral administration may be by intravenous injection, subcutaneous injection, intramuscular injection, intra-arterial injection, intrathecal injection, intraperitoneal injection or direct injection or other administration to one or more specific sites. Injectable forms of administration are sometimes preferred for maximal effect in, for example, bone marrow. When long-term administration by injection is necessary, venous access devices such as medi-ports, in-dwelling catheters, or automatic pumping mechanisms are also preferred wherein direct and immediate access is provided to the arteries in and around the heart and other major organs and organ systems.

[0070] Another effective method of administering the composition is by direct contact with, for example, bone marrow through an incision or some other artificial opening into the body. Compositions may also be administered to the nasal passages as a spray. Arteries of the nasal area provide a rapid and efficient access to the bloodstream and immediate access to the pulmonary system. Access to the gastrointestinal tract, which can also rapidly introduce substances to the blood stream, can be gained using oral, enema, suppository or injectable forms of administration. Compositions may be administered as a bolus injection or spray as appropriate. Compositions may be given sequentially over time (episodically) such as every two, four, six or eight hours, every day (QD) or every other day (QOD), or over longer periods of time such as weeks to months. Compositions may also be administered in a timed-release fashion such as by using slow-release resins and other timed or delayed release materials and devices. Orally active compositions are more preferred as oral administration is usually the safest, most convenient and economical mode of drug delivery. However, direct injection may be preferred when immediate access to the blood system is desired.

[0071] Treatments to the patient may be therapeutic or prophylactic.

Therapeutic treatment involves administration of one or more compositions of the invention to a patient suffering from one or more symptoms of the disorder. Symptoms typically associated with gtobin disorders include, for example, anemia tissue hypoxia, organ dysfunction, abnormal hematocrit values, ineffective erythropoiesis, abnormal reticulocyte count, abnormal iron load, splenomegaly, hepatomegaly, impaired peripheral blood flow, dyspnea, increased hemolysis, jaundice, anemic crises and pain such as angina pectoris. Relief and even partial relief from one or more of these symptoms corresponds to an increased life span or simply an increased quality of life. Further, treatments that alleviate a pathological symptom can allow for other treatments to be administered.

[0072] Prophylactic treatments involve pulsed administration of a composition to a patient having a confirmed or suspected blood disorder without having any overt symptoms. For example, otherwise healthy patients who have been genetically screened and determined to be at high risk for the future development of a blood disorder may be administered compositions of the invention prophylactically. Administration can begin at birth and continue, if necessary, for life. Both prophylactic and therapeutic uses are readily acceptable because these compounds are generally safe and non-toxic.

[0073] It will be appreciated that the amount of the ecPNA-containing compositions of the invention required for use in treatment will vary with the route of administration, the nature of the condition for which treatment is required, and the age, body weight and condition of the patient and will be ultimately at the discretion of the attendant physician or veterinarian. These compositions will typically comprise a therapeutically effective amount of the compositions of the invention. Preliminary doses can be determined according to animal tests, and the scaling of dosages for human administration can be performed according to art-accepted practices.

[0074] Keeping the above description in mind, typical dosages of an ecPNA molecule of the invention for ex vivo or in vitro use range from about 1 μM to about 10 μΜ. Typical dosages of an ecPNA molecule of the invention for in vivo use range from about 0.1 mg/kg to about 100 mg kg.

[0075] Keeping the above description in mind, typical dosages of cells (e.g., bone marrow cells) that have been treated with an ecPNA composition of the invention ex vivo for subsequent administration to a patient range from about 1 x 10 ⁵ to about 1 x 10 ⁷ cells per kg of body weight. In a preferred embodiment, the dosage is about 1 x 10 ⁶ cells kg body weight

Combination Treatments

[0076] The present invention also encompasses combination treatments, wherein an additional agent or agents can be administered to a patient conjointly with an ecPNA of the invention (e.g., in the same composition as the ecPNA or in separate compositions, at the same or different sites, at the same or different times, and for the same or different duration of time). In some embodiments, the additional agent or agents are effective for enhancing the desired effect of the ecPNA on gene expression.

[0077] Non- limiting examples of such combination treatments for β-globin disorders include conjoint administration of a γ-globin-specific ecPNA with a second agent such as, for example, hydroxyurea, a short chain fatty acid (SCFA) inducer (e.g., butyrate), 5-azacytidine, or a histone deactylase inhibitor (e.g., suberoylanilide hydroxamic acid [SAHA]). For example, a pulsed butyrate regimen was shown to work best to effectively increase O-globin levels in patients who already exhibited a slightly higher baseline level (> 2%) of fetal hemoglobin (HbF) production. [See, Atweh, G.F. et al. Sustained induction of fetal hemoglobin by pulse butyrate therapy in sickle cell disease. Blood 93, 1790- 1797 (1999).] Examples

[0078] The following examples are included to demonstrate certain embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1

Design, Synthesis, and Storage of PN A

[0079] A previous study that used PNA to alter γ-globin expression relied on a triplex-forming 'clamp" design that forms a Hoogstein PNA/DNA/PNA triple helix. [See Wang, G. et al., Peptide nucleic acid (PNA) binding-mediated induction of human gamma- globin gene expression. Nucleic Acids Res 27, 2806-2813 (1999)· and Pooga, M., Land, T., Bartfai, T. & Langel, U., PNA oligomers as tools for specific modulation of gene expression. Biomol Eng 17, 183-192 (2001).] However, this design has significant limitations, as it requires the use of base analogues that are not all commercially available and the presence of homopurine homopyrimidine sequences at the target site. [See Kaihatsu, K., Janowski, B.A. & Corey, D.R., Recognition of chromosomal DNA by PNAs. Chem Biol 11, 749-758 (2004). J Further, the possible number of DNA targets was significantly limited using this approach.

[0080] In the present invention, it was desired to streamline this approach by designing short, complementary single stranded PNAs which function via strand invasion and which can accommodate a wider range of DNA targets. By dividing the proximal γ- globin promoter (-202 to +33) into fifteen segments, four (4) 12- to 15-mer sequences suitable for PNA synthesis were identified that correspond to the γ-globin promoter at positions -150, -116, -78, -7, relative to the transcription start site (Figure 1(a)). All PNAs were synthesized by Biosynthesis Inc. (Lewisville, Texas) and have the following structures: PNA7: Biotin-OO-TGTGGAACTGCTGAA-O-k; PNA78: Biotin-OO- TACTCTAAGACTATT-O-k; PNA116: Biotin-OO-GGCTATTGGTCAAGGC-k; PNA 150: Biotin-OO- GAGTTTAGCCAGG-O-k; PNA78/NLS: Biotin-OO- TACTCTAAGACTATT-O-PKKKRKV; PNA78/TAT: Biotin-OO-

TACTCTAAG ACT ATT-O- YGRKKRRQRRR ; MUT PNA78/TAT: Biotin-OO- TACTATAAAACTATT-O-YGRKKRRQRRR; VP2/PNA78/TAT: Bio-OO-

DFDlDMLGFDLDML3-0-TACTCTAAGACTATT-0- YGRKKRRQRRR; Bio- ATF/PNA78/TAT: Bio-OO-CGSDALDDFDLDML-O-TACTCTAAGACTATT-O- YGRKKRRQRRR; and ATF-Bio/PNA78/TAT: N ₂ H-CGSDALDDFDLDML-Biotin-0- TACTCTAAGACTATT-O- YGRKKRRQRRR. In the above structures, "O" represents the stable polyether linker, AEEA (2-aminoethoxy-2-ethoxyacetic acid). As used throughout the present disclosure, ' Bio" is the abbreviated form of "biotin" are used interchangeably. All TAT amino acids were the D- isomer form. PNAs were dissolved in sterile distilled/deionized water and stored at 4°C, and heated at 50°C for 3 minutes just before any application to prevent aggregation.

Example 2

Magnetic Pull-down Assay

[0081] A novel assay was developed to monitor specific binding of PNA molecules to their cognate DNA site (Figure 1(b)). 2 ng of ³² P-labelled double-stranded DNA oligonucleotide (designed at regions of the γ-promoter centered at each of the four PNA target sites) was incubated with various molar excess (Ox to lOOOx) of PNA at 37°C overnight in a final volume of 15 uL with 10 mM Tris and 1 raM of EDTA. The resultant material was incubated with equal amount of Dynabeads M280-streptavidin (DYNAL biotech) for 15 seconds at 25°C. Beads had been prepared by washing twice with BW buffer (lOmM Tris, pH7.5, lmM EDTA, 2.0M NaCl). The beads were pulled down with a Dynal MPC-S magnetic particle concentrator and washed twice with BW buffer. Samples were finally suspended in 50 uL BW buffer, and radioactivity was counted for 10 minutes in a scintillation counter to yield the average counts per minute (cpm) taken for each sample, which was then divided by the cpm of the input (1 uL of the radio-labeled oligo). [0082] It was found that the ability of each PNA to bind and discriminate between wild type and mutant oligos varied considerably. In the tests. PNA ISO or PNA7 did not discriminate or bind well to their target sites under these conditions. However. PNA 116 and PNA78 bound their target DNA oligo in proportion to their input. Even though PNA116 has the best recovery rate of its wild type DNA target, it also bound to the mutant DNA target to an unacceptable extent. On the other hand, PNA78 exhibited the best discrimination (~25-fold) and specificity in recognizing its target sequence.

Example 3

KM11O ₄ Probing and Footprinting

[0083] To further confirm the observation in the magnetic-pull down assay, permanganate (KMnO. probing of the PNA78/DNA interaction in solution was carried out (Figure 1(c)). In a modification of a published protocol [Bentin, T. Hansen, G.I. & Nielsen, P.E. Measurement of PNA binding to double-stranded DNA. Methods Mol Biol 208 91- 109 (2002)] 1 ng of a singly ³² P-end-labeled gel purified Rsal/Xhol fragment derived from the p250 plasmid that contains the γ-globin promoter region -299 to +37 was incubated in a volume of 15 uL with a final concentration of 10 mM Tris and 1 mM EDTA for 30 minutes at 37°C. PNA was added at a final concentration of 50 uM for further incubation overnight at 37°C. 1 uL 20 mM KMn0 ₄ was added to the reaction, and after 15 s 10 uL stop buffer (1.5 M sodium acetate 1 M 2-mercaptoethanol Ph 7.0) was added. 2.5X vol of 96% ethanol was added and the DNA collected by centrifugation at 13000 rpm for 15 minutes. The supernatant was removed and the DNA pellet dissolved in lOOuL 10% piperidine followed by incubation at 90°C for 20 minutes. The lympholized DNA sample was resuspended in 8 μL· FA buffer (80% deionized fomamide lOmM EDTA, 0.25% xylenecyanol FF 0.25 % bromphenol blue). DNA was denatured at 90°C for 2 minutes chilled on ice, and then electrophoresed on thin 10% polyacrylamide gels containing 7 M urea. The dried gel was then exposed for autoradiography. Mn0 ₄ probing gives single-base pair resolution and makes it easy to map the PNA78 target site on the γ- promoter when co-electrophoresed adjacent to a separate reaction that generates a partial sequence ladder. [0084] The results demonstrated that PNA78 binds specifically to its target region within the γ-globin promoter fragment, with residues at the 5 ^* end of its interaction most accessible to chemical cleavage. Based on the results of the in vitro studies, PNA78 was used for the remainder of the experiments described in the present Examples.

Example 4

Cellular and Nuclear Localization of PNA

[0085] hi order to alter transcription, it is necessary for the PNA to reach and enter the cell nucleus. The basis for cellular uptake of PNA is not yet fully understood, although it may be related to passive diffusion [see e.g., Pooga. M. Land T., Bartfai, T. & Langel. U. PNA oligomers as tools for specific modulation of gene expression. Biomol Eng 17, 183-192 (2001)], particularly as unmodified and slightly modified PNAs have been successfully delivered directly to cells without the use of transfection reagents and protocols. [See Nielsen, P.E., Peptide nucleic acid: a versatile tool in genetic diagnostics and molecular biology. Curr Opin Biotechnol 12, 16-20 (2001); and Sei, S. et al.. Identification of a key target sequence to block human immunodeficiency virus type 1 replication within the gag-pol transframe domain. J Virol 74, 4621-4633 (2000).] In order to ascertain whether the PNA molecules efficiently enter an erythroid cell, varying amounts of PNA78 were incubated for different lengths of time with erythroleukemic K562 cells (ATCC deposit number CCL243) and entry and localization was monitored by fluorescent microscopy after incubation with streptavidin-FITC. 562 cells are human erythroid leukemia cells that display similar characteristics of fetal erythrocytes, because they express high levels of γ-globin and no or very low levels of beta globin. [See Stamatoyannopoulos, J.A. & Nienhuis, A.W., Therapeutic approaches to hemoglobin switching in treatment of hemoglobinopathies. Annu Rev Med 43, 497-521 (1992).]

[0086] PNA efficiently entered cells after a simple overnight incubation in growth media with an optimal concentration of 10 uM (Figure 2(a)). Based on published studies, concentrations of PNA as high as 30 uM are not toxic to cultured cells. [See Cutrona G. et al.. Effects in live cells of a c-myc anti-gene PNA linked to a nuclear localization signal. Nat Biotechnol 18, 300-303 (2000).] Although the efficiency of cell entry was high (over 90%) after an overnight incubation, in all cases no evidence of nuclear entry was observed, as all of the PNA molecules remained visible as punctate spots in the cytoplasm.

Cell culture

[0087] COS7 cells were maintained in DMEM (Cellgro®, Mediatech Inc.

(Manassas, VA)) and K562 cells were maintained in RPMI 1640 medium (Gibco®, Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum, penicillin and streptomycin. C ID-dependent wild type β-YAC mouse bone marrow cells (MBCs) [see Blau. C.A. et al., γ-Globin gene expression in chemical inducer of dimerization (CID)- dependent multipotential cells established from human β-globtn locus yeast artificial chromosome (β-YAC) transgenic mice. J Biol Chem 280, 36642-36647 (2005)] were cultured in the presence of AP20187 dimerizer (ΙΟΟηΜ; Ariad Pharmaceuticals, Cambridge, MA) in Isocove's modified Dulbecco's medium containing 10% fetal bovine serum, penicillin, and streptomycin.

Delivery off P A to cells

[0088] PNA was introduced into actively growing COS7 cells. For suspension cells, 3x10 ⁵ 562 cells or β-YAC MBCs were resuspended with 200uL OPTI- MEM, and plated on poly-lysine coated 8-well chambered cover glass slides (Nunc) and allowed to attach to the slide overnight. In all cases, PNA was added to a final concentration of 5uM or lOuM along with Strepavidin-FITC to a final concentration of 5ug/mL. Based on published studies, concentrations of PNA as high as 30 uM are not toxic to cultured cells. [See Cutrona, G. et al.. Effects in live cells of a c-myc anti-gene PNA linked to a nuclear localization signal. Nat Biotechnol 18, 300-303 (2000).] After 4 hours, 300uL RPMI 1640 and FBS was added to a final concentration of 10%.

Confocal analysis and live cell imaging of cellular localization of PNAs

[0089] A live cell imaging protocol was developed to monitor cellular localization of chimeric PNA molecules. At 16 hours post PNA-treatment, DRAQS (Biostatus Limited, UK, [see Martin, R.M., Leonhardt, H. & Cardoso, M.C., DNA labeling in living cells. Cytometry A 67, 45-52 (2005)]) was added to cells to a final concentration of 1 μΜ, and incubated at room temperature for 30 minutes; media was discarded and cells were gently washed twice with lx PBS. lOOuL lx PBS was then added to each chamber for further observation for confocal microscopy. DIC, DRAQ5 (staining cell nucleus), and FTTC images were collected on the ZEISS LSM-510 META Confocal Microscope.

[0090] To help more easily detect subcellular localization of the

PNA78 TAT or PNA78/NLS, initial tests were performed with COS7 cells (Figure 2(b)). The results showed that, after only a 2-hour incubation, the PNA/TAT molecules not only entered the cells but also proceeded to the nucleus.

[0091] This procedure was then performed with the 562 erythrotd cell line.

However, because these normally grow in suspension, they were first attached to poly-L- lysine coated plates to enable easier visualization of the signal localization. As before, the PNA molecules were directly added to K562 cells in serum-free media along with streptavidin-FITC, and their localization was monitored by confocal microscopy without fixation (Figure 2(c)). Easily visible bright positive signals were present in the cell nucleus both in PNA78 TAT and PNA78 NLS treated cells: however, the distribution and efficiency was significantly higher in PNA78 TAT treated cells. These results were highly encouraging, as efficiencies of cell entry were always greater than 50% and sometimes approached 95%. Based on these experiments, it was concluded that PNA molecules attached to a TAT sequence at its 3' end provide a consistent and efficient means of directing the PNA cargo to the nucleus.

Example 5

In Vivo Target Binding of PNA78 in K562 Cells Using a Novel Chromatin Association Assay

[0092] To determine whether PNA78 can interact with its target site in vivo. a modification of the chromatin immunoprecipitation assay was developed. This assay took advantage of the biotin label already incorporated into the 5' end of the PNA molecule and was based on the standard ChIP assay whereby streptavidin- magnetic beads (rather than antibodies) are used to pull-down the PNA78 DNA complex after the normal series of PNA/cell incubation, formaldehyde cross-linking, and chromosomal DNA shearing steps (Figure 3(a)). Along with the "no addition" negative control, a variant of PNA78 that contain two point mutations (MUT PNA78) was synthesized and used it as an additional negative control along with a "TAT alone" control.

[0093] As the PNA/DNA interaction is not protein-based, a novel chromatin association assay that does not utilize antibodies, but rather relies on the biotin moiety in the PNA molecule as a tag for its isolation with streptavidin beads was developed. This assay incorporates elements of chromatin [see Siatecka, M., Xue, L. & Bieker, J.J., Sumoylation of EKLF promotes transcriptional repression and is involved in inhibition of megakaryopoiesis. Mol Cell Biol 27, 8547-8560 (2007)] and biotinylated protein [see Viens. A., Mechold. U., Lehrmann, H. Harel-Bellan, A. & Ogryzko, V., Use of protein biotinylation in vivo for chromatin immunoprecipitation. Anal Biochem 325, 68-76 (2004)] immunoprecipitation protocols. PNA 78/TAT or mutant (MUT) PNA 78/TAT were added to 10 ⁷ 562 cells to a final concentration of 10 uM. After 36 hours, a portion of the cells were analyzed and quantified for PNA incorporation efficiency by confocal analysis of live cells (as above), and the rest were harvested and cross-linked with 0.4% formaldehyde (Fisher Scientific, F79-500) for 10 minutes in room temperature then quenched by addition of glycine in the final concentration of 0.125 M for another 10 minutes. Cells were then washed twice in cold PBS, pelleted at 3000 rpm and suspended in 500 μΐ- SDS buffer (50 mM Tris at pH 8.1 0.5% SDS, 100 ttiM NaCl, 5 mM EDTA and protease inhibitors) and incubated 10 minutes on ice. Cells were then pelleted by centrifugation and resuspended in 1.6 mL IP buffer (0.3% SDS, 1.1% Triton X-100, 1.2mM EDTA, 16.7mM Tris pH8.1, 167mM NaCl and protease inhibitor) and disrupted by sonication (power 4, 21% duty, 30 seconds each time for 10 times with 2 minute-pulse on ice) yielding genomic DNA fragments of -1000 bp. Lysates were collected at 13000 rpm for 30 minutes and diluted 1:5 in IP buffer and incubated with 40uL streptavidin MagneSphere Paramagnetic particles (Promega Cat# Z5481) for 2 hours. Magnetic Particle Concentrator (Dynal MPCS) was used to collect the MagneSphere and its bound material, which was washed five times with LiCl buffer (lOOmM Tris pH8.0, 500mM LiCl, 1% NP-40, 1% deoxycholic acid). Beads were finally resuspended in 150 uL 300mM NaCl at 65°C overnight followed by incubation at 42°C for 2 hours with 20 uL protein kinase K (20 mg/mL). DNA was purified by phenol/chloroform extraction and resuspended in 20 μΙ~ ddH20.

[0094] After 16 hours of incubation of K.562 cells, any formed PNA- chromatin complex was cross-linked and cells were disrupted and chromatin was sheared by sonication. Since all the PNAs were linked to biotin, the complex formed between PNA and the bound chromatin was pulled down and isolated by binding to streptavidin-conjugated magnetic beads. The DNA was purified for sequence identification by quantitative PCR To more precisely quantitate the extent of PNA entry into the nucleus a portion of the cells from each treatment was collected, stained with the DRAQ5 live-cell nuclear stain, attached to chamber-slides, and inspected under confocal microscopy to compare PNA and TAT nuclear entry efficiency. The results (Figure 3(b)) show that while the two PNAs and TAT were taken up by cells with a similar efficiency, only wild-type (WT) PNA78 interacted with its target sequence within the γ-globin promoter to a level greater than the non-specific MUT PNA78 and TAT alone, which had values that are not higher than the 'no addition control. This data confirmed that WT PNA78 has target binding specificity with the endogenous γ-globin promoter in vivo.

Example 6

PNA Conjugated with a Minimum Activation Domain (AD) Alters Transcriptional Activity in K562 Cells

[0095] The above experiments established that WT PNA78 is able to enter the cell nucleus and bind to its target sequence at the γ-globin promoter site in vivo. To assess whether WT PNA78 can alter transcription, a transient assay was used A γ-globin luciferase reporter was transfected into K562 cells followed by exposure of the cells to TAT, PNA78/NLS, or PNA78/TAT. Although TAT alone had no effect on the basal activity of the reporter, PNA78/NLS exerted a mild, and PNA78/TAT a stronger, superactivation effect on its expression (Figure 4(a)). As predicted, mutation of the PNA78 target site on the γ-promoter negated its ability to be superactivated by PNA78/TAT. However, the basal activity of the γ-promoter was dramatically impaired (Figure 4(b)), suggesting that the DNA sequence complementary to PNA78 encodes an important γ- promoter element.

[0096] It was then examined whether the potential of WT PNA78 to alter transcriptional activity in vivo can be enhanced by linking it to a transcriptional activator motif. The VP2 minimal activation domain (AD) was chosen for the first set of experiments. The VP2 minimal activation domain is a highly acidic 16 amino acid sequence (MLGDFDLDMLGDFDLD) (SEQ ID NO: 30) derived from the herpes simplex virus C- terminus transactivation domain of VP16. [See Ansari, A.Z., Mapp, A.K., Nguyen, D.H., Dervan P.B. & Ptashne, M., Towards a minimal motif for artificial transcriptional activators. Chem Biol 8, 583-592 (2001). ] VP2 is a very potent artificial transactivator in vitro when linked to a DNA-binding protein domain. [See Arora, P.S., Ansari, A.Z., Best, T.P., Ptashne, M. & Dervan, P.B., Design of artificial transcriptional activators with rigid poly-L-proline linkers. J Am Chem Soc 124, 13067-13071 (2002).] This amino acid sequence was linked to PNA78 TAT at its 5 ^* domain to yield VP2/PNA78 TAT. This molecule retains the biotin label at the beginning of the VP2 sequence for monitoring purposes. The ability to superactivate the γ-globin luciferase reporter in transfected 562 cells was tested, but there was little effect beyond that seen with PNA78/TAT. Then an artificial luciferase reporter plasmid was constructed with four tandem repeats of PNA78 target sequence upstream of the minimal SV40 promoter and its activity in erythroid K562 cell line and in the non-erythroid 293T cell line (ATCC deposit number CRL11268) was tested. Three hours post-transfection, PNA78/TAT or VP2/PNA78/TAT were added to cells in serum-free media. A portion of the cells were also inspected under confocal microscopy after streptavidin-FITC treatment to normalize PNA uptake efficiency. It was found that the luciferase activity increased by 2.3-fold in cells treated with VP2 PNA78 TAT compared to non-treated cells, a stronger effect than seen with PNA78/TAT and not observed at all in 293T cells (Figure 4(c)). This demonstrated that the attachment of a transactivation domain to the PNA78 TAT molecule enhances its ability to activate transcription in the K562 fetal- like erythroid environment Plasmids

[0097] The γ-globin reporter contained a 1.5 Kb Human HS2 fragment upstream of the -299 to +37 human γ-globin promoter in Promega's (Madison, WI) pGL2 Basic plasmid. [See Caterina, J.J., Donze, D., Sun. C.W., Ciavatta, D.J. & Townes, T.M., Cloning and functional characterization of LCR-F1: a bZIP transcription factor that activates erythroid-specific. human globin gene expression. Nucleic Acids Res 22, 2383- 2391 (1994).] A modified pGL2-promoter vector from Promega with one or four copies of the PNA78 target sequence (CCGGTTGACCAATAGTCTTAGAGTATCC) (SEQ ID NO:40) upstream of the minimal SV40 promoter was also generated by synthesis of the oligonucleotide followed by ligation into the Xmal and Xhol sites of the vector.

Transfection

[0098] Transfection of K562 cells was performed using the DMRIE lipofection method (Gibco, Invitrogen, Carlsbad, CA). 562 cells were counted and 1x10 ^s cells were resuspended in 0.1 mL OPTI-MEM in a 12- well plate (BD Falcon, Franklin Lakes, NJ). Experiments were all done in triplicates. 1.5 ng Renilla plasmid (Promega Corporation, Madison, WI) and 0.5 μg reporter construct were added to each well. After 2 hours of transfection, PNAs were added to a final concentration of 10 uM. 2 hours later, 1 ml RPMI and a final concentration of 10% FBS were added to each well. This was followed by incubation at 37°C for 36 hours.

Luciferase reporter assay

[0099] 36 hours after PNA addition, K562 cells were collected by centrifugation at 2500 rpm. Media was discarded and cells were washed once with lx PBS and resuspended in 250 μL lx passive lysis buffer (PLB) (Promega). Tubes that contained cells were put on a rocker platform for 10 minutes at room temperature. Cells were then centrifuged at 13000 rpm and resuspended in 200 L, 1x PLB, loaded on a 96 well plate and further analyzed with a Turner Biosystem Microplate Luminometer. [See Siatecka, M., Xue, L. & Bieker, J. J., Sumoylation of EKLF promotes transcriptional repression and is involved in inhibition of megakaryopoiesis. Mol Cell Biol 27, 8547-8560 (2007).] Luciferase values were divided by the Renilla value from the same sample after it had been divided by 1000. To minimize the differences in between loading, each sample was loaded in three different wells and the average of three was calculated.

Example 7

PNA-AD changes the globin expression pattern in mouse bone marrow cells

[00100] The above tests had been directed at a non-chromatinized DNA target promoter that is already highly active prior to any treatment. It was thus important to test whether the PNA constructs can activate a dormant γ-globin promoter in an erythroid cell that has already switched off its expression in favor of adult β-globin. To test this, a line of mouse bone marrow cells engineered to carry a yeast artificial chromosome that contains the complete human B-like globin locus (B-YAC) was used. [See Blau, C.A. et al. y - Globin gene expression in chemical inducer of dimerization (CID)-dependent multipotential cells established from human β- globin locus yeast artificial chromosome (β-YAC) transgenic mice. J Biol Chem 280, 36642-36647 (2005).) These adult bone marrow cells have shut off human fetal γ-globin expression and solely express human adult B-globin (in addition to the endogenous mouse adult β-globin). The large B-YAC is properly controlled during murine development in that human γ-globin is expressed in the yolk sac but switches expression to human β-globin at the same time as the endogenous mouse B-globin. [See Peterson, K.R. et al. ,Use of yeast artificial chromosomes (YACs) for studying control of gene expression: correct regulation of the genes of a human beta-globin locus YAC following transfer to mouse erythroleukemia cell lines. Proc Natl Acad Sci U S A 90, 11207- 11211 (1993).] The fact that these cells are responsive to a known γ-globin inducer (5-azacytidine) makes them particularly clinically relevant. [See Blau, C.A. et al., supra.]

[00101] To investigate whether PNA78 attached to an activation domain can reactivate the endogenous γ-globin gene in these mouse bone marrow cells, its expression was compared in cells treated with two different minimal AD attached to WT PNA78: VP2, and another minimal activation domain, ATF14 (CGSDALDDFDLDML) (SEQ ID NO:27), which is also highly acidic and has been shown to be a promising candidate as an artificial transcription factor in vitro and in vivo. [See Qiu, C, Olivier, E.N., Velho, M. & Bouhassira, E E, Globin switches in yolk sac-like primitive and fetal-like definitive red blood cells produced from human embryonic stem cells. Blood 111, 2400-2408 (2008). | In addition, to address whether the relative position of the AD and biotin may affect its transactivational ability or even the ability to visually monitor its presence, two PNA chimeric variants with ATF14 were generated: one (analogous to PNANP2 construct) with the biotin at the 5' end (amino terminus) of the molecule (B io- ATF/PN A78/TAT), and one with the biotin inserted between ATF and PNA78 ( ATF-B io/PN A78/TAT).

[00102] After treatment, cells were harvested and mRNA was extracted for reverse transcriptase (RT> quantitative polymerase chain reaction (qPCR) using two different sets of published primers that recognize sequences within the γ-globin gene. [See Blau, C.A. et al., supra and Qiu, C , et al., supra]. The results (Figure SA and B) show that although VP2/PNA78/TAT increased γ-globin levels about 2-fold, ATF-B io/PNA78/TAT is the most effective at increasing γ- globin expression, attaining a 7-fold increase in comparison to cells treated with PNA78/TAT alone. It is of interest that placement of the biotin is critical, as placing it at the extreme 5'end (Bio- ATF/PN A78/TAT) precluded its ability to activate γ-globin expression. It was also found that γ-globtn protein, which is not detectable in the untreated cells, is expressed in ATF-Bio/PNA78/TAT cells in the cytoplasm at levels approaching that seen in uninduced K562 cells (Figure 5C). From these studies it was concluded that it is possible to design a chimeric molecule containing peptide nucleic acids linked to cell entry/localization and activation amino acid sequences that can bind and directly activate transcription of a specific endogenous target gene in the cell.

[00103] The ability of the chimeric PNA molecule's ability to stimulate□- globin expression was further tested in human mobilized peripheral blood CD34+ cells. When incubated in a two-step serum-free culture system, these progenitors differentiated to phenotypically and morphologically mature erythroid cells (Figure 6(a), Figure 6(b)). Changes in cell surface expression were already apparent between days 2-4 of culture (Figure 7(a)), and the onset of adult B-globin RNA and protein expression begins by day 2 (Figure 7(b), Figure 7(c)). As a result, peripheral blood CD34+ cells cultured for two days were used for further testing. [00104] A mismatched PNA78 variant ( ATF- B io/MUT-PN A78/TAT) was used that was designed based on MUT-PNA as an additional negative control. Purified CD34+ cells were cultured, treated at the end of day 2 with the various PNA chimeras or with no PNA, and harvested 16 hours later for analysis. The results show that only cells treated with ATF-Bio/WT-PNA78/TAT increase v -globin expression (by almost 5-fold compared to untreated cells) (Figure 8(a)), and that this leads to Y -globin protein accumulation solely in those cells (Figure 8(b)). Visual analysis of > 100 cells per field from the same experiment indicates that 22% of cells treated with ATF-Bio/WT-PNA78/TAT were positive; all other samples were 0% positive (Figure 8(b)). Use of a novel flow cytometric analysis of streptavidin-FITC-positive (i.e., PNA-positive) cells not only demonstrated a high level of PNA incorporation into the cultured CD34+ cells, but also confirmed that cell death caused by PNA (measured by dual monitoring of F1TC+/7-AAD+) was always below 0.5% (Figure 8(c)). In Figure 8(c), the percent dead cells (7-AAD- positive) among all PNA-positive cells (>98% FITC+ as boxed in the PNA78/TAT example on the left) is indicated above the 7-AAD gate and was no higher than untreated cells. In Figure 8(c), FITC+/7-AAD+ (i.e., percent dead cell) results are also shown for CD34+ cells treated with ATF-Bio/MUT-PNA78/TAT or ATF-Bio/WT-PNA78/TAT. Expression profiles of phenotypic markers (CD34, CD36, CD235a, and CD71) were unaltered as well (Figures 9(a)-(c)), demonstrating that there was no erythroid differentiation abnormality caused by PNA treatment. As shown in Figures 9(a)- 9(c), all cells has similar forward scatter ("FSC") and side scatter ("SSC") profiles (Figure 9(a)) and expressed the same level of CD34, CD36, and CD235a markers (Figure 9(b)) even after exposure to the different PNA78 variants. In a separate experiment, H-PB cells from day 4 of culture were analyzed by flow cytometry 24 hours after treatment and show the same level of CD235a and CD71 expression irrespective of variant PNA78 exposure (Figure 9(c)).

Quantitative PCR Analysis

[00105] One microliter of DNA was used in 20 μΐ of quantitative PCR reaction using a Quantitect SYBR Green PCR kit from Qiagen (Valenica, CA) and the presence of γ-globin promoter sequences was quantified with an ABI PRISM 7900HT and SDS software. [See Lohmann, F. & Bieker, J J. Activation of Eklf expression during hematopoiesis by Gata2 and SmadS prior to erythroid commitment. Development 135, 2071-2082 (2008).] Each reaction was performed in triplicate. Primers used for the analysis were: Gamma-promo-fwd: AGCCTTGACAAGGCAAACTTGA (SEQ ID NO:31); Gamma-promo-rev: CCCTGGCCTCACTGGATACTCT (SEQ ID NO:32).

Quantitative Globin Expression Analysis

[00106] Total RNA from β-YAC MBCs were isolated with a RNeasy micro kit (Qiagen) following the manufacturer's instructions. RNA concentrations were quantified using a Nanodrop. Reverse transcription of RNA was performed with Promega's Reverse Transcription System and expression levels were quantified using a Quantitect SYBR Green PCR kit from Qiagen in conjunction with an ABI PRISM 7900HT and SDS software [see Lohmann, F. & Bieker, J.J. supra] Q-PCR results for globin expression were normalized by 2-[(Ct γ - Ct β) - (Ct γ [no PNA| - Ct β (noPNAJ)l [see Livak, K.J. & Schmittgen, T.D., Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25, 402-408 (2001)] divided by the P A incorporation efficiency (determined from the live cell image data). Each reaction was performed in triplicate.

[00107] The PCR primer sequences were derived from two studies [see Blau, C.A. et al. and Qiu, C, Olivier, E.N., Velho, M. & Bouhassira. E.E. Globin switches in yolk sac-like primitive and fetal- like definitive red blood cells produced from human embryonic stem cells. Blood 111, 2400-2408 (2008)]: for globin expression in MBCs: (primer set 1) human gamma-globin forward: ACCGTTTTGGCAATCCATTTC (SEQ ID NO: 33), reverse: TATTGCTTGCAGAATAAGCC (SEQ ID NO:34); (primer set 2) human gamma- globin forward: GACCGTTTTGGCAATCCATTTC (SEQ ID NO:35), reverse: TATTGCTTGCAGAATAAGCC (SEQ ID NO:36). Both studies used the same set of human β-globin primers: forward: AC AC A ACTGTGTTC ACT AGC AACCTC A (SEQ ID NO:37), reverse: GGTTGCCCATAACAGCATCAGGAGT (SEQ ID NO:38).

Immunofluorescent detection of γ-globin protein

[00108] At 48 hours post-PNA treatment, 100K MBC- βΥ AC cells were collected and spun onto frosted slides (Fisher) in a Shandon Cytospin 2 centrifuge at 500 rpm for 5 minutes. All steps were carried out at room temperature except when indicated. Cells were Fixed for 4 minutes in ice-cold acetone methanol (1 :1) on ice and rinsed twice with 2% BSA/PBS. Samples were then incubated with blocking solution (10 FCS, 0.05% NP40/PBS) for 30 minutes followed by a two-hour incubation with hemoglobin γ antibody (Santa Cruz Biotechnology, sc-21756) diluted 1:100 in blocking solution. Slides were washed 3x with 29%BSA. 0.05% NP40/PBS and then incubated with goat anti-mouse secondary antibody conjugated to Alexa 568 (Invitrogen) at a 1:50 dilution for one hour in a dark chamber. Slides were then washed 3x with 2% BSA/PBS with a final rinse in PBS and mounted with D API-containing Vectashield. DAPI and Alexa568 images were visualized on a Zeiss spinning disc confocal microscope (PerkinElmer).

PNA persistence test

[00109] 30000 562 cells were suspended in OPTI-MEM and attached to the bottom of a 8- well L-polylysine coated cover glass chamber slide (Nunc) and treated with ATF-Bio/PNA78/TAT in a final concentration of lOuM and incubation at 37°C. 2 hours after PNA addition, Streptavidin-FTTC was added to cells with a final concentration of 5ug/mL followed by another 2 hours of incubation at 37°C. Cells were inspected every day after 48 hours post- PNA treatment under ZEISS LSM-510 META confocal microscopy. DRAQ5 was used to stain cell nucleus 30 minutes prior to each confocal observation. Images of DRAQ5 and FITC were collected and compared with data collected during previous days.

Example 8

Use of Animal Model for Testing ecPNAs for Treatment of a β-globin Disorder

[00110] Efficacy of the γ-globin specific ecPNAs of the invention can be tested in a humanized mouse model of a β-globin disorder. The mice used in this model have been described. One suitable example is a humanized mouse model of Cooley's Anemia (CA) which has been generated by targeted gene replacement in embryonic stem (ES) cells. In these mice, a delayed switching human γ to βθ globin gene cassette (γβ0) is inserted directly into the murine β globin locus replacing both adult mouse β globin genes. The inserted human βθ globin allele has a mutation in the splice donor site that produces the same aberrant transcripts in mice as described in human cells. No functional human β globin polypeptide chains are produced. Heterozygous γβθ mice suffer from microcytic anemia. Homozygous γβθ mice switch from mouse embryonic globin chains to human fetal γ-globin during fetal life. When bred with human a globin knockin mice, homozygous CA mice survive solely upon human fetal hemoglobin at birth. [See. Yongliang Huo et al. et al.

[2009] J. Biol. Chem. 284: 4889-4896.]

[00111] Bone marrow cells can be isolated from these humanized mice (expressing the human globin genes) and cultured as described above. The γ- globin specific ecPNAs of the invention can be transfected into these cultured bone marrow cells. The transfected ceils (1 to 10 x 10 ⁶ ) can be then administered to the transgenic mice via tail vein injection in 200 ml PBS and the efficacy of the transfected cells for treating the β- globin disorder can be assessed by determining the red blood cell count.

*************************

[00112] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

[00113] While the compositions and methods of this invention have been described in terms of specific embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the scope of the invention as defined by the appended claims.

[00114] It is further to be understood that all values are approximate, and are provided for description.