WO2017079269A1

WO2017079269A1 - Igf2bp1 and/or igf2bp3 for treatment

Info

Publication number: WO2017079269A1
Application number: PCT/US2016/060084
Authority: WO
Inventors: Jeffery Miller; Yuanwei Terry LEE; Colleen BYRNES; Laxminath TUMBURU
Original assignee: The United States Of America, As Represented By The Secretary, Department Of Health And Human Services
Priority date: 2015-11-04
Filing date: 2016-11-02
Publication date: 2017-05-11

Abstract

In embodiments of the invention, the present invention provides a method of increasing the amount of fetal hemoglobin in a cell and/or decreasing the amount of adult type hemoglobin in a cell, wherein the cell is optionally ex vivo, the method comprising administering to a stem cell or an erythroid cell an effective amount of a substance and/or 5-azacytidine, wherein the administration of the substance and/or 5-azacytidine increases the amount of fetal hemoglobin in the cell and/or decreases the amount of adult type hemoglobin in the cell. In embodiments of the invention, the present invention provides a substance and/or 5-azacytidine for use in preventing or treating sickle cell disease and/or a beta-thalassemia in a mammal. The substance comprises or encodes the protein IGF2BP1 and/or comprises or encodes the protein IGF2BP3, or any combination thereof.

Description

IGF2BP1 AND/OR IGF2BP3 FOR TREATMENT

CROSS-REFERENCE TO A RELATED APPLICATION

[0001] This patent application claims the benefit of U.S. Provisional Patent Application No. 62/250,815, filed November 4, 2015, which is incorporated by reference in its entirety.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED

ELECTRONICALLY

[0002] Incorporated by reference in its entirety herein is a computer-readable

nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: One 69,169 Byte ASCII (Text) file named "726739SeqListing_ST25.txt" created on November 2, 2016.

BACKGROUND OF THE INVENTION

[0003] Sickle-cell anemia and the β -thalassemias are among the most common genetic diseases worldwide. They are caused by mutated or reduced expression of the adult-stage β- globin gene and manifest when gene expression in the β-globin locus sequentially switches from fetal-to-adult types around the time of birth.

[0004] Multicellular organisms have networks of temporally regulated genes, known as heterochronic genes, responsible for the control of developmental timing. Heterochronic gene pathways were initially investigated in Caenorhabditis elegans, where the RNA-binding protein Lin28 and its major target, the let-7 miRNA, were first described as having an important role in early development, including the regulation of the transition from larva-to- adulthood. During vertebrate development, the Lin28-let-7 axis is also functionally involved in the maternal-to-zygotic transition during early zebrafish embryogenesis, suggesting that the heterochronic Lin28-let-7 axis and its role in the larval- or fetal-to-adult transition is conserved across evolution.

[0005] In human reticulocytes, changes in the levels of the let-7 miRNAs were associated with the fetal-to-adult developmental transition. Increased LIN28 or suppressed let-7 were shown to be developmentally regulated, and both cause increased gamma- globin gene expression in cultured human adult erythroblasts. However, a complete reversal of the fetal- to-adult hemoglobin profiles was not detected. [0006] There is a need for a more complete reversal of the fetal-to-adult hemoglobin profile in adults for the prevention and treatment of hemoglobinopathies, including sickle-cell anemia and the β-thalassemias.

BRIEF SUMMARY OF THE INVENTION

[0007] In embodiments of the invention, the present invention provides a method of increasing the amount of fetal hemoglobin in a cell, wherein the cell can be ex vivo, the method comprising, consisting essentially of, or consisting of increasing the expression of IGF2BP1 and/or IGF2BP3 in the cell, wherein increasing the expression of IGF2BP1 and/or IGF2BP3 increases the amount of fetal hemoglobin in the cell.

[0008] In embodiments of the invention, the present invention provides a method of decreasing the amount of adult type hemoglobin in a cell, wherein the cell can be ex vivo, the method comprising, consisting essentially of, or consisting of increasing the expression of IGF2BP1 and/or IGF2BP3 in the cell, wherein increasing the expression of IGF2BP1 and/or IGF2BP3 decreases the amount of adult type hemoglobin, optionally including sickle cell hemoglobin, in the cell.

[0009] In embodiments of the invention, the present invention provides a method of preventing or treating sickle cell disease in a mammal, the method comprising, consisting essentially of, or consisting of increasing the expression of IGF2BP1 and/or IGF2BP3 in the mammal, wherein the increasing the expression of IGF2BP1 and/or IGF2BP3 prevents or treats sickle cell disease in the mammal.

[0010] In embodiments of the invention, the present invention provides a method of preventing or treating beta-thalassemia in a mammal, the method comprising, consisting essentially of, or consisting of increasing the expression of IGF2BP1 and/or IGF2BP3 in the mammal, wherein the increasing the expression of IGF2BP1 and/or IGF2BP3 prevents or treats beta-thalassemia in the mammal.

[0011] In embodiments of the invention, the present invention provides a method of increasing the amount of fetal hemoglobin in a cell, wherein the cell is optionally ex vivo, the method comprising, consisting essentially of, or consisting of administering to a stem cell or an erythroid cell an effective amount of a substance, the substance comprising, consisting essentially of, or consisting of the protein IGF2BP1 or comprising, consisting essentially of, or consisting of nucleic acid encoding the protein IGF2BP1 or the substance comprising, consisting essentially of, or consisting of the protein IGF2BP3 or comprising, consisting essentially of, or consisting of nucleic acid encoding the protein IGF2BP3, or any combination thereof, wherein the administration of the substance increases the amount of fetal hemoglobin in the cell.

[0012] In embodiments of the invention, the present invention provides a method of decreasing the amount of adult type hemoglobin in a cell, wherein the cell is optionally ex vivo, the method comprising, consisting essentially of, or consisting of administering to a stem cell or an erythroid cell an effective amount of a substance, the substance comprising, consisting essentially of, or consisting of the protein IGF2BP1 or comprising, consisting essentially of, or consisting of nucleic acid encoding the protein IGF2BP1 or the substance comprising, consisting essentially of, or consisting of the protein IGF2BP3 or comprising, consisting essentially of, or consisting of nucleic acid encoding the protein IGF2BP3, or any combination thereof, wherein the administration of the substance decreases the amount of adult type hemoglobin, optionally including sickle cell hemoglobin, in the cell.

[0013] In embodiments of the invention, the present invention provides a substance for use in preventing or treating sickle cell disease in a mammal, the substance comprising, consisting essentially of, or consisting of the protein IGF2BP1 or comprising, consisting essentially of, or consisting of nucleic acid encoding the protein IGF2BP1 or the substance comprising, consisting essentially of, or consisting of the protein IGF2BP3 or comprising, consisting essentially of, or consisting of nucleic acid encoding the protein IGF2BP3, or any combination thereof.

[0014] In embodiments of the invention, the present invention provides a substance for preventing or treating a β-thalassemia in a mammal, the substance comprising, consisting essentially of, or consisting of the protein IGF2BP1 or comprising, consisting essentially of, or consisting of nucleic acid encoding the protein IGF2BP1 or the substance comprising, consisting essentially of, or consisting of the protein IGF2BP3 or comprising, consisting essentially of, or consisting of nucleic acid encoding the protein IGF2BP3, or any combination thereof.

[0015] In another embodiment of the invention, the present invention provides plasmids encoding the protein IGF2BP1 and/or IGF2BP3.

[0016] In embodiments of the invention, the present invention provides a method of increasing the amount of fetal hemoglobin in a cell, wherein the cell is optionally ex vivo, the method comprising, consisting essentially of, or consisting of administering to a stem cell or an erythroid cell an effective amount of 5-azacytidine, wherein the administration of 5- azacytidine increases the amount of fetal hemoglobin in the cell.

[0017] In embodiments of the invention, the present invention provides a method of decreasing the amount of adult type hemoglobin in a cell, wherein the cell is optionally ex vivo, the method comprising, consisting essentially of, or consisting of administering to a stem cell or an erythroid cell an effective amount of 5-azacytidine, wherein the administration of 5-azacytidine decreases the amount of adult type hemoglobin, optionally including sickle cell hemoglobin, in the cell.

[0018] In embodiments of the invention, the present invention provides 5-azacytidine for use in preventing or treating sickle cell disease in a mammal.

[0019] In embodiments of the invention, the present invention provides 5-azacytidine for preventing or treating a β-thalassemia in a mammal.

[0020] Further embodiments of the invention are disclosed below and are incorporated into this section.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 presents a developmental RNA-binding cascade for the regulation of fetal hemoglobin.

[0022] Figures 2A-2C show expression patterns of IGF2BP1 and IGF2BP3 mRNAs and proteins in fetal and adult human tissues. IGF2BP1 and IGF2BP3 mRNA expression in (A) human fetal liver and adult bone marrow samples and (B) cultured cord blood and adult blood erythroblasts. Mean value ± SD are shown. (C) IGF2BP1 and IGF2BP3 protein expression in human fetal liver protein medley, adult bone marrow, cord blood and adult blood erythroblasts. K562 cell extracts were used as a positive control and Beta-Actin as a loading control. Molecular weight shown in kilodaltons (kDa). FL, fetal liver; BM, adult bone marrow; CB, cord blood erythroblasts; AB, adult blood erythroblasts.

[0023] Figures 3A-3D show IGF2BP1 and IGF2BP3 over-expression, in accordance with embodiments of the invention. (A) IGF2BP1 over-expression in adult human erythroblasts confirmed by Q-RT-PCR at culture day 14. Mean value ± SD of three independent donors for each condition. (B) Western blot analysis confirmation of IGF2BP1 over-expression in CD34(+) cells versus empty vector control from three healthy adult donors. K562 cells were used as a positive control and Beta-Actin as a loading control. (C) IGF2BP3 over-expression in adult human erythroblasts was confirmed by Q-RT-PCR at culture day 14. Mean value ± SD of four independent donors for each condition. (D) Western analyses confirmation of IGF2BP3 over-expression in CD34(+) cells versus empty vector control from three healthy adult donors (the additional bands present in the IGF2BP3 lanes are due to an artifact of the antibody used). K562 cells were used as a positive control and Beta-Actin as a loading control. C, empty vector control; OE, over-expression. Molecular weight shown in kilodaltons (kDa). P-values were calculated using two-tailed Student's t-test. Asterisk denotes p<0.05.

[0024] Figures 4A-4D show IGF2BP1 and IGF2BP3 over-expression effects upon hemoglobin expression pattern, in accordance with embodiments of the invention. HPLC analyses of hemoglobin from (A) empty vector control, (B) IGF2BP1 over-expression (IGF2BP1-OE), (C) empty vector control and (D) IGF2BP3 over-expression samples were performed at culture day 21. Datasets are representative of three independent donors for each condition. HbF and HbA peaks are labeled on each graph (y-axis, mVolts; x-axis, elution time in minutes).

[0025] Figures 5A-5F show the effects of IGF2BP1 over-expression upon differentiation, maturation, enucleation and cellular morphology of CD34(+) cells, in accordance with embodiments of the invention. Representative flow dot plots at culture day 21 stained for CD71 and GPA from (A) empty vector control cells and (B) IGF2BP1 over-expression. Thiazole orange staining was utilized to assess enucleation for (C) empty vector control and (D) IGF2BP1 over-expression at culture day 21. Sorted enucleated cells were imaged in panels (E) empty vector control and (F) IGF2BP1 over-expression after Wright-Giemsa staining. Datasets are representative of three independent donors for each condition. CD71 , anti-transferrin receptor; GPA, anti-glycophorin A; HbF, fetal hemoglobin.

[0026] Figures 6A-6F show quantitative analyses of the percentage of

(A) CD71(+)/GPA(+) Day 14, (B) CD71 (+)/GPA(-) Day 14, (C) CD71 (+)/GPA(+) Day 21 , and (D) CD71(-)/GPA(+) Day 21 measured by fluorescence-activated cell sorter analysis of control and IGF2BP1-OE at culture days 14 and 21 , in accordance with embodiments of the invention. Mean value ± SD of three independent donors for each condition. IGF2BP1-OE increases in fetal hemoglobin are maintained after terminal differentiation. HPLC analyses of hemoglobin at culture day 21 from sorted IGF2BP1 -OE (E) enucleated cells and (F) non- enucleated cells. HbF and HbA peaks are labeled on each graph (y-axis: mVolts; x-axis: elution time in minutes). CD71, anti-transferrin receptor; GPA, anti-glycophorin A. C = empty vector control and OE = IGF2BP1 over-expression. [0027] Figures 7A-7J show IGF2BP1 -OE and control transductions investigated for (A) epsilon-globin, (B) gamma-globin, (C) delta-globin, (D) beta-globin, (E) zeta-globin, (F) mu-globin, (G) alpha-globin, (H) theta-globin, (I) CA1 and (J) GCNT2, in accordance with embodiments of the invention. Q-RT-PCR analyses were performed at culture day 14. Open bars represent control transductions and black bars represent IGF2BP1-OE. Mean value ± SD of three independent donors for each condition. P-values were calculated using two-tailed Student's t-test. Asterisks indicate p<0.05. C = empty vector control transduction; OE = IGF2BP1 over-expression.

[0028] Figures 8A-8I show IGF2BP1-OE and control transductions investigated for (A) let-7a, (B) let-7b, (C) let-7c, (D) let-7d, (E) let-7e, (F) let-7f, (G) let-7g, (H) let-7i, and (I) miR-98, in accordance with embodiments of the invention. Q-RT-PCRs were performed at culture day 14. Open bars represent empty vector control and black bars represent IGF2BP1 - OE. Mean value ± SD of three independent donors for each condition. P-values were calculated using two-tailed Student's t-test. Asterisk indicate p<0.05. C = empty vector control transduction; OE = IGF2BP 1 over-expression.

[0029] Figures 9 A-9D show IGF2BP 1 -OE and control transductions investigated for the mRNA levels of (A) c-MYC, (B) IGF2 and (C) Beta-Actin, in accordance with embodiments of the invention. Q-RT-PCRs were performed at culture day 14. Open bars represent empty vector control and black bars represent IGF2BP1-OE. Mean value ± SD of three independent donors for each condition. (D) Western analyses of c-MYC, IGF2 and Beta-Actin expression using protein extracts at culture day 14 of empty vector control and IGF2BP 1 over-expressed CD34(+) cells. Alpha tubulin was used as a loading control. Molecular weight shown in kilodaltons (kDa). C = empty vector control and OE = IGF2BP1 over- expression.

[0030] Figures 10A-10E show IGF2BP1 -OE and control transductions wre investigated for the mRNA levels of (A) BCL11A and (B) HMGA2, in accordance with embodiments of the invention. Q-RT-PCRs were performed at culture day 14. Open bars represent empty vector control and black bars represent 1GF2BP1 -OE. Mean value ± SD of three independent donors for each condition. (C) Western analyses of BCL1 1 A and HMGA2 expression using protein extracts at culture day 14 of empty vector control and IGF2BP1 over-expressed CD34(+) cells. Histone H3 and Lamin Bl were used as loading controls. RNA

immunoprecipitation (RIP) using antibody against IGF2BP1 was performed to assess direct binding of RNA to IGF2BP1 protein followed by Q-RT-PCR quantitation for (D) BCL11A and (E) HMGA2 transcripts. RIP was performed at culture day 14. Mean value ± SD of three independent donors for each condition. P-value was calculated using one- or two-tailed Student's t-test. Asterisks indicate p<0.05. C = empty vector control, OE = IGF2BP1 over- expression, Input = RNA sample before immunoprecipitation, IgG = immunoprecipitation with isotype control, BP 1 -RIP = IGF2BP1 RNA immunoprecipitation.

DETAILED DESCRIPTION OF THE INVENTION

[0031] In an embodiment of the invention, the present invention provides a method of increasing the amount of fetal hemoglobin in a cell, wherein the cell can be ex vivo, the method comprising, consisting essentially of, or consisting of increasing the expression of IGF2BP1 and/or IGF2BP3 in the cell, wherein increasing the expression of IGF2BP1 and/or IGF2BP3 increases the amount of fetal hemoglobin in the cell.

[0032] In an embodiment of the invention, the present invention provides a method of decreasing the amount of adult type hemoglobin in a cell, wherein the cell can be ex vivo, the method comprising, consisting essentially of, or consisting of increasing the expression of IGF2BP1 and/or IGF2BP3 in the cell, wherein increasing the expression of IGF2BP1 and/or IGF2BP3 decreases the amount of adult type hemoglobin, optionally including sickle cell hemoglobin, in the cell.

[0033] In an embodiment of the invention, the present invention provides a method of preventing or treating sickle cell disease in a mammal, the method comprising, consisting essentially of, or consisting of increasing the expression of IGF2BP1 and/or IGF2BP3 in the mammal, wherein the increasing the expression of IGF2BP1 and/or IGF2BP3 prevents or treats sickle cell disease in the mammal.

[0034] In an embodiment of the invention, the present invention provides a method of preventing or treating beta-thalassemia in a mammal, the method comprising, consisting essentially of, or consisting of increasing the expression of IGF2BP1 and/or IGF2BP3 in the mammal, wherein the increasing the expression of IGF2BP1 and/or IGF2BP3 prevents or treats beta-thalassemia in the mammal.

[0035] Any method of increasing the expression of IGF2BP1 and/or IGF2BP3 is contemplated. These methods include, for example, gene therapy techniques. The gene therapy techniques can include those as described herein, e.g., providing transcripts of IGF2BP1 and/or IGF2BP3 that are resistant to degradation, e.g., by lacking let-7 sites. Also, increasing expression through the use of small molecule compounds is contemplated. For example, it has been found that 5-azacytidine reactivates IGF2BP1 expression (Ioannidis et al., J. Biol. Chem. 280:20086-20093 (2005), incorporated herein by reference in its entirety).

[0036] In an embodiment of the invention, the present invention provides a method of increasing the amount of fetal hemoglobin in a cell, wherein the cell is optionally ex vivo, the method comprising, consisting essentially of, or consisting of administering to a stem cell or an erythroid cell an effective amount of a substance, the substance comprising, consisting essentially of, or consisting of the protein IGF2BP1 or comprising, consisting essentially of, or consisting of nucleic acid encoding the protein IGF2BP 1 or the substance comprising, consisting essentially of, or consisting of the protein IGF2BP3 or comprising, consisting essentially of, or consisting of nucleic acid encoding the protein IGF2BP3, or any

combination thereof, wherein the administration of the substance increases the amount of fetal hemoglobin in the cell. In an embodiment of the invention, the present invention provides a method of decreasing the amount of adult type hemoglobin in a cell, wherein the cell is optionally ex vivo, the method comprising, consisting essentially of, or consisting of administering to a stem cell or an erythroid cell an effective amount of a substance, the substance comprising, consisting essentially of, or consisting of the protein IGF2BP1 or comprising, consisting essentially of, or consisting of nucleic acid encoding the protein IGF2BP1 or the substance comprising, consisting essentially of, or consisting of the protein IGF2BP3 or comprising, consisting essentially of, or consisting of nucleic acid encoding the protein IGF2BP3, or any combination thereof, wherein the administration of the substance decreases the amount of adult type hemoglobin, optionally including sickle cell hemoglobin, in the cell.

[0037] In an embodiment of the invention, the present invention provides a method of preventing or treating sickle cell disease in a mammal, the method comprising, consisting essentially of, or consisting of administering to a mammal in need thereof an effective amount of a substance, the substance comprising, consisting essentially of, or consisting of the protein IGF2BP1 or comprising, consisting essentially of, or consisting of nucleic acid encoding the protein IGF2BP1 or the substance comprising, consisting essentially of, or consisting of the protein IGF2BP3 or comprising, consisting essentially of, or consisting of nucleic acid encoding the protein IGF2BP3, or any combination thereof, wherein the administration of the substance prevents or treats sickle cell disease in the mammal. In an embodiment of the invention, the present invention provides a method of preventing or treating a beta-thalassemia in a mammal, the method comprising, consisting essentially of, or consisting of administering to a mammal in need thereof an effective amount of a substance, the substance comprising, consisting essentially of, or consisting of the protein IGF2BP1 or comprising, consisting essentially of, or consisting of nucleic acid encoding the protein IGF2BP1 or the substance comprising, consisting essentially of, or consisting of the protein IGF2BP3 or comprising, consisting essentially of, or consisting of nucleic acid encoding the protein IGF2BP3, or any combination thereof, wherein the administration of the substance prevents or treats the beta-thalassemia in the mammal.

[0038] The following paragraph is provided without wishing to be bound by theory. Insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1, IMP1, CRDBP, ZP1, VICKZl) is a multifunctional RNA-binding protein that is believed to control the fate of target mRNAs in the cytoplasm, possibly by means of controlling the localization or stability of mRNA transcripts or preventing the decay of target mRNAs. Multiple transcripts have been identified as potential or validated targets of IGF2BP1. Insulin-like growth factor 2 mRNA-binding protein3 (IGF2BP3, IMP3, OC1 , VICKZ3) is a KH domain-containing protein and is oversexpressed in cancer. 1GF2BP1 and IGF2BP3 have 73% identity at the protein level, and both manifest highly regulated mRNA and protein expression patterns during human ontogeny with silencing during the transition from fetal-to-adult life, and hemoglobin switching occurs during the same developmental period. Developmental silencing can occur through LIN28B and activation of let-7 in erythroid cells. Both of these molecules function in the binding and post-transcriptional regulation of multiple RNA targets, and as a consequence have multiple biological functions, including pluripotency of stem cells and regulation of glucose metabolism, let-7 miRNAs are downstream targets of LIN28B, and the IGF2BP mRNAs are targets of let-7. Figure 1 presents a developmental regulatory pathway for fetal hemoglobin expression during ontogeny. IGF2BP1 may act to increase the expression of some proteins by competition with let-7, and IGF2BP1 may act to suppress the expression of other proteins via multiple separate mechanisms including translational repression, protein compartmentalization, or increased mRNA decay.

[0039] An advantage of the embodiments of the present invention includes use of a naturally-occurring protein and not a foreign protein. The adult body has previously encountered fetal hemoglobin as self protein, and therefore there normally would be no immune response against the protein. Also, increased expression of IGF2BP1 and/or IGF2BP3 in humans is predicted to be non-immunogenic. [0040] Low level expression of IGF2BP1 and/or IGF2BP3 is expected to provide therapeutic levels of fetal hemoglobin expression. Erythroid-specific expression of IGF2BP1 and/or IGF2BP3 is sufficient for its therapeutic effects in order to provide for increased safety. The increased expression of fetal hemoglobin is balanced by a reduction in adult hemoglobin. The methods of the present invention are contemplated to be useful with all hematopoietic gene delivery methods that cause erythroid-specific IGF2BP1 and/or IGF2BP3 expression in humans of any age, including the lentivirus examples provided herein.

[0041] Thalassemia is an autosomal recessive disorder that is caused by mutations in the a-globin gene (a-thalassemia), the β-globin gene (β -thalassemia), or less commonly, the δ- globin gene (δ-thalassemia). Both a- and β-thalassemia can occur in two forms: thalassemia major or thalassemia minor. Inheritance of two mutant globin genes, one from each parent, results in thalassemia major. Inheritance of only one mutant globin gene from one parent results in thalassemia minor. Humans with thalassemia minor are earners of the disease and typically do not exhibit disease symptoms.

[0042] In any of the above embodiments of the invention, the substance can be a plasmid, nucleic acid, or derivative thereof encoding the protein IGF2BP1 and/or encoding the protein IGF2BP3, wherein the plasmid, nucleic acid, or derivative thereof can be administered to the cell or mammal. In any of the above embodiments, the plasmid, nucleic acid, or derivative thereof can be for a virus or other nucleic acid delivery technique. In any of the above embodiments, the plasmid, nucleic acid, or derivative thereof can include at least one erythroid-specific element (e.g., erythroid-specific promoter, enhancer, polyadenylation, etc.). In any of the above embodiments, the plasmid, nucleic acid, or derivative thereof can encode a let-7 resistant transcript of the protein IGF2BP1 and/or of the protein IGF2BP3. For example, the 3' untranslated region (3' UTR) of IGF2BP1 and IGF2BP3 contain binding sites for let-7. In an embodiment of the invention, the protein coding region for IGF2BP1 and/or IGF2BP3 is included, but the 3 'UTR is not included or is removed. The plasmid can be introduced into the cell by any suitable means understood by one of ordinary skill in the art, for example, by electroporation of, e.g., mature cells. In any of the embodiments, the substance can comprise, consist essentially of, or consist of the protein IGF2BP1 and/or the protein IGF2BP3, and the substance can be administered, e.g., directly, to the cell. In any of the above embodiments of the invention, the substance can comprise, consist essentially of, or consist of the protein IGF2BP1 and/or the protein IGF2BP3, and the substance can be administered, e.g., directly, to the mammal. For example, administration may be through use of protein encapsulated for delivery. Direct administration may be by any suitable means understood to one of ordinary skill in the art. Such methods include, for example, micropipette injection of the substance into the cell. The plasmid can be a viral vector plasmid. In any of the above embodiments of the invention, the substance can be a viral nucleic acid encoding the protein IGF2BP1 and/or encoding the protein IGF2BP3, wherein the viral nucleic acid can be administered to the cell through viral transduction. In any of the above embodiments, the viral nucleic acid can comprise, consist essentially of, or consist of lentiviral nucleic acid, e.g., administered using a lentiviral vector.

[0043] In embodiments of the invention, the methods can further comprise, consist essentially of, or consist of administering to a stem cell or an erythroid cell the substance and/or 5-azacytidine and administering to the mammal the cell having been administered the substance and/or 5-azacytidine. In embodiments of the invention, the methods can further comprise, consist essentially of, or consist of extracting from the mammal a stem cell or an erythroid cell, administering to the cell the substance and/or 5-azacytidine and administering to the mammal the cell having been administered the substance and/or 5-azacytidine.

[0044] Lentiviruses are a subclass of Retroviruses. Lentiviruses resemble γ -retro viruses (γ-RV) in their ability to stably integrate into the target cell genome, resulting in persistent expression of the gene of interest. However, in contrast to γ-retroviruses, lentiviruses also can transduce nondividing cells, which has led to their wide use as gene transfer vectors. The lentivirus genome is monopartite, linear, dimeric, positive-strand single-stranded RNA ("ssRNA(+)") of 9.75 kb, with a 5'-cap and a 3'poly-A tail. The lentiviral genome is flanked by the 5' and 3' long terminal repeat (LTR) sequences which have promoter/enhancer activity and are essential for the correct expression of the full-length lentiviral vector transcript. The LTRs also have an important role in reverse transcription and integration of the vector into the target cell genome. Upon viral entry into a cell, the RNA genome is reverse-transcribed into double-stranded DNA, which is then inserted into the genome at a random position by the viral integrase enzyme. The lentivirus, now called a provirus, remains in the genome and is passed on to the progeny of the cell when it divides. Species of lentivirus include, for example, human immunodeficiency virus 1 (HIV-1 ), human immunodeficiency virus 2 (HIV-2), simian immunodeficiency virus (SIV), bovine immunodeficiency virus (BIV), and feline immunodeficiency virus (FIV). The lentiviral vector can be based on any lentivirus species. [0045] Lentiviral vectors typically are generated by trans-complementation in packaging cells that are co-transfected with a plasmid containing the vector genome and the packaging constructs that encode only the proteins essential for lentiviral assembly and function. A self- inactivating (SIN) lentiviral vector can be generated by abolishing the intrinsic

promoter/enhancer activity of the HIV-1 LTR, which reduces the likelihood of aberrant expression of cellular coding sequences located adjacent to the vector integration site (see, e.g., Vigna et al., J. Gene Med., 2: 308-316 (2000); Naldini et al., Science, 272: 263-267 (1996); and Matrai et al, Molecular Therapy, 18(3): 477-490 (2010)). The most common procedure to generate lentiviral vectors is to co-transfect cell lines (e.g., 293T human embryonic kidney cells) with a lentiviral vector plasmid and three packaging constructs encoding the viral Gag-Pol, Rev-Tat, and envelope (Env) proteins. The lentiviral vector particle can integrate its genome into a host cell genome. In some applications, however, it may be desirable to avoid potential insertional mutagenesis induced by an integrating lentivirus vector. In such cases, the lentiviral vector particle does not integrate its genome into a host cell genome (also referred to as a "non-integrating" vector). Non-integrating lentiviral vectors typically are generated by mutating the lentiviral integrase gene or by modifying the attachment sequences of the LTRs (see, e.g., Sarkis et al, Curr. Gene. Ther., 6: 430-437 (2008)).

[0046] Methods for generating lentiviral vectors are well-known in the art, and the inventive lentiviral vector can be constructed using any suitable such method. As discussed above, lentiviral vectors typically are produced by co-transfecting 293 T human embryonic kidney cells with several different plasmid constructs, which separately contain the lentiviral cis-acting sequences and trans-acting factors that are required for viral particle production, infection, and integration. Lentiviral vector production systems typically include four plasmids. The transfer vector contains the transgene be delivered in a lentiviral backbone containing all of the cis-acting sequences required for genomic RNA production and packaging. Three additional provide the trans-acting factors required for packaging, namely Gag-Pol, Rev-Tat, and the envelope protein VSVG, respectively. When these four plasmids are transfected into 293T human embryonic kidney cells, viral particles accumulate in the supernatant, and the viral product can be concentrated by ultracentrifugation. Lentiviral production protocols are further described in, for example, Tiscornia et al., Nature Protocols, 1 : 241 -245 (2006); Stevenson, M., Curr. Top. Microbiol. Immunol., 261 : 1 -30 (2002); Cronin et al., Curr. Gene Ther., 5: 387-398 (2005); Sandrin et al., Curr. Top. Microbiol. Immunol., 281 : 137-178 (2003); Zufferey, R., Curr. Top. Microbiol. Immunol., 261 : 107-121 (2002); Sinn et al., Gene Ther., 12: 1089-1098 (2005); and Saenz, D.T. and Poeschla, E.M., J. Gene Med., 6: S95-S104 (2004). Other methods for producing lentiviral vectors are known in the art and described in, for example, U.S. Patent Application Publications 2008/0254008 and 2010/0003746; and Yang et al., Hum Gene Ther. Methods, 23(2): 73-83 (2012).

[0047] With appropriate packaging vectors assisting in viral transduction, the vectors described herein overexpress IGF2BP1 and/or IGF2BP3 and provide expression that is tissue specific or restricted to erythroid cells (erythroblasts and erythrocytes), where the packaging vectors have tropism toward human hematopoietic cells or other cells that are capable of differentiation into the erythroid lineage (e.g., erythroblasts and erythrocytes). In general, the viral tropism is achieved by usage of appropriate helper/packaging plasmids in combination with the appropriate signal to package (the signal being present in the vectors of the present invention). Determination of which packaging vectors to use is routine in the art using standard techniques. Suitable packing vectors include the pCL-10A1 packaging vector for retroviruses. The pCL-lOAl vector is a part of the RetroMax expression system from

Imgenex (San Diego, CA, USA). Packaging vectors suitable for lentiviruses include pAG4- RTR2-1 , pCAGGS-VSVG-1, and pCAG-kGPl-lR-1. Also, the Clontech Lenti-X HTX Packaging system (VSV-G lentiviral packaging) that produces VSV-G pseudotyped lentivirus, which readily infects virtually all types of cells (Catalog Number 631247) and the ViraPower™ Lentiviral Packaging Mix (Catalog Number K4975-00) are two systems that may be used. Examples of plasmids are provided at SEQ ID NO: 1 (backbone); SEQ ID NOs: 2-4 (packaging); SEQ ID NO: 5 (having IGF2BP1); and SEQ ID NO: 6 (having IGF2BP3). Thus, any suitable gene therapy method is contemplated to perform the methods of the invention, including use of the CRISPR/Cas9 system.

[0048] In embodiments of the present invention, the methods and transgenes (i.e., IGF2BP1 and/or IGF2BP3) of the present invention may be used with other vector systems as would be understood by one of ordinary skill in the art. Exemplary systems and additional plasmids/vectors include, for example, those described in Perumbeti and Malik, Ann. NY Acad. Sci., 2010, 1202:36-44; Papanikolaou and Anagnou, Curr. Gene Ther., 2010, 10:404- 12; Persons, Hematology Am. Soc. Hematol. Educ. Program, 2009:690-7; Breda et al., Mediterr. J. Hematol. Infect. Dis., 2009, l :e2009008; Sadelain et al., Curr. Mol. Med., 2008, 8:690-7; Lebensburger and Persons, Cun-. Opin. Drug Discov. Devel., 2008, 1 1 :225-32; Bank et al., Ann. NY Acad. Sci., 2005, 1054:308-16; Hanawa, et al., Mol. Ther., 2002, 5:242-251 ; Aiuti et al., Science, 2013, 341 :1233151 ; and Biffi et al, Science, 2013, 341 : 1233158; each of which is incorporated herein by reference in its entirety. The plasmids/vectors of the present invention may be isolated and/or purified using standard techniques.

[0049] In embodiments of the invention where the substance is nucleic acid, the nucleic acid may be, e.g., RNA, or nucleic acid derivatives (e.g., protein nucleic acid (PNA), morpholino and locked nucleic acid (LNA), glycol nucleic acid (GNA), threose nucleic acid (TNA), or other synthesized derivatives).

[0050] In any embodiment of the invention described herein, the suitable cells used in accordance with the present invention can include, for example, erythroid cells, or its progenitors, precursors, or stem cells, including, for example, hematopoietic stem cells, induced pluripotent stem cells, or other cells with erythroid potential (e.g., any cell that has the capability to undergo erythroid differentiation or that can be reprogrammed toward erythroid differentiation). The methods as described herein, including ex vivo methods, are applicable to these cells. It is desirable to have, e.g., erythroid specificity, e.g., to provide targeted increase in expression of IGF2BP1 and/or IGF2BP3. A targeted approach can decrease the possibility of having off-target effects when expression is modulated in, e.g., non-erythroid cells.

[0051] In any embodiment, the cell can be in or transplanted into a mammal. In any embodiment, the mammal can be a human. In an embodiment of the invention, the cell can be in a mammal. For purposes of the present invention, mammals include, but are not limited to, mice, rats, rabbits, cats, dogs, cows, pigs, horses, monkeys, apes, and humans. The mammal may be an adult mammal, e.g., wherein the fetal-to-adult transition of hemoglobin has already occurred, e.g., the mammal may be an adult human.

[0052] In embodiments of the invention, the present invention provides a method of increasing the amount of fetal hemoglobin in a cell, wherein the cell is optionally ex vivo, the method comprising, consisting essentially of, or consisting of administering to a stem cell or an erythroid cell an effective amount of 5-azacytidine, wherein the administration of 5- azacytidine increases the amount of fetal hemoglobin in the cell.

[0053] In embodiments of the invention, the present invention provides a method of decreasing the amount of adult type hemoglobin in a cell, wherein the cell is optionally ex vivo, the method comprising, consisting essentially of, or consisting of administering to a stem cell or an erythroid cell an effective amount of 5-azacytidine, wherein the administration of 5-azacytidine decreases the amount of adult type hemoglobin, optionally including sickle cell hemoglobin, in the cell.

[0054] In embodiments of the invention, the present invention provides 5-azacytidine for use in preventing or treating sickle cell disease in a mammal. In embodiments of the invention, the present invention provides 5-azacytidine for preventing or treating a β- thalassemia in a mammal.

[0055] In an embodiment of the invention, the present invention provides a method of preventing or treating sickle cell disease in a mammal, the method comprising, consisting essentially of, or consisting of administering to a mammal in need thereof an effective amount of 5-azacytidine, wherein the administration of 5-azacytidine prevents or treats sickle cell disease in the mammal. In an embodiment of the invention, the present invention provides a method of preventing or treating a beta-thalassemia in a mammal, the method comprising, consisting essentially of, or consisting of administering to a mammal in need thereof an effective amount of 5-azacytidine, wherein the administration of the 5-azacytidine prevents or treats the beta-thalassemia in the mammal.

[0056] The substance comprising, consisting essentially of, or consisting of the protein IGF2BP1 or comprising, consisting essentially of, or consisting of nucleic acid encoding the protein IGF2BP1 or the substance comprising, consisting essentially of, or consisting of the protein IGF2BP3 or comprising, consisting essentially of, or consisting of nucleic acid encoding the protein IGF2BP3, or any combination thereof, and/or 5-azacytidine, can be formulated into a composition, such as a pharmaceutical composition with a carrier. The pharmaceutical compositions can comprise more than one of the substances and/or 5- azacytidine. Alternatively, the pharmaceutical composition can comprise a single substance or 5-azacytidine in combination with other pharmaceutically active agents or drugs.

[0057] In an embodiment, the carrier is a pharmaceutically acceptable carrier. With respect to pharmaceutical compositions, the carrier can be any of those conventionally used and is limited only by chemico-physical considerations, such as solubility and lack of reactivity with the active compound(s), and by the route of administration. The

pharmaceutically acceptable carriers described herein, for example, vehicles, adjuvants, excipients, and diluents, are well-known to those skilled in the art and are readily available to the public. It is preferred that the pharmaceutically acceptable carrier be one which is chemically inert to the active agent(s) and one which has no detrimental side effects or toxicity under the conditions of use. [0058] The choice of carrier will be determined in part by the particular substance comprising, consisting essentially of, or consisting of the protein IGF2BP1 or comprising, consisting essentially of, or consisting of nucleic acid encoding the protein IGF2BP1 or the substance comprising, consisting essentially of, or consisting of the protein IGF2BP3 or comprising, consisting essentially of, or consisting of nucleic acid encoding the protein IGF2BP3, or any combination thereof, and/or 5-azacytidine, as well as by the particular method used to administer the substance and/or 5-azacytidine. Accordingly, there are a variety of suitable formulations of the pharmaceutical composition. The formulations for, e.g., oral, parenteral, subcutaneous, intravenous, intramuscular, intraarterial, intrathecal, and interperitoneal administration are exemplary and are in no way limiting. More than one route can be used to administer the substance and/or 5-azacytidine, and in certain instances, a particular route can provide a more immediate and more effective response than another route.

[0059] Suitable compositions include aqueous and non-aqueous isotonic sterile solutions, which can contain anti-oxidants, buffers, and bacteriostats, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The composition can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water, immediately prior to use. Extemporaneous solutions and suspensions can be prepared from sterile powders, granules, and tablets. Preferably, the carrier is a buffered saline solution. More preferably, the inventive system is part of a composition formulated to protect the lentiviral vector from damage prior to administration. For example, the composition can be formulated to reduce loss of the lentiviral vector on devices used to prepare, store, or administer lentiviral vector, such as glassware, syringes, or needles. The composition can be formulated to decrease the light sensitivity and/or temperature sensitivity of the system. To this end, the composition preferably comprises a pharmaceutically acceptable liquid carrier, such as, for example, those described above, and a stabilizing agent selected from the group consisting of polysorbate 80, L-arginine, polyvinylpyrrolidone, trehalose, and combinations thereof. Use of such a composition will extend the shelf life of the system, and facilitate its administration. Formulations for lentiviral-containing compositions are further described in, for example, Ausubel et al, Bioprocess Int., 10(2): 32-43 (2012), U.S. Patent 7,575,924, and International Patent Application Publication WO 2013/139300. [0060] Topical formulations are well-known to those of skill in the art. Such formulations are particularly suitable in the context of the invention for application to the skin, including absorption into the capillaries of the skin.

[0061] Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the substance comprising, consisting essentially of, or consisting of the protein IGF2BP1 or comprising, consisting essentially of, or consisting of nucleic acid encoding the protein IGF2BP1 or the substance comprising, consisting essentially of, or consisting of the protein IGF2BP3 or comprising, consisting essentially of, or consisting of nucleic acid encoding the protein IGF2BP3, or any combination thereof, and/or 5-azacytidine dissolved in diluents, such as water, saline, or orange juice; (b) capsules, sachets, tablets, lozenges, and troches, each containing a predetermined amount of the active ingredient, as solids or granules; (c) powders; (d) suspensions in an appropriate liquid; and (e) suitable emulsions. Liquid formulations may include diluents, such as water and alcohols, for example, ethanol, benzyl alcohol, and the polyethylene alcohols, either with or without the addition of a pharmaceutically acceptable surfactant. Capsule forms can be of the ordinary hard- or soft-shelled gelatin type containing, for example, surfactants, lubricants, and inert fillers, such as lactose, sucrose, calcium phosphate, and corn starch. Tablet forms can include one or more of lactose, sucrose, mannitol, corn starch, potato starch, alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, disintegrating agents, moistening agents, preservatives, flavoring agents, and other pharmacologically compatible excipients. Lozenge forms can comprise the substance and/or 5-azacytidine in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the substance and/or 5-azacytidine in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to, such excipients as are known in the art.

[0062] Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The substance comprising, consisting essentially of, or consisting of the protein IGF2BP1 or comprising, consisting essentially of, or consisting of nucleic acid encoding the protein IGF2BP1 or the substance comprising, consisting essentially of, or consisting of the protein IGF2BP3 or comprising, consisting essentially of, or consisting of nucleic acid encoding the protein IGF2BP3, or any combination thereof, and/or 5-azacytidine, can be administered in a physiologically acceptable diluent in a pharmaceutical carrier, such as a sterile liquid or mixture of liquids, including water, saline, aqueous dextrose and related sugar solutions, an alcohol, such as ethanol or hexadecyl alcohol, a glycol, such as propylene glycol or polyethylene glycol, dimethylsulfoxide, glycerol, ketals such as 2,2-dimethyl-l ,3-dioxolane- 4-methanol, ethers, poly(ethyleneglycol) 400, oils, fatty acids, fatty acid esters or glycerides, or acetylated fatty acid glycerides with or without the addition of a pharmaceutically acceptable surfactant, such as a soap or a detergent, suspending agent, such as pectin, carbomers, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying agents and other pharmaceutical adjuvants.

[0063] Oils, which can be used in parenteral formulations, include petroleum, animal, vegetable, or synthetic oils. Specific examples of oils include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral. Suitable fatty acids for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.

[0064] Injectable formulations are in accordance with the invention. The requirements for effective pharmaceutical carriers for injectable compositions are well-known to those of ordinary skill in the art (see, e.g., Pharmaceutics and Pharmacy Practice, J.B. Lippincott Company, Philadelphia, PA, Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630 (1986), each of which is incorporated by reference herein in its entirety).

[0065] It will be appreciated by one of skill in the art that, in addition to the above- described pharmaceutical compositions, the substance comprising, consisting essentially of, or consisting of the protein IGF2BP1 or comprising, consisting essentially of, or consisting of nucleic acid encoding the protein IGF2BP1 or the substance comprising, consisting essentially of, or consisting of the protein IGF2BP3 or comprising, consisting essentially of, or consisting of nucleic acid encoding the protein IGF2BP3, or any combination thereof, and/or 5-azacytidine, can be formulated as inclusion complexes, such as cyclodextrin inclusion complexes, or liposomes.

[0066] For purposes of the invention, the amount or dose of the substance comprising, consisting essentially of, or consisting of the protein IGF2BP1 or comprising, consisting essentially of, or consisting of nucleic acid encoding the protein IGF2BP1 or the substance comprising, consisting essentially of, or consisting of the protein IGF2BP3 or comprising, consisting essentially of, or consisting of nucleic acid encoding the protein IGF2BP3, or any combination thereof, and/or 5-azacytidine, administered should be sufficient to effect, e.g., a therapeutic or prophylactic response, in the subject or mammal over a reasonable time frame. For example, the dose of the substance and/or 5-azacytidine should be sufficient to treat or prevent a condition as described herein in a period of from about 2 hours or longer, e.g., 12 to 24 or more hours, from the time of administration. In certain embodiments, the time period could be even longer. The dose will be determined by the efficacy of the particular substance and/or 5-azacytidine and the condition of the mammal (e.g., human), as well as the body weight of the mammal (e.g., human) to be treated. The effect of IGF2BP1 appears to be greater than IGF2BP3; thus, for example, appropriate dosing may be achieved by using one instead of the other or a ratio of one to the other. Thus, a treatment regimen may be tailored based on the effects of IGF2BP1, IGF2BP3, and/or 5-azacytidine.

[0067] The dose of the substance comprising, consisting essentially of, or consisting of the protein IGF2BP1 or comprising, consisting essentially of, or consisting of nucleic acid encoding the protein IGF2BP1 or the substance comprising, consisting essentially of, or consisting of the protein IGF2BP3 or comprising, consisting essentially of, or consisting of nucleic acid encoding the protein IGF2BP3, or any combination thereof, and/or 5- azacytidine, will be determined by the existence, nature and extent of any adverse side effects that might accompany the administration of a particular substance and/or 5-azacytidine. Typically, the attending physician will decide the dosage of the substance and/or 5- azacytidine with which to treat each individual patient, taking into consideration a variety of factors, such as age, body weight, general health, diet, sex, substance and/or 5-azacytidine to be administered, route of administration, and the severity of the condition being treated. By way of example and not intending to limit the invention, the dose of the substance and/or 5- azacytidine can be about 0.001 to about 1000 mg/kg body weight of the subject being treated/day, from about 0.01 to about 10 mg/kg body weight/day, from about 0.01 mg to about 1 mg/kg body weight/day.

10068] The substance comprising, consisting essentially of, or consisting of the protein IGF2BP1 or comprising, consisting essentially of, or consisting of nucleic acid encoding the protein IGF2BP1 or the substance comprising, consisting essentially of, or consisting of the protein IGF2BP3 or comprising, consisting essentially of, or consisting of nucleic acid encoding the protein IGF2BP3, or any combination thereof, and/or 5-azacytidine, can be modified into a depot form, such that the manner in which the substance and/or 5-azacytidine is released into the body to which it is administered is controlled with respect to time and location within the body (see, for example, U.S. Patent 4,450,150, which is incorporated by reference herein in its entirety). Depot forms of the substance and/or 5-azacytidine can be, for example, an implantable composition comprising the substance and/or 5-azacytidine and a porous or non-porous material, such as a polymer, wherein the substance and/or 5-azacytidine is encapsulated by or diffused throughout the material and/or degradation of the non-porous material. The depot is then implanted into the desired location within the body and the substance and/or 5-azacytidine is released from the implant at a predetermined rate.

[0069] In an embodiment, the inventive system comprising a lentivirus vector particle, or composition comprising the inventive system, may be introduced into a host cell by

"transfection," "transformation," or "transduction." The terms "transfection,"

"transformation," or "transduction," as used herein, refer to the introduction of one or more exogenous polynucleotides into a host cell by using physical or chemical methods. Many transfection techniques are known in the art and include, for example, calcium phosphate DNA co-precipitation (see, e.g., Murray E.J. (ed.), Methods in Molecular Biology, Vol. 7, Gene Transfer and Expression Protocols, Humana Press (1991)); DEAE-dextran;

electroporation; cationic liposome-mediated transfection; tungsten particle-facilitated microparticle bombardment (Johnston, Nature, 346: 776-777 (1990)); and strontium phosphate DNA co-precipitation (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)).

Lentiviral vectors typically are introduced into host cells after growth of infectious particles in suitable packaging cells.

[0070] The terms "treat" and "prevent," as well as words stemming therefrom, as used herein, do not necessarily imply 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. In this respect, the inventive methods can provide any amount of any level of treatment or prevention, and an "effective amount" provides any such level of treatment or prevention. Furthermore, the treatment or prevention provided by the inventive methods can include treatment or prevention of one or more conditions or symptoms being treated or prevented. Also, for purposes herein, "prevention" can encompass delaying the onset of a disease described herein, e.g., sickle cell disease, or a symptom or condition thereof, e.g., cell sickling. [0071] The following includes certain aspects of the invention.

[0072] Aspect 1 . A method of increasing the amount of fetal hemoglobin in a cell, wherein the cell can be ex vivo, the method comprising, consisting essentially of, or consisting of administering to a stem cell or an erythroid cell an effective amount of a substance, the substance comprising, consisting essentially of, or consisting of the protein IGF2BP 1 or comprising, consisting essentially of, or consisting of nucleic acid encoding the protein IGF2BP 1 , wherein the administration of the substance increases the amount of fetal hemoglobin in the cell.

[0073] Aspect 2. The method of aspect 1 , wherein the substance is a plasmid, nucleic acid, or derivative thereof encoding the protein IGF2BP1 , wherein the plasmid, nucleic acid, or derivative thereof is administered to the cell.

[0074] Aspect 3. The method of aspect 2, wherein the plasmid, nucleic acid, or derivative thereof is for a virus or other nucleic acid delivery technique.

[0075] Aspect 4. The method of aspect 2 or 3, wherein the plasmid, nucleic acid, or derivative thereof includes at least one erythroid-specific element.

[0076] Aspect 5. The method of any one of aspects 2-4, wherein the plasmid, nucleic acid, or derivative thereof encodes a let-7 resistant transcript of the protein IGF2BP 1 .

[0077] Aspect 6. The method of aspect 1 , wherein the substance is viral nucleic acid encoding the protein IGF2BP1 , wherein the viral nucleic acid is administered to the cell through viral transduction.

[0078] Aspect 7. The method of aspect 6, wherein the viral nucleic acid includes at least one erythroid-specific element.

[0079] Aspect 8. The method of aspect 6 or 7, wherein the viral nucleic acid encodes a let-7 resistant transcript of the protein IGF2BP1.

[0080] Aspect 9. The method of any one of aspects 6-8, wherein the viral nucleic acid comprises, consists essentially of, or consists of lentiviral nucleic acid.

[0081] Aspect 10. The method of aspect 1 , wherein the substance comprises, consists essentially of, or consists of the protein IGF2BP1 , and the substance is administered directly to the cell.

[0082] Aspect 1 1 . The method of any one of aspects 1 -10, wherein the cell is in or transplanted into a mammal.

[0083] Aspect 12. The method of aspect 1 1 , wherein the mammal is a human. [0084] Aspect 13. A method of decreasing the amount of adult type hemoglobin in a cell, the method comprising, consisting essentially of, or consisting of administering to a stem cell or an erythroid cell an effective amount of a substance, the substance comprising, consisting essentially of, or consisting of the protein IGF2BP1 or comprising, consisting essentially of, or consisting of nucleic acid encoding the protein IGF2BP1 , wherein the administration of the substance decreases the amount of adult type hemoglobin, optionally including sickle cell hemoglobin, in the cell.

[0085] Aspect 14. The method of aspect 13, wherein the substance is a plasmid, nucleic acid, or derivative thereof encoding the protein IGF2BP1 , wherein the plasmid, nucleic acid, or derivative thereof is administered to the cell.

[0086] Aspect 15. The method of aspect 14, wherein the plasmid, nucleic acid, or derivative thereof is for a virus or other nucleic acid delivery technique.

[0087] Aspect 16. The method of aspect 14 or 15, wherein the plasmid, nucleic acid, or derivative thereof includes at least one erythroid-specific element.

[0088] Aspect 17. The method of any one of aspects 14-16, wherein the plasmid, nucleic acid, or derivative thereof encodes a let-7 resistant transcript of the protein IGF2BP1.

[0089] Aspect 18. The method of aspect 13, wherein the substance is viral nucleic acid encoding the protein IGF2BP1 , wherein the viral nucleic acid is administered to the cell through viral transduction.

[0090] Aspect 19. The method of aspect 18, wherein the viral nucleic acid includes at least one erythroid-specific element.

[0091] Aspect 20. The method of aspect 18 or 19, wherein the viral nucleic acid encodes a let-7 resistant transcript of the protein IGF2BP1.

[0092] Aspect 21. The method of any one of aspects 18-20, wherein the viral nucleic acid comprises, consists essentially of, or consists of lentiviral nucleic acid.

[0093] Aspect 22. The method of aspect 13, wherein the substance comprises, consists essentially of, or consists of the protein IGF2BP1 , and the substance is administered directly to the cell.

[0094] Aspect 23. The method of any one of aspects 13-22, wherein the cell is in or transplanted into a mammal.

[0095] Aspect 24. The method of aspect 23, wherein the mammal is a human.

[0096] Aspect 25. A substance for use in preventing or treating sickle cell disease in a mammal, the substance comprising, consisting essentially of, or consisting of the protein IGF2BP1 or comprising, consisting essentially of, or consisting of nucleic acid encoding the protein IGF2BP1.

[0097] Aspect 26. The substance of aspect 25, wherein the substance is a plasmid, nucleic acid, or derivative thereof encoding the protein IGF2BP1 , wherein the plasmid, nucleic acid, or derivative thereof is for direct administration to the mammal.

[0098] Aspect 27. The substance of aspect 25, wherein the substance is a plasmid, nucleic acid, or derivative thereof encoding the protein IGF2BP 1 , wherein the substance is for administering to a stem cell or erythroid cell, and the cell is for administering to the mammal.

[0099] Aspect 28. The substance of aspect 25, wherein the substance is a plasmid, nucleic acid, or derivative thereof encoding the protein IGF2BP1 , wherein the substance is for administering to a stem cell or erythroid cell extracted from the mammal, and the cell is for administering to the mammal.

[0100] Aspect 29. The substance of any one of aspects 26-28, wherein the plasmid, nucleic acid, or derivative thereof is for a virus or other nucleic acid delivery technique.

[0101] Aspect 30. The substance of any one of aspects 26-29, wherein the plasmid, nucleic acid, or derivative thereof includes at least one erythroid-specific element.

[0102] Aspect 31. The substance of any one of aspects 26-30, wherein the plasmid, nucleic acid, or derivative thereof encodes a let-7 resistant transcript of the protein IGF2BP1.

[0103] Aspect 32. The substance of aspect 25, wherein the substance is viral nucleic acid encoding the protein IGF2BP1 , wherein the viral nucleic acid is for

administration to the mammal through viral transduction.

[0104] Aspect 33. The substance of aspect 25, wherein the substance is viral nucleic acid encoding the protein IGF2BP1 , wherein the substance is for administering to a stem cell or an erythroid cell through viral transduction, and the cell is for administering to the mammal.

[0105] Aspect 34. The substance of aspect 25, wherein the substance is viral nucleic acid encoding the protein IGF2BP1 , wherein the substance is for administering through viral transduction to a stem cell or an erythroid cell extracted from the mammal, and the cell is for administering to the mammal. [0106] Aspect 35. The substance of any one of aspects 32-34, wherein the viral nucleic acid includes at least one erythroid-specific element.

[0107] Aspect 36. The substance of any one of aspects 32-35, wherein the viral nucleic acid encodes a let-7 resistant transcript of the protein IGF2BP1.

[0108] Aspect 37. The substance of any one of aspects 32-36, wherein the viral nucleic acid comprises, consists essentially of, or consists of lentiviral nucleic acid.

[0109] Aspect 38. The substance of aspect 25, wherein the substance comprises, consists essentially of, or consists of the protein IGF2BP1 , and the substance is for direct administration to the mammal.

[0110] Aspect 39. The substance of any one of aspects 25-38, wherein the mammal is a human.

[0111] Aspect 40. A substance for preventing or treating a beta-thalassemia in a mammal, the substance comprising, consisting essentially of, or consisting of the protein IGF2BP1 or comprising, consisting essentially of, or consisting of nucleic acid encoding the protein IGF2BP1.

[0112] Aspect 41. The substance of aspect 40, wherein the substance is a plasmid, nucleic acid, or derivative thereof encoding the protein IGF2BP1, wherein the plasmid, nucleic acid, or derivative thereof is for direct administration to the mammal.

[0113] Aspect 42. The substance of aspect 40, wherein the substance is a plasmid, nucleic acid, or derivative thereof encoding the protein IGF2BP1 , wherein the substance is for administering to a stem cell or erythroid cell, and the cell is for administering to the mammal.

[0114] Aspect 43. The substance of aspect 40, wherein the substance is a plasmid, nucleic acid, or derivative thereof encoding the protein IGF2BP1 , wherein the substance is for administering to a stem cell or erythroid cell extracted from the mammal, and the cell is for administering to the mammal.

[0115] Aspect 44. The substance of any one of aspects 41-43, wherein the plasmid, nucleic acid, or derivative thereof is for a virus or other nucleic acid delivery technique.

[0116] Aspect 45. The substance of any one of aspects 41 -44, wherein the plasmid, nucleic acid, or derivative thereof includes at least one erythroid-specific element. |0117] Aspect 46. The substance of any one of aspects 41-45, wherein the plasmid, nucleic acid, or derivative thereof encodes a let-7 resistant transcript of the protein IGF2BP1.

[0118] Aspect 47. The substance of aspect 40, wherein the substance is viral nucleic acid encoding the protein IGF2BP1 , wherein the viral nucleic acid is for

administration to the mammal through viral transduction.

[0119] Aspect 48. The substance of aspect 40, wherein the substance is viral nucleic acid encoding the protein 1GF2BP1 , wherein the substance is for administering to a stem cell or an erythroid cell through viral transduction, and the cell is for administering to the mammal.

[0120] Aspect 49. The substance of aspect 40, wherein the substance is viral nucleic acid encoding the protein IGF2BP1 , wherein the substance is for administering through viral transduction to a stem cell or an erythroid cell extracted from the mammal, and the cell is for administering to the mammal.

[0121] Aspect 50. The substance of any one of aspects 47-49, wherein the viral nucleic acid includes at least one erythroid-specific element.

[0122] Aspect 51. The substance of any one of aspects 47-50, wherein the viral nucleic acid encodes a let-7 resistant transcript of the protein IGF2BP1.

[0123] Aspect 52. The substance of any one of aspects 47-51 , wherein the viral nucleic acid comprises, consists essentially of, or consists of lentiviral nucleic acid.

[0124] Aspect 53. The substance of aspect 40, wherein the substance comprises, consists essentially of, or consists of the protein IGF2BP1 , and the substance is for direct administration to the mammal.

[0125] Aspect 54. The substance of any one of aspects 40-53, wherein the mammal is a human.

[0126] Aspect 55. A method of preventing or treating sickle cell disease in a mammal, the method comprising, consisting essentially of, or consisting of administering to a mammal in need thereof an effective amount of a substance, the substance comprising, consisting essentially of, or consisting of the protein IGF2BP1 or comprising, consisting essentially of, or consisting of nucleic acid encoding the protein IGF2BP1 , wherein the administration of the substance prevents or treats sickle cell disease in the mammal. [0127] Aspect 56. The method of aspect 55, wherein the substance is a plasmid, nucleic acid, or derivative thereof encoding the protein IGF2BP1 , wherein the plasmid, nucleic acid, or derivative thereof is administered to the mammal.

[0128] Aspect 57. The method of aspect 55, further comprising administering to a stem cell or an erythroid cell the substance, wherein the substance is a plasmid, nucleic acid, or derivative thereof encoding the protein IGF2BP1 , and administering to the mammal the cell having been administered the plasmid, nucleic acid, or derivative thereof.

[0129] Aspect 58. The method of aspect 55, further comprising extracting from the mammal a stem cell or an erythroid cell, administering to the cell the substance, wherein the substance is a plasmid, nucleic acid, or derivative thereof encoding the protein IGF2BP1, and administering to the mammal the cell having been administered the plasmid, nucleic acid, or derivative thereof.

[0130] Aspect 59. The method of any one of aspects 56-58, wherein the plasmid, nucleic acid, or derivative thereof is for a virus or other nucleic acid delivery technique.

[0131] Aspect 60. The method of any one of aspects 56-59, wherein the plasmid, nucleic acid, or derivative thereof includes at least one erythroid-specific element.

[0132] Aspect 61. The method of any one of aspects 56-60, wherein the plasmid, nucleic acid, or derivative thereof encodes a let-7 resistant transcript of the protein IGF2BP1.

[0133] Aspect 62. The method of aspect 55, wherein the substance is viral nucleic acid encoding the protein IGF2BP1 , wherein the viral nucleic acid is administered to the mammal through viral transduction.

[0134] Aspect 63. The method of aspect 55, further comprising administering to a stem cell or an erythroid cell the substance through viral transduction, wherein the substance is viral nucleic acid encoding the protein IGF2BP1 , and administering to the mammal the cell having been administered the viral nucleic acid.

[0135] Aspect 64. The method of aspect 55, further comprising extracting from the mammal a stem cell or an erythroid cell, administering to the cell the substance through viral transduction, wherein the substance is viral nucleic acid encoding the protein IGF2BP1, and administering to the mammal the cell having been administered the viral nucleic acid.

[0136] Aspect 65. The method of any one of aspects 62-64, wherein the viral nucleic acid includes at least one erythroid-specific element.

[0137] Aspect 66. The method of any one of aspects 62-65, wherein the viral nucleic acid encodes a let-7 resistant transcript of the protein IGF2BP1. [0138] Aspect 67. The method of any one of aspects 62-66, wherein the viral nucleic acid comprises, consists essentially of, or consists of lentiviral nucleic acid.

[0139] Aspect 68. The method of aspect 55, wherein the substance comprises, consists essentially of, or consists of the protein IGF2BP1 , and the substance is administered directly to the mammal.

[0140] Aspect 69. The method of any one of aspects 55-68, wherein the mammal is a human.

[0141] Aspect 70. A method of preventing or treating a beta-thalassemia in a mammal, the method comprising, consisting essentially of, or consisting of administering to a mammal in need thereof an effective amount of a substance, the substance comprising, consisting essentially of, or consisting of the protein IGF2BP1 or comprising, consisting essentially of, or consisting of nucleic acid encoding the protein IGF2BP1, wherein the administration of the substance prevents or treats the beta-thalassemia in the mammal.

[0142] Aspect 71. The method of aspect 70, wherein the substance is a plasmid, nucleic acid, or derivative thereof encoding the protein IGF2BP1 , wherein the plasmid, nucleic acid, or derivative thereof is administered to the mammal.

[0143] Aspect 72. The method of aspect 70, further comprising administering to a stem cell or an erythroid cell the substance, wherein the substance is a plasmid, nucleic acid, or derivative thereof encoding the protein IGF2BP1 , and administering to the mammal the cell having been administered the plasmid, nucleic acid, or derivative thereof.

[0144] Aspect 73. The method of aspect 70, further comprising extracting from the mammal a stem cell or an erythroid cell, administering to the cell the substance, wherein the substance is a plasmid, nucleic acid, or derivative thereof encoding the protein IGF2BP1 , and administering to the mammal the cell having been administered the plasmid, nucleic acid, or derivative thereof.

[0145] Aspect 74. The method of any one of aspects 71-73, wherein the plasmid, nucleic acid, or derivative thereof is for a virus or other nucleic acid delivery technique.

[0146] Aspect 75. The method of any one of aspects 71-74, wherein the plasmid, nucleic acid, or derivative thereof includes at least one erythroid-specific element.

[0147] Aspect 76. The method of any one of aspects 71-75, wherein the plasmid, nucleic acid, or derivative thereof encodes a let-7 resistant transcript of the protein IGF2BP1. [0148] Aspect 77. The method of aspect 70, wherein the substance is viral nucleic acid encoding the protein IGF2BP1 , wherein the viral nucleic acid is administered to the mammal through viral transduction.

[0149] Aspect 78. The method of aspect 70, further comprising administering to a stem cell or an erythroid cell the substance through viral transduction, wherein the substance is viral nucleic acid encoding the protein IGF2BP1 , and administering to the mammal the cell having been administered the viral nucleic acid.

[0150] Aspect 79. The method of aspect 70, further comprising extracting from the mammal a stem cell or an erythroid cell, administering to the cell the substance through viral transduction, wherein the substance is viral nucleic acid encoding the protein IGF2BP 1 , and administering to the mammal the cell having been administered the viral nucleic acid.

[0151] Aspect 80. The method of any one of aspects 77-79, wherein the viral nucleic acid includes at least one erythroid-specific element.

[0152] Aspect 81. The method of any one of aspects 77-80, wherein the viral nucleic acid encodes a let-7 resistant transcript of the protein IGF2BP1.

[0153] Aspect 82. The method of any one of aspects 77-81 , wherein the viral nucleic acid comprises, consists essentially of, or consists of lentiviral nucleic acid.

[0154] Aspect 83. The method of aspect 70, wherein the substance comprises, consists essentially of, or consists of the protein IGF2BP1 , and the substance is administered directly to the mammal.

[0155] Aspect 84. The method of any one of aspects 70-83, wherein the mammal is a human.

[0156] Aspect 85. A method of increasing the amount of fetal hemoglobin in a cell, wherein the cell can be ex vivo, the method comprising, consisting essentially of, or consisting of administering to a stem cell or an erythroid cell an effective amount of a substance, the substance comprising, consisting essentially of, or consisting of the protein IGF2BP3 or comprising, consisting essentially of, or consisting of nucleic acid encoding the protein IGF2BP3, wherein the administration of the substance increases the amount of fetal hemoglobin in the cell.

[0157] Aspect 86. The method of aspect 85, wherein the substance is a plasmid, nucleic acid, or derivative thereof encoding the protein IGF2BP3, wherein the plasmid, nucleic acid, or derivative thereof is administered to the cell. [0158] Aspect 87. The method of aspect 86, wherein the plasmid, nucleic acid, or derivative thereof is for a virus or other nucleic acid delivery technique.

[0159] Aspect 88. The method of aspect 86 or 87, wherein the plasmid, nucleic acid, or derivative thereof includes at least one erythroid-specific element.

[0160] Aspect 89. The method of any one of aspects 86-88, wherein the plasmid, nucleic acid, or derivative thereof encodes a let-7 resistant transcript of the protein IGF2BP3.

[0161] Aspect 90. The method of aspect 85, wherein the substance is viral nucleic acid encoding the protein IGF2BP3, wherein the viral nucleic acid is administered to the cell through viral transduction.

[0162] Aspect 91. The method of aspect 90, wherein the viral nucleic acid includes at least one erythroid-specific element.

[0163] Aspect 92. The method of aspect 90 or 91 , wherein the viral nucleic acid encodes a let-7 resistant transcript of the protein IGF2BP3.

[0164] Aspect 93. The method of any one of aspects 90-92, wherein the viral nucleic acid comprises, consists essentially of, or consists of lentiviral nucleic acid.

[0165] Aspect 94. The method of aspect 85, wherein the substance comprises, consists essentially of, or consists of the protein IGF2BP1, and the substance is administered directly to the cell.

[0166] Aspect 95. The method of any one of aspects 85-94, wherein the cell is in or transplanted into a mammal.

[0167] Aspect 96. The method of aspect 95, wherein the mammal is a human.

[0168] Aspect 97. A method of decreasing the amount of adult type hemoglobin in a cell, the method comprising, consisting essentially of, or consisting of administering to a stem cell or an erythroid cell an effective amount of a substance, the substance comprising, consisting essentially of, or consisting of the protein IGF2BP3 or comprising, consisting essentially of, or consisting of nucleic acid encoding the protein IGF2BP3, wherein the administration of the substance decreases the amount of adult type hemoglobin, optionally including sickle cell hemoglobin, in the cell.

[0169] Aspect 98. The method of aspect 97, wherein the substance is a plasmid, nucleic acid, or derivative thereof encoding the protein IGF2BP3, wherein the plasmid, nucleic acid, or derivative thereof is administered to the cell.

[0170] Aspect 99. The method of aspect 98, wherein the plasmid, nucleic acid, or derivative thereof is for a virus or other nucleic acid delivery technique. [0171] Aspect 100. The method of aspect 98 or 99, wherein the plasmid, nucleic acid, or derivative thereof includes at least one erythroid-specific element.

[0172] Aspect 101. The method of any one of aspects 98-100, wherein the plasmid, nucleic acid, or derivative thereof encodes a let-7 resistant transcript of the protein IGF2BP3.

[0173] Aspect 102. The method of aspect 97, wherein the substance is viral nucleic acid encoding the protein IGF2BP3, wherein the viral nucleic acid is administered to the cell through viral transduction.

[0174] Aspect 103. The method of aspect 102, wherein the viral nucleic acid includes at least one erythroid-specific element.

[0175] Aspect 104. The method of aspect 102 or 103, wherein the viral nucleic acid encodes a let-7 resistant transcript of the protein IGF2BP3.

[0176] Aspect 105. The method of any one of aspects 102-104, wherein the viral nucleic acid comprises, consists essentially of, or consists of lentiviral nucleic acid.

[0177] Aspect 106. The method of aspect 97, wherein the substance comprises, consists essentially of, or consists of the protein IGF2BP1 , and the substance is administered directly to the cell.

[0178] Aspect 107. The method of any one of aspects 97-106, wherein the cell is in or transplanted into a mammal.

[0179] Aspect 108. The method of aspect 107, wherein the mammal is a human.

[0180] Aspect 109. A substance for use in preventing or treating sickle cell disease in a mammal, the substance comprising, consisting essentially of, or consisting of the protein IGF2BP3 or comprising, consisting essentially of, or consisting of nucleic acid encoding the protein IGF2BP3.

[0181] Aspect 1 10. The substance of aspect 109, wherein the substance is a plasmid, nucleic acid, or derivative thereof encoding the protein IGF2BP3, wherein the plasmid, nucleic acid, or derivative thereof is for direct administration to the mammal.

[0182] Aspect 1 1 1 . The substance of aspect 109, wherein the substance is a plasmid, nucleic acid, or derivative thereof encoding the protein IGF2BP3, wherein the substance is for administering to a stem cell or erythroid cell, and the cell is for administering to the mammal.

[0183] Aspect 1 12. The substance of aspect 109, wherein the substance is a plasmid, nucleic acid, or derivative thereof encoding the protein IGF2BP3, wherein the substance is for administering to a stem cell or erythroid cell extracted from the mammal, and the cell is for administering to the mammal.

[0184] Aspect 1 13. The substance of any one of aspects 1 10-1 12, wherein the plasmid, nucleic acid, or derivative thereof is for a virus or other nucleic acid delivery technique.

[0185] Aspect 1 14. The substance of any one of aspects 1 10-1 13, wherein the plasmid, nucleic acid, or derivative thereof includes at least one erythroid-specific element.

[0186] Aspect 1 15. The substance of any one of aspects 1 10-1 14, wherein the plasmid, nucleic acid, or derivative thereof encodes a let-7 resistant transcript of the protein IGF2BP3.

[0187] Aspect 1 16. The substance of aspect 109, wherein the substance is viral nucleic acid encoding the protein IGF2BP3, wherein the viral nucleic acid is for

administration to the mammal through viral transduction.

[0188] Aspect 1 17. The substance of aspect 109, wherein the substance is viral nucleic acid encoding the protein IGF2BP3, wherein the substance is for administering to a stem cell or an erythroid cell through viral transduction, and the cell is for administering to the mammal.

[0189] Aspect 1 18. The substance of aspect 109, wherein the substance is viral nucleic acid encoding the protein IGF2BP3, wherein the substance is for administering through viral transduction to a stem cell or an erythroid cell extracted from the mammal, and the cell is for administering to the mammal.

[0190] Aspect 1 19. The substance of any one of aspects 1 16-1 1 8, wherein the viral nucleic acid includes at least one erythroid-specific element.

[0191] Aspect 120. The substance of any one of aspects 1 16-1 19, wherein the viral nucleic acid encodes a let-7 resistant transcript of the protein IGF2BP3.

[0192] Aspect 121. The substance of any one of aspects 1 16-120, wherein the viral nucleic acid comprises, consists essentially of, or consists of lentiviral nucleic acid.

[0193] Aspect 122. The substance of aspect 109, wherein the substance comprises, consists essentially of, or consists of the protein IGF2BP 1 , and the substance is for direct administration to the mammal.

[0194] Aspect 123. The substance of any one of aspects 109-122, wherein the mammal is a human. [0195] Aspect 124. A substance for preventing or treating a beta-thalassemia in a mammal, the substance comprising, consisting essentially of, or consisting of the protein IGF2BP3 or comprising, consisting essentially of, or consisting of nucleic acid encoding the protein IGF2BP3.

[0196] Aspect 125. The substance of aspect 124, wherein the substance is a plasmid, nucleic acid, or derivative thereof encoding the protein IGF2BP3, wherein the plasmid, nucleic acid, or derivative thereof is for direct administration to the mammal.

[0197] Aspect 126. The substance of aspect 124, wherein the substance is a plasmid, nucleic acid, or derivative thereof encoding the protein IGF2BP3, wherein the substance is for administering to a stem cell or erythroid cell, and the cell is for administering to the mammal.

[0198] Aspect 127. The substance of aspect 124, wherein the substance is a plasmid, nucleic acid, or derivative thereof encoding the protein IGF2BP3, wherein the substance is for administering to a stem cell or erythroid cell extracted from the mammal, and the cell is for administering to the mammal.

[0199] Aspect 128. The substance of any one of aspects 125-127, wherein the plasmid, nucleic acid, or derivative thereof is for a virus or other nucleic acid delivery technique.

[0200] Aspect 129. The substance of any one of aspects 125-128, wherein the plasmid, nucleic acid, or derivative thereof includes at least one erythroid-specific element.

[0201] Aspect 130. The substance of any one of aspects 125-129, wherein the plasmid, nucleic acid, or derivative thereof encodes a let- 7 resistant transcript of the protein IGF2BP3.

[0202] Aspect 131. The substance of aspect 124, wherein the substance is viral nucleic acid encoding the protein IGF2BP3, wherein the viral nucleic acid is for

administration to the mammal through viral transduction.

[0203] Aspect 132. The substance of aspect 124, wherein the substance is viral nucleic acid encoding the protein IGF2BP3, wherein the substance is for administering to a stem cell or an erythroid cell through viral transduction, and the cell is for administering to the mammal.

[0204] Aspect 133. The substance of aspect 124, wherein the substance is viral nucleic acid encoding the protein IGF2BP3, wherein the substance is for administering through viral transduction to a stem cell or an erythroid cell extracted from the mammal, and the cell is for administering to the mammal.

[0205] Aspect 134. The substance of any one of aspects 131-133, wherein the viral nucleic acid includes at least one erythroid-specific element.

[0206] Aspect 135. The substance of any one of aspects 131 -134, wherein the viral nucleic acid encodes a let-7 resistant transcript of the protein IGF2BP3.

[0207] Aspect 136. The substance of any one of aspects 131- 135, wherein the viral nucleic acid comprises, consists essentially of, or consists of lentiviral nucleic acid.

[0208] Aspect 137. The substance of aspect 40, wherein the substance comprises, consists essentially of, or consists of the protein IGF2BP3, and the substance is for direct administration to the mammal.

[0209] Aspect 138. The substance of any one of aspects 124-137, wherein the mammal is a human.

[0210] Aspect 139. A method of preventing or treating sickle cell disease in a mammal, the method comprising, consisting essentially of, or consisting of administering to a mammal in need thereof an effective amount of a substance, the substance comprising, consisting essentially of, or consisting of the protein IGF2BP3 or comprising, consisting essentially of, or consisting of nucleic acid encoding the protein IGF2BP3, wherein the administration of the substance prevents or treats sickle cell disease in the mammal.

[0211] Aspect 140. The method of aspect 139, wherein the substance is a plasmid, nucleic acid, or derivative thereof encoding the protein IGF2BP3, wherein the plasmid, nucleic acid, or derivative thereof is administered to the mammal.

[0212] Aspect 141. The method of aspect 139, further comprising administering to a stem cell or an erythroid cell the substance, wherein the substance is a plasmid, nucleic acid, or derivative thereof encoding the protein IGF2BP3, and administering to the mammal the cell having been administered the plasmid, nucleic acid, or derivative thereof.

[0213] Aspect 142. The method of aspect 139, further comprising extracting from the mammal a stem cell or an erythroid cell, administering to the cell the substance, wherein the substance is a plasmid, nucleic acid, or derivative thereof encoding the protein 1GF2BP3, and administering to the mammal the cell having been administered the plasmid, nucleic acid, or derivative thereof. [0214] Aspect 143. The method of any one of aspects 140-142, wherein the plasmid, nucleic acid, or derivative thereof is for a virus or other nucleic acid delivery technique.

[0215] Aspect 144. The method of any one of aspects 140-143, wherein the plasmid, nucleic acid, or derivative thereof includes at least one erythroid-specific element.

[0216] Aspect 145. The method of any one of aspects 140-144, wherein the plasmid, nucleic acid, or derivative thereof encodes a let-7 resistant transcript of the protein IGF2BP3.

[0217] Aspect 146. The method of aspect 139, wherein the substance is viral nucleic acid encoding the protein IGF2BP3, wherein the viral nucleic acid is administered to the mammal through viral transduction.

[0218] Aspect 147. The method of aspect 139, further comprising administering to a stem cell or an erythroid cell the substance through viral transduction, wherein the substance is viral nucleic acid encoding the protein IGF2BP3, and administering to the mammal the cell having been administered the viral nucleic acid.

[0219] Aspect 148. The method of aspect 139, further comprising extracting from the mammal a stem cell or an erythroid cell, administering to the cell the substance through viral transduction, wherein the substance is viral nucleic acid encoding the protein IGF2BP3, and administering to the mammal the cell having been administered the viral nucleic acid.

[0220] Aspect 149. The method of any one of aspects 146-148, wherein the viral nucleic acid includes at least one erythroid-specific element.

[0221] Aspect 150. The method of any one of aspects 146-149, wherein the viral nucleic acid encodes a let-7 resistant transcript of the protein IGF2BP3.

[0222] Aspect 151. The method of any one of aspects 146-150, wherein the viral nucleic acid comprises, consists essentially of, or consists of lentiviral nucleic acid.

[0223] Aspect 152. The method of aspect 139, wherein the substance comprises, consists essentially of, or consists of the protein IGF2BP3, and the substance is administered directly to the mammal.

[0224] Aspect 153. The method of any one of aspects 139-152, wherein the mammal is a human.

[0225] Aspect 154. A method of preventing or treating a beta-thalassemia in a mammal, the method comprising, consisting essentially of, or consisting of administering to a mammal in need thereof an effective amount of a substance, the substance comprising, consisting essentially of, or consisting of the protein IGF2BP3 or comprising, consisting essentially of, or consisting of nucleic acid encoding the protein IGF2BP3, wherein the administration of the substance prevents or treats the beta-thalassemia in the mammal.

[0226] Aspect 155. The method of aspect 154, wherein the substance is a plasmid, nucleic acid, or derivative thereof encoding the protein 1GF2BP3, wherein the plasmid, nucleic acid, or derivative thereof is administered to the mammal.

[0227] Aspect 156. The method of aspect 154, further comprising administering to a stem cell or an erythroid cell the substance, wherein the substance is a plasmid, nucleic acid, or derivative thereof encoding the protein IGF2BP3, and administering to the mammal the cell having been administered the plasmid, nucleic acid, or derivative thereof.

[0228] Aspect 157. The method of aspect 154, further comprising extracting from the mammal a stem cell or an erythroid cell, administering to the cell the substance, wherein the substance is a plasmid, nucleic acid, or derivative thereof encoding the protein IGF2BP3, and administering to the mammal the cell having been administered the plasmid, nucleic acid, or derivative thereof.

[0229] Aspect 158. The method of any one of aspects 155-157, wherein the plasmid, nucleic acid, or derivative thereof is for a virus or other nucleic acid delivery technique.

[0230] Aspect 159. The method of any one of aspects 155-158, wherein the plasmid, nucleic acid, or derivative thereof includes at least one erythroid-specific element.

[0231] Aspect 160. The method of any one of aspects 155-159, wherein the plasmid, nucleic acid, or derivative thereof encodes a let-7 resistant transcript of the protein IGF2BP3.

[0232] Aspect 161. The method of aspect 154, wherein the substance is viral nucleic acid encoding the protein IGF2BP3, wherein the viral nucleic acid is administered to the mammal through viral transduction.

[0233] Aspect 162. The method of aspect 154, further comprising administering to a stem cell or an erythroid cell the substance through viral transduction, wherein the substance is viral nucleic acid encoding the protein IGF2BP3, and administering to the mammal the cell having been administered the viral nucleic acid.

[0234] Aspect 163. The method of aspect 154, further comprising extracting from the mammal a stem cell or an erythroid cell, administering to the cell the substance through viral transduction, wherein the substance is viral nucleic acid encoding the protein IGF2BP3, and administering to the mammal the cell having been administered the viral nucleic acid.

[0235] Aspect 164. The method of any one of aspects 161 -79, wherein the viral nucleic acid includes at least one erythroid-specific element.

[0236] Aspect 165. The method of any one of aspects 161-80, wherein the viral nucleic acid encodes a let-7 resistant transcript of the protein IGF2BP3.

[0237] Aspect 166. The method of any one of aspects 161-81 , wherein the viral nucleic acid comprises, consists essentially of, or consists of lentiviral nucleic acid.

[0238] Aspect 167. The method of aspect 154, wherein the substance comprises, consists essentially of, or consists of the protein IGF2BP3, and the substance is administered directly to the mammal.

[0239] Aspect 168. The method of any one of aspects 154-167, wherein the mammal is a human.

[0240] Aspect 169. A method of increasing the amount of fetal hemoglobin in a cell, wherein the cell is optionally ex vivo, the method comprising, consisting essentially of, or consisting of administering to a stem cell or an erythroid cell an effective amount of 5- azacytidine, wherein the administration of 5-azacytidine increases the amount of fetal hemoglobin in the cell.

[0241] Aspect 170. The method of aspect 169, wherein the cell is in or transplanted into a mammal.

[0242] Aspect 171 . The method of aspect 170, wherein the mammal is a human.

[0243] Aspect 172. A method of decreasing the amount of adult type hemoglobin in a cell, wherein the cell is optionally ex vivo, the method comprising, consisting essentially of, or consisting of administering to a stem cell or an erythroid cell an effective amount of 5- azacytidine, wherein the administration of 5-azacytidine decreases the amount of adult type hemoglobin, optionally including sickle cell hemoglobin, in the cell.

[0244] Aspect 173. The method of aspect 172, wherein the cell is in or transplanted into a mammal.

[0245] Aspect 174. The method of aspect 173, wherein the mammal is a human.

[0246] Aspect 175. 5-Azacytidine for use in preventing or treating sickle cell disease in a mammal. [0247] Aspect 176. The 5-azacytidine of aspect 175, wherein the 5-azacytidine is for administering to a stem cell or erythroid cell, and the cell is for administering to the mammal.

[0248] Aspect 177. The 5-azacytidine of aspect 175, wherein the 5-azacytidine is for administering to a stem cell or erythroid cell extracted from the mammal, and the cell is for administering to the mammal.

[0249] Aspect 178. The 5-azacytidine of any one of aspects 175-177, wherein the mammal is a human.

[0250] Aspect 179. 5-azacytidine for use in preventing or treating a β-thalassemia in a mammal.

[0251] Aspect 180. The 5-azacytidine of aspect 179, wherein the 5-azacytidine is for administering to a stem cell or erythroid cell, and the cell is for administering to the mammal.

[0252] Aspect 181. The 5-azacytidine of aspect 179, wherein the 5-azacytidine is for administering to a stem cell or erythroid cell extracted from the mammal, and the cell is for administering to the mammal.

[0253] Aspect 182. The substance of any one of aspects 179-181 , wherein the mammal is a human.

[0254] Aspect 183. A method of preventing or treating sickle cell disease in a mammal, the method comprising, consisting essentially of, or consisting of administering to a mammal in need thereof an effective amount of 5-azacytidine, wherein the administration of 5-azacytidine prevents or treats sickle cell disease in the mammal.

[0255] Aspect 184. The method of aspect 183, further comprising administering to a stem cell or an erythroid cell the 5-azacytidine, and administering to the mammal the cell having been administered the 5-azacytidine.

[0256] Aspect 185. The method of aspect 183, further comprising extracting from the mammal a stem cell or an erythroid cell, administering to the cell the 5-azacytidine, and administering to the mammal the cell having been in the contact with the 5-azacytidine.

[0257] Aspect 186. A method of preventing or treating a beta-thalassemia in a mammal, the method comprising, consisting essentially of, or consisting of administering to a mammal in need thereof an effective amount of 5-azacytidine, wherein the administration of the 5-azacytidine prevents or treats the beta-thalassemia in the mammal. [0258] Aspect 187. The method of aspect 186, further comprising administering to a stem cell or an erythroid cell the 5-azacytidine, and administering to the mammal the cell having been administered the 5-azacytidine.

[0259] Aspect 188. The method of aspect 186, further comprising extracting from the mammal a stem cell or an erythroid cell, administering to the cell the 5-azacytidine, and administering to the mammal the cell having been in the contact with the 5-azacytidine.

[0260] Aspect 189. A method of increasing the amount of fetal hemoglobin in a cell, wherein the cell can be ex vivo, the method comprising, consisting essentially of, or consisting of increasing the expression of IGF2BP1 and/or IGF2BP3 in the cell, wherein increasing the expression of 1GF2BP1 and/or IGF2BP3 increases the amount of fetal hemoglobin in the cell.

[0261] Aspect 190. A method of decreasing the amount of adult type hemoglobin in a cell, , wherein the cell can be ex vivo, the method comprising, consisting essentially of, or consisting of increasing the expression of IGF2BP1 and/or IGF2BP3 in the cell, wherein increasing the expression of IGF2BP1 and/or IGF2BP3 decreases the amount of adult type hemoglobin, optionally including sickle cell hemoglobin, in the cell.

[0262] Aspect 191. A method of preventing or treating sickle cell disease in a mammal, the method comprising, consisting essentially of, or consisting of increasing the expression of IGF2BP1 and/or IGF2BP3 in the mammal, wherein the increasing the expression of IGF2BP1 and/or IGF2BP3 prevents or treats sickle cell disease in the mammal.

[0263] Aspect 192. A method of preventing or treating beta-thalassemia in a mammal, the method comprising, consisting essentially of, or consisting of increasing the expression of IGF2BP1 and/or IGF2BP3 in the mammal, wherein the increasing the expression of IGF2BP1 and/or IGF2BP3 prevents or treats beta-thalassemia in the mammal.

[0264] It shall be noted that the preceding are merely examples of embodiments. Other exemplary embodiments are apparent from the entirety of the description herein. It will also be understood by one of ordinary skill in the art that each of these embodiments may be used in various combinations with the other embodiments provided herein.

[0265] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.

EXAMPLES

[0266] The following protocols were used for the Examples. [0267] Cell culture. Written informed consent was obtained from all research subjects prior to participation in this study, and approval for the research protocol and consent documents using primary erythroblasts and peripheral blood samples was granted by the National Institute of Diabetes and Digestive and Kidney Diseases Institutional Intramural Review Board. CD34(+) cells from adult human healthy volunteers were studied ex vivo using a serum-free 21 -day culture system as previously described (Lee et al., Blood,

122(6): 1034-1041 (2013)). Human cord blood CD34(+) erythroblasts were purchased from All Cells (Emeryville, CA) and ReachBio LLC (Seattle, WA, USA) and grown in the same conditions as previously described (Lee et al., Blood, 122(6): 1034-1041 (2013)).

[0268] Lentiviral vector construction, production and transduction. The empty lentiviral vector (control) with the human spectrin alpha gene (SPTAl) promoter was constructed as previously described (Lee et al, PLoS One 10(12):e0144977 (2015)). An erythroid-specific IGF2BP1 over-expression (IGF2BP1-OE) vector with human SPTAl promoter encoding the 1GF2BP1 open reading frame was produced by conventional RT-PCR with high fidelity Taq from cultured cord blood CD34(+) mRNA with primers excluding the 5' and 3' untranslated (UTR) regions and containing Xhol and BamHI restriction sites for cloning as follows: 5'- ACCCTCGAGTATGAACAAGCTTTACATCGGCAACCTCAACGA-3' (SEQ ID NO: 7) and 5'-CCGGATCCTCACTTCCTCCGTGCCTGGGCCT-3' (SEQ ID NO: 8). The consensus let-7 binding sequence encoded in the 3 'UTR region of IGF2BP1 mRNA was excluded from the IGF2BP1-OE lentivirus transgene to prevent degradation of the transcripts in the adult CD34(+) cells. Human IGF2BP3 open reading frame was synthesized by Eurofins MWG Operon LLC (Louisville, KY, USA) and cloned into SPTAl promoter empty vector using Xhol and Notl restriction sites (IGF2BP3-OE). Lentivirus was packaged using HEK293T cells (Thermo Scientific, Waltham, MA, USA) co-transfected with packaging helper virus plasmids (SEQ ID NOs: 2-4) (a gift from Dr. Derek Persons and Dr. Arthur Nienhuis, St. Jude Children's Research Hospital, Memphis, TN, USA) and the vector plasmid (SPTAl -empty vector control or SFTAI-IGF2BP1 (SEQ ID NO: 5) over-expression vector or SPTAl -IGF2BP3 over-expression vector (SEQ ID NO: 6)) as previously described (Lee et al, PLoS One 10(12):e0144977 (2015); Niwa et al., Gene, 108(2): 193-199 (1991); Hanawa et al., Mol. Ther., 5(3):242-251 (2002)). Following lentiviral production, CD34(+) cells were transduced on day 3 of phase I as previously described (Lee et al, PLoS One

10(12):e0144977 (2015)). [0269] Flow cytometry analysis. To evaluate the differentiation stages of maturation in CD34(+) cells, antibodies directed against transferrin receptor (CD71) and glycophorin A (Life Technologies, Grand Island, NY, USA) were utilized on culture days 14 and 21 and analyzed using the BD FACSAria I flow cytometer (BD Biosciences, San Jose, CA, USA) as previously described (Krivega et al., Blood, 126(5):665-672 (2015)). Thiazole orange (Sigma Aldrich, St. Louis, MO, USA) was used to assess enucleation on culture day 21.

[0270] Cytospin preparation and Wright-Giemsa staining. Cytospins were prepared by centrifugation of the cytoslides at 1000 rpm for 2 minutes using the Shandon Cytospin 4 (Thermo Fisher Scientific, Waltham, MA, USA). Cytoslides were then stained with Wright- Giemsa (Sigma-Aldrich, St. Louis, MO, USA) for 50 seconds and then washed twice in distilled water for 1 minute.

[0271] Quantitative PCR for mRNAs. Q-RT-PCR assays and conditions were performed as previously described (Lee et al., Blood, 122(6): 1034-1041 (2013)) with additional primers and probes for analysis of gene expression using the following Assay-on-Demand Gene Expression Products (Applied Biosystems, Grand Island, NY, USA): IGF2BP1

(Hs00977566_ml), IGF2BP3 (HsOl 122560_gl), c-MYC (Hs00153408_ml), ACTB

(Hs99999903_ml). Comparison to a standard curve from plasmid DNA encoding each target gene was used to calculate individual sample gene copy numbers.

[0272] Quantitative PCR analysis for the let-7 family of miRNAs. Q-RT-PCR assays and conditions were performed as previously described (Noh et al., J. Transl. Med., England, Vol. 7, p. 98 (2009)). Known concentrations of a synthetic targeted mature miRNA

oligonucleotide (1 : 10 serial dilutions, n = 6) were used for quantification of each let-7 family in the biological samples.

[0273] RNA-binding protein immunoprecipitation. RNA immunoprecipitation (RIP) was performed on day 14 cells cultured from three independent donors transduced with IGF2BP1 lentiviral particles with antibody against IGF2BP1 (MBL, Woburn, MA, USA, number RN007P) or Rabbit IgG using RiboCluster Profiler RIP-Assay Kit (MBL, catalog number RN1001) following manufacturer's protocol. Briefly, 10 to 20 million cells were washed three times in cold PBS and lysed in 500 μΐ lysis buffer supplemented with HALT Protease Inhibitor Cocktail (Thermo Fisher Scientific/Pierce Biotechnology, Rockford, IL, USA), 1.5 mM Dithiothreitol (Life Technologies), and 200 U/ml RNAseOut (Life Technologies). Cell lysates were pre-cleared using 50 μΐ Dynabeads Protein A (Life Technologies) for one hour at 4°C. Antibody immobilized beads were prepared using 15 μg of IGF2BP1 antibody or Rabbit IgG with 50 μΐ of Dynabeads Protein A for 1 hour at 4°C. Pre-cleared cell lysates were then incubated with either IGF2BP 1 -Protein A beads or Rabbit IgG-Protein A beads at 4°C overnight. Magnet was used to capture the antibody-immobilized beads and the supernatant was discarded. Beads were then washed four times with 1 ml cold washing buffer supplemented with 1.5 mM Dithiothreitol. In the last wash, 100 μΐ was removed for Western Blot analysis. RNA isolation was performed according to manufacturer's protocol with overnight precipitation at -20°C and reconstitution in 10 μΐ of RNase-free water.

[0274] HPLC analysis. Three million cultured cells at day 21 were pelleted and lysed in distilled water with two repeat freeze thaw cycles in a dry ice ethanol bath. Cell debris was removed by filtration through Ultrafree-MC devices (Millipore, Billerica, MA, USA).

Hemoglobin content was analyzed for fetal globin (HbF) and adult globin (HbA) using a 20x4 mm PolyCAT A column (PolyLC Incorporated, Columbia, MD, USA) fitted to a Shimadzu Prominence HPLC system (Shimadzu Scientific Instruments, Columbia, MD, USA). The hemoglobins were eluted during 15 minutes in 20 mM Bis-Tris, 2 mM KCN, pH 6.90 buffer with a gradient of 0% to 25% in a buffer consisting of 20 mM Bis-Tris, 2 mM KCN, 200 mM NaCl, pH 6.45. Variant Hemoglobin Control AF (Analytical Control Systems Incorporated, Fishers, IN, USA), which has values for HbA and HbF of approximately 60% and 40%), respectively, was used as a standard control. Hemoglobin proteins were detected by absorbance measurements at 415 nm. The HbA and HbF peaks were quantitated using the LabSolutions software (Shimadzu Scientific Instruments). Total areas under the HbA and HbF peaks were used for ratio comparisons.

[0275] Western analyses. Cytoplasmic and nuclear extracts were prepared using the NEPER Nuclear Protein Extraction Kit (Pierce Biotechnology) as previously described (Lee et al, Blood, 122(6): 1034-1041 (2013)), and protein extracts were separated on a NuPAGE NOVEX 4-12% Bis-Tris gel as described previously (Lee et al., Blood, 122(6): 1034-1041 (2013)). The nitrocellulose membranes were probed with the following antibodies: BCLl 1A (Abeam, Cambridge, MA, USA), IGF2BP1 (Cell Signaling Technology, Danvers, MA, USA), IGF2BP3 (Abeam), HMGA2 (Abeam), c-Myc (Cell Signaling Technology) and IGF2 (NeoScientific, Woburn, MA). Histone H3 (Abeam), Lamin Bl (Abeam), Alpha tubulin (ProSci Incorporated, Poway, CA, USA) and Beta-Actin (Abeam) were used as loading controls. To assess IGF2BP1 expression in fetal liver and adult bone marrow, human Fetal Liver Protein Medley (catalog number: 635342) was purchased from Clontech Laboratories (Mountain View, CA, USA), and the mononuclear cells from adult bone marrow were separated using Ficoll-Paque PREMIUM (GE Healthcare Life Sciences, Pittsburgh, PA, USA).

[0276] Confocal microscopy. Slides containing 70,000 sorted live cells on culture day 12 were fixed with 3.7% paraformaldehyde in PBS (Electron Microscopy Sciences, Hatfield, PA, USA) and permeabilized with 2% BSA/PBS containing 0.1% Triton X (Thermo Fisher Scientific, Grand Island, NY, USA). After cell permeabilization, slides were probed with antibody against human IGF2BP1 (1 :500, 8482S, Cell Signaling, Danvers, MA, USA) overnight at 4 °C in 2% BSA/PBS solution. Slides were incubated with secondary antibody against rabbit IgG (H+L) conjugated with CF-555 (1 :500, Sigma) in 2% BSA/PBS solution for one hour at room temperature. After secondary antibody incubation, slides were washed three times in PBS and once in distilled water then mounted with Pro Long Diamond Antifade Mountant with DAPI (Thermo Fisher Scientific). Laser scanned confocal images were obtained from LSM 5 Live Duoscan (Carl Zeiss, Oberkochen, Germany) and analyzed with Zen2007 software.

[0277] Statistical analysis. R eplicates are expressed as mean ± SD values and significance was calculated by one- or two-tailed Student's t-test.

EXAMPLE 1

[0278] This example demonstrates IGF2BP1 and IGF2BP3 are differentially expressed in fetal and adult human erythroid tissues.

[0279] In vivo expression levels of IGF2BP1 and IGF2BP3 were determined using human erythroid tissues. Both IGF2BP1 and IGF2BP3 are expressed in fetal liver

(IGF2BP1 : fetal liver: 3.5.E+02 ± 5.7.E+01 , adult bone marrow: below detection limits and IGF2BP3: fetal liver: 2.0.E+01 ± 2.7.E+00) but undetectable in bone marrow samples (Figure 2A). Among cultured primary erythroblasts, both IGF2BP1 and IGF2BP3 are highly expressed in cord blood erythroblasts (IGF2BP1 : cord blood: 1.3.E+03 ± 4.3.E+02 and adult blood: below detection limits; IGF2BP3: cord blood: 5.8.E+02 ± 2.4.E+02) but undetectable in adult blood erythroblasts (Figure 2B). At the mRNA level, IGF2BP1 was detected at higher levels in both fetal liver and cord blood samples compared to IGF2BP3.

[0280] To investigate the expression pattern of IGF2BP1 and IGF2BP3 at the protein level, Western blot analyses were performed for fetal liver, adult bone marrow, and erythroblasts cultured from cord and adult blood. IGF2BP1 and IGF2BP3 were detected in fetal liver and cord blood erythroblasts, with low or undetectable levels in adult bone marrow and adult blood erythroblasts (Figure 2C).

[0281] These results indicate that loss of IGF2BP1 and IGF2BP3 correlates with the fetal-to-adult developmental transition.

EXAMPLE 2

[0282] This example demonstrates IGF2BP1 and IGF2BP3 reverse adult erythroblasts to a more fetal-like pattern of globin genes and fetal hemoglobin expression, in accordance with embodiments of the invention.

[0283] To investigate the effects of IGF2BP1 and IGF2BP3 expression in cultured adult erythroblasts, an erythroid-specific IGF2BP1 over-expression (IGF2BP1-OE) or IGF2BP3 over-expression (IGF2BP3-OE) lentiviral vector was utilized in direct comparison with an empty vector control. Transductions were performed in CD34(+) cells from adult healthy volunteers cultivated ex vivo in erythropoietin-supplemented serum-free media for 21 days. IGF2BP1 and IGF2BP3 over-expression were confirmed by Q-RT-PCR and Western blot analyses at culture day 14 (Figures 3A-3D). Over-expression of IGF2BP3 gave two bands by Western analysis (Fig. 3D) with a strong 50 kDa band detected from the transgene that was not observed in non-transduced fetal liver, cord blood or adult blood samples.

[0284] Hemoglobin profiles (HPLC) were used to determine the effects of IGF2BP1 and IGF2BP3 over-expression on HbF levels at culture day 21. IGF2BP3-OE caused moderate effects on the HbF levels [HbF/(HbF + HbA)] (18.6 ± 1.0% in IGF2BP3-OE cells compared to 4.0 ± 2.1 in donor matched control cells, p= 0.0021 ). IGF2BP1 -OE caused a much more robust increase in the level of HbF [HbF/(HbF + HbA)] reaching 68.6 ± 3.9% in IGF2BP1- OE cells compared to 5.0 ± 1.8%) in donor matched control cells (p=0.0004). See Figure 4.

[0285] For protein localization studies, confocal imaging of sorted cells was utilized. IGF2BP1-OE caused IGF2BP 1 protein expression throughout the cytoplasm and localized to small granules. Granules were unevenly distributed and more abundantly observed in the perinuclear regions. Nuclear detection of IGF2BP1 protein remained at background control levels.

[0286] IGF2BP 1 -related changes in the differentiation, maturation and enucleation of the cultivated erythroblasts were assessed by flow cytometry. Detection of transferrin receptor (CD71 ) and glycophorin A (GPA) were performed at culture days 14 and 21 (Figures 5 and 6). The reduction in the loss of surface CD71 in IGF2BP 1 -OE cells compared to empty vector control suggests altered maturation kinetics over the 21 -day culture period. IGF2BP1 - OE did not prevent hemoglobin accumulation or terminal maturation of the cells.

Enucleation, as indicated by the lack of thiazole orange staining, was detected in

approximately 30% of the IGF2BP1 -OE and empty vector control samples (Figure 5C and 5D). Sorted IGF2BP1 -OE enucleated cells were imaged and demonstrated equivalent cellular morphology compared to empty vector control samples (Figures 5E and 5F).

Terminal maturation and enucleation did not cause a major reduction in HbF (Figures 6E and 6F). Pancellular distribution of HbF was detected for IGF2BP1-OE cells, with the HbF content of the enucleated cells remaining elevated at 70% compared with 5% HbF in the enucleated empty vector control cells.

[0287] The effects of IGF2BP 1 -OE on the expression of globin genes were characterized by Q-RT-PCR analyses at culture day 14. The changes observed in the globin genes, including high-levels of gamma-globin mRNA accompanied by suppression of the adult delta- and beta-globin genes, reflected a more fetal-like pattern in the IGF2BP1-OE cells compared to control transductions (Figures 7A-7H). IGF2BP 1-OE caused a dominance

[89.8% of all beta-like transcripts] of gamma-globin mRNA [control (empty vector) = 3.2E+06 ± 8.2E+05 copies/ng; IGF2BP1-OE = 2.0E+07 ± 5.9E+06 copies/ng; p=0.046], while beta-globin mRNA decreased to minor levels of about 10.1 % of all beta-like transcripts [control (empty vector) = 2.2E+07 ± 4.0E+06 copies/ng; IGF2BP1-OE = 2.2E+06 ± 6.2E+05 copies/ng; p=0.019] compared with the empty vector control transductions. In addition, delta-globin mRNA was also reduced to 0.03% of all the beta-like transcripts in IGF2BP1 - OE samples [control (empty vector) = 2.4E+05 ± 5.9E+04 copies/ng; IGF2BP1-OE = 7.4E+03 ± 1.8E+03 copies/ng; p=0.020]. The total output of transcripts from the beta- and alpha-gene clusters remained unchanged [Beta-cluster total output: control (empty vector) = 2.5E+07 ± 1.0E+07 copies/ng; IGF2BP 1 -OE = 2.2E+07 ± 9.6E+06 copies/ng; p>0.05;

Alpha-cluster total output: control (empty vector) = 1.4E+07 ± 6.8E+06 copies/ng; IGF2BP1 - OE = 1.0E+07 ± 4.8E+06 copies/ng; p>0.05].

[0288] In addition to the globin genes, the human fetal-to-adult developmental transition is identified by increased expression of the carbonic anhydrase I (CA ) gene and

carbohydrate modification due to the increased expression of the GCNT2 gene. Both CAl and GCNT2 were significantly down-regulated in IGF2BP1 -OE cells compared to empty vector control transductions (Figures 71 and 7J). [0289] IGF2BP3-OE cells maintained their ability to differentiate and enucleate ex vivo compared to donor-matched controls. The expression levels of globin genes were evaluated at culture day 14 by qRT-PCR and there was significantly increased gamma-globin expression levels compared to control transductions (control: 7.7.E+05 ± 1.7.E+05;

IGF2BP3-OE: 8.4.E+06 ± 3.2.E+06; p=0.018). Consistent with increased gamma-globin, HbF rose to moderately high levels upon IGF2BP3-OE (control: 4.0 ± 2.1%; IGF2BP3-OE: 18.6 ± 1.0%; p=0.0021). In addition, the expression pattern of the erythroid transcription factor BCL11A was investigated by qRT-PCR at culture day 14 and no significant changes were observed (control: 5.6.E+02 ± 2.7.E+02; IGF2BP3-OE: 6.7.E+02 ± 3.5.E+02; p=0.694). Minor decreases in BCL11 A protein levels were detected by Western analysis.

[0290] Overall, these results demonstrate that IGF2BP1 and IFG2BP3 regulate HbF and other markers of the fetal -to-adult developmental transition, suggesting that IGF2BP1 and IFG2BP3 reverse adult erythroblasts towards a more fetal-like phenotype. The effects of IGF2BP3-OE on HbF levels were less robust when compared to IGF2BP1-OE in cultured adult erythroblasts.

EXAMPLE 3

[0291] This example demonstrates IGF2BP1 expression regulates BCL1 1 A and HMGA2 in adult erythroblasts, in accordance with embodiments of the invention.

[0292] Potential causes for the effects of IGF2BP1 upon developmentally-related erythroid genes were explored. Initially the potential for IGF2BP1 over-expression to reduce the expression levels of the let-7 miRNAs in adult CD34(+) erythroblasts via a feedback-loop was investigated. IGF2BP1 -OE had little effect on the expression of the let-7 family of miRNAs (Figure 8). These results essentially exclude the interpretation that IGF2BP 1 acts mainly through a regulatory feedback loop in the let-7 cascade.

[0293] Additionally, known IGF2BP1 targets that are expressed in erythroid cells were investigated by Q-RT-PCR and Western blot analyses. In the three analyzed targets: c-MYC, IGF2 and Beta-Actin, no significant changes were observed at the mRNA or protein levels in the IGF2BP1 -OE samples compared to empty vector control transductions (Figure 9). This demonstrates that several known IGF2BP1 downstream targets remain unaffected by

IGF2BP1 expression in adult erythroblasts. There was no increase in the expression of LIN28. [0294] IGF2BP1-OE effects on the expression of BCL1 1 A, a transcription factor that has been reported to regulate HbF levels, and HMGA2, a downstream target of the let-7 family of miPvNAs, which has also been associated with the regulation of HbF, were investigated. While the mRNA levels of BCLllA and HMGA2 were not significantly changed (Figures 10A and 10B), IGF2BP1 over-expression caused profound effects at the protein level with decreased BCLl lA and increased HMGA2 (Figure I OC) {BCLllA mRNA levels among IGF2BP1-OE cells - control: 6.5.E+03 ± 3.8.E+03, IGF2BP1 -OE: 4.1.E+03 ± 1.2.E+02, p=0.403; BCL11 A protein detection was reduced to background levels by Western analysis compared to transduction control cell lysates).

[0295] The differences in mRNA and protein levels of BCL11 A and HMGA2 suggest post-transcriptional regulation of both proteins by IGF2BP1. Hence, BCLllA and HMGA2 transcripts were examined further as to whether they interact with IGF2BP1 by RNA- immunoprecipitation followed by Q-RT-PCR assays. Strikingly, both BCLllA and HMGA2 transcripts were enriched when compared to input and IgG control RNA samples (Figures 10D and 10E), suggesting that BCLllA and HMGA2 mRNAs bind to IGF2BP1 protein. RIP- enrichment of BCLllA mRNA was 4.6 ± 1.2 fold compared to input sample, and there was enrichment of mRNA encoding other modifiers of the globin gene, including KLF1 and ZBTB7A.

[0296] Overall, these results suggest that IGF2BP1 binds and post-transcriptionally increases HMGA2 levels. In addition, binding and post-transcriptional suppression of BCLl 1 A, a known epigenetic regulator of erythroblast fetal hemoglobin were identified.

[0297] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0298] The use of the terms "a" and "an" and "the" and "at least one" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term "at least one" followed by a list of one or more items (for example, "at least one of A and B") is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0299] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

CLAIM(S):

1. A method of increasing the amount of fetal hemoglobin in an ex vivo cell, the method comprising administering to a stem cell or an erythroid cell an effective amount of a substance, the substance comprising or encoding the protein IGF2BP1 or comprising or encoding the protein IGF2BP3, or a combination thereof, wherein the administration of the substance increases the amount of fetal hemoglobin in the cell.

2. The method of claim 1 , wherein the substance is a plasmid, nucleic acid, or derivative thereof encoding the protein, wherein the plasmid, nucleic acid, or derivative thereof is administered to the cell.

3. The method of claim 2, wherein the plasmid, nucleic acid, or derivative thereof is for a virus or other nucleic acid delivery technique.

4. The method of claim 2 or 3, wherein the plasmid, nucleic acid, or derivative thereof includes at least one erythroid-specific element.

5. The method of any one of claims 2-4, wherein the plasmid, nucleic acid, or derivative thereof encodes a let-7 resistant transcript of the protein.

6. The method of claim 1 , wherein the substance is viral nucleic acid encoding the protein, wherein the viral nucleic acid is administered to the cell through viral transduction.

7. The method of claim 6, wherein the viral nucleic acid includes at least one erythroid-specific element.

8. The method of claim 6 or 7, wherein the viral nucleic acid encodes a let-7 resistant transcript of the protein.

9. The method of any one of claims 6-8, wherein the viral nucleic acid comprises lentiviral nucleic acid.

10. The method of claim 1 , wherein the substance comprises the protein, and the substance is administered directly to the cell.

11. The method of any one of claims 1 -10, wherein the cell is in or transplanted into a mammal.

12. The method of claim 1 1 , wherein the mammal is a human.

13. A method of decreasing the amount of adult type hemoglobin in an ex vivo cell, the method comprising administering to a stem cell or an erythroid cell an effective amount of a substance, the substance comprising or encoding the protein IGF2BP1 or comprising or encoding the protein IGF2BP3, or a combination thereof, wherein the administration of the substance decreases the amount of adult type hemoglobin, optionally including sickle cell hemoglobin, in the cell.

14. The method of claim 13, wherein the substance is a plasmid, nucleic acid, or derivative thereof encoding the protein, wherein the plasmid, nucleic acid, or derivative thereof is administered to the cell.

15. The method of claim 14, wherein the plasmid, nucleic acid, or derivative thereof is for a virus or other nucleic acid delivery technique.

16. The method of claim 14 or 15, wherein the plasmid, nucleic acid, or derivative thereof includes at least one erythroid-specific element.

17. The method of any one of claims 14-16, wherein the plasmid, nucleic acid, or derivative thereof encodes a let-7 resistant transcript of the protein.

18. The method of claim 13, wherein the substance is viral nucleic acid encoding the protein, wherein the viral nucleic acid is administered to the cell through viral transduction.

19. The method of claim 18, wherein the viral nucleic acid includes at least one erythroid-specific element.

20. The method of claim 18 or 19, wherein the viral nucleic acid encodes a let-7 resistant transcript of the protein.

21. The method of any one of claims 18-20, wherein the viral nucleic acid comprises lentiviral nucleic acid.

22. The method of claim 13, wherein the substance comprises the protein, and the substance is administered directly to the cell.

23. The method of any one of claims 13-22, wherein the cell is in or transplanted into a mammal.

24. The method of claim 23, wherein the mammal is a human.

25. A substance for use in preventing or treating sickle cell disease in a mammal, the substance comprising or encoding the protein IGF2BP1 or comprising or encoding the protein IGF2BP3, or a combination thereof.

26. The substance of claim 25, wherein the substance is a plasmid, nucleic acid, or derivative thereof encoding the protein, wherein the plasmid, nucleic acid, or derivative thereof is for direct administration to the mammal.

27. The substance of claim 25, wherein the substance is a plasmid, nucleic acid, or derivative thereof encoding the protein, wherein the substance is for administering to a stem cell or erythroid cell, and the cell is for administering to the mammal.

28. The substance of claim 25, wherein the substance is a plasmid, nucleic acid, or derivative thereof encoding the protein, wherein the substance is for administering to a stem cell or erythroid cell extracted from the mammal, and the cell is for administering to the mammal.

29. The substance of any one of claims 26-28, wherein the plasmid, nucleic acid, or derivative thereof is for a virus or other nucleic acid delivery technique.

30. The substance of any one of claims 26-29, wherein the plasmid, nucleic acid, or derivative thereof includes at least one erythroid-specific element.

31. The substance of any one of claims 26-30, wherein the plasmid, nucleic acid, or derivative thereof encodes a let-7 resistant transcript of the protein.

32. The substance of claim 25, wherein the substance is viral nucleic acid encoding the protein, wherein the viral nucleic acid is for administration to the mammal through viral transduction.

33. The substance of claim 25, wherein the substance is viral nucleic acid encoding the protein, wherein the substance is for administering to a stem cell or an erythroid cell through viral transduction, and the cell is for administering to the mammal.

34. The substance of claim 25, wherein the substance is viral nucleic acid encoding the protein, wherein the substance is for administering through viral transduction to a stem cell or an erythroid cell extracted from the mammal, and the cell is for administering to the mammal.

35. The substance of any one of claims 32-34, wherein the viral nucleic acid includes at least one erythroid-specific element.

36. The substance of any one of claims 32-35, wherein the viral nucleic acid encodes a let-7 resistant transcript of the protein.

37. The substance of any one of claims 32-36, wherein the viral nucleic acid comprises lentiviral nucleic acid.

38. The substance of claim 25, wherein the substance comprises the protein, and the substance is for direct administration to the mammal.

39. The substance of any one of claims 25-38, wherein the mammal is a human.

40. A substance for preventing or treating a β-thalassemia in a mammal, the substance comprising or encoding the protein IGF2BP1 or comprising or encoding the protein IGF2BP3, or a combination thereof.

41. The substance of claim 40, wherein the substance is a plasmid, nucleic acid, or derivative thereof encoding the protein, wherein the plasmid, nucleic acid, or derivative thereof is for direct administration to the mammal.

42. The substance of claim 40, wherein the substance is a plasmid, nucleic acid, or derivative thereof encoding the protein, wherein the substance is for administering to a stem cell or erythroid cell, and the cell is for administering to the mammal.

43. The substance of claim 40, wherein the substance is a plasmid, nucleic acid, or derivative thereof encoding the protein, wherein the substance is for administering to a stem cell or erythroid cell extracted from the mammal, and the cell is for administering to the mammal.

44. The substance of any one of claims 41-43, wherein the plasmid, nucleic acid, or derivative thereof is for a virus or other nucleic acid delivery technique.

45. The substance of any one of claims 41 -44, wherein the plasmid, nucleic acid, or derivative thereof includes at least one erythroid-specific element.

46. The substance of any one of claims 41-45, wherein the plasmid, nucleic acid, or derivative thereof encodes a let-7 resistant transcript of the protein.

47. The substance of claim 40, wherein the substance is viral nucleic acid encoding the protein, wherein the viral nucleic acid is for administration to the mammal through viral transduction.

48. The substance of claim 40, wherein the substance is viral nucleic acid encoding the protein IGF2BP1 and/or the protein IGF2BP3, wherein the substance is for administering to a stem cell or an erythroid cell through viral transduction, and the cell is for administering to the mammal.

49. The substance of claim 40, wherein the substance is viral nucleic acid encoding the protein, wherein the substance is for administering through viral transduction to a stem cell or an erythroid cell extracted from the mammal, and the cell is for administering to the mammal.

50. The substance of any one of claims 47-49, wherein the viral nucleic acid includes at least one erythroid-specific element.

51. The substance of any one of claims 47-50, wherein the viral nucleic acid encodes a let-7 resistant transcript of the protein.

52. The substance of any one of claims 47-51 , wherein the viral nucleic acid comprises lentiviral nucleic acid.

53. The substance of claim 40, wherein the substance comprises the protein, and the substance is for direct administration to the mammal.

54. The substance of any one of claims 40-53, wherein the mammal is a human.

55. A plasmid comprising SEQ ID NO: 5 or 6.

56. A method of increasing the amount of fetal hemoglobin in an ex vivo cell, the method comprising administering to a stem cell or an erythroid cell an effective amount of 5- azacytidine, wherein the administration of 5-azacytidine increases the amount of fetal hemoglobin in the cell.

57. The method of claim 56, wherein the cell is in or transplanted into a mammal.

58. The method of claim 57, wherein the mammal is a human.

59. A method of decreasing the amount of adult type hemoglobin in a cell, wherein the cell is optionally ex vivo, the method comprising administering to a stem cell or an erythroid cell an effective amount of 5-azacytidine, wherein the administration of 5- azacytidine decreases the amount of adult type hemoglobin, optionally including sickle cell hemoglobin, in the cell.

60. The method of claim 59, wherein the cell is in or transplanted into a mammal.

61. The method of claim 60, wherein the mammal is a human.

62. 5-Azacytidine for use in preventing or treating sickle cell disease in a mammal.

63. The 5-azacytidine of claim 62, wherein the 5-azacytidine is for administering to a stem cell or erythroid cell, and the cell is for administering to the mammal.

64. The 5-azacytidine of claim 62, wherein the 5-azacytidine is for administering to a stem cell or erythroid cell extracted from the mammal, and the cell is for administering to the mammal.

65. The 5-azacytidine of any one of claims 62-64, wherein the mammal is a human.

66. 5-azacytidine for use in preventing or treating a beta-thalassemia in a mammal.

67. The 5-azacytidine of claim 66, wherein the 5-azacytidine is for administering to a stem cell or erythroid cell, and the cell is for administering to the mammal.

68. The 5-azacytidine of claim 66, wherein the 5-azacytidine is for administering to a stem cell or erythroid cell extracted from the mammal, and the cell is for administering to the mammal.

69. The 5-azacytidine of any one of aspects 66-68, wherein the mammal is a human.

70. A method of increasing the amount of fetal hemoglobin in an ex vivo cell, the method comprising increasing the expression of IGF2BP1 and/or IGF2BP3 in the cell, wherein increasing the expression of IGF2BP1 and/or IGF2BP3 increases the amount of fetal hemoglobin in the cell.

71. A method of decreasing the amount of adult type hemoglobin in an ex vivo cell, the method comprising increasing the expression of IGF2BP1 and/or IGF2BP3 in the cell, wherein increasing the expression of IGF2BP1 and/or IGF2BP3 decreases the amount of adult type hemoglobin, optionally including sickle cell hemoglobin, in the cell.