WO2025096404A1 - Compositions and methods for hemoglobin production - Google Patents

Abstract

Methods and compositions for increasing hemoglobin levels (e.g., fetal hemoglobin) and/or gamma-globin in a cell or subject by the administration of at least one FTP A inhibitor and/or at least one TIPRL inhibitor to the cell or subject is disclosed. In another aspect methods of treating and/or preventing a hemoglobinopathy (e.g., sickle cell disease) or thalassemia in a subject by the administration at least one PTPA inhibitor and/or at least one TIPRL inhibitor to the subject in need thereof are further disclosed.

Description

COMPOSITIONS AND METHODS FOR HEMOGLOBIN PRODUCTION

By Gerd Blobel Elizabeth Traxler Junwei Shi

This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63/595,005, filed November 1, 2023. The foregoing application is incorporated by reference herein.

Incorporated herein by reference in its entirety is the Sequence Listing being concurrently submitted as a XML file named SeqList, created October 29, 2024, and having a size of 16,916 bytes.

This invention was made with government support under grant numbers CA258904 and HL119479 awarded by the National Institutes of Health. The government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to the field of hematology. More specifically, the invention provides compositions and methods for the production of various forms of hemoglobin, including adult and fetal type hemoglobin.

BACKGROUND OF THE INVENTION

Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated herein by reference as though set forth in full.

Sickle cell disease and thalassemia cause significant worldwide morbidity and mortality (Modell et al. (2008) Bull. World Health Org., 86:480-487; Modell et al. (2008) J. Cardiovasc. Magn. Reson., 10:42). However, effective drugs do not exist for these illnesses. One goal in the treatment of these diseases is to reactivate the expression of fetal hemoglobin (HbF). HbF reduces the propensity of sickle cell disease red blood cells for undergoing sickling. Indeed, high fetal globin levels are associated with improved outcomes for sickle cell anemia patients (Platt et al. (1994) N. Engl. J. Med., 330: 783-784). Elevating HbF also reduces the globin chain imbalance in thalassemia, thereby improving symptoms. There is an enormous unmet need to identify compounds that ameliorate the course of these diseases. SUMMARY OF THE INVENTION

In accordance with the present invention, compositions and methods are provided for increasing hemoglobin levels (e.g., fetal hemoglobin) and/or y-globin in a cell or subject. In certain embodiments, the method comprises administering at least one PTPA inhibitor and/or at least one TIPRL inhibitor to the cell or subject. In certain embodiments, the subject has a hemoglobinopathy or thalassemia. In certain embodiments, the subject has sickle cell disease. In certain embodiments, the cell is an erythroid cell or erythroblast. In certain embodiments, the PTPA and/or TIPRL inhibitor is a small molecule. The PTPA and/or TIPRL inhibitor may be, for example, a phosphatase binding domain inhibitor. The PTPA and/or TIPRL inhibitor may be an inhibitory nucleic acid molecule. In certain embodiments, the PTPA and/or TIPRL inhibitor is CRISPR based and targets the PTPA and/or TIPRL gene. In certain embodiments, the PTPA and/or TIPRL inhibitor is an siRNA or shRNA targeting a nucleic acid encoding PTPA and/or TIPRL. In certain embodiments, the PTPA and/or TIPRL inhibitor is a proteolysis-targeting chimera (PROTAC) targeting the PTPA and/or TIPRL protein for degradation. The method may further comprise delivering at least one fetal hemoglobin inducer to the cell or subject. The method may exploit additive or synergistic effects with other fetal hemoglobin inducing methods based on pharmacologic compounds or various forms of gene therapy.

In accordance with another aspect of the instant invention, methods of inhibiting, treating, and/or preventing a hemoglobinopathy (e.g., sickle cell disease) or thalassemia in a subject are provided. In certain embodiments, the method comprises administering at least one PTPA inhibitor and/or at least one TIPRL inhibitor to a subject in need thereof. The PTPA and/or TIPRL inhibitor may be in a composition with a pharmaceutically acceptable carrier. In certain embodiments, the subject has a P-chain hemoglobinopathy. In certain embodiments, the subject has sickle cell anemia. In certain embodiments, the PTPA and/or TIPRL inhibitor is a small molecule. The PTPA and/or TIPRL inhibitor may be, for example, a phosphatase binding domain inhibitor. The PTPA and/or TIPRL inhibitor may be an inhibitory nucleic acid molecule. In certain embodiments, the PTPA and/or TIPRL inhibitor is CRISPR based and targets the PTPA and/or TIPRL gene. In certain embodiments, the PTPA and/or TIPRL inhibitor is an siRNA or shRNA targeting a nucleic acid encoding PTPA and/or TIPRL. In certain embodiments, the PTPA and/or TIPRL inhibitor is a proteolysis-targeting chimera (PROTAC) targeting the PTPA and/or TIPRL protein for degradation. The method may further comprise delivering at least one other fetal hemoglobin inducer to the subject.

BRIEF DESCRIPTIONS OF THE DRAWINGS

Figures 1 A and IB provide a schematic of the CRISPR genetic screening strategy. Figure 1C provides a scatter plot of the screen results showing the median HbF enrichment and fitness scores for each gene, highlighting TIPRL and PTPA. Figure ID provides a validation scatter plot showing median HbF enrichment from two experiments. Human umbilical cord blood-derived erythroid progenitor cells (HUDEP-2) expressing Cas9 were transduced with one of the 3 sgRNAs targeting TIPRL or PTPA, a positive control sgRNA targeting +58 kb enhancer of BCL11 A, or 2 negative control of non -targeting sgRNA. Figure IE provides a graph of the percentages of cells positive for HbF expression. Figure IF provides a graph of y- globin mRNA levels. Figures 1G and 1H provide images of the TIPRL and PTPA protein levels, respectively, in cells with BCL11 A controls.

Figures 2A-2G show HbF induction with TIPRL or PTPA loss of function in primary human derived CD34+ erythroblasts. Figure 2A provides a schematic of the primary erythroblast culture system, with PTPA or TIPRL gene knockout with CRISPR-Cas9. Figures 2B and 2C provide graphs of the y-globin expression by mRNA levels and protein levels determined by high performance liquid chromatography (HPLC), respectively, compared to positive and negative controls for PTPA. Figure 2D provides an image of protein expression of PTPA and y-globin by Western blot. Figures 2E and 2F provide graphs of the y-globin expression by mRNA levels and protein levels determined by high performance liquid chromatography (HPLC), respectively, compared to positive and negative controls for TIPRL. Figure 2G provides an image of the protein expression of TIPRL and y-globin by Western blot.

Figure 3 A provides an image of protein expression of PTPA and BCL11A in HUDEP2 cells with the indicated gRNA by Western blot. Figure 3B provides a graph of relative BCL11 A expression by mRNA levels compared to positive and negative controls.

Figure 4A provides a schematic of a rescue experiment with overexpression of HA-tagged BCL11 A. Figure 4B provides a graph of HBG mRNA levels in HUDEP2 cells with the indicated PTPA gRNA with or without BCL11 A overexpression (OE). Figure 4C provides Western blot images of the expression of BCL11 A, HA, PTPA, actin, and y-globin in HUDEP2 cells with the indicated PTPA gRNA with or without BCL11 A overexpression.

Figure 5 A provides an example of an amino acid sequence of TIPRL (SEQ ID NO: 1). Figure 5B provides an example of a nucleotide sequence encoding TIPRL (SEQ ID NO: 2).

Figure 6 A provides an example of an amino acid sequence of PTPA (SEQ ID NO: 3). Figure 6B provides an example of a nucleotide sequence encoding PTPA (SEQ ID NO: 4).

DETAILED DESCRIPTION OF THE INVENTION

A major goal in the treatment of a hemoglobinopathy, such as sickle cell disease and thalassemia, is the reactivation of fetal type globin expression in cells of the adult red blood cell lineage. In two unbiased genetic screens, TIPRL and PTPA were identified as repressors of fetal globin production. TIPRL (TOR signaling pathway regulator, see, e.g., PubMed GenelD: 261726) and PTPA (protein phosphatase 2 phosphatase activator; PPP2R4, see, e.g., PubMed GenelD: 5524) are regulators of the PP2A phosphatase complex. It is shown herein that the depletion of TIPRL and/or PTPA raises fetal hemoglobin levels. The genetic screens described herein for HbF inducers in human cells indicate that the loss of PTPA and/or TIPRL function increases HbF levels. Additional experiments show that the loss of PTPA and/or TIPRL increases fetal hemoglobin production in human erythroid cells, including primary cells. Without being bound by theory, the mechanism by which this occurs likely involves the transcriptional regulation of HbF regulators or coregulators which modulate the transcriptional and/or posttranscriptional regulation of fetal hemoglobin production. This role is exploited herein to treat hemoglobinopathies such as sickle cell anemia and thalassemia.

In certain embodiments, the TIPRL of the instant invention is human TIPRL. Amino acid and nucleotide sequences of TIPRL are provided, for example, in PubMed Gene ID: 261726 and GenBank Nos. NM_001031800.3; NP_001026970.1; NM_152902.5; and NP_690866.1. Figure 5 A provides an example of an amino acid sequence of TIPRL. Figure 5B provides an example of a nucleotide sequence encoding TIPRL, particularly nucleotides 117-935 of the provided sequence encode TIPRL. In certain embodiments, the PTPA of the instant invention is human PTPA. Amino acid and nucleotide sequences of PTPA are provided, for example, in PubMed GenelD: 5524 and GenBank Nos. NM_001193397.2; NP_001180326.1; NM_001271832.2; NP_001258761.1; NM_021131.5; NP_066954.2; NM_178000.3; NP_821067.1; NM_178001.3; NP_821068.1; NM_178003.3 ; and NP_821070.1. In certain embodiments, the human PTPA is isoform a (isoform beta) (358 amino acids) or isoform b (isoform alpha) (323 amino acids, which lacks amino acids 71-105 of isoform a (e.g., SEQ ID NO: 3)). Figure 6A provides an example of an amino acid sequence of PTPA. Figure 6B provides an example of a nucleotide sequence encoding PTPA, particularly nucleotides 187-1263 of the provided sequence encode PTPA.

Gene editing has emerged as a promising gene therapy for genetic diseases including P-hemoglobinopathies (Canver, et al. (2016) Blood 127(21):2536-2545). In addition, PROTAC has been recently developed to be an effective technology for targeted protein degradation (Li, et al. (2020) J. Hematol. Oncol., 13(1): 50). For example, a PROTAC molecule may comprise a ligand or targeting moiety (e.g., anti- PTPA antibody or antigen binding fragment thereof or anti-TIPRL antibody or antigen binding fragment thereof) and a covalently linked ligand of an E3 ubiquitin ligase (E3), which will recruit E3 for ubiquitination and proteasome-mediated degradation of PTPA or TIPRL. Hence, PTPA or TIPRL targeting by gene editing or PROTAC in human CD34+ hematopoietic stem and progenitor cells (HSPCs) will effectively increase and/or reactivate HbF levels in these cells, which will benefit patients with a hemoglobinopathy such as sickle cell anemia or thalassemia.

In accordance with the instant invention, compositions and methods are provided for increasing hemoglobin production in a cell or subject. Compositions and methods are also provided for increasing y-globin production in a cell or subject. In certain embodiments, the method increases fetal hemoglobin and/or embryonic globin expression. In certain embodiments, the method increases fetal hemoglobin. In certain embodiments, the method increases y-globin. In certain embodiments, the method increases protein levels (e.g., fetal hemoglobin and/or y-globin). In accordance with the instant invention, PTPA inhibitors and compositions comprising the same are provided. In accordance with the instant invention, TIPRL inhibitors and compositions comprising the same are provided. In certain embodiments, the compositions and/or methods comprise at least one PTPA inhibitor and/or at least one TTPRL inhibitor.

The methods of the instant invention comprise administering at least one PTPA inhibitor and/or at least one TIPRL inhibitor to a cell, particularly an erythroid precursor cell (e.g., erythroblast or hematopoietic stem cell (HSC)) or erythroid cell, or subject. While the instant invention describes PTPA inhibitors throughout, the instant invention also encompasses inhibitors of the PP2A complex or the PP2A/PTPA complex (e.g., in place of the PTPA inhibitor or in combination with the PTPA inhibitor). When more than one inhibitor is administered, the PTPA and/or TIPRL inhibitors can be delivered to the cell or subject sequentially or consecutively (e.g., in different compositions) and/or at the same time (e.g., in the same composition). In a particular embodiment, the subject has a hemoglobinopathy (such as sickle cell disease) or thalassemia. In a particular embodiment, the subject has sickle cell anemia. In a particular embodiment, the subject has thalassemia, particularly P-thalassemia, and more particularly major P-thalassemia.

The PTPA and/or TIPRL inhibitor may be administered in a composition further comprising at least one pharmaceutically acceptable carrier. In certain embodiments, the composition comprises at least one PTPA inhibitor and at least one TIPRL inhibitor. In certain embodiments, the composition comprises at least one PTPA inhibitor. In certain embodiments, the composition comprises at least one TIPRL inhibitor.

In certain embodiments, the method further comprises any means by which to induce fetal hemoglobin, such as administering at least one other fetal hemoglobin inducer. In certain embodiments, the fetal hemoglobulin inducer yields a synergistic effect with the PTPA and/or TIPRL inhibitor. Fetal hemoglobin inducers include, without limitation, a lysine-specific demethylase 1 (LSD1) inhibitor (e.g., RN-1 and tranylcypromine (TCP) (Cui et al. (2015) Blood 126(3):386-96; Shi et al. (2013) Nat. Med., 19(3): 291-294; Sun et al. (2016) Reprod. Biol. Endocrinol., 14: 17)), pomalidomide (Moutouh-de Parseval et al. (2008) J. Clin. Invest., 118(l):248-258; Dulmovits et al., Blood (2016) 127(11): 1481-92), hydroxyurea (Charache et al., NEJM (1995) 332(20): 1317-22), 5-azacytidine (Humphries et al., J. Clin. Invest. (1985) 75(2) : 547-57), sodium butyrate, activators or inducers of the FOXO3 pathway (e.g., metformin, phenformin, or resveratrol; Zhang et al., Blood (2018) 132(3): 321- 333), L-glutamine, histone methyltransferase (HMT) inhibitors (e.g., a histone lysine methyltransferase inhibitor, euchromatic histone-lysine N-methyltransferase 2 (EHMT2; G9a) inhibitor, euchromatic histone-lysine N-methyltransferase 1 (EHMT1; G9a-like protein (GLP)) inhibitor, UNC0638 (2-cyclohexyl-N-(l-isopropylpiperidin- 4-yl)-6-methoxy-7-(3-(pyrrolidin-l-yl)propoxy) quinazolin-4-amine) (Renneville et al., Blood (2015) 126(16): 1930-9; Krivega et al., Blood (2015) 126(5):665-72), chaetocin, BIX-01294, UNC 0224, UNC 0642, UNC 0631, UNC 0646, A-366 (Sweis et al. (2014) ACS Med. Chem. Lett., 5(2):205-209), etc.), histone deacetylase (HD AC) inhibitors (e.g., entinostat; Bradner et al., PNAS (2010) 107(28): 12617-22), and eIF2aKl inhibitors (see, e.g., PCT/US18/15918). In certain embodiments, the fetal hemoglobin inducer is selected from the group consisting of lysine-specific demethylase 1 (LSD1) inhibitor, pomalidomide, hydroxyurea, 5-azacytidine, sodium butyrate, activators of the Foxo3 pathway (e.g., metformin, phenformin, and resveratrol), L-Glutamine, or histone deacetylase (HD AC) inhibitor. In certain embodiments, the fetal hemoglobin inducer is pomalidomide or related Imid (immunomodulatory imide drug) compounds, hydroxyurea, or a EHMT1/2 inhibitor such as UNC0638 or related compounds. In certain embodiments, the fetal hemoglobin inducer is pomalidomide or hydroxyurea, particularly pomalidomide or similar Imid. The PTPA and/or TIPRL inhibitor and the fetal hemoglobin inducer can be delivered to the cell or subject sequentially or consecutively (e.g., in different compositions) and/or at the same time (e.g., in the same composition).

In accordance with another aspect of the instant invention, compositions and methods for inhibiting (e.g., reducing or slowing), treating, and/or preventing a hemoglobinopathy or thalassemia in a subject are provided. In certain embodiments, the methods comprise administering to a subject in need thereof a therapeutically effective amount of at least one PTPA inhibitor and/or at least one TIPRL inhibitor. When more than one inhibitor is administered, the PTPA and/or TIPRL inhibitors can be administered sequentially or consecutively (e.g., in different compositions) and/or at the same time (e.g., in the same composition). The PTPA and/or TIPRL inhibitor may be administered in a composition further comprising at least one pharmaceutically acceptable carrier. In certain embodiments, the hemoglobinopathy is thalassemia or sickle cell anemia. In certain embodiments, the subject has sickle cell anemia. In certain embodiments, the subject has thalassemia, particularly P- thalassemia, and more particularly major P-thalassemia. The methods of the instant invention may comprise administering at least two different PTPA and/or TIPRL inhibitors (e.g., two different mechanisms of action). In certain embodiment, the method further comprises administering at least one other fetal hemoglobin inducer to the subject as described hereinabove. In certain embodiments, the fetal hemoglobin inducer is pomalidomide or related Imid compound, hydroxyurea, or a EHMT1/2 inhibitor such as UNC0638 or related compound. In certain embodiments, the fetal hemoglobin inducer is pomalidomide or hydroxyurea, particularly pomalidomide. The PTPA and/or TIPRL inhibitor and the fetal hemoglobin inducer can be administered to the subject sequentially or consecutively (e.g., in different compositions) and/or at the same time (e.g., in the same composition).

In certain embodiments, PTPA inhibitors are complexes (e.g., molecular complexes) or compounds which edit the PTPA gene, diminish PTPA expression, and/or target the protein for degradation. In certain embodiments, PTPA inhibitors are compounds which reduce PTPA activity, inhibit or reduce PTPA-substrate/partner interaction, and/or the expression of PTPA. The PTPA inhibitor may inhibit any or all isoforms of PTPA.

In certain embodiments, PTPA inhibitors can edit the PTPA gene, diminish PTPA expression, and/or target PTPA protein for degradation. In certain embodiments, the PTPA inhibitor is specific to PTPA. Examples of PTPA inhibitors include, without limitation, proteins, polypeptides, peptides, antibodies, small molecules, and nucleic acid molecules. In certain embodiments, the PTPA inhibitor is a small molecule. In certain embodiments, the PTPA inhibitor is a PROTAC. The PTPA inhibitor may be a synthetic or non-natural compound. In certain embodiments, the PTPA inhibitor is a phosphatase binding domain inhibitor (e.g., a small molecule inhibitor which binds the phosphatase binding domain). Examples of PTPA inhibitors are provided in U.S. Patent Application Publication Nos. 20080153773 and 20040023906 (each incorporated by reference herein). In certain embodiments, the PTPA inhibitor is an inhibitory nucleic acid molecule, such as an antisense, miRNA, siRNA, or shRNA molecule (or a nucleic acid molecule encoding the inhibitory nucleic acid molecule). In certain embodiments, the inhibitory nucleic acid molecule targets a sequence (e.g., is the complement of) or comprises a sequence (inclusive of RNA version of DNA molecules) as set forth in the Example provided herein (e.g., SEQ ID NO: 9, 10, or 11). In certain embodiments, the inhibitory nucleic acid molecule targets a sequence or comprises a sequence (e.g., RNA version) which has at least 80%, 85%, 90%, 95%, 97%, 99%, or 100% homology or identity to a sequence set forth in the Example (e.g., SEQ ID NO: 9, 10, or 11). The sequences may be extended or shortened by 1, 2, 3, 4, or 5 nucleotides at the end of the sequence (e.g., the extended sequence may correspond to the genomic sequence). In certain embodiments, the PTPA inhibitor is a CRISPR based targeting of the PTPA gene (e.g., with a guide RNA targeting the PTPA gene). In certain embodiments, the PTPA inhibitor comprises an sgRNA comprising or targeting SEQ ID NO: 9, 10, or 11 or the indicated nucleotides provided in Table 1.

In certain embodiments, TIPRL inhibitors are complexes (e.g., molecular complexes) or compounds which edit the TIPRL gene, diminish TIPRL expression, and/or target the protein for degradation. In certain embodiments, TIPRL inhibitors are compounds which reduce TIPRL activity, inhibit or reduce TIPRL- substrate/partner interaction, and/or the expression of TIPRL. The TIPRL inhibitor may inhibit any or all isoforms of TIPRL.

In certain embodiments, TIPRL inhibitors can edit the TIPRL gene, diminish TIPRL expression, and/or target TIPRL protein for degradation. In certain embodiments, the TIPRL inhibitor is specific to TIPRL. Examples of TIPRL inhibitors include, without limitation, proteins, polypeptides, peptides, antibodies, small molecules, and nucleic acid molecules. In certain embodiments, the TIPRL inhibitor is a small molecule. In certain embodiments, the TIPRL inhibitor is a PROTAC. The TIPRL inhibitor may be a synthetic or non-natural compound. In certain embodiments, the TIPRL inhibitor is a phosphatase binding domain inhibitor (e.g., a small molecule inhibitor which binds the phosphatase binding domain). In certain embodiments, the TIPRL inhibitor is an inhibitory nucleic acid molecule, such as an antisense, siRNA, or shRNA molecule (or a nucleic acid molecule encoding the inhibitory nucleic acid molecule). U.S. Patent Application Publication No. 20120315284 (incorporated herein by reference) provides examples of TIPRL inhibitory nucleic acid molecules (e.g., SEQ ID NO: 2). In certain embodiments, the inhibitory nucleic acid molecule targets a sequence (e.g., is the complement of) or comprises a sequence (inclusive of RNA version of DNA molecules) as set forth in the Example provided herein (e.g., SEQ ID NO: 6, 7, or 8). In certain embodiments, the inhibitory nucleic acid molecule targets a sequence or comprises a sequence (e.g., RNA version) which has at least 80%, 85%, 90%, 95%, 97%, 99%, or 100% homology or identity to a sequence set forth in the Example (e.g., SEQ ID NO: 6, 7, or 8). The sequences may be extended or shortened by 1, 2, 3, 4, or 5 nucleotides at the end of the sequence (e.g., the extended sequence may correspond to the genomic sequence). In certain embodiments, the TIPRL inhibitor is a CRISPR based targeting of the TIPRL gene (e.g., with a guide RNA targeting the TIPRL gene). In certain embodiments, the TIPRL inhibitor comprises an sgRNA comprising or targeting SEQ ID NO: 6, 7, or 8 or the indicated nucleotides provided in Table 1.

Clustered, regularly interspaced, short palindromic repeat (CRISPR)/Cas9 (e.g., from Streptococcus pyogenes) technology and gene editing are well known in the art (see, e.g., Sander et al. (2014) Nature Biotech., 32:347-355; Jinek et al. (2012) Science, 337:816-821; Cong et al. (2013) Science 339:819-823; Ran et al. (2013) Nature Protocols 8:2281-2308; Mali et al. (2013) Science 339:823-826; addgene.org/crispr/guide/). The RNA-guided CRISPR/Cas9 system involves expressing Cas9 along with a guide RNA molecule (gRNA). When coexpressed, gRNAs bind and recruit Cas9 to a specific genomic target sequence where it mediates a double strand DNA (dsDNA) break. The binding specificity of the CRISPR/Cas9 complex depends on two different elements. First, the binding complementarity between the targeted genomic DNA (genDNA) sequence and the complementary recognition sequence of the gRNA (e.g., -18-22 nucleotides, particularly about 20 nucleotides). Second, the presence of a protospacer-adjacent motif (PAM) juxtaposed to the genDNA/gRNA complementary region (Jinek et al. (2012) Science 337:816- 821; Hsu et al. (2013) Nat. Biotech., 31 :827-832; Sternberg et al. (2014) Nature 507:62-67). The PAM motif for S. Pyogenes Cas9 has been fully characterized, and is NGG or NAG (Jinek et al. (2012) Science 337:816-821; Hsu et al. (2013) Nat. Biotech., 31 :827-832). Other PAMs of other Cas9 are also known (see, e.g., addgene. org/crispr/guide/#pam-table). Guidelines and computer-assisted methods for generating gRNAs are available (see, e.g, CRISPR Design Tool (crispr.mit.edu/); Hsu et al. (2013) Nat. Biotechnol. 31 :827-832; addgene.org/CRISPR; and CRISPR gRNA Design tool - DNA2.0 (dna20.com/eCommerce/startCas9)). Typically, the PAM sequence is 3’ of the DNA target sequence in the genomic sequence.

In certain embodiments, the method comprises administering at least one Cas9 (e.g., the protein and/or a nucleic acid molecule encoding Cas9) and at least one gRNA (e.g., a nucleic acid molecule encoding the gRNA) to the cell or subject. In certain embodiments, the Cas9 is S. pyogenes Cas9. In certain embodiments, the gRNA is a single guide RNA (sgRNA). In certain embodiments, the targeted PAM is in the 5’UTR, promoter, or first intron. In certain embodiments, the targeted PAM is in an exon. When present, a second gRNA may be provided which targets anywhere from the 5’UTR to the 3’UTR of the gene, particularly within the first intron. The nucleic acids of the instant invention may be administered consecutively (before or after) and/or at the same time (concurrently). The nucleic acid molecules may be administered in the same composition or in separate compositions. In certain embodiments, the nucleic acid molecules are delivered in a single vector (e.g., a viral vector).

In certain embodiments, the nucleic acid molecules of the instant invention are delivered (e.g., via infection, transfection, electroporation, etc.) and expressed in cells via a vector (e.g., a plasmid), particularly a viral vector. The expression vectors of the instant invention may employ a strong promoter, a constitutive promoter, and/or a regulated promoter. In certain embodiments, the nucleic acid molecules are expressed transiently. Examples of promoters are well known in the art and include, but are not limited to, RNA polymerase II promoters, the T7 RNA polymerase promoter, and RNA polymerase III promoters (e.g., U6 and Hl; see, e.g., Myslinski et al. (2001) Nucl. Acids Res., 29:2502-09). Examples of expression vectors for expressing the molecules of the invention include, without limitation, plasmids and viral vectors (e.g., adeno-associated viruses (AAVs), adenoviruses, retroviruses, and lentiviruses).

In certain embodiment, the guide RNA of the instant invention may comprise separate nucleic acid molecules. For example, one RNA may specifically hybridize to a target sequence (crRNA) and another RNA (trans-activating crRNA (tracrRNA)) specifically hybridizes with the crRNA. In a particular embodiment, the guide RNA is a single molecule (sgRNA) which comprises a sequence which specifically hybridizes with a target sequence (crRNA; complementary sequence) and a sequence recognized by Cas9 (e.g., a tracrRNA sequence; scaffold sequence). Examples of gRNA scaffold sequences are well known in the art (e.g., 5’-GUUUUAGAGC UAGAAAUAGC AAGUUAAAAU AAGGCUAGUC CGUUAUCAAC UUGAAAAAGU GGCACCGAGU CGGUGCUUUU; SEQ ID NO: 5). As used herein, the term “specifically hybridizes” or “targets” does not mean that the nucleic acid molecule needs to be 100% complementary to the target sequence, although it may be preferred. Rather, the sequence may be at least 80%, 85%, 90%, 95%, 97%, 99%, or 100% complementary to the target sequences (e.g., the complementary between the gRNA and the genomic DNA). The greater the complementarity reduces the likelihood of undesired cleavage events at other sites of the genome. In certain embodiments, the region of complementarity (e.g., between a guide RNA and a target sequence) is at least about 10, at least about 12, at least about 15, at least about 17, at least about 20, at least about 25, at least about 30, at least about 35, or more nucleotides. In certain embodiments, the region of complementarity (e.g., between a guide RNA and a target sequence) is about 15 to about 25 nucleotides, about 15 to about 23 nucleotides, about 16 to about 23 nucleotides, about 17 to about 21 nucleotides, about 18 to about 22 nucleotides, or about 20 nucleotides. In certain embodiments, the guide RNA targets a sequence or comprises a sequence (inclusive of RNA version of DNA molecules) as set forth in the Example provided herein. In a particular embodiment, the guide RNA targets a sequence or comprises a sequence (e.g., RNA version) which has at least 80%, 85%, 90%, 95%, 97%, 99%, or 100% homology or identity to a sequence set forth in the Example (e.g., SEQ ID NO: 6, 7, 8, 9, 10, or 11). The sequences may be extended or shortened by 1, 2, 3, 4, or 5 nucleotides at the end of the sequence opposite from the PAM (e.g., at the 5’ end). When the sequence is extended the added nucleotides should correspond to the genomic sequence.

The above methods also encompass ex vivo methods. In certain embodiments, the methods of the instant invention use autologous cells. For example, the methods of the instant invention can comprise isolating hematopoietic cells (e.g., erythroid precursor cells or erythroblasts) or erythroid cells from a subject, delivering at least one TIPRL and/or PTPA inhibitor to the cells, and administering the treated cells to the subject. The isolated cells (e.g., erythroid precursor cells or erythroblasts or erythroid cells) may also be treated with other reagents in vitro, such as at least one fetal hemoglobin inducer, prior to administration to the subject.

The methods of the instant invention may further comprise monitoring the disease or disorder in the subject after administration of the composition(s) of the instant invention to monitor the efficacy of the method. For example, the subject may be monitored for characteristics of low hemoglobin or a hemoglobinopathy or thalassemia. For example, the subject may be monitored for cell sickling.

When an inhibitory nucleic acid molecule is delivered to a cell or subject, the inhibitory nucleic acid molecule may be administered directly or an expression vector may be used. In certain embodiments, the inhibitory nucleic acid molecules are delivered (e.g., via infection, transfection, electroporation, etc.) and expressed in cells via a vector (e.g., a plasmid), particularly a viral vector. The expression vectors of the instant invention may employ a strong promoter, a constitutive promoter, and/or a regulated promoter. In certain embodiments, the inhibitory nucleic acid molecules are expressed transiently. In certain embodiments, the promoter is cell-type specific (e.g., erythroid cells). Examples of promoters are well known in the art and include, but are not limited to, RNA polymerase II promoters, the T7 RNA polymerase promoter, and RNA polymerase III promoters (e.g., U6 and Hl; see, e.g., Myslinski et al. (2001) Nucl. Acids Res., 29:2502-09). Examples of expression vectors for expressing the molecules of the invention include, without limitation, plasmids and viral vectors (e.g., adeno-associated viruses (AAVs), adenoviruses, retroviruses, and lentiviruses).

As explained hereinabove, the compositions of the instant invention are useful for increasing hemoglobin production and for treating hemoglobinopathies and thalassemias. A therapeutically effective amount of the composition may be administered to a subject in need thereof. The dosages, methods, and times of administration are readily determinable by persons skilled in the art, given the teachings provided herein.

The components as described herein will generally be administered to a patient as a pharmaceutical preparation. The term “patient” or “subject” as used herein refers to human or animal subjects. The components of the instant invention may be employed therapeutically, under the guidance of a physician for the treatment of the indicated disease or disorder.

The pharmaceutical preparation comprising the components of the invention may be conveniently formulated for administration with an acceptable medium (e.g., pharmaceutically acceptable carrier) such as water, buffered saline, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like), dimethyl sulfoxide (DMSO), oils, detergents, suspending agents or suitable mixtures thereof. The concentration of the agents in the chosen medium may be varied and the medium may be chosen based on the desired route of administration of the pharmaceutical preparation. Except insofar as any conventional media or agent is incompatible with the agents to be administered, its use in the pharmaceutical preparation is contemplated.

The compositions of the present invention can be administered by any suitable route, for example, by injection (e.g., for local (direct) or systemic administration), oral, pulmonary, topical, nasal or other modes of administration. The composition may be administered by any suitable means, including parenteral, intramuscular, intravenous, intraarterial, intraperitoneal, subcutaneous, topical, inhalatory, transdermal, intrapulmonary, intraarterial, intrarectal, intramuscular, and intranasal administration. In a particular embodiment, the composition is administered directly to the blood stream (e.g., intravenously). In general, the pharmaceutically acceptable carrier of the composition is selected from the group of diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers. The compositions can include diluents of various buffer content (e.g., Tris HC1, acetate, phosphate), pH and ionic strength; and additives such as detergents and solubilizing agents (e.g., polysorbate 80), anti oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol). The compositions can also be incorporated into particulate preparations of polymeric compounds such as polyesters, polyamino acids, hydrogels, polylactide/glycolide copolymers, ethylenevinylacetate copolymers, polylactic acid, polyglycolic acid, etc., or into liposomes. Such compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of components of a pharmaceutical composition of the present invention. See, e.g., Remington: The Science and Practice of Pharmacy, 21st edition, Philadelphia, PA. Lippincott Williams & Wilkins. The pharmaceutical composition of the present invention can be prepared, for example, in liquid form, or can be in dried powder form (e.g., lyophilized for later reconstitution).

As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media and the like which may be appropriate for the desired route of administration of the pharmaceutical preparation, as exemplified in the preceding paragraph. The use of such media for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the molecules to be administered, its use in the pharmaceutical preparation is contemplated.

Pharmaceutical compositions containing a compound of the present invention as the active ingredient in intimate admixture with a pharmaceutical carrier can be prepared according to conventional pharmaceutical compounding techniques. The carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., intravenous. Injectable suspensions may be prepared, in which case appropriate liquid carriers, suspending agents and the like may be employed. Pharmaceutical preparations for injection are known in the art. If injection is selected as a method for administering the therapy, steps should be taken to ensure that sufficient amounts of the molecules reach their target cells to exert a biological effect.

A pharmaceutical preparation of the invention may be formulated in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form, as used herein, refers to a physically discrete unit of the pharmaceutical preparation appropriate for the patient undergoing treatment. Each dosage should contain a quantity of active ingredient calculated to produce the desired effect in association with the selected pharmaceutical carrier. Procedures for determining the appropriate dosage unit are well known to those skilled in the art. Dosage units may be proportionately increased or decreased based on the weight of the patient. Appropriate concentrations for alleviation of a particular pathological condition may be determined by dosage concentration curve calculations, as known in the art. The appropriate dosage unit for the administration of the molecules of the instant invention may be determined by evaluating the toxicity of the molecules in animal models. Various concentrations of pharmaceutical preparations may be administered to mice, and the minimal and maximal dosages may be determined based on the results and side effects as a result of the treatment. Appropriate dosage unit may also be determined by assessing the efficacy of the treatment in combination with other standard therapies.

The pharmaceutical preparation comprising the molecules of the instant invention may be administered at appropriate intervals, for example, at least twice a day or more until the pathological symptoms are reduced or alleviated, after which the dosage may be reduced to a maintenance level. The appropriate interval in a particular case would normally depend on the condition of the patient.

Definitions

The singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

The terms “isolated” is not meant to exclude artificial or synthetic mixtures with other compounds or materials, or the presence of impurities that do not interfere with the fundamental activity, and that may be present, for example, due to incomplete purification, or the addition of stabilizers.

“Pharmaceutically acceptable” indicates approval 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 animals, and more particularly in humans.

A “carrier” refers to, for example, a diluent, adjuvant, preservative (e.g., Thimersol, benzyl alcohol), anti-oxidant (e.g., ascorbic acid, sodium metabisulfite), solubilizer (e.g., polysorbate 80), emulsifier, buffer (e.g., Tris HC1, acetate, phosphate), antimicrobial, bulking substance (e.g., lactose, mannitol), excipient, auxiliary agent or vehicle with which an active agent of the present invention is administered. Pharmaceutically acceptable carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin. Water or aqueous saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in Remington: The Science and Practice of Pharmacy, (Lippincott, Williams and Wilkins); Liberman, et al., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y.; and Rowe, et al., Eds., Handbook of Pharmaceutical Excipients, Pharmaceutical Pr.

The term “treat” as used herein refers to any type of treatment that imparts a benefit to a patient suffering from an injury, including improvement in the condition of the patient (e.g., in one or more symptoms), delay in the progression of the condition, etc.

As used herein, the term “prevent” refers to the prophylactic treatment of a subject who is at risk of developing a condition and/or sustaining an injury, resulting in a decrease in the probability that the subject will develop conditions associated with the hemoglobinopathy or thalassemia.

A “therapeutically effective amount" of a compound or a pharmaceutical composition refers to an amount effective to prevent, inhibit, or treat a particular injury and/or the symptoms thereof. For example, “therapeutically effective amount” may refer to an amount sufficient to modulate the pathology associated with a hemoglobinopathy or thalassemia.

As used herein, the term “subject” refers to an animal, particularly a mammal, particularly a human.

A “vector” is a genetic element, such as a plasmid, cosmid, bacmid, phage, transposon, or virus, to which another genetic sequence or element (either DNA or RNA) may be attached so as to bring about the replication and/ or expression of the attached sequence or element. A vector may be either RNA or DNA and may be single or double stranded. A vector may comprise expression operons or elements such as, without limitation, transcriptional and translational control sequences, such as promoters, enhancers, translational start signals, polyadenylation signals, terminators, and the like, and which facilitate the expression of a polynucleotide or a polypeptide coding sequence in a host cell or organism.

As used herein, the term “small molecule” refers to a substance or compound that has a relatively low molecular weight (e.g., less than 4,000, less than 2,000, particularly less than 1 kDa or 800 Da). Typically, small molecules are organic, but are not proteins, polypeptides, amino acids, or nucleic acids.

An “antibody” or “antibody molecule” is any immunoglobulin, including antibodies and fragments thereof, that binds to a specific antigen. As used herein, antibody or antibody molecule contemplates intact immunoglobulin molecules, immunologically active portions/fragment (e.g., antigen binding portion/fragment) of an immunoglobulin molecule, and fusions of immunologically active portions of an immunoglobulin molecule. Antibody fragments include, without limitation, immunoglobulin fragments including, without limitation: single domain (Dab; e.g., single variable light or heavy chain domain), Fab, Fab', F(ab')2, and F(v); and fusions (e.g., via a linker) of these immunoglobulin fragments including, without limitation: scFv, SCFV2, SCFV-FC, minibody, diabody, triabody, and tetrabody.

As used herein, the term “immunologically specific” refers to proteins/polypeptides, particularly antibodies, that bind to one or more epitopes of a protein or compound of interest, but which do not substantially recognize and bind other molecules in a sample containing a mixed population of antigenic biological molecules.

The phrase “small, interfering RNA (siRNA)” refers to a short (typically less than 30 nucleotides long, particularly 12-30 or 20-25 nucleotides in length) double stranded RNA molecule. Typically, the siRNA modulates the expression of a gene to which the siRNA is targeted. Methods of identifying and synthesizing siRNA molecules are known in the art (see, e.g., Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Inc). Short hairpin RNA molecules (shRNA) typically consist of short complementary sequences (e.g., an siRNA) separated by a small loop sequence (e.g., 6-15 nucleotides, particularly 7-10 nucleotides) wherein one of the sequences is complimentary to the gene target. shRNA molecules are typically processed into an siRNA within the cell by endonucleases. Exemplary modifications to siRNA molecules are provided in U.S. Application Publication No. 20050032733. For example, siRNA and shRNA molecules may be modified with nuclease resistant modifications (e.g., phosphorothioates, locked nucleic acids (LNA), 2'-O-methyl modifications, or morpholino linkages). Expression vectors for the expression of siRNA or shRNA molecules may employ a strong promoter which may be constitutive or regulated. Such promoters are well known in the art and include, but are not limited to, RNA polymerase II promoters, the T7 RNA polymerase promoter, and the RNA polymerase III promoters U6 and Hl (see, e.g., Myslinski et al. (2001) Nucl. Acids Res., 29:2502-09).

“Antisense nucleic acid molecules” or “antisense oligonucleotides” include nucleic acid molecules (e.g., single stranded molecules) which are targeted (complementary) to a chosen sequence (e.g., to translation initiation sites and/or splice sites) to inhibit the expression of a protein of interest. Such antisense molecules are typically between about 15 and about 50 nucleotides in length, more particularly between about 15 and about 30 nucleotides, and often span the translational start site of mRNA molecules. Antisense constructs may also be generated which contain the entire sequence of the target nucleic acid molecule in reverse orientation. Antisense oligonucleotides targeted to any known nucleotide sequence can be prepared by oligonucleotide synthesis according to standard methods. Antisense oligonucleotides may be modified as described above to comprise nuclease resistant modifications.

The following example is provided to illustrate various embodiments of the present invention. It is not intended to limit the invention in any way.

EXAMPLE

Reversal of the postnatal hemoglobin switch in red blood cells is a promising approach to therapies for sickle cell disease and P-thalassemia, as reactivating fetal hemoglobin (HbF, a2y2) in adult red blood cells ameliorates disease complications. Studies over several decades have illuminated the paradigm of this fetal-to-adult hemoglobin transition, including central roles of transcriptional repressors BCL11 A, LRF, and NFIA/X, which recruit repressive chromatin modifiers to the y-globin locus. To elucidate undescribed regulatory mechanisms of these molecules and reveal new targets for therapies, the first genome-wide genetic screen to pinpoint specific pathways or protein complexes that regulate HbF expression in adult erythroid cells was performed.

A clustered, regularly interspaced, short palindromic repeat (CRISPR) screening strategy was employed to identify regulators of fetal globin expression (Shi, et al. (2015) Nat. Biotechnol., 33(6):661-667). CRISPR/Cas9 technology is well known in the art (see, e.g., Jinek, et al. (2012) Science 337(6096):816-821; Cong, et al. (2013) Science 339(6121):819-823; Mali, et al. (2013) Science 339(6121):823- 826; Ran, et al. (2013) Nat. Protoc., 8(11):2281-2308; Sander, et al. (2014) Nat. Biotechnol., 32(4):347-355). Cas9 possesses two nuclease domains - a RuvC-like nuclease domain and a HNH-like nuclease domain - and is responsible for the destruction of the target DNA (Sapranauskas, et al. (2011) Nucleic Acids Res., 39(21): 9275-9282; Jinek, et al. (2012) Science 337(6096):816-821). The two nucleases generate double-stranded breaks. The double-stranded endonuclease activity of Cas9 requires a target sequence (e.g., ~20 nucleotides) and a short conserved sequence (~2-5 nucleotides; e.g., 3 nucleotides) known as protospacer- associated motif (PAM), which follows immediately 3' - of the CRISPR RNA (crRNA) complementary sequence (Jinek, et al. (2012) Science 337(6096): 816-821; Nishimasu, et al. (2014) Cell 156(5):935-949; Sternberg, et al. (2014) Nature 507(7490):62-67). The double strand break can be repaired by non-homologous end joining (NHEJ) pathway yielding an insertion and/or deletion or, in the presence of a donor template, by homology-directed repair (HDR) pathway for replacement mutations (Gong, et al. (2005) Nat. Struct. Mol. Biol., 12(4):304-312; Overballe- Petersen, et al. (2013) Proc. Natl. Acad. Sci., 110(49): 19860-19865). The RNA- guided CRISPR/Cas9 system involves expressing Cas9 along with a guide RNA molecule (gRNA). When coexpressed, sgRNAs bind and recruit Cas9 to a specific genomic target sequence where it mediates a double strand DNA (dsDNA) break and activates the dsDNA break repair machinery. Specific DNA fragments can be deleted when two sgRNA/Cas9 complexes generate dsDNA breaks at relative proximity, and the genomic DNA is repaired by nonhomologous end joining.

Because Cas9 technology is limiting at the genome-wide scale, a CRISPR- Casl2a genetic screen was performed targeting all known coding genes in the human genome using a novel custom array directed towards 18,292 genes with 2 sgRNAs per gene. The single CRISPR RNA (crRNA) library was cloned into a lentivirus scaffold and transduced into the adult-type erythroid cell line, HUDEP2, engineered to stably express Casl2a. HUDEP2 cells express little HbF, allowing for positive phenotypic selection (HbF+) by fluorescence-activated cell-sorting (Figure 1 A). By isolating the top 10% and bottom 10% of HbF-expressing cells and determining the representation of crRNAs via deep sequencing, a crRNA subset was identified enriched in the high- HbF population encompassing 211 candidate genes, representing potential regulators of HbF (Figure 1 A). To prioritize these genes for follow up studies, a domain- focused, CRISPR-Cas9 screen of all 211 candidate genes was performed involving 6 sgRNAs targeting each domain of each gene. Using 2,184 sgRNAs, this validation Cas9 screen in HUDEP2 cells nominated 18 high-confidence targets (Figure IB). The results show that TIPRL and PTPA targeting sgRNAs result in enriched fetal globin expression (Figures 1C and ID).

These results were confirmed in HUDEP-2 stably expressing Cas9 cells by individually expressing 3 different sgRNAs targeting either TIPRL or PTPA, a positive control sgRNA targeting the +58 kb enhancer of BCL11 A, or negative control non-targeting sgRNAs (Figures 1E-1H). Sequences within TIPRL and PTPA used for the sgRNA are provided in Table 1. Fluorescence-activated cell sorting (FACS) with an HbF antibody shows that the TIPRL or PTPA sgRNAs significantly increased expression of HbF compared to the negative controls (Figure IE). Quantitative reverse transcription PCR (RT-qPCR) analysis was also performed on the above cells to measure mRNA levels (Figure IF). As seen in Figure IF, the TIPRL or PTPA sgRNAs significantly increased mRNA levels compared to the negative control. Figures 1G and 1H provide Western blots of the TIPRL and PTPA protein levels, respectively, in cells with BCL11 A controls.

In additional experiments, TIPRL or PTPA was depleted in primary, human CD34+ hematopoietic stem and progenitor cells (HSPCs) erythroblasts by electroporation of RNP Cas9: sgRNA complexes with 3 independent TIPRL or PTPA sgRNAs or control sgRNAs (Figure 2A). Fetal hemoglobin was found to be significantly upregulated. As seen in Figures 2B-2C, PTPA-directed sgRNAs increased HBG (also called y-globin, the fetal beta-like globin chain) expression and HbF percentage as determined by high performance liquid chromatography (HPLC) in primary human erythroid cells. Figure 2D shows protein expression of the g- globin, BCL11 A, and PTPA for these samples. Figures 2E-2G show HBG mRNA expression and HPLC and Western blot showing protein levels in TIPRL-depleted primary erythroblasts.

Table 1

In further experiments, it was determined whether PTPA depletion reduces BCL11 A mRNA. In HUDEP-2 stably expressing Cas9, cells individually expresed 3 different sgRNAs targeting either PTPA, a positive control sgRNA targeting the +58 kb enhancer of BCL11 A, or negative control non-targeting sgRNAs. As seen in Figures 3A, BCL11 A protein levels were reduced with the depletion of PTPA. As seen in Figure 3B, BCL11 A mRNA was reduced with the depletion of PTPA.

Figure 4A provides a schematic of a rescue experiment. As seen in Figures 4B and 4C, BCL11 A overexpression rescues the loss of PTPA.

Thus, two of the top candidate genes were identified as modulators of the phosphatase PP2A, PPP2R4 and TIPRL. PP2A is a multi-subunit complex with diverse cellular functions as a serine/threonine phosphatase. It has no previously known role on globin gene production. In validation experiments, disruption of either PPP2R4 or TIPRL in HUDEP2 cells significantly increased y-globin mRNA and protein expression. Using sgRNA-Cas9 ribonucleoprotein complex nucleofection, PPP2R4 gene editing in primary human CD34+ cell derived erythroblasts significantly increased y-globin mRNA and protein expression without overtly affecting erythroid maturation. Notably, the degree of effect is similar to that of BCL11 A +58 enhancer disruption. Depletion of either PP2A regulator in HUDEP2 cells resulted in markedly reduced BCL11 A protein levels. Furthermore, BCL11 A overexpression in PPP2R4 or TIPRL-targeted cells reduced y-globin mRNA commensurate to parental control cells, thereby demonstrating both TIPRL and PPP2R4 mediate y-globin silencing predominantly via BCL11 A. RNA-sequencing of TIPRL and PPP2R4-edited HUDEP2 cells revealed strong overlap of differentially expressed genes, consistent with convergent function. Altogether, the studies utilizing a novel Casl2a-based, genome-wide screen identify a new pathway mediating y-globin silencing via regulation of BCL11 A.

While certain of the preferred embodiments of the present invention have been described and specifically exemplified above, it is not intended that the invention be limited to such embodiments. Various modifications may be made thereto without departing from the scope and spirit of the present invention, as set forth in the following claims.

Claims

What is claimed is:

1. A method of increasing the level of human fetal hemoglobin and/or y -globin in a cell or subject, the method comprising administering at least one PTPA inhibitor and/or at least one TIPRL inhibitor to the cell or subject.

2. The method of claim 1, comprising administering at least one PTPA inhibitor to the cell or subject.

3. The method of claim 1, wherein the subject has a P-chain hemoglobinopathy.

4. The method of claim 1, wherein the subject has thalassemia.

5. The method of claim 1, wherein the subject has sickle cell disease.

6. The method of any one of claims 1-5, wherein the PTPA and/or TIPRL inhibitor is an inhibitory nucleic acid molecule.

7. The method of claim 6, wherein said inhibitory nucleic acid molecule is an siRNA, shRNA, or an antisense molecule.

8. The method of any one of claims 1-5, wherein the PTPA and/or TIPRL inhibitor is a phosphatase binding domain inhibitor.

9. The method of claim 1, wherein the cell is an erythroid cell, erythroblast, progenitor cell, or stem cell.

10. The method of claim 1, further comprising administering at least one fetal hemoglobin inducer to the cell or subject.

11. The method of claim 10, wherein said fetal hemoglobin inducer is pomalidomide.

12. A method of treating a hemoglobinopathy in a subject in need thereof, the method comprising administering a composition comprising at least one PTPA inhibitor and/or at least one TIPRL inhibitor and a pharmaceutically acceptable carrier to the subject.

13. The method of claim 12, said method comprising administering a composition comprising at least one PTPA inhibitor and a pharmaceutically acceptable carrier to the subject.

14. The method of claim 12, wherein the subject has a P-chain hemoglobinopathy.

15. The method of claim 12, wherein the subject has thalassemia.

16. The method of claim 12, wherein the subject has sickle cell anemia.

17. The method of any one of claims 12-16, wherein the PTPA and/or TIPRL inhibitor is an inhibitory nucleic acid molecule.

18. The method of claim 17, wherein said inhibitory nucleic acid molecule is an siRNA, shRNA, or an antisense molecule.

19. The method of any one of claims 12-16, wherein the PTPA and/or TIPRL inhibitor is a phosphatase binding domain inhibitor.

20. The method of claim 12, further comprising administering at least one fetal hemoglobin inducer to the subject.

21. The method of claim 20, wherein said fetal hemoglobin inducer is pomalidomide.

22. The method of claim 12, wherein the PTPA and/or TIPRL inhibitor is contained within a cell administered to the subject.