WO2014200557A1 - Method of increasing the amount of fetal hemoglobin in a cell and/or mammal - Google Patents

Abstract

In embodiments of the invention, the invention provides a method of increasing the amount of fetal hemoglobin or decreasing the amount of adult hemoglobin in a cell. In other embodiments of the invention, the invention provides a method of preventing or treating sickle cell disease or a β-thalassemia in a mammal. In another embodiment of the invention, the invention provides plasmids encoding the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof.

Description

METHOD OF INCREASING THE AMOUNT OF FETAL HEMOGLOBIN

IN A CELL AND/OR MAMMAL

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims the benefit of U.S. Provisional Patent Application No. 61/833,275, filed June 10, 2013 and U.S. Provisional Patent Application No. 61/837,840, filed June 21 , 2013, each of which is incorporated herein 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 82,192 Byte ASCII (Text) file named "714367ST25.txt," created on October 30, 2013.

BACKGROUND OF THE INVENTION

[0003] In humans and some other mammals, the composition of hemoglobin tetramers in erythrocytes switch from fetal hemoglobin (HbF, having two alpha globin chains and two gamma globin chains - α2γ2) to adult hemoglobin (HbA, having two alpha globin chains and two beta globin chains - α2β2) during the last stages of fetal development until early infancy. HbF is an important known modifier of the clinical symptoms for patients with sickle cell disease (SCD) and β-thalassemias, which are among the most common genetic disorders worldwide.

[0004] In patients with SCD, the polymerization of sickle hemoglobin results in erythrocyte deformation and hemolysis. SCD patient clinical outcomes are largely improved by inhibition of the polymerization of sickle hemoglobin, where HbF is resistant to such polymerization.

[0005] In β-thalassemias, decreased production of beta-globin causes imbalanced globin polypeptide chain synthesis, and leads to severe effects on erythroid cell maturation and survival. The loss of beta-globin expression may be compensated by an increase in HbF production that leads to improvement of the clinical phenotype. [0006] Genome-wide association studies (GWAS) in both normal individuals and patients with β-hemoglobinopathies have identified BCLl 1A, HSB IL-MYB, and HBB clusters as having an association with the persistence of HbF in adults. Suppression of the BCLl 1 A transcription factor causes an increase in HbF levels. Lin28 is a highly conserved gene that is expressed in the early stages of development of multicellular organisms. In the nematode, Caenorhabditis elegans, lin28 regulates developmental timing at the larvae L2 stage. Mammals express two homologs of the C. elegans lin28 gene known as LIN28A and LIN28B. Human LIN28 genetic variation correlates with developmental timing characteristics such as variation in height, timing of puberty and age at natural menopause. The effects of LIN28 proteins depend, in part, on the cell type in which they are expressed, as evidenced by its role in promoting the pluripotency of stem cells, as well as the differentiation of skeletal muscle cells.

[0007] Upregulation of let-7 miRNAs have been suggested to be involved with hemoglobin switching during the fetal-to-adult developmental transition. Let-7 and LIN28B have been suggested to be differentially expressed in lymphoid progenitors originating from fetal and adult hematopoietic stem cells, where LIN28B represses let-7 and induces hematopoietic

stem/progenitor cells to revert to a fetal-like phenotype. However, there is a need for the development of compositions and methods for increasing the amount of fetal hemoglobin in an adult and for the prevention and treatment of hemoglobinopathies, including SCD and β- thalassemias.

BRIEF SUMMARY OF THE INVENTION

[0008] In embodiments of the invention, the invention provides plasmids encoding the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof

[0009] In another embodiment of the invention, the invention provides a method of increasing the amount of fetal hemoglobin in a cell, e.g., an erythroid cell, the method comprising administering to the cell an effective amount of a substance, the substance comprising or encoding the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof, wherein the administration of the substance increases the amount of fetal hemoglobin in the cell. [0010] In another embodiment of the invention, the invention provides a method of decreasing the amount of adult hemoglobin in a cell, e.g., an erythroid cell, the method comprising administering to the cell an effective amount of a substance, the substance comprising or encoding the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof, wherein the administration of the substance decreases the amount of adult hemoglobin in the cell.

[0011] In another embodiment of the invention, the invention provides a method of preventing or treating sickle cell disease in a mammal, the method comprising administering to a mammal in need thereof an effective amount of a substance, the substance comprising or encoding the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof, wherein the administration of the substance prevents or treats sickle cell disease in the mammal.

[0012] In another embodiment of the invention, the invention provides a method of preventing or treating a β-thalassemia in a mammal, the method comprising administering to a mammal in need thereof an effective amount of a substance, the substance comprising or encoding the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof, wherein the administration of the substance prevents or treats the β -thalassemia in the mammal.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 is a vector map of showing certain features of MSCV-IRES-Puro-LIN28B.

[0014] Figure 2 is a bar graph showing LIN28B knockdown (LIN28B-KD) confirmation by Q-RT-PCR quantitation of copy number per nanogram cDNA (Copies/ng cDNA), in accordance with embodiments of the invention. LIN28B-KD and control samples were evaluated. C: empty vector control; D: LIN28B knockdown.

[0015] Figure 3 is a bar graph showing the relative expression levels of the let-7 family of miRNAs as determined by Q-RT-PCR, in accordance with embodiments of the invention. Open bars represent control samples and black bars represent LIN28B- D. Standard deviation bars are shown, and asterisks denote p<0.05 in triplicate experiments.

[0016] Figure 4 presents chromatograms showing hemoglobin profile demonstrated by HPLC analysis for control (A), and LIN28B-KD (B) cultures, in accordance with embodiments of the invention. HbF and HbA peaks are labeled on each graph (y-axis, mVolts; x-axis, elution time in minutes). Data are representative of more than three independent experiments.

[0017] Figure 5 is a bar graph showing LIN28B over-expression (OE) in adult human erythroblasts as confirmed by Q-RT-PCR quantitation of copy number per nanogram cDNA (Copies/ng cDNA), in accordance with embodiments of the invention. Analyses were performed at culture day 14. Mean value ± SD of three independent donors for each condition.

[0018] Figure 6 presents flow cytometry plots showing (A) control and (B) LIN28B overexpression (LIN28B-OE) at culture day 14, and (C) control and (D) LIN28B-OE at culture day 21 stained with anti-transferrin receptor (CD71) and anti-glycophorin A (GPA) antibodies, in accordance with embodiments of the invention. Data are representative of more than three independent experiments.

[0019] Figure 7 presents flow cytometry plots showing enucleation is represented by staining of culture day 21 cells with thiazole orange for (A) control and (B) LIN28B-OE, in accordance with embodiments of the invention. Data are representative of more than three independent experiments.

[0020] Figure 8 presents chromatograms showing HPLC analysis of hemoglobin from (A) control and (B) LIN28B-OE samples performed at culture day 21 , in accordance with embodiments of the invention. HbF and HbA peaks are labeled on each graph (y-axis, mVolts; x-axis, elution time in minutes). Data are representative of more than three independent experiments.

[0021] Figure 9 is a vector map showing certain features of DNM-106, a mammalian expression, lentivirus plasmid used for human LIN28A fused with a DYKDDDDK epitope tag, under the control of a CMV promoter.

[0022] Figure 10 presents chromatograms showing HPLC analysis of hemoglobin from (A) control and (B) LI 28A-OE samples performed at culture day 21, in accordance with embodiments of the invention. HbF and HbA peaks are labeled on each graph (y-axis, mVolts; x-axis, elution time in minutes). Data are representative of more than three independent experiments.

[0023] Figure 1 1 is a bar graph showing alpha, mu, theta and zeta globins expression analysis of LI 28B-OE compared to control samples, in accordance with embodiments of the invention. Open bars represent control and black bars represent LIN28B-OE. Q-RT-PCR analyses were performed at culture day 14. Mean value ± SD of three independent donors for each condition.

[0024] Figure 12 is a bar graph showing beta, delta, gamma and epsilon globins expression analysis of LIN28B-OE compared to control samples, in accordance with embodiments of the invention. Open bars represent control and black bars represent LIN28B-OE. Q-RT-PCR analyses were performed at culture day 14. Mean value ± SD of three independent donors for each condition. P values were calculated using two-tailed Student's t-test. *p<0.05.

[0025] Figure 13 presents bar graphs showing (A) CA1 and (B) GCNT2 mRNA expression analysis of LIN28B-OE compared to control samples, in accordance with embodiments of the invention. Open bars represent control and black bars represent LIN28B-OE. Q-RT-PCR analyses were performed at culture day 14. Mean value ± SD of three independent donors for each condition. P values were calculated using two-tailed Student's t-test. C: empty vector control; OE: LIN28B over-expression. *p<0.05.

[0026] Figure 14 is a bar graph showing LIN28B-OE compared to control samples in the relative expression levels of the let-7 family of miRNAs (open bars represent control and black bars represent LIN28B-OE), in accordance with embodiments of the invention. Q-RT-PCR analyses were performed at culture day 14. miRNAs relative expression levels (y-axis) in the control cells were defined as a level of one for comparison. Error bars denote ± SD of three independent donors for each condition. P values were calculated using two-tailed Student's t- test. *p<0.05.

[0027] Figure 15 is a bar graph showing LIN28B-OE compared to control samples in the mRNA expression levels of HMGA2, in accordance with embodiments of the invention. Q-RT- PCR analyses were performed at culture day 14. Error bars denote ± SD of three independent donors for each condition. C: empty vector control; OE: LIN28B over-expression.

[0028] Figure 16 is a bar graph showing LIN28B-OE compared to control samples in the mRNA expression levels of IGF2, in accordance with embodiments of the invention. Q-RT- PCR analyses were performed at culture day 14. Error bars denote ± SD of three independent donors for each condition. C: empty vector control; OE: LIN28B over-expression. [0029] Figure 17 is a bar graph showing LIN28B-OE compared to control samples in the relative expression levels of miR-96, miR-29c, miR-451, miR-144 and miR-142, in accordance with embodiments of the invention. Q-RT-PCR analyses were performed at culture day 14. miRNAs relative expression levels (y-axis) in the control cells were defined as a level of one for comparison. Error bars denote ± SD of three independent donors for each condition. P values were calculated using two-tailed Student's t-test. *p<0.05.

[0030] Figure 18 is a bar graph showing LIN28B-OE compared to control samples in the mRNA expression of the transcription factors (A) BCL11A, (B) GATA1 , (C) LF1 and

(D) SOX6, in accordance with embodiments of the invention. Q-RT-PCR analyses were performed at culture day 14. Error bars denote ± SD of three independent donors for each condition. P values were calculated using two-tailed Student's t-test. *p<0.05.

[0031] Figure 19 is a bar graph showing relative expression levels of the let-7 family of miRNAs (open bars represent control, black bars represent LIN28B-OE, and hatch-marked bars represent let-7 sponge) after transduction of the let-7 sponge or LIN28B-OE encoding retrovirus. Q-RT-PCR analyses were performed at culture day 14, and compared with control transductions, in accordance with embodiments of the invention. miRNAs relative expression levels (y-axis) in the control cells were defined as a level of one for comparison. Error bars denote ± SD of three independent donors for each condition. P values were calculated using two-tailed Student's t- test.*p<0.05.

[0032] Figure 20 presents chromatograms showing HPLC analysis of hemoglobin from (A) control, (B) LIN28B-OE, and (C) let-7 sponge samples performed at culture day 21, in accordance with embodiments of the invention. HbF and HbA peaks are labeled on each graph (y-axis, mVolts; x-axis, elution time in minutes). Data are representative of three independent experiments.

[0033] Figure 21 is a vector map showing certain features of a clinical vector (CL20c Ins400R mLAR bv5Dgm3) where the sequence encoding LIN28A, LIN28B, or a let-7 sponge is inserted in the place of the globin protein encoding sequence (exl, ex2, ex3), in accordance with embodiments of the invention.

[0034] Figure 22 is a vector map showing certain features of a clinical vector where the sequence encoding LIN28A is inserted, in accordance with embodiments of the invention. [0035] Figure 23 is a vector map showing certain features of a clinical vector where the sequence encoding a let-7 sponge is inserted, in accordance with embodiments of the invention.

[0036] Figure 24 is a vector map showing certain features of a clinical vector where the sequence encoding LIN28B is inserted, in accordance with embodiments of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0037] Embodiments of the present invention are predicated on the surprising discovery that in adults the expression of fetal hemoglobin can be increased to levels that can provide therapeutic effects due to the expression of one or more transgenes of LIN28 A, LIN28B, and/or a let-7 sponge, where the transgene expression levels are significantly below the levels previously thought to be required for transgene expression. Previous transgenic studies, e.g., those using transgenes to directly increase the expression of globins, have shown that high levels of expression of the transgenes were required in order to achieve a therapeutic effect. High levels of transgene expression can be problematic in that untoward effects also may be observed, including improper integration of the transgene into genomic DNA, which may lead to, for example, unintentionally high levels of expression of neighboring genes. Such effects may lead to, for example, leukogenesis.

[0038] In one embodiment of the invention, the invention provides a method of increasing the amount of fetal hemoglobin in a cell, e.g., an erythroid cell, the method comprising administering to the cell an effective amount of a substance, the substance comprising or encoding the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof, wherein the administration of the substance increases the amount of fetal hemoglobin in the cell.

[0039] As provided in the Examples below, examination of LIN28B mRNA in cord blood erythroblasts (cord blood cells do not naturally express high levels of Lin28B) suggests that low- level expression is sufficient to regulate let-7 and fetal hemoglobin in cord blood erythroblasts. Significant reduction in fetal hemoglobin expression in cord blood erythroblasts was mediated by reductions in the erythroblast LIN28B mRNA levels (105 copies/ng reduced to 21 copies/ng). The effects of reducing LIN28B expression are thus amplified to regulate gamma-globin RNA levels that are normally detected at 5-log higher levels (10⁷ copies/ng). In the adult

erythroblasts, LIN28A or LIN28B expression was below the detection limit in the control cultures. LIN28B overexpression in the adult cells reached levels that were approximately 100- fold higher than the cord blood erythroblasts (10⁴ copies/ng). Higher-level expression of the transduced LIN28B in the adult cells caused increases in fetal hemoglobin that approached, but did not exceed, that of cord blood derived erythroblasts. The lack of increases in fetal hemoglobin above the cord blood levels, despite higher LIN28B expression, suggests saturation of the globin gene regulating effects in adult cells.

[0040] Additionally, it was surprisingly discovered that the increase in fetal hemoglobin in accordance with embodiments of the present invention decreases adult hemoglobin. Increasing expression of fetal hemoglobin without decreasing expression of adult hemoglobin has disadvantages. For example, previous treatments of sickle cell disease in a subject by overexpressing globins that are not susceptible to sickling (e.g., globin for increasing fetal hemoglobin) have not decreased sickling of red blood cells in the subject since the susceptible globin (e.g., the subject's own mutated globin) is still expressed at normal levels in the subject.

[0041] In embodiments of the invention, 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.

[0042] In an embodiment of the invention, the invention provides a method of decreasing the amount of adult hemoglobin in a cell, e.g., an erythroid cell, the method comprising

administering to the cell an effective amount of a substance, the substance comprising or encoding the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof, wherein the administration of the substance decreases the amount of adult hemoglobin in the cell.

[0043] Thus, in accordance with embodiments of the present invention, the hemoglobin susceptible to sickling is decreased, whereas the non-susceptible hemoglobin is increased.

Importantly, this feature has not been previously achieved with other vector strategies, including globin-encoding lentivirus vectors. [0044] It also was surprisingly discovered that embodiments of the present invention do not adversely affect differentiation of cells, even though BCL1 1 A is also found to be regulated. Previous attempts to indirectly increase gamma globin expression by decreasing BCL11 A expression, which regulates HbF, have adversely affected differentiation of the modified cells. Since LIN28A, LIN28B, or let-7 sponge transgenic expression does not cause significant effects upon erythroid differentiation, the opportunity for successful differentiation into erythrocytes is greatly increased. This feature is predicted to be advantageous when compared to vector designs aimed exclusively toward the manipulation of chromatin structure or specific transcription factors (BCL1 1A, KLF1, LSD1, histone modifying enzymes, etc.). As provided in the Examples below and without wishing to be bound by any theory, LIN28B over-expression, as well as let-7 suppression in the absence of LIN28A or LIN28B, caused decreased expression of BCL11A as a predominant mechanism for increasing gamma-globin mRNA in the adult erythroblasts.

Without wishing to be bound by any theory, since neither BCL11 A knockdown nor direct let-7 suppression had effects upon LIN28A or LIN28B expression, the data suggest that LIN28B affects the expression of fetal hemoglobin primarily by targeting let-7 to act as an upstream regulator of BCL1 1A. In addition to binding and inhibiting let-7, LIN28 proteins may affect non-let-7 RNA species; however, the let-7 sponge studies suggest that the let-7 miRNAs are targets of LI 28 proteins.

[0045] Additional advantages of embodiments of the present invention include 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. Increased expression of LIN28 A or LIN28B proteins, as well as decreased expression of let-7 micro RNA in humans is predicted to be non-immunogenic. Regulated expression of the host's endogenous genes encoding fetal (gamma) globin protein is superior to delivery of a globin transgenes that may not encode a globin protein that is identical to the globin protein in the host's genome. Additionally, LIN28A, LIN28B, or let-7 sponge transgenes are not predicted to cause an immune response or cause resulting death of cells transduced.

[0046] A let-7 inhibitor (or "sponge") is any substance that reduces the amount of let-7 available to down-regulate fetal hemoglobin expression, the reduction of let-7 resulting, for example, from down-regulation of let-7 or inhibition of let-7. Examples of let-7 inhibitors include the LIN28A protein, the LIN28B protein, or suitable natural or. synthetic anti-miRNA, including, without limitation, 5'-TccTAgAAa-GAgtAgA-3', uppercase: LNA, lowercase: DNA. Other suitable miRNAs would be understood by one of ordinary skill in the art based on the sequences of the let-7 miRNAs. The let-7 miRNA family includes let-7a, -7b, 7c, -7d, -7e, -7f2, -7g, and -7i.

[0047] In an embodiment of the invention, the invention provides the substance comprising the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof, where the substance is administered directly to the cell. 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.

[0048] In an embodiment of the invention, the invention provides the substance as a plasmid encoding the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof, wherein the plasmid is administered to the cell. The plasmid can be a viral vector plasmid. 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.

[0049] In an embodiment of the invention, the invention provides the substance as viral DNA encoding the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof, wherein the viral DNA is administered to the cell through viral transduction. The transgenes described herein are small, and can be inserted into other vectors previously developed for transgene therapy using globin genes. For example, globin coding regions can be replaced with LIN28A, LIN28B, and/or a let-7 sponge coding region. Whereas the vector with globin genes may have produced untoward effects due to overexpression and may not have reduced, e.g., sickling in SCD, replacing the globin coding regions with the transgenes of the present invention is expected to provide the advantages as described above.

[0050] In embodiments of the present invention, the methods and transgenes of the present invention may be used with other vector systems. The lentiviral systems are one of the vector systems predicted to be sufficient for clinical success. Several lentiviral systems exist. The approach as described herein for delivery and expression of LIN28A, LIN28B, or let-7 sponge DNA may be applied to alternate viral vector technologies as would be understood by one of ordinary sill in the art. Exemplary systems are described in Perumbeti and Malik, Ann. NY Acad. ScL, 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, Curr. Opin. Drug Discov. Devel, 2008, 1 1 :225-32; Bank et al, Ann. NY Acad. ScL, 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.

[0051] In an embodiment of the invention, the cell is 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] With appropriate packaging vectors assisting in viral transduction, the vectors of the present invention can provide the benefits as described herein when the vectors of the present invention overexpress human Lin28A, Lin28B, and/or knock-down let-7 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-lOAl packaging vector for retroviruses. The pCL-lOAl vector is a part of the etroMax expression system from Imgenex (San Diego, CA, USA). Packaging vectors suitable for lentiviruses include pAG4-RTR2-l, 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.

[0053] In another embodiment of the invention, the invention provides a method of preventing or treating sickle cell disease in a mammal, the method comprising administering to a mammal in need thereof an effective amount of a substance, the substance comprising or encoding the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof, wherein the administration of the substance prevents or treats sickle cell disease in the mammal. In an embodiment, the invention provides a substance for use in preventing or treating sickle cell disease in a mammal, the substance comprising or encoding the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof.

[0054] In another embodiment of the invention, the invention provides a method of preventing or treating a β-thalassemia in a mammal, the method comprising administering to a mammal in need thereof an effective amount of a substance, the substance comprising or encoding the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof, wherein the administration of the substance prevents or treats the β -thalassemia in the mammal. In an embodiment, the invention provides a substance for preventing or treating a β-thalassemia in a mammal, the substance comprising or encoding the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof.

[0055] In an embodiment of the invention, the invention provides the substance comprising the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof, and the substance is administered directly to the mammal, e.g., in vivo. Direct administration to the mammal is by any suitable means as understood in the art, as outlined below.

[0056] The substance comprising the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof 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. Alternatively, the pharmaceutical composition can comprise a single substance 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 the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof, as well as by the particular method used to administer the substance. 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 in certain instances, a particular route can provide a more immediate and more effective response than another route.

[0059] 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.

[0060] Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the substance comprising the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof 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, macrocrystalline 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 in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the substance 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.

[0061] 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 the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof 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.

[0062] 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.

[0063] 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).

[0064] It will be appreciated by one of skill in the art that, in addition to the above-described pharmaceutical compositions, the substance comprising the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof can be formulated as inclusion complexes, such as cyclodextrin inclusion complexes, or liposomes.

[0065] For purposes of the invention, the amount or dose of the substance comprising the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof 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 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 the condition of the mammal (e.g., human), as well as the body weight of the mammal (e.g., human) to be treated.

[0066] The dose of the substance comprising the protein LIN28A, the protein LIN28B, a let- 7 sponge, or any combination thereof will be determined by the existence, nature and extent of any adverse side effects that might accompany the administration of a particular substance. Typically, the attending physician will decide the dosage of the substance with which to treat each individual patient, taking into consideration a variety of factors, such as age, body weight, general health, diet, sex, substance 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 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.

[0067] Alternatively, the substance comprising the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof can be modified into a depot form, such that the manner in which the substance 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 can be, for example, an implantable composition comprising the substance and a porous or non-porous material, such as a polymer, wherein the substance 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 is released from the implant at a predetermined rate.

[0068] In an embodiment of the invention, the invention provides the substance as a plasmid encoding the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof, wherein the plasmid is administered to the mammal. The plasmid can be a viral vector plasmid. In another embodiment of the invention, the invention provides the substance as viral DNA encoding the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof, wherein the viral DNA is administered to the mammal through viral transduction. In an embodiment of the invention, the invention further provides administering to a cell, e.g., an erythroid cell, the substance, wherein the substance is a plasmid encoding the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof, and administering to the mammal the cell having the plasmid. In an embodiment of the invention, the invention further provides extracting from the mammal a cell, e.g., an erythroid cell, administering to the cell the substance, wherein the substance is a plasmid encoding the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof, and administering to the mammal the cell having the plasmid. Extraction from a mammal and administration to the mammal would provide an autologous cell transplant, which can be advantageous, e.g., in preventing an immune response to the cells. The plasmid can be a viral vector plasmid. Administration may be through gene transfer techniques, as would be understood by one of ordinary skill in the art, for example 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, 1 :e2009008; Sadelain et al, Curr. Mol. Med., 2008, 8:690-7; Lebensburger and Persons, Curr. Opin. Drug Discov. Devel., 2008, 11 :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.

[0069] In an embodiment of the invention, the invention further provides administering to a cell, e.g., an erythroid cell, the substance through viral transduction, wherein the substance is viral DNA encoding the protein LIN28A, the protein LIN28B, a let-7 sponge, or any

combination thereof, and administering to the mammal the cell having the viral DNA. The cell may be, e.g., grown using cells foreign from the mammal. In an embodiment of the invention, the invention further provides extracting from the mammal a cell, e.g., an erythroid cell, administering to the cell the substance through viral transduction, wherein the substance is viral DNA encoding the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof, and administering to the mammal the cell having the viral DNA.

[0070] Such ex vivo methods are advantageous in that it is desirable to have, e.g., erythroid specificity, e.g., to provide targeted increase in expression of LIN28A, LIN28B, and/or a let-7 sponge. A targeted approach can decrease the possibility of having off-target effects when expression is modulated in, e.g., non-erythroid cells.

[0071] 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. 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., SCD, or a symptom or condition thereof, e.g., cell sickling.

[0072] In another embodiment of the invention, the invention provides plasmids encoding the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof. SEQ ID NOS: 1-3 are translated portions of certain vectors described herein. In an embodiment, the plasmid is suitable for clinical use, e.g., in humans. In an embodiment, the plasmid is a vector with the sequence at SEQ ID NO: 4. In another embodiment, the vector at SEQ ID NO: 4 has the LIN28A open reading frame and noncoding linker DNA inserted, as provided at SEQ ID NO: 5. Additional plasmids/vectors may be used, as discussed above, wherein globin coding regions are replaced with the coding regions of LIN28A, LIN28B, and/or a let-7 sponge. Thus, the plasmid of SEQ ID NO: 4 may be used to form a new plasmid by inserting any one or more of LIN28A, LIN28B, and/or let-7 sponge into an appropriate site within SEQ ID NO: 4 using standard molecular biology techniques. In another embodiment, the vector has a sequence encoding a let-7 sponge, as in SEQ ID NO: 6. In another embodiment, the vector has a sequence encoding LIN28B, as in SEQ ID NO: 7. In another embodiment, the vector is at SEQ ID NO: 8. In another embodiment, the vector is at SEQ ID NO: 9. Additional plasmids/vectors would be understood by one of ordinary skill in the art to include, for example, those 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, Curr. Opin. Drug Discov. Devel., 2008, 11 :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.

[0073] In an embodiment, the LIN28A sequence can be replaced with LIN28B or a let-7 sponge. Also, the promoter sequence can be replaced with other erythroid promoters, the LCR (Locus Control Region) can be replaced with other erythroid regulatory elements. For example, the sequences can be replaced to provide appropriate levels of transgene expression at appropriate stages of erythroid differentiation and/or to prevent/reduce expression in other cell types to provide targeted expression.

[0074] 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.

[0075] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.

EXAMPLE 1

[0076] This example provides techniques used herein for culturing cells.

[0077] For ex vivo culture, a 21 day serum-free system consisting of three phases was utilized. During phase I of culture (day 0 to day 7): adult or cord blood CD34+ cells were placed in media containing StemPro-34 complete media (2mM L-glutamine, lU/ml pen-strep, and StemPro-34 nutrient supplement) (Invitrogen, Carlsbad, CA, USA) with 50 ng/ml SCF (HumanZyme, Chicago, IL, USA), 50 ng/ml FLT3 Ligand (HumanZyme) and 10 ng/ml IL-3 (HumanZyme). After phase I, the cells were transferred to phase II media (day 7 to day 14). During this expansion phase, the cell count was monitored on days 9, 1 1 and 14 to maintain cell counts under 2x10⁶ cells per ml. Phase II medium is the following: StemPro-34 complete medium, 4 U/ml EPO (Amgen, Thousand Oaks, CA, USA), 10 ng/ml SCF, 10 μg/ml insulin (Sigma Aldrich, St. Louis, MO, USA), 3 U/ml heparin (Hospira, Lake Forest, IL, USA) and 0.8 mg/ml total holo transferrin (Sigma Aldrich). On culture day 14, cells were counted and transferred to phase III media at 8xl0⁵ cells per ml for the remaining 7 days of culture. Phase III medium is the following: StemPro-34 complete medium, 4 U/ml EPO, 3 μΜ RU486 (Sigma Aldrich), 10 μg/ml insulin, 3 U/ml heparin and 0.8 mg/ml total holo transferrin.

EXAMPLE 2

[0078] This example demonstrates LIN28B binds primary let-7 transcripts and regulates fetal hemoglobin in human cord blood CD34+ cells.

[0079] The methods of Example 1 were generally followed. Human cord blood CD34+ cells were obtained from All Cells (Emeryville, CA, USA) and ReachBio LLC (Seattle, WA, USA).

[0080] RNA immunoprecipitation was performed following the manufacturer's instructions (Magna RIP RNA-Binding Protein Immunoprecipitation Kit, Millipore, Billerica, MA, USA) with equal amounts of lysates from human cord blood cells using antibodies against LIN28B (Cell Signaling, Danvers, MA, USA, catalog #4196) or IgG (Millipore, catalog #PP64). The immunoprecipitated RNA was purified before RT-PCR with primers to primary let-7d miRNA (Assays-on-Demand Gene Expression Product # Hs03302562_pri, Applied Biosystems, Grand Island, NY, USA).

[0081] Q-RT-PCR assays and PCR conditions were performed as described (Sripichai et al, Blood, 2009, 1 14:2299-2306, incorporated by reference herein in its entirety). Additional primers and probes used in this study are described in the table below. Table 1

Gene Probe Sequence Information* (Applied Biosystems)

LIN28B Assays-on-Demand Gene Expression Products # HsOl 013729_ml

pri-Let-7d Assays-on-Demand Gene Expression Products # Hs03302562_pri

*Also see Noh et al., J. Transl. Med, 2009, 7:98 and Sripichai et al, Blood, 2009, 114:2299-2306 for previously reported Q-PCR reagents.

[0082] To evaluate miR A expression, reactions and data normalization were performed as described (Noh et al., J. Transl. Med, 2009, 7:98, incorporated by reference herein in its entirety).

[0083] For recombinant viral transduction, human LIN28B was subcloned into an MSCV- IRES-Puro based retroviral vector as described (Yuan et al., Science, 2012, 335: 1 195-1200, incorporated by reference herein in its entirety). A vector map is at Figure 1. A possible sequence for the MSCV-IRES-Puro LIN28B vector was constructed and is at SEQ ID NO: 8. MSCV-IRES-Puro is a retroviral vector and will transduce most mammalian cells due to the 10A1 envelop from the pCL-lOAl packaging vector. MSCV-IRES-Puro has the following characteristics: LI 28B Cloning site 5' : Bglll; LIN28B Cloning site 3' : Xhol; Vector type: Mammalian Expression, Retrovirus; Bacterial resistance(s): Ampicillin; Growth strain(s):

DH5alpha; Growth temperature (°C): 37.

[0084] Details of the vector:

Table 2

Additional putative details of the vector are as follows:

Table 3

* Potential for blockage by DAM methylation of the DNA

[0086] The pCL-lOAl packaging vector is a part of the RetroMax expression system from Imgenex (San Diego, CA, USA) (Cat#10040K and 1004 IK) and contains a ampicillin resistance gene (Naviaux et al., J. Virol, 1996, 70: 5701-5705; Rasheed et al., Int. J.Cancer, 1982, 29: 345- 350; and Ott et al., J. Virol., 1990, 64: 757-766; each of which is incorporated herein by reference in its entirety). Human LIN28A over-expression lentiviral particles were purchased from Qiagen (Valencia, CA, USA), and used for comparison studies. Human LIN28B knockdown clones were purchased from Sigma Aldrich. A micro-RNA sponge experimental approach was utilized for suppression of let-7 (Ebert et al., Nat. Methods, 2007, 4:721-726, incorporated by reference herein in its entirety). The let-7 sponge plasmid (MSCV puro let-7 sponge) was purchased from Addgene (Cambridge, MA, USA, plasmid, catalog #29766) and used with the pCL-lOAl vector. A possible sequence for the MSCV puro let-7 sponge vector was constructed and is at SEQ ID NO: 9. Putative details of the vector are as follows:

Table 4

MSCV_rev_primer 1496 1473

mPGK_F_primer 1870 1889

puro (variant) 1958 2557

M 13_reverse_primer 3354 3336

M 13 jpUC_rev_primer 3375 3353

lac_promoter 3418 3389

Ampicillin 5361 4501

AmpR_promoter 5431 5403

pGEX 3_primer 5612 5590

lacZ a 5942 5811

M 13__pUC_fwd_primer 5925 5947

pB RrevB am_pr imer 6075 6094

Table 5

[0087] Retroviral supernatants were produced by transient Lipofectamine 2000 (Invitrogen) cotransfection of 293T cells with the RetroMax packaging vector pCL-Eco (Imgenex, San Diego, CA, USA). Seventy-two hours post-transfection, cell supernatant was harvested and concentrated with Retro-X concentrator solution (Clontech, Mountain View, CA, USA) following the manufacturer's instructions. Empty vectors were used as controls. [0088] For all viral transductions, the methods of Example 1 were modified: On culture day 3 of phase I, the cells were transduced with either LIN28 or let-7 sponge viral particles

(estimated multiplicity of infection, MOI, is 12). After 24 hours, puromycin (Sigma) was added to the culture. On culture day 7, cells were transferred to phase II medium containing EPO and puromycin until culture day 9. After culture day 9, cells were cultivated at the conditions described above without puromycin.

[0089] Samples for HPLC were prepared and analyzed as described (Tanno et al., Blood, 2009, 114: 181-186, incorporated by reference herein in its entirety).

[0090] Statistical analysis. Replicate data are expressed as mean value ± SD with significance calculated by two-tailed Student's t test.

[0091] Initial studies were performed to determine if LIN28B binds endogenous let-7 primary transcripts in cord blood derived erythroblasts. RNA immunoprecipitation in CD34+ cord blood lysates was performed and quantitated by RT-PCR for primary let-7d. Primary let-7d was enriched by LIN28B immunoprecipitation compared to the IgG control.

[0092] To investigate a potential role for cord blood LIN28B expression in fetal hemoglobin regulation, CD34+ cord blood cells were transduced with lentivirus encoding a short-hairpin RNA (shRNA) against LIN28B and cultured ex vivo in erythropoietin-supplemented serum-free media for 21 days. Two shRNA clones (TRCN0000122191 and TRCN0000122599) demonstrated equivalent results; the results using clone TRCN0000122191 are shown. LIN28B knockdown (LIN28B-KD) was confirmed by Q-RT-PCR (control: 105 + 24 copies/ng, LIN28B- KD: 20.7 + 0.9 copies/ng, p=0.02), as demonstrated in Figure 2. Let-7 microRNAs were measured by Q-RT-PCR in LIN28B- D cells. As shown in Figure 3, LIN28B-KD caused increased expression of several let-7 species (let-7a, let-7b, let-7c, let-7d and let-7e), and the increased levels of let-7g and let-7i reached statistical significance compared to the controls (p=0.05 and p=0.01, respectively). Hemoglobin expression profiles for control and LIN28B-KD were determined by standard HPLC (Figure 4). Triplicate experiments demonstrated a significant reduction in HbF expression in LIN28B-KD cells compared to the controls (HbF: LIN28B-KD: 31.0 ± 5.3%; control: 54.2 ± 7.7% p=0.03). EXAMPLE 3

[0093] This example demonstrates LIN28B expression activates fetal hemoglobin during adult human erythropoiesis.

[0094] The methods of the above Examples were generally followed.

[0095] For Western blot analysis, nuclear and cytoplasmic extracts were prepared from culture day 14 erythroblasts using NE-PER Nuclear Protein Extraction Kit (Pierce

Biotechnology, Rockford, IL, USA) according to the manufacturer's protocol. Equal amounts of protein (20-30 μg) were separated by NuPAGE Novex 4-12% Bis-Tris gel electrophoresis in MOPS buffer and transferred using the iBlot Blotting System onto nitrocellulose membranes (Invitrogen). Blots were probed with antibodies against human LIN28B (Cell Signaling) and the appropriate horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology). Immunoreactive proteins were detected and visualized using ECL Plus Western blotting detection reagents (GE Healthcare, Waukesha, WI, USA). All blots were probed with β-actin (Abeam) antibody as a loading control.

[0096] For confocal microscopy, slides containing culture day 14 cells were probed against human LIN28B (Cell Signaling). Laser scanned confocal images were obtained from LSM 5 Live Duoscan (Carl Zeiss, Oberkochen, Germany) and analyzed with Zen2007 software.

[0097] For flow cytometry analyses, immunostaining with antibodies directed against CD71 and glycophorin A (Invitrogen) were performed to assay erythroid differentiation on culture day 21 as described (Tanno et al., Blood, 2009, 1 14:181-186, incorporated by reference herein in its entirety) using the BD FACSAria I flow cytometer (BD Biosciences, San Jose, CA, USA). Enucleation was quantitated using thiazole orange staining. Enucleated erythroid cell populations were isolated by filtering day 21 cells through Purecell Neo Leukocyte Reduction filters (Pall, Covina, CA, USA).

[0098] When compared to the cord blood erythroblasts, adult erythroblasts demonstrated a major reduction in LIN28B expression (0.1 + 0.4 copies/ng). To increase LIN28B expression, the cells were transduced with a retrovirus encoding LIN28B. LIN28B over-expression

(LIN28B-OE) was confirmed by Q-RT-PCR analysis (LIN28B-OE: 1.8E+04 + 3.5E+02 copies/ng, p=0.01) (Figure 5) and Western analysis. In transduced erythroblasts, LIN28B was expressed predominantly in the cytoplasm without distinct nuclear localization as shown by confocal microscopy.

[0099] Erythroblast differentiation and the patterns of hemoglobin expression were compared between control and LIN28B-OE cells. Flow cytometric analysis of erythroblast plasma membrane markers, transferrin receptor (CD71) and glycophorin A (GPA), were performed at culture days 14 and 21 for erythroblast differentiation (Figure 6). Surprisingly, LIN28B-OE did not overtly affect erythroid cell maturation when compared to empty vector controls. Furthermore, thiazole orange staining of nuclei on culture day 21 in LIN28B-OE and control cells showed comparable results (Figure 7; LIN28B-OE enucleation 38 + 2.1% compared to control 25 + 9.3%), thus indicating the ability of LIN28B-OE erythroblasts to undergo terminal maturation.

[0100] The culture-generated erythrocytes were purified and further studied. The LIN28B- OE erythrocytes were morphologically equivalent to controls for all analyzed samples.

Hemoglobin profiles (HPLC) showed markedly increased HbF levels in the LIN28B-OE cells (Figure 8; LIN28B-OE: 33.6 ± 9.4%; control: 5.8 + 4.5% p=0.01). For comparison, a different lentiviral vector encoding LIN28A was utilized. LIN28A was inserted into vector DNM-106 from Qiagen, cat. no. 337402 (Figure 9). (DNM-106 also has the following characteristics: Bacterial resistance(s):Ampicillin; Growth strain(s): DH5alpha; Growth temperature (°C): 37; Selective Marker: Puromycin.) A robust increase in fetal hemoglobin expression was also detected in LIN28A transduced cells (Figure 10). Taken together, these data demonstrate that over-expression of LIN28A or LIN28B is sufficient for the manifestation of high-level fetal hemoglobin expression in cultured adult human erythroblasts.

EXAMPLE 4

[0101] This example demonstrates adult erythroid cells expressing LIN28B manifest a more fetal-like phenotype.

[0102] The methods of the above Examples were generally followed.

[0103] Additional primers used are as listed in the table below. Table 7

Gene Probe Sequence Information* (Applied Biosystems)

CA1 Assays-on-Demand Gene Expression Products # HsO 1 100176_ml

GCNT2 Assays-on-Demand Gene Expression Products # Hs00377334_ml

*Also see Noh et al., J. Transl. Med, 2009, 7:98 and Sripichai et al., Blood, 2009, 1 14:2299-2306 for previously reported Q-PCR reagents.

[0104] Additional antibodies used for Western blots were human CA1 (Abeam, Cambridge, MA, USA) and GCNT2 (Santa Cruz Biotechnology, Dallas, TX, USA).

[0105] The fetal-to-adult hemoglobin transition in erythrocytes encompasses a reduction in the expression of the fetal (^Ay/^Gy-globin) genes among erythroblasts coinciding with an increased expression of the adult (β- and δ-globin) genes. This process begins during the later stages of fetal development and continues through early infancy. Based upon the high-level of fetal hemoglobin expression in LIN28B-OE cells, globin gene expression patterns were quantitated by Q-RT-PCR. The expression levels of alpha, mu, theta, zeta, beta, delta, gamma and epsilon globin genes were evaluated for LIN28B-OE and compared to controls. As shown in Figures 11 and 12, only the fetal gamma-globin mRNA expression is significantly heightened with LIN28B- OE (control: 5.1E+06 ± 2.6E+06 copies/ng, LIN28B-OE: 1.8E+07 ± 5.8E+06 copies/ng, p=0.04). The increased expression of gamma-globin was balanced by reductions in beta-globin and delta-globin. No changes were observed in alpha, mu and theta globins, and the low-level expression patterns of zeta and epsilon globins indicated only minor increases.

[0106] The fetal-to-adult transition in humans is further characterized by an increase in the carbonic anhydrase I (CA1) gene expressed in erythrocytes, as well as the carbohydrate modification due to the augmented expression of glucosaminyl (N-acetyl) transferase 2

(GCNT2). Expression of CA1 and GCNT2 were therefore investigated by Q-RT-PCR with LIN28B-OE. As shown in Figure 13, both CA1 and GCNT2 were significantly reduced upon LIN28B-OE (CA1 - control: 5E+03 ± 4E+03 copies/ng, LIN28B-OE: 2E+03 ± 5E+02 copies/ng; p=0.01 ; GCNT2 - control: 1E+03 ± 3E+02 copies/ng, LIN28B-OE: 1E+02 + 4E+01 copies/ng; p=0.01). Western analysis using anti-human CA1 antibody in LIN28B-OE compared to control samples at culture day 21 confirmed a reduction in CA1 levels; however, a major change in GCNT2 protein expression was not detected.

EXAMPLE 5

[0107] This example demonstrates LIN28B suppresses let-7 and miR-96 miRNAs.

[0108] The methods of the above Examples were generally followed.

[0109] Additional primers used are as listed in the table below.

Table 8

Gene Probe Sequence Information* (Applied Biosystems)

HMGA2 Assays-on-Demand Gene Expression Products # Hs00971724_ml

IGF2 Assays-on-Demand Gene Expression Products # Hs01005963_ml

*Also see Noh et al, J. Transl. Med, 2009, 7:98 and Sripichai et al., Blood, 2009, 1 14:2299-2306 for previously reported Q-PCR reagents.

[0110] The expression patterns for several let-7 family members (let-7a, let-7b, let-7c, let-7d, let-7e, let-7f-2, let-7g and let-7i) were investigated in LIN28B-OE erythroblasts. The let-7 family members demonstrated robust and consistent suppression with a greater than 70% reduction in LIN28B-OE compared to the control (Figure 14). The chromatin modifier HMGA2 is a validated target of let-7 that also modulates growth and differentiation. Retroviral integration of globin encoding vectors into the HMGA2 locus resulted in hematopoietic clonal dominance at the integration site as well as increased fetal hemoglobin expression. Lin28 protein also increases the efficiency of insulin like growth factor 2 (IGF2) protein translation in muscle cells. IGF2 is expressed at high levels in fetal hepatocytes and supports expansion of hematopoietic stem cells in fetal liver. Despite the strong suppression of the let-7 family, HMGA2 was not significantly up-regulated in LIN28B-OE samples (Figure 15). IGF2 expression was slightly increased in the LIN28B-OE samples (Figure 16).

[0111] Three erythroid miRNAs that are not developmentally regulated (miR-451 , miR- 144 and miR- 142) were tested as controls. Among the group, a significant reduction was observed only in miR-96 (Figure 17), suggesting possible targeting of this miRNA by LIN28B. EXAMPLE 6

[0112] This example demonstrates LIN28B regulates BCLl 1 A expression.

[0113] The methods of the above Examples were generally followed.

[0114] Additional primers used are as listed in the table below.

Table 9

Gene Probe Sequence Information* (Applied Biosystems)

GATAl Assays-on-Demand Gene Expression Products # Hs01085821_ml

KLF1 Assays-on-Demand Gene Expression Products # Hs00610592_ml

[0115] BCLl 1 A knockdown clones were selected as described (Sankaran et al., Science, 2008, 322: 1839-1842, incorporated by reference herein in its entirety).

[0116] For recombinant viral transduction: On culture day 3 of phase I, the cells were transduced with either LIN28, BCLl 1 A or let-7 sponge viral particles (estimated MOI 12).

[0117] Additional antibodies used: BCLl 1A (Abeam).

[0118] The expression patterns of the transcription factors BCLl 1A, KLF1 and SOX6 were investigated, along with the erythroid transcription regulator, GATAl (Figure 18). Among the group, only BCLl 1 A was affected by LIN28B-OE. LIN28B-OE caused a 65% reduction in BCLl 1A expression (control: 3.07E+03 ± 1.5 E+02 copies/ng, LIN28B-OE: 1.07E+03 ± 18 copies/ng; p=0.02).

[0119] In accordance with the mRNA expression data, LIN28B-OE down-regulated BCLl 1 A at the protein level. To investigate whether BCLl 1 A modulates LIN28B expression, primary CD34+ cells were transduced with the shRNA lentivirus knockdown vector of BCLl 1A and lentiviral controls. BCLl 1 A knockdown was evaluated and the results showed no effect on LIN28B expression.

EXAMPLE 7

[0120] This example demonstrates let-7 miRNAs regulate BCLl 1 A expression and fetal hemoglobin.

[0121] The methods of the above Examples were generally followed. [0122] To investigate if the main targets of LIN28B, the let-7 family of miRNAs, are responsible for the regulation of fetal hemoglobin, adult CD34+ erythroid cells were transduced with a let-7 sponge retrovirus. LIN28A and LIN28B expression remained below the QPCR detection threshold after transduction. Transduction with the sponge retrovirus resulted in significant suppression of let-7 (Figure 19). However, the level of let-7 suppression was more robust with LIN28B-OE than the let-7 sponge. Comparison of LIN28B-OE and let-7 sponge experiments also demonstrated greater effects of LIN28B-OE upon BCL11A and fetal hemoglobin than achieved after let-7 suppression. The hemoglobin profiles (HPLC)

demonstrated significantly increased HbF levels in adult erythroid cells with either LIN28B-OE or let-7 sponge compared to controls (control: 3.5 ± 0.3%; LIN28B-OE: 31.1 + 2.9% p = 0.0029; let-7 sponge: 19.1 + 0.2%> p=0.0003, Figure 20). These results demonstrate that suppression of let-7 miRNAs is sufficient to reduce BCL1 1A and increase fetal hemoglobin in adult human erythroblasts, even in the absence of LIN28 A or LIN28B expression.

EXAMPLE 8

[0123] This example demonstrates the use of LIN28A, LIN28B, and/or a let-7 sponge in gene transfer technologies or vectors that are designed for delivery or expression into erythroid cells, their progenitors, or stem cells, including hematopoietic stem cells.

[0124] The sequence encoding LIN28A, LIN28B, or a let-7 sponge is inserted into a clinical vector CL20c Ins400R mLAR bv5Dgm3 (SEQ ID NO: 4; Figure 21) where the LIN28A, LIN28B, or let-7 sponge sequence is inserted in the place of the globin protein encoding sequence (exl, ex2, ex3). A vector of SEQ ID NO: 4 with LIN28A is SEQ ID NO: 5 (Figure 22). The plasma is then utilized to produce recombinant viral particles for ex vivo or in vivo transduction of human cells.

[0125] Details of the vector:

Table 10

Feature Location Size (bp)

4..383 380

CMV enhancer

Notes: human cytomegalovirus immediate early enhancer

462..642 181

5' LTR

Notes: truncated 5' long terminal repeat (LTR) from HIV-1

Notes: gene is bla

Table 11

Enzymes Sites Location

Acc65I 1 7345

Agel 1 4080

Bbsl 1 7447

Bell 1 * 6824*

* Potential for blockage by DAM methylation of the DNA [0126] The sequence encoding a let-7 sponge (SEQ ID NO: 10) is inserted into clinical vector of SEQ ID NO: 4 to produce a vector (Figure 23; SEQ ID NO: 6). The plasmid is then utilized to produce recombinant viral particles for ex vivo or in vivo transduction of human cells. Details of the vector:

Table 12

[0127] The sequence encoding LIN28B is inserted into clinical vector of SEQ ID NO: 4 to produce a vector (Figure 24; SEQ ID NO: 7). The plasmid is then utilized to produce recombinant viral particles for ex vivo or in vivo transduction of human cells.

[0128] Details of the vector:

Table 13

T U 2013/067811

37

Tthl l ll 1 5314

* Potential for blockage by DAM methylation of the DNA

EXAMPLE 9

[0129] This Example demonstrates LIN28A expression in sickle cell donor cells.

[0130] After obtaining consent and assent, CD34(+) cells from five pediatric research subjects with HbSS genotype (ages 9-16 years old) were harvested from discarded whole blood following partial manual exchange transfusions. Transgenic expression of LIN28A was accomplished using lentiviral transduction (using vector DNM-106 from Qiagen (cat. no. 337402)) of human CD34(+) sickle cells cultivated ex vivo in serum-free medium for a total of 21 days. On culture day 14, LIN28A over-expression (LIN28A-OE) was confirmed by Q-RT- PCR (control: non-transduced, no delivered Lin28A): 8.6E+00 ± 8.1E+00 copies/ng, LIN28A- OE: 2.3E+05 ± 2.1E+05 copies/ng) and Western blot analyses. Erythroblast differentiation and terminal maturation were not affected by LIN28 A-OE. Enucleation, as assessed by thiazole orange (TO) staining, was equivalent between the LIN28A-OE cells and control transductions (LIN28A-OE enucleation 40.8 ± 17.0% compared to control 49.9 ± 23.4%, p=0.19). LIN28A- OE strongly suppressed all members of the let-7 family of miRNAs, with average reductions from 66% to 96% for let-7a, let-7b, let-7c, let-7d, let-7e, let-7f-2, let-7g and let-7i. LIN28A-OE caused reduced expression of BCL11A, a known repressor of gamma-globin gene expression. Gamma-, beta (sickle)- and alpha-globin mRNA levels were also investigated by Q-RT-PCR. Gamma-globin mRNA expression levels were significantly increased in LIN28A-OE samples (control: 2.0E+06 ± 7.0E+05 copies/ng, LIN28A-OE: 2.0E+07 ± 6.0E+06 copies/ng, p=0.006), and beta (sickle)-globin mRNA significantly decreased in LIN28A-OE samples (control:

2.0E+07 ± 5.2E+06 copies/ng, LIN28A-OE: 1.6E+07 ± 6.3E+06 copies/ng, p=0.024).

Differences in alpha-globin mRNA expression were not statistically significant. Hemoglobin chromatography (HPLC) demonstrated that LIN28A-OE significantly increased the proportion of fetal hemoglobin (HbF control: 10.8 ± 7.1%, LIN28A-OE: 40.1 ± 14.0%; p=0.003) that was balanced by a significant decrease in the proportion of sickle hemoglobin. HbA was not detected. [0131] For investigation of the sickling phenotype, enucleated [TO(-)] sickle erythrocytes from LIN28A-OE and control transductions of two subjects' cells were sorted at the end of the culture period into duplicate tissue culture wells. The sorted erythrocytes were incubated in hypoxia (2% oxygen) for 16 hours, and imaged using inverted microscopy within three minutes after removal from the hypoxia incubator. Four random microscopic field images from each well were acquired. Blinded observers then scored the images from the control and LIN28A-OE transductions according to non-sickled versus sickled morphologies. Cultured erythrocytes from the control transductions demonstrated 86.3 ± 9.5% with sickled morphologies. By comparison, a significant reduction in sickling morphology was observed in the LIN28A-OE cells (56.2 ± 23.1% sickled morphologies; p=0.000009).

[0132] These results demonstrate that transgenic expression of LI 28A during ex vivo erythropoiesis causes increased gamma-globin gene and protein expression balanced with decreased beta (sickle)-globin at levels that are sufficient to ameliorate hypoxia-related sickling of mature erythrocytes.

[0133] 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.

[0134] 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. Also, everywhere "comprising" (or its equivalent) is recited, the "comprising" is considered to incorporate "consisting essentially of and "consisting of." Thus, an embodiment "comprising" (an) element(s) supports embodiments "consisting essentially of and "consisting of the recited element(s). Everywhere "consisting essentially of is recited is considered to incorporate "consisting of." Thus, an embodiment "consisting essentially of (an) element(s) supports embodiments "consisting of the recited element(s). 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.

[0135] 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 plasmid formed from the sequence of SEQ ID NO: 4, wherein the plasmid has a sequence encoding LIN28A protein, LIN28B protein, or let-7 sponge.

2. A plasmid comprising the sequence of SEQ ID NO: 5.

3. A plasmid comprising the sequence of SEQ ID NO: 6.

4. A plasmid comprising the sequence of SEQ ID NO: 7.

5. The plasmid of any of claims 1-4, further comprising sequence encoding an additional LIN28A protein, LIN28B protein, or let-7 sponge.

6. A method of increasing the amount of fetal hemoglobin in an erythroid cell, the method comprising administering to the erythroid cell an effective amount of a substance, the substance comprising or encoding the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof, wherein the administration of the substance increases the amount of fetal hemoglobin in the erythroid cell.

7. The method of claim 6, wherein the substance comprises the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof, and the substance is administered directly to the cell.

8. The method of claim 6, wherein the substance is a plasmid encoding the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof, wherein the plasmid is administered to the cell.

9. The method of claim 8, wherein the plasmid is a viral vector plasmid.

10. The method of claim 6, wherein the substance is viral DNA encoding the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof, wherein the viral DNA is administered to the cell through viral transduction.

11. The method of any one of claims 6-10, wherein the cell is in a mammal.

12. The method of claim 11, wherein the mammal is an adult mammal.

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

14. A method of decreasing the amount of adult hemoglobin in an erythroid cell, the method comprising administering to the erythroid cell an effective amount of a substance, the substance comprising or encoding the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof, wherein the administration of the substance decreases the amount of adult hemoglobin in the erythroid cell.

15. The method of claim 14, wherein the substance comprises the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof, and the substance is

administered directly to the cell.

16. The method of claim 14, wherein the substance is a plasmid encoding the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof, wherein the plasmid is administered to the cell.

17. The method of claim 16, wherein the plasmid is a viral vector plasmid.

18. The method of claim 14, wherein the substance is viral DNA encoding the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof, wherein the viral DNA is administered to the cell through viral transduction.

19. The method of any one of claims 14-18, wherein the cell is in a mammal.

20. The method of claim 19, wherein the mammal is an adult mammal.

21. The method of claim 19 or 20, wherein the mammal is a human.

22. A substance for use in preventing or treating sickle cell disease in a mammal, the substance comprising or encoding the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof.

23. The substance of claim 22, wherein the substance comprises the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof, and the substance is for direct administration to the mammal.

24. The substance of claim 22, wherein the substance is a plasmid encoding the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof, wherein the plasmid is for direct administration to the mammal.

25. The substance of claim 24, wherein the plasmid is a viral vector plasmid.

26. The substance of claim 22, wherein the substance is viral DNA encoding the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof, wherein the viral DNA is for administration to the mammal through viral transduction.

27. The substance of claim 22, wherein the substance is a plasmid encoding the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof, wherein the substance is for administering to an erythroid cell, and the erythroid cell is for administering to the mammal.

28. The substance of claim 22, wherein the substance is a plasmid encoding the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof, wherein the substance is for administering to an erythroid cell extracted from the mammal, and the erythroid cell is for administering to the mammal.

29. The substance of claim 27 or 28, wherein the plasmid is a viral vector plasmid.

30. The substance of claim 22, wherein the substance is viral DNA encoding the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof, wherein the substance is for administering to an erythroid cell through viral transduction, and the erythroid cell is for administering to the mammal.

31. The substance of claim 22, wherein the substance is viral DNA encoding the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof, wherein the substance is for administering through viral transduction to an erythroid cell extracted from the mammal, and the erythroid cell is for administering to the mammal.

32. The substance of any of claims 22-31 , wherein the mammal is an adult.

33. The substance of any of claims 22-32, wherein the mammal is a human.

34. A substance for preventing or treating a β-thalassemia in a mammal, the substance comprising or encoding the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof.

35. The substance of claim 34, wherein the substance comprises the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof, and the substance is for direct administration to the mammal.

36. The substance of claim 34, wherein the substance is a plasmid encoding the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof, wherein the plasmid is for direct administration to the mammal.

37. The substance of claim 36, wherein the plasmid is a viral vector plasmid.

38. The substance of claim 34, wherein the substance is viral DNA encoding the protein LI 28A, the protein LIN28B, a let-7 sponge, or any combination thereof, wherein the viral DNA is for administration to the mammal through viral transduction.

39. The substance of claim 34, wherein the substance is a plasmid encoding the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof, wherein the substance is for administering to an erythroid cell, and the erythroid cell is for administering to the mammal.

40. The substance of claim 34, wherein the substance is a plasmid encoding the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof, wherein the substance is for administering to an erythroid cell extracted from the mammal, and the erythroid cell is for administering to the mammal.

41. The substance of claim 39 or 40, wherein the plasmid is a viral vector plasmid.

42. The substance of claim 34, wherein the substance is viral DNA encoding the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof, wherein the substance is for administering to an erythroid cell through viral transduction, and the erythroid cell is for administering to the mammal.

43. The substance of claim 34, wherein the substance is viral DNA encoding the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof, wherein the substance is for administering through viral transduction to an erythroid cell extracted from the mammal, and the erythroid cell is for administering to the mammal.

44. The substance of any of claims 34-43, wherein the mammal is an adult.

45. The substance of any of claims 34-44, wherein the mammal is a human.

46. A method of preventing or treating sickle cell disease in a mammal, the method comprising administering to a mammal in need thereof an effective amount of a substance, the substance comprising or encoding the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof, wherein the administration of the substance prevents or treats sickle cell disease in the mammal.

47. The method of claim 46, wherein the substance comprises the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof, and the substance is

administered directly to the mammal.

48. The method of claim 46, wherein the substance is a plasmid encoding the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof, wherein the plasmid is administered to the mammal.

49. The method of claim 48, wherein the plasmid is a viral vector plasmid.

50. The method of claim 46, wherein the substance is viral DNA encoding the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof, wherein the viral DNA is administered to the mammal through viral transduction.

51. The method of claim 46, further comprising administering to an erythroid cell the substance, wherein the substance is a plasmid encoding the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof, and administering to the mammal the erythroid cell having the plasmid.

52. The method of claim 46, further comprising extracting from the mammal an erythroid cell, administering to the erythroid cell the substance, wherein the substance is a plasmid encoding the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof, and administering to the mammal the erythroid cell having the plasmid.

53. The method of claim 51 or 52, wherein the plasmid is a viral vector plasmid.

54. The method of claim 46, further comprising administering to an erythroid cell the substance through viral transduction, wherein the substance is viral DNA encoding the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof, and administering to the mammal the erythroid cell having the viral DNA.

55. The method of claim 46, further comprising extracting from the mammal an erythroid cell, administering to the erythroid cell the substance through viral transduction, wherein the substance is viral DNA encoding the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof, and administering to the mammal the erythroid cell having the viral DNA.

56. The method of any of claims 46-55, wherein the mammal is an adult.

57. The method of any of claims 46-56, wherein the mammal is a human.

58. A method of preventing or treating a β-thalassemia in a mammal, the method comprising administering to a mammal in need thereof an effective amount of a substance, the substance comprising or encoding the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof, wherein the administration of the substance prevents or treats the β- thalassemia in the mammal.

59. The method of claim 58, wherein the substance comprises the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof, and the substance is

administered directly to the mammal.

60. The method of claim 58, wherein the substance is a plasmid encoding the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof, wherein the plasmid is administered to the mammal.

61. The method of claim 60, wherein the plasmid is a viral vector plasmid.

62. The method of claim 58, wherein the substance is viral DNA encoding the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof, wherein the viral DNA is administered to the mammal through viral transduction.

63. The method of claim 58, further comprising administering to an erythroid cell the substance, wherein the substance is a plasmid encoding the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof, and administering to the mammal the erythroid cell having the plasmid.

64. The method of claim 58, further comprising extracting from the mammal an erythroid cell, administering to the erythroid cell the substance, wherein the substance is a plasmid encoding the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof, and administering to the mammal the erythroid cell having the plasmid.

65. The method of claim 63 or 64, wherein the plasmid is a viral vector plasmid.

66. The method of claim 58, further comprising administering to an erythroid cell the substance through viral transduction, wherein the substance is viral DNA encoding the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof, and administering to the mammal the erythroid cell having the viral DNA.

67. The method of claim 58, further comprising extracting from the mammal an erythroid cell, administering to the erythroid cell the substance through viral transduction, wherein the substance is viral DNA encoding the protein LIN28A, the protein LIN28B, a let-7 sponge, or any combination thereof, and administering to the mammal the erythroid cell having the viral DNA.

68. The method of any of claims 58-67, wherein the mammal is an adult.

69. The method of any of claims 58-68, wherein the mammal is a human.